Publications by authors named "Jennifer R Cochran"

85 Publications

LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation.

Nat Chem Biol 2021 Mar 25. Epub 2021 Mar 25.

Department of Chemistry and Stanford ChEM-H, Stanford University, Stanford, CA, USA.

Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome-targeting chimeras (LYTACs) targeted extracellular and membrane proteins for degradation by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosome-targeting receptor, to degrade extracellular proteins in a cell-type-specific manner. We conjugated binders to a triantenerrary N-acetylgalactosamine (tri-GalNAc) motif that engages ASGPR to drive the downregulation of proteins. Degradation of epidermal growth factor receptor (EGFR) by GalNAc-LYTAC attenuated EGFR signaling compared to inhibition with an antibody. Furthermore, we demonstrated that a LYTAC consisting of a 3.4-kDa peptide binder linked to a tri-GalNAc ligand degrades integrins and reduces cancer cell proliferation. Degradation with a single tri-GalNAc ligand prompted site-specific conjugation on antibody scaffolds, which improved the pharmacokinetic profile of GalNAc-LYTACs in vivo. GalNAc-LYTACs thus represent an avenue for cell-type-restricted protein degradation.
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http://dx.doi.org/10.1038/s41589-021-00770-1DOI Listing
March 2021

CD52 Is Elevated on B cells of SLE Patients and Regulates B Cell Function.

Front Immunol 2020 4;11:626820. Epub 2021 Feb 4.

VA Palo Alto Healthcare System, Palo Alto, CA, United States.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by B cell dysregulation and breaks in tolerance that lead to the production of pathogenic autoantibodies. We performed single-cell RNA sequencing of B cells from healthy donors and individuals with SLE which revealed upregulated CD52 expression in SLE patients. We further demonstrate that SLE patients exhibit significantly increased levels of B cell surface CD52 expression and plasma soluble CD52, and levels of soluble CD52 positively correlate with measures of lupus disease activity. Using CD52-deficient JeKo-1 cells, we show that cells lacking surface CD52 expression are hyperresponsive to B cell receptor (BCR) signaling, suggesting an inhibitory role for the surface-bound protein. In healthy donor B cells, antigen-specific BCR-activation initiated CD52 cleavage in a phospholipase C dependent manner, significantly reducing cell surface levels. Experiments with recombinant CD52-Fc showed that soluble CD52 inhibits BCR signaling in a manner partially-dependent on Siglec-10. Moreover, incubation of unstimulated B cells with CD52-Fc resulted in the reduction of surface immunoglobulin and CXCR5. Prolonged incubation of B cells with CD52 resulted in the expansion of IgD+IgM anergic B cells. In summary, our findings suggest that CD52 functions as a homeostatic protein on B cells, by inhibiting responses to BCR signaling. Further, our data demonstrate that CD52 is cleaved from the B cell surface upon antigen engagement, and can suppress B cell function in an autocrine and paracrine manner. We propose that increased expression of CD52 by B cells in SLE represents a homeostatic mechanism to suppress B cell hyperactivity.
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http://dx.doi.org/10.3389/fimmu.2020.626820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917337PMC
February 2021

Neutralizing antibodies targeting the SARS-CoV-2 receptor binding domain isolated from a naïve human antibody library.

Protein Sci 2021 04 24;30(4):716-727. Epub 2021 Feb 24.

Stanford ChEM-H, Stanford University, Stanford, California, USA.

Infection with SARS-CoV-2 elicits robust antibody responses in some patients, with a majority of the response directed at the receptor binding domain (RBD) of the spike surface glycoprotein. Remarkably, many patient-derived antibodies that potently inhibit viral infection harbor few to no mutations from the germline, suggesting that naïve antibody libraries are a viable means for discovery of novel SARS-CoV-2 neutralizing antibodies. Here, we used a yeast surface-display library of human naïve antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: one that blocks interaction with angiotensin-converting enzyme 2 (ACE2), the human receptor for SARS-CoV-2, and two that target other epitopes on the RBD. These three antibodies neutralized SARS-CoV-2 spike-pseudotyped lentivirus with IC values as low as 60 ng/ml in vitro. Using a biolayer interferometry-based binding competition assay, we determined that these antibodies have distinct but overlapping epitopes with antibodies elicited during natural COVID-19 infection. Taken together, these analyses highlight how in vitro selection of naïve antibodies can mimic the humoral response in vivo, yielding neutralizing antibodies and various epitopes that can be effectively targeted on the SARS-CoV-2 RBD.
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http://dx.doi.org/10.1002/pro.4044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980507PMC
April 2021

Identification of N-Terminally Diversified GLP-1R Agonists Using Saturation Mutagenesis and Chemical Design.

ACS Chem Biol 2021 01 14;16(1):58-66. Epub 2020 Dec 14.

Department of Bioengineering, Stanford University, Shriram Center, 443 Via Ortega, Stanford, California 94305, United States.

