Publications by authors named "K Dane Wittrup"

189 Publications

High-throughput phenotypic screen and transcriptional analysis identify new compounds and targets for macrophage reprogramming.

Nat Commun 2021 02 3;12(1):773. Epub 2021 Feb 3.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA.

Macrophages are plastic and, in response to different local stimuli, can polarize toward multi-dimensional spectrum of phenotypes, including the pro-inflammatory M1-like and the anti-inflammatory M2-like states. Using a high-throughput phenotypic screen in a library of ~4000 FDA-approved drugs, bioactive compounds and natural products, we find ~300 compounds that potently activate primary human macrophages toward either M1-like or M2-like state, of which ~30 are capable of reprogramming M1-like macrophages toward M2-like state and another ~20 for the reverse repolarization. Transcriptional analyses of macrophages treated with 34 non-redundant compounds identify both shared and unique targets and pathways through which the tested compounds modulate macrophage activation. One M1-activating compound, thiostrepton, is able to reprogram tumor-associated macrophages toward M1-like state in mice, and exhibit potent anti-tumor activity. Our compound-screening results thus help to provide a valuable resource not only for studying the macrophage biology but also for developing therapeutics through modulating macrophage activation.
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http://dx.doi.org/10.1038/s41467-021-21066-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7858590PMC
February 2021

Therapy of Myeloid Leukemia using Novel Bispecific Fusion Proteins Targeting CD45 and Y-DOTA.

Mol Cancer Ther 2020 12 20;19(12):2575-2584. Epub 2020 Oct 20.

Swedish Cancer Institute, Seattle, Washington.

Pretargeted radioimmunotherapy (PRIT) has been investigated as a multi-step approach to decrease relapse and toxicity for high-risk acute myeloid leukemia (AML). Relevant factors including endogenous biotin and immunogenicity, however, have limited the use of PRIT with an anti-CD45 antibody streptavidin conjugate and radiolabeled DOTA-biotin. To overcome these limitations we designed anti-murine and anti-human CD45 bispecific antibody constructs using 30F11 and BC8 antibodies, respectively, combined with an anti-yttrium (Y)-DOTA single-chain variable fragment (C825) to capture a radiolabeled ligand. The bispecific construct targeting human CD45 (BC8-Fc-C825) had high uptake in leukemia HEL xenografts [7.8 ± 0.02% percent injected dose/gram of tissue (% ID/g)]. Therapy studies showed that 70% of mice with HEL human xenografts treated with BC8-Fc-C825 followed by 44.4 MBq (1,200 μCi) of Y-DOTA-biotin survived at least 170 days after therapy, while all nontreated controls required euthanasia because of tumor progression by day 32. High uptake at sites of leukemia (spleen and bone marrow) was also seen with 30F11-IgG1-C825 in a syngeneic disseminated SJL murine leukemia model (spleen, 9.0 ± 1.5% ID/g and bone marrow, 8.1 ± 1.2% ID/g), with minimal uptake in all other normal organs (<0.5% ID/g) at 24 hours after Y-DOTA injections. SJL leukemia mice treated with the bispecific 30F11-IgG1-C825 and 29.6 MBq (800 μCi) of Y-DOTA-biotin had a survival advantage compared with untreated leukemic mice (median, 43 vs. 30 days, respectively; < 0.0001). These data suggest bispecific antibody-mediated PRIT may be highly effective for leukemia therapy and translation to human studies.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718432PMC
December 2020

Pharmacokinetic tuning of protein-antigen fusions enhances the immunogenicity of T-cell vaccines.

Nat Biomed Eng 2020 06 1;4(6):636-648. Epub 2020 Jun 1.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.