The glucagon-like peptide 1 receptor (GLP-1R) is a class B G-protein coupled receptor (GPCR) and diabetes drug target expressed mainly in pancreatic β-cells that, when activated by its agonist glucagon-like peptide 1 (GLP-1) after a meal, stimulates insulin secretion and β-cell survival and proliferation. The N-terminal region of GLP-1 interacts with membrane-proximal residues of GLP-1R, stabilizing its active conformation to trigger intracellular signaling. The best-studied agonist peptides, GLP-1 and exendin-4, share sequence homology at their N-terminal region; however, modifications that can be tolerated here are not fully understood. In this work, a functional screen of GLP-1 variants with randomized N-terminal domains reveals new GLP-1R agonists and uncovers a pattern whereby a negative charge is preferred at the third position in various sequence contexts. We further tested this sequence-structure-activity principle by synthesizing peptide analogues where this position was mutated to both canonical and noncanonical amino acids. We discovered a highly active GLP-1 analogue in which the native glutamate residue three positions from the N-terminus was replaced with the sulfo-containing amino acid cysteic acid (GLP-1-CYA). The receptor binding and downstream signaling properties elicited by GLP-1-CYA were similar to the wild type GLP-1 peptide. Computational modeling identified a likely mode of interaction of the negatively charged side chain in GLP-1-CYA with an arginine on GLP-1R. This work highlights a strategy of combinatorial peptide screening coupled with chemical exploration that could be used to generate novel agonists for other receptors with peptide ligands.
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http://dx.doi.org/10.1021/acschembio.0c00722DOI Listing
January 2021

Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome.

Sci Immunol 2020 12;5(54)

Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals' SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection.
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http://dx.doi.org/10.1126/sciimmunol.abe0240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857392PMC
December 2020

PET Reporter Gene Imaging and Ganciclovir-Mediated Ablation of Chimeric Antigen Receptor T Cells in Solid Tumors.

Cancer Res 2020 11 21;80(21):4731-4740. Epub 2020 Sep 21.

Department of Bioengineering, Stanford University School of Medicine, Stanford, California.

Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. In addition, the potential adverse effects of CAR T cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a PET reporter gene for imaging of T-cell trafficking in patients with brain tumor. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir. Here we report the molecular engineering, imaging, and ganciclovir-mediated destruction of B7H3 CAR T cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for ganciclovir. The sr39tk gene did not affect B7H3 CAR T-cell functionality and and studies in osteosarcoma models showed no significant effect on B7H3 CAR T-cell antitumor activity. PET/CT imaging with 9-(4-[F]-fluoro-3-[hydroxymethyl]butyl)guanine ([F]FHBG) of B7H3-sr39tk CAR T cells in an orthotopic model of osteosarcoma revealed tumor homing and systemic immune expansion. Bioluminescence and PET imaging of B7H3-sr39tk CAR T cells confirmed complete tumor ablation with intraperitoneal ganciclovir administration. This imaging and suicide ablation system can provide insight into CAR T-cell migration and proliferation during clinical trials while serving as a suicide switch to limit potential toxicities. SIGNIFICANCE: This study showcases the only genetically engineered system capable of serving the dual role both as an effective PET imaging reporter and as a suicide switch for CAR T cells.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-3579DOI Listing
November 2020

An engineered antibody binds a distinct epitope and is a potent inhibitor of murine and human VISTA.

Sci Rep 2020 09 16;10(1):15171. Epub 2020 Sep 16.

Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA.

V-domain immunoglobulin (Ig) suppressor of T cell activation (VISTA) is an immune checkpoint that maintains peripheral T cell quiescence and inhibits anti-tumor immune responses. VISTA functions by dampening the interaction between myeloid cells and T cells, orthogonal to PD-1 and other checkpoints of the tumor-T cell signaling axis. Here, we report the use of yeast surface display to engineer an anti-VISTA antibody that binds with high affinity to mouse, human, and cynomolgus monkey VISTA. Our anti-VISTA antibody (SG7) inhibits VISTA function and blocks purported interactions with both PSGL-1 and VSIG3 proteins. SG7 binds a unique epitope on the surface of VISTA, which partially overlaps with other clinically relevant antibodies. As a monotherapy, and to a greater extent as a combination with anti-PD1, SG7 slows tumor growth in multiple syngeneic mouse models. SG7 is a promising clinical candidate that can be tested in fully immunocompetent mouse models and its binding epitope can be used for future campaigns to develop species cross-reactive inhibitors of VISTA.
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http://dx.doi.org/10.1038/s41598-020-71519-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494997PMC
September 2020

SARS-CoV-2 Antibody Responses Correlate with Resolution of RNAemia But Are Short-Lived in Patients with Mild Illness.

medRxiv 2020 Aug 17. Epub 2020 Aug 17.

SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, could offer protective immunity, and may affect clinical outcomes of COVID-19 patients. We analyzed 625 serial plasma samples from 40 hospitalized COVID-19 patients and 170 SARS-CoV-2-infected outpatients and asymptomatic individuals. Severely ill patients developed significantly higher SARS-CoV-2-specific antibody responses than outpatients and asymptomatic individuals. The development of plasma antibodies was correlated with decreases in viral RNAemia, consistent with potential humoral immune clearance of virus. Using a novel competition ELISA, we detected antibodies blocking RBD-ACE2 interactions in 68% of inpatients and 40% of outpatients tested. Cross-reactive antibodies recognizing SARS-CoV RBD were found almost exclusively in hospitalized patients. Outpatient and asymptomatic individuals' serological responses to SARS-CoV-2 decreased within 2 months, suggesting that humoral protection may be short-lived.
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http://dx.doi.org/10.1101/2020.08.15.20175794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7444305PMC
August 2020

Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals.

Nat Methods 2020 08 13;17(8):852-860. Epub 2020 Jul 13.

Department of Neurobiology, Stanford University, Stanford, CA, USA.

Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.
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http://dx.doi.org/10.1038/s41592-020-0889-6DOI Listing
August 2020

Engineering a potent receptor superagonist or antagonist from a novel IL-6 family cytokine ligand.

Proc Natl Acad Sci U S A 2020 06 10;117(25):14110-14118. Epub 2020 Jun 10.

Department of Bioengineering, Stanford University, Stanford, CA 94305;

Interleukin-6 (IL-6) family cytokines signal through multimeric receptor complexes, providing unique opportunities to create novel ligand-based therapeutics. The cardiotrophin-like cytokine factor 1 (CLCF1) ligand has been shown to play a role in cancer, osteoporosis, and atherosclerosis. Once bound to ciliary neurotrophic factor receptor (CNTFR), CLCF1 mediates interactions to coreceptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). By increasing CNTFR-mediated binding to these coreceptors we generated a receptor superagonist which surpassed the potency of natural CNTFR ligands in neuronal signaling. Through additional mutations, we generated a receptor antagonist with increased binding to CNTFR but lack of binding to the coreceptors that inhibited tumor progression in murine xenograft models of nonsmall cell lung cancer. These studies further validate the CLCF1-CNTFR signaling axis as a therapeutic target and highlight an approach of engineering cytokine activity through a small number of mutations.
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http://dx.doi.org/10.1073/pnas.1922729117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322068PMC
June 2020

Multi-phase catheter-injectable hydrogel enables dual-stage protein-engineered cytokine release to mitigate adverse left ventricular remodeling following myocardial infarction in a small animal model and a large animal model.

Cytokine 2020 03 21;127:154974. Epub 2020 Jan 21.

Department of Bioengineering, Stanford University, Stanford, CA 94305, United States; Department of Cardiothoracic Surgery, Stanford University, Stanford, CA 94304, United States. Electronic address:

Although ischemic heart disease is the leading cause of death worldwide, mainstay treatments ultimately fail because they do not adequately address disease pathophysiology. Restoring the microvascular perfusion deficit remains a significant unmet need and may be addressed via delivery of pro-angiogenic cytokines. The therapeutic effect of cytokines can be enhanced by encapsulation within hydrogels, but current hydrogels do not offer sufficient clinical translatability due to unfavorable viscoelastic mechanical behavior which directly impacts the ability for minimally-invasive catheter delivery. In this report, we examine the therapeutic implications of dual-stage cytokine release from a novel, highly shear-thinning biocompatible catheter-deliverable hydrogel. We chose to encapsulate two protein-engineered cytokines, namely dimeric fragment of hepatocyte growth factor (HGFdf) and engineered stromal cell-derived factor 1α (ESA), which target distinct disease pathways. The controlled release of HGFdf and ESA from separate phases of the hyaluronic acid-based hydrogel allows extended and pronounced beneficial effects due to the precise timing of release. We evaluated the therapeutic efficacy of this treatment strategy in a small animal model of myocardial ischemia and observed a significant benefit in biological and functional parameters. Given the encouraging results from the small animal experiment, we translated this treatment to a large animal preclinical model and observed a reduction in scar size, indicating this strategy could serve as a potential adjunct therapy for the millions of people suffering from ischemic heart disease.
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http://dx.doi.org/10.1016/j.cyto.2019.154974DOI Listing
March 2020

Antitumor activity of an engineered decoy receptor targeting CLCF1-CNTFR signaling in lung adenocarcinoma.

Nat Med 2019 11 7;25(11):1783-1795. Epub 2019 Nov 7.

Division of Hematology and Oncology, Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA.