The formulations of peptide-based antitumour vaccines being tested in clinical studies are generally associated with weak potency. Here, we show that pharmacokinetically tuning the responses of peptide vaccines by fusing the peptide epitopes to carrier proteins optimizes vaccine immunogenicity in mice. In particular, we show in immunized mice that the carrier protein transthyretin simultaneously optimizes three factors: efficient antigen uptake in draining lymphatics from the site of injection, protection of antigen payloads from proteolytic degradation and reduction of antigen presentation in uninflamed distal lymphoid organs. Optimizing these factors increases vaccine immunogenicity by up to 90-fold and maximizes the responses to viral antigens, tumour-associated antigens, oncofetal antigens and shared neoantigens. Protein-peptide epitope fusions represent a facile and generalizable strategy for enhancing the T-cell responses elicited by subunit vaccines.
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http://dx.doi.org/10.1038/s41551-020-0563-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7575059PMC
June 2020

Enhanced CAR-T cell activity against solid tumors by vaccine boosting through the chimeric receptor.

Science 2019 07;365(6449):162-168

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Chimeric antigen receptor-T cell (CAR-T) therapy has been effective in the treatment of hematologic malignancies, but it has shown limited efficacy against solid tumors. Here we demonstrate an approach to enhancing CAR-T function in solid tumors by directly vaccine-boosting donor cells through their chimeric receptor in vivo. We designed amphiphile CAR-T ligands (amph-ligands) that, upon injection, trafficked to lymph nodes and decorated the surfaces of antigen-presenting cells, thereby priming CAR-Ts in the native lymph node microenvironment. Amph-ligand boosting triggered massive CAR-T expansion, increased donor cell polyfunctionality, and enhanced antitumor efficacy in multiple immunocompetent mouse tumor models. We demonstrate two approaches to generalizing this strategy to any chimeric antigen receptor, enabling this simple non-human leukocyte antigen-restricted approach to enhanced CAR-T functionality to be applied to existing CAR-T designs.
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http://dx.doi.org/10.1126/science.aav8692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800571PMC
July 2019

Anchoring of intratumorally administered cytokines to collagen safely potentiates systemic cancer immunotherapy.

Sci Transl Med 2019 06;11(498)

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.

The clinical application of cytokine therapies for cancer treatment remains limited due to severe adverse reactions and insufficient therapeutic effects. Although cytokine localization by intratumoral administration could address both issues, the rapid escape of soluble cytokines from the tumor invariably subverts this effort. We find that intratumoral administration of a cytokine fused to the collagen-binding protein lumican prolongs local retention and markedly reduces systemic exposure. Combining local administration of lumican-cytokine fusions with systemic immunotherapies (tumor-targeting antibody, checkpoint blockade, cancer vaccine, or T cell therapy) improves efficacy without exacerbating toxicity in syngeneic tumor models and the / genetically engineered melanoma model. Curative abscopal effects on noncytokine-injected tumors were also observed as a result of a protective and systemic CD8 T cell response primed by local therapy. Cytokine collagen-anchoring constitutes a facile, tumor-agnostic strategy to safely potentiate otherwise marginally effective systemic immunotherapies.
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http://dx.doi.org/10.1126/scitranslmed.aaw2614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7811803PMC
June 2019

Combining the Specific Anti-MUC1 Antibody TAB004 and Lip-MSA-IL-2 Limits Pancreatic Cancer Progression in Immune Competent Murine Models of Pancreatic Ductal Adenocarcinoma.

Front Oncol 2019 30;9:330. Epub 2019 Apr 30.

Department of Biological Sciences, UNC Charlotte, Charlotte, NC, United States.