Proinflammatory cytokines in the tumor microenvironment can promote tumor growth, yet their value as therapeutic targets remains underexploited. We validated the functional significance of the cardiotrophin-like cytokine factor 1 (CLCF1)-ciliary neurotrophic factor receptor (CNTFR) signaling axis in lung adenocarcinoma (LUAD) and generated a high-affinity soluble receptor (eCNTFR-Fc) that sequesters CLCF1, thereby inhibiting its oncogenic effects. eCNTFR-Fc inhibits tumor growth in multiple xenograft models and in an autochthonous, highly aggressive genetically engineered mouse model of LUAD, driven by activation of oncogenic Kras and loss of Trp53. Abrogation of CLCF1 through eCNTFR-Fc appears most effective in tumors driven by oncogenic KRAS. We observed a correlation between the effectiveness of eCNTFR-Fc and the presence of KRAS mutations that retain the intrinsic capacity to hydrolyze guanosine triphosphate, suggesting that the mechanism of action may be related to altered guanosine triphosphate loading. Overall, we nominate blockade of CLCF1-CNTFR signaling as a novel therapeutic opportunity for LUAD and potentially for other tumor types in which CLCF1 is present in the tumor microenvironment.
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http://dx.doi.org/10.1038/s41591-019-0612-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7087454PMC
November 2019

Structure and Functional Binding Epitope of V-domain Ig Suppressor of T Cell Activation.

Cell Rep 2019 09;28(10):2509-2516.e5

Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA. Electronic address:

V-domain immunoglobulin (Ig) suppressor of T cell activation (VISTA) is an immune checkpoint protein that inhibits the T cell response against cancer. Similar to PD-1 and CTLA-4, a blockade of VISTA promotes tumor clearance by the immune system. Here, we report a 1.85 Å crystal structure of the elusive human VISTA extracellular domain, whose lack of homology necessitated a combinatorial MR-Rosetta approach for structure determination. We highlight features that make the VISTA immunoglobulin variable (IgV)-like fold unique among B7 family members, including two additional disulfide bonds and an extended loop region with an attached helix that we show forms a contiguous binding epitope for a clinically relevant anti-VISTA antibody. We propose an overlap of this antibody-binding region with the binding epitope for V-set and Ig domain containing 3 (VSIG3), a purported functional binding partner of VISTA. The structure and functional epitope presented here will help guide future drug development efforts against this important checkpoint target.
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http://dx.doi.org/10.1016/j.celrep.2019.07.073DOI Listing
September 2019

Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVβ3 and α5β1.

Structure 2019 09 25;27(9):1443-1451.e6. Epub 2019 Jul 25.

Leukocyte Biology and Inflammation Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; Structural Biology Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; Division of Nephrology/Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA. Electronic address:

Targeting both integrins αVβ3 and α5β1 simultaneously appears to be more effective in cancer therapy than targeting each one alone. The structural requirements for bispecific binding of ligand to integrins have not been fully elucidated. RGD-containing knottin 2.5F binds selectively to αVβ3 and α5β1, whereas knottin 2.5D is αVβ3 specific. To elucidate the structural basis of this selectivity, we determined the structures of 2.5F and 2.5D as apo proteins and in complex with αVβ3, and compared their interactions with integrins using molecular dynamics simulations. These studies show that 2.5D engages αVβ3 by an induced fit, but conformational selection of a flexible RGD loop accounts for high-affinity selective binding of 2.5F to both integrins. The contrasting binding of the highly flexible low-affinity linear RGD peptides to multiple integrins suggests that a "Goldilocks zone" of conformational flexibility of the RGD loop in 2.5F underlies its selective binding promiscuity to integrins.
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http://dx.doi.org/10.1016/j.str.2019.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726563PMC
September 2019

Engineering a potent inhibitor of matriptase from the natural hepatocyte growth factor activator inhibitor type-1 (HAI-1) protein.

J Biol Chem 2018 04 31;293(14):4969-4980. Epub 2018 Jan 31.

From the Departments of Bioengineering and

Dysregulated matriptase activity has been established as a key contributor to cancer progression through its activation of growth factors, including the hepatocyte growth factor (HGF). Despite its critical role and prevalence in many human cancers, limitations to developing an effective matriptase inhibitor include weak binding affinity, poor selectivity, and short circulating half-life. We applied rational and combinatorial approaches to engineer a potent inhibitor based on the hepatocyte growth factor activator inhibitor type-1 (HAI-1), a natural matriptase inhibitor. The first Kunitz domain (KD1) of HAI-1 has been well established as a minimal matriptase-binding and inhibition domain, whereas the second Kunitz domain (KD2) is inactive and involved in negative regulation. Here, we replaced the inactive KD2 domain of HAI-1 with an engineered chimeric variant of KD2/KD1 domains and fused the resulting construct to an antibody Fc domain to increase valency and circulating serum half-life. The final protein variant contains four stoichiometric binding sites that we showed were needed to effectively inhibit matriptase with a of 70 ± 5 pm, an increase of 120-fold compared with the natural HAI-1 inhibitor, to our knowledge making it one of the most potent matriptase inhibitors identified to date. Furthermore, the engineered inhibitor demonstrates a protease selectivity profile similar to that of wildtype KD1 but distinct from that of HAI-1. It also inhibits activation of the natural pro-HGF substrate and matriptase expressed on cancer cells with at least an order of magnitude greater efficacy than KD1.
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http://dx.doi.org/10.1074/jbc.M117.815142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892588PMC
April 2018

CAR T-cell immunotherapy of MET-expressing malignant mesothelioma.

Oncoimmunology 2017;6(12):e1363137. Epub 2017 Aug 14.

King's College London, Division of Cancer Studies, Guy's Hospital, Great Maze Pond, London SE1 9RT, UK.

Mesothelioma is an incurable cancer for which effective therapies are required. Aberrant MET expression is prevalent in mesothelioma, although targeting using small molecule-based therapeutics has proven disappointing. Chimeric antigen receptors (CARs) couple the HLA-independent binding of a cell surface target to the delivery of a tailored T-cell activating signal. Here, we evaluated the anti-tumor activity of MET re-targeted CAR T-cells against mesothelioma. Using immunohistochemistry, MET was detected in 67% of malignant pleural mesotheliomas, most frequently of epithelioid or biphasic subtype. The presence of MET did not influence patient survival. Candidate MET-specific CARs were engineered in which a CD28+CD3ζ endodomain was fused to one of 3 peptides derived from the N and K1 domains of hepatocyte growth factor (HGF), which represents the minimum MET binding element present in this growth factor. Using an NIH3T3-based artificial antigen-presenting cell system, we found that all 3 candidate CARs demonstrated high specificity for MET. By contrast, these CARs did not mediate T-cell activation upon engagement of other HGF binding partners, namely CD44v6 or heparan sulfate proteoglycans, including Syndecan-1. NK1-targeted CARs demonstrated broadly similar potency, indicated by destruction of MET-expressing mesothelioma cell lines, accompanied by cytokine release. anti-tumor activity was demonstrated following intraperitoneal delivery to mice with an established mesothelioma xenograft. Progressive tumor regression occurred without weight loss or other clinical indicators of toxicity. These data confirm the frequent expression of MET in malignant pleural mesothelioma and demonstrate that this can be targeted effectively and safely using a CAR T-cell immunotherapeutic strategy.
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http://dx.doi.org/10.1080/2162402X.2017.1363137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5706532PMC
August 2017

Development of a Protease Biosensor Based on a Dimerization-Dependent Red Fluorescent Protein.

ACS Chem Biol 2018 01 12;13(1):66-72. Epub 2017 Dec 12.

Department of Bioengineering, Stanford University , Stanford, California 94305, United States.

Dysregulated activity of the protease matriptase is a key contributor to aggressive tumor growth, cancer metastasis, and osteoarthritis. Methods for the detection and quantification of matriptase activity and inhibition would be useful tools. To address this need, we developed a matriptase-sensitive protein biosensor based on a dimerization-dependent red fluorescent protein (ddRFP) reporter system. In this platform, two adjoining protein domains, connected by a protease-labile linker, produce fluorescence when assembled and are nonfluorescent when the linker is cleaved by matriptase. A panel of ddRFP-based matriptase biosensor designs was created that contained different linker lengths between the protein domains. These constructs were characterized for linker-specific cleavage, matriptase activity, and matriptase selectivity; a biosensor containing a RSKLRVGGH linker (termed B4) was expressed at high yields and displayed both high catalytic efficiency and matriptase specificity. This biosensor detects matriptase inhibition by soluble and yeast cell surface expressed inhibitor domains with up to a 5-fold dynamic range and also detects matriptase activity expressed by human cancer cell lines. In addition to matriptase, we highlight a strategy that can be used to create effective biosensors for quantifying activity and inhibition of other proteases of interest.
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http://dx.doi.org/10.1021/acschembio.7b00715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453536PMC
January 2018

Heterochiral Knottin Protein: Folding and Solution Structure.

Biochemistry 2017 10 17;56(43):5720-5725. Epub 2017 Oct 17.

Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.

Homochirality is a general feature of biological macromolecules, and Nature includes few examples of heterochiral proteins. Herein, we report on the design, chemical synthesis, and structural characterization of heterochiral proteins possessing loops of amino acids of chirality opposite to that of the rest of a protein scaffold. Using the protein Ecballium elaterium trypsin inhibitor II, we discover that selective β-alanine substitution favors the efficient folding of our heterochiral constructs. Solution nuclear magnetic resonance spectroscopy of one such heterochiral protein reveals a homogeneous global fold. Additionally, steered molecular dynamics simulation indicate β-alanine reduces the free energy required to fold the protein. We also find these heterochiral proteins to be more resistant to proteolysis than homochiral l-proteins. This work informs the design of heterochiral protein architectures containing stretches of both d- and l-amino acids.
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http://dx.doi.org/10.1021/acs.biochem.7b00722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818992PMC
October 2017

High-throughput screening technologies for enzyme engineering.

Curr Opin Biotechnol 2017 12 15;48:196-202. Epub 2017 Jun 15.