Immunotherapy regimens have shown success in subsets of cancer patients; however, their efficacy against pancreatic ductal adenocarcinoma (PDA) remain unclear. Previously, we demonstrated the potential of TAB004, a monoclonal antibody targeting the unique tumor-associated form of MUC1 (tMUC1) in the early detection of PDA. In this study, we evaluated the therapeutic benefit of combining the TAB004 antibody with Liposomal-MSA-IL-2 in immune competent and human MUC1 transgenic (MUC1.Tg) mouse models of PDA and investigated the associated immune responses. Treatment with TAB004 + Lip-MSA-IL-2 resulted in significantly improved survival and slower tumor growth compared to controls in MUC1.Tg mice bearing an orthotopic PDA.MUC1 tumor. Similarly, in the spontaneous model of PDA that expresses human MUC1, the combination treatment stalled the progression of pancreatic intraepithelial pre-neoplastic (PanIN) lesion to adenocarcinoma. Treatment with the combination elicited a robust systemic and tumor-specific immune response with (a) increased percentages of systemic and tumor infiltrated CD45+CD11b+ cells, (b) increased levels of myeloperoxidase (MPO), (c) increased antibody-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP), (d) decreased percentage of immune regulatory cells (CD8+CD69+ cells), and (e) reduced circulating levels of immunosuppressive tMUC1. We report that treatment with a novel antibody against tMUC1 in combination with a unique formulation of IL-2 can improve survival and lead to stable disease in appropriate models of PDA by reducing tumor-induced immune regulation and promoting recruitment of CD45+CD11b+ cells, thereby enhancing ADCC/ADCP.
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http://dx.doi.org/10.3389/fonc.2019.00330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503151PMC
April 2019

Order of administration of combination cytokine therapies can decouple toxicity from efficacy in syngeneic mouse tumor models.

Oncoimmunology 2019;8(5):e1558678. Epub 2019 Feb 19.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.

In combination cancer immunotherapies, consideration should be given to designing treatment schedules that harmonize with the immune system's natural timing. An efficacious temporally programmed combination therapy of extended half-life interleukin 2 (eIL2), tumor targeting antibody, and interferon (IFN) α was recently reported; however, tumor-ablative efficacy was associated with significant toxicity. In the current work, altering the order and timing of the three agents is shown to decouple toxicity from efficacy. Delaying the administration of eIL2 to be concurrent with or after IFNα eliminates toxicity without affecting efficacy in multiple syngeneic tumor models and mouse strains. The toxicity resulting from eIL2 administration before IFNα is dependent on multiple systemic inflammatory cytokines including IL6, IL10, IFNγ, and tumor necrosis factor α. Natural killer (NK) cells are the main cellular contributor to toxicity, but are not essential for tumor control in this system. When pre-conditioned with eIL2, splenic NK cells became hyper-activated and upregulate IFNα signaling proteins that cause an excessive, toxic response to subsequent IFNα exposure. This work illustrates an example where accounting for the temporal dynamics of the immune system in combination therapy treatment schedule can favorably decouple efficacy and toxicity.
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http://dx.doi.org/10.1080/2162402X.2018.1558678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492973PMC
February 2019

What, Why, Where, and When: Bringing Timing to Immuno-Oncology.

Trends Immunol 2019 01 10;40(1):12-21. Epub 2018 Dec 10.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address:

A plethora of new cancer immunotherapies are under clinical development individually and in combination for a wide variety of indications, but optimizing therapeutic outcomes will require precise consideration of timing in treatment schedule design. In this review, we summarize the current understanding of the temporal rhythms of the anticancer immune response. Lessons learned in preclinical and clinical studies begin to define a framework for incorporating duration and sequencing into immunotherapy. We also discuss key challenges and opportunities for translation of temporally programmed treatment schedules to the clinic, including alignment of immunological timescales in preclinical models and humans, and the use of current and emerging biomarkers.
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http://dx.doi.org/10.1016/j.it.2018.11.003DOI Listing
January 2019

A Raf-Competitive K-Ras Binder Can Fail to Functionally Antagonize Signaling.

Mol Cancer Ther 2018 08 2;17(8):1773-1780. Epub 2018 May 2.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.

Mutated in approximately 30% of human cancers, Ras GTPases are the most common drivers of oncogenesis and render tumors unresponsive to many standard therapies. Despite decades of research, no drugs directly targeting Ras are currently available. We have previously characterized a small protein antagonist of K-Ras, R11.1.6, and demonstrated its direct competition with Raf for Ras binding. Here we evaluate the effects of R11.1.6 on Ras signaling and cellular proliferation in a panel of human cancer cell