Department of Bioengineering, Stanford University, United States; Department of Chemical Engineering, Stanford University, United States. Electronic address:

Emerging technologies are enabling ultra-high-throughput screening of combinatorial enzyme libraries to identify variants with improved properties such as increased activity, altered substrate specificity, and increased stability. Each of these enzyme engineering platforms relies on compartmentalization of reaction components, similar to microtiter plate-based assays which have been commonly used for testing the activity of enzyme variants. The technologies can be broadly divided into three categories according to their spatial segregation strategy: (1) cells as reaction compartments, (2) in vitro compartmentalization via synthetic droplets, and (3) microchambers. Here, we discuss these emerging platforms, which in some cases enable the screening of greater than 10 million enzyme variants, and highlight benefits and limitations of each technology.
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http://dx.doi.org/10.1016/j.copbio.2017.05.012DOI Listing
December 2017

A novel protein-engineered hepatocyte growth factor analog released via a shear-thinning injectable hydrogel enhances post-infarction ventricular function.

Biotechnol Bioeng 2017 10 29;114(10):2379-2389. Epub 2017 Jun 29.

Department of Cardiothoracic Surgery, Stanford University, Stanford, California, 94305.

In the last decade, numerous growth factors and biomaterials have been explored for the treatment of myocardial infarction (MI). While pre-clinical studies have demonstrated promising results, clinical trials have been disappointing and inconsistent, likely due to poor translatability. In the present study, we investigate a potential myocardial regenerative therapy consisting of a protein-engineered dimeric fragment of hepatocyte growth factor (HGFdf) encapsulated in a shear-thinning, self-healing, bioengineered hydrogel (SHIELD). We hypothesized that SHIELD would facilitate targeted, sustained intramyocardial delivery of HGFdf thereby attenuating myocardial injury and post-infarction remodeling. Adult male Wistar rats (n = 45) underwent sham surgery or induction of MI followed by injection of phosphate buffered saline (PBS), 10 μg HGFdf alone, SHIELD alone, or SHIELD encapsulating 10 μg HGFdf. Ventricular function, infarct size, and angiogenic response were assessed 4 weeks post-infarction. Treatment with SHIELD + HGFdf significantly reduced infarct size and increased both ejection fraction and borderzone arteriole density compared to the controls. Thus, sustained delivery of HGFdf via SHIELD limits post-infarction adverse ventricular remodeling by increasing angiogenesis and reducing fibrosis. Encapsulation of HGFdf in SHIELD improves clinical translatability by enabling minimally-invasive delivery and subsequent retention and sustained administration of this novel, potent angiogenic protein analog. Biotechnol. Bioeng. 2017;114: 2379-2389. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/bit.26345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947314PMC
October 2017

Engineered ligand-based VEGFR antagonists with increased receptor binding affinity more effectively inhibit angiogenesis.

Bioeng Transl Med 2017 03 17;2(1):81-91. Epub 2017 Feb 17.

Dept. of Bioengineering Stanford University Stanford CA 94303.

Pathologic angiogenesis is mediated by the coordinated action of the vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling axis, along with crosstalk contributed by other receptors, notably αβ integrin. We build on earlier work demonstrating that point mutations can be introduced into the homodimeric VEGF ligand to convert it into an antagonist through disruption of binding to one copy of VEGFR2. This inhibitor has limited potency, however, due to loss of avidity effects from bivalent VEGFR2 binding. Here, we used yeast surface display to engineer a variant with VEGFR2 binding affinity approximately 40-fold higher than the parental antagonist, and 14-fold higher than the natural bivalent VEGF ligand. Increased VEGFR2 binding affinity correlated with the ability to more effectively inhibit VEGF-mediated signaling, both in vitro and in vivo, as measured using VEGFR2 phosphorylation and Matrigel implantation assays. High affinity mutations found in this variant were then incorporated into a dual-specific antagonist that we previously designed to simultaneously bind to and inhibit VEGFR2 and αβ integrin. The resulting dual-specific protein bound to human and murine endothelial cells with relative affinities of 120 ± 10 pM and 360 ± 50 pM, respectively, which is at least 30-fold tighter than wild-type VEGF (3.8 ± 0.5 nM). Finally, we demonstrated that this engineered high-affinity dual-specific protein could inhibit angiogenesis in a murine corneal neovascularization model. Taken together, these data indicate that protein engineering strategies can be combined to generate unique antiangiogenic candidates for further clinical development.
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http://dx.doi.org/10.1002/btm2.10051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412928PMC
March 2017

Integrin-targeted cancer immunotherapy elicits protective adaptive immune responses.

J Exp Med 2017 06 4;214(6):1679-1690. Epub 2017 May 4.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139

Certain RGD-binding integrins are required for cell adhesion, migration, and proliferation and are overexpressed in most tumors, making them attractive therapeutic targets. However, multiple integrin antagonist drug candidates have failed to show efficacy in cancer clinical trials. In this work, we instead exploit these integrins as a target for antibody Fc effector functions in the context of cancer immunotherapy. By combining administration of an engineered mouse serum albumin/IL-2 fusion with an Fc fusion to an integrin-binding peptide (2.5F-Fc), significant survival improvements are achieved in three syngeneic mouse tumor models, including complete responses with protective immunity. Functional integrin antagonism does not contribute significantly to efficacy; rather, this therapy recruits both an innate and adaptive immune response, as deficiencies in either arm result in reduced tumor control. Administration of this integrin-targeted immunotherapy together with an anti-PD-1 antibody further improves responses and predominantly results in cures. Overall, this well-tolerated therapy achieves tumor specificity by redirecting inflammation to a functional target fundamental to tumorigenic processes but expressed at significantly lower levels in healthy tissues, and it shows promise for translation.
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http://dx.doi.org/10.1084/jem.20160831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460993PMC
June 2017

Targeting ligand-receptor interactions for development of cancer therapeutics.

Curr Opin Chem Biol 2017 Jun 31;38:62-69. Epub 2017 Mar 31.

Department of Bioengineering, Stanford University, United States; Department of Chemical Engineering, Stanford University, United States; Stanford Cancer Institute, United States. Electronic address:

The biological importance and druggable properties of receptors and their cognate ligands have designated them as especially useful clinical targets. This significance continues to expand as new molecular insights underlying disease pathophysiology are uncovered. While both ligands and receptors have been exploited as drug targets, their differing biochemical properties require nuanced considerations for drug development, including where in the body they are located and how they are regulated on a cellular and molecular level. In this review we will discuss ligands and receptors as therapeutics targets, including their biodistribution and biological function. We provide examples of monoclonal antibodies (mAbs) used to modulate the activity of these targets, and discuss approaches for using engineered versions of ligands and receptors themselves for therapeutic intervention in cancer.
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http://dx.doi.org/10.1016/j.cbpa.2017.03.010DOI Listing
June 2017

Measurements of translation initiation from all 64 codons in E. coli.

Nucleic Acids Res 2017 04;45(7):3615-3626

Joint Initiative for Metrology in Biology, Stanford, CA 94305, USA.

Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the 'start codons' for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.
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http://dx.doi.org/10.1093/nar/gkx070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397182PMC
April 2017

Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions.

Biotechnol J 2017 May 20;12(5). Epub 2017 Apr 20.

Dept. of Bioengineering, Schools of Engineering and Medicine, Stanford University, Stanford, California, USA.

Yeast surface display, a well-established technology for protein analysis and engineering, involves expressing a protein of interest as a genetic fusion to either the N- or C-terminus of the yeast Aga2p mating protein. Historically, yeast-displayed protein variants are flanked by peptide epitope tags that enable flow cytometric measurement of construct expression using fluorescent primary or secondary antibodies. Here, we built upon this technology to develop a new yeast display strategy that comprises fusion of two different proteins to Aga2p, one to the N-terminus and one to the C-terminus. This approach allows an antibody fragment, ligand, or receptor to be directly coupled to expression of a fluorescent protein readout, eliminating the need for antibody-staining of epitope tags to quantify yeast protein expression levels. We show that this system simplifies quantification of protein-protein binding interactions measured on the yeast cell surface. Moreover, we show that this system facilitates co-expression of a bioconjugation enzyme and its corresponding peptide substrate on the same Aga2p construct, enabling enzyme expression and catalytic activity to be measured on the surface of yeast.
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http://dx.doi.org/10.1002/biot.201600696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708543PMC
May 2017

Engineering High Affinity Protein-Protein Interactions Using a High-Throughput Microcapillary Array Platform.

ACS Chem Biol 2017 02 20;12(2):336-341. Epub 2016 Dec 20.

Department of Bioengineering, ‡Institute for Stem Cell Biology and Regenerative Medicine, §Stanford Photonics Research Center, ∥Chemical Engineering, Stanford University , 450 Serra Mall, Stanford, California 94305, United States.

Affinity maturation of protein-protein interactions requires iterative rounds of protein library generation and high-throughput screening to identify variants that bind with increased affinity to a target of interest. We recently developed a multipurpose protein engineering platform, termed μSCALE (Microcapillary Single Cell Analysis and Laser Extraction). This technology enables high-throughput screening of libraries of millions of cell-expressing protein variants based on their binding properties or functional activity. Here, we demonstrate the first use of the μSCALE platform for affinity maturation of a protein-protein binding interaction. In this proof-of-concept study, we engineered an extracellular domain of the Axl receptor tyrosine kinase to bind tighter to its ligand Gas6. Within 2 weeks, two iterative rounds of library generation and screening resulted in engineered Axl variants with a 50-fold decrease in kinetic dissociation rate, highlighting the use of μSCALE as a new tool for directed evolution.
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http://dx.doi.org/10.1021/acschembio.6b00794DOI Listing
February 2017

Inhibition of the GAS6/AXL pathway augments the efficacy of chemotherapies.

J Clin Invest 2017 01 28;127(1):183-198. Epub 2016 Nov 28.

The AXL receptor and its activating ligand, growth arrest-specific 6 (GAS6), are important drivers of metastasis and therapeutic resistance in human cancers. Given the critical roles that GAS6 and AXL play in refractory disease, this signaling axis represents an attractive target for therapeutic intervention. However, the strong picomolar binding affinity between GAS6 and AXL and the promiscuity of small molecule inhibitors represent important challenges faced by current anti-AXL therapeutics. Here, we have addressed these obstacles by engineering a second-generation, high-affinity AXL decoy receptor with an apparent affinity of 93 femtomolar to GAS6. Our decoy receptor, MYD1-72, profoundly inhibited disease progression in aggressive preclinical models of human cancers and induced cell killing in leukemia cells. When directly compared with the most advanced anti-AXL small molecules in the clinic, MYD1-72 achieved superior antitumor efficacy while displaying no toxicity. Moreover, we uncovered a relationship between AXL and the cellular response to DNA damage whereby abrogation of AXL signaling leads to accumulation of the DNA-damage markers γH2AX, 53BP1, and RAD51. MYD1-72 exploited this relationship, leading to improvements upon the therapeutic index of current standard-of-care chemotherapies in preclinical models of advanced pancreatic and ovarian cancer.
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http://dx.doi.org/10.1172/JCI85610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5199716PMC
January 2017

Emerging Strategies for Developing Next-Generation Protein Therapeutics for Cancer Treatment.

Trends Pharmacol Sci 2016 12 9;37(12):993-1008. Epub 2016 Nov 9.

Department of Bioengineering, Stanford University, Stanford, CA, USA; Stanford Cancer Institute, Stanford, CA, USA; Department of Chemical Engineering, Stanford University, Stanford, CA, USA. Electronic address:

Protein-based therapeutics have been revolutionizing the oncology space since they first appeared in the clinic two decades ago. Unlike traditional small-molecule chemotherapeutics, protein biologics promote active targeting of cancer cells by binding to cell-surface receptors and other markers specifically associated with or overexpressed on tumors versus healthy tissue. While the first approved cancer biologics were monoclonal antibodies, the burgeoning field of protein engineering is spawning research on an expanded range of protein formats and modifications that allow tuning of properties such as target-binding affinity, serum half-life, stability, and immunogenicity. In this review we highlight some of these strategies and provide examples of modified and engineered proteins under development as preclinical and clinical-stage drug candidates for the treatment of cancer.
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http://dx.doi.org/10.1016/j.tips.2016.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6238641PMC
December 2016

Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses.

Nat Med 2016 12 24;22(12):1402-1410. Epub 2016 Oct 24.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.

Checkpoint blockade with antibodies specific for cytotoxic T lymphocyte-associated protein (CTLA)-4 or programmed cell death 1 (PDCD1; also known as PD-1) elicits durable tumor regression in metastatic cancer, but these dramatic responses are confined to a minority of patients. This suboptimal outcome is probably due in part to the complex network of immunosuppressive pathways present in advanced tumors, which are unlikely to be overcome by intervention at a single signaling checkpoint. Here we describe a combination immunotherapy that recruits a variety of innate and adaptive immune cells to eliminate large tumor burdens in syngeneic tumor models and a genetically engineered mouse model of melanoma; to our knowledge tumors of this size have not previously been curable by treatments relying on endogenous immunity. Maximal antitumor efficacy required four components: a tumor-antigen-targeting antibody, a recombinant interleukin-2 with an extended half-life, anti-PD-1 and a powerful T cell vaccine. Depletion experiments revealed that CD8 T cells, cross-presenting dendritic cells and several other innate immune cell subsets were required for tumor regression. Effective treatment induced infiltration of immune cells and production of inflammatory cytokines in the tumor, enhanced antibody-mediated tumor antigen uptake and promoted antigen spreading. These results demonstrate the capacity of an elicited endogenous immune response to destroy large, established tumors and elucidate essential characteristics of combination immunotherapies that are capable of curing a majority of tumors in experimental settings typically viewed as intractable.
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http://dx.doi.org/10.1038/nm.4200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5209798PMC
December 2016

Engineered knottin peptides as diagnostics, therapeutics, and drug delivery vehicles.

Curr Opin Chem Biol 2016 10 16;34:143-150. Epub 2016 Sep 16.

Department of Bioengineering, Stanford University, United States; Department of Chemical Engineering, Stanford University, United States. Electronic address:

Inhibitor cystine-knots, also known as knottins, are a structural family of ultra-stable peptides with diverse functions. Knottins and related backbone-cyclized peptides called cyclotides contain three disulfide bonds connected in a particular arrangement that endows these peptides with high thermal, proteolytic, and chemical stability. Knottins have gained interest as candidates for non-invasive molecular imaging and for drug development as they can possess the pharmacological properties of small molecules and the target affinity and selectively of protein biologics. Naturally occurring knottins are clinically approved for treating chronic pain and GI disorders. Combinatorial methods are being used to engineer knottins that can bind to other clinically relevant targets in cancer, and inflammatory and cardiac disease. This review details recent examples of engineered knottin peptides; their use as molecular imaging agents, therapeutics, and drug delivery vehicles; modifications that can be introduced to improve peptide folding and bioactivity; and future perspectives and challenges in the field.
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http://dx.doi.org/10.1016/j.cbpa.2016.08.022DOI Listing
October 2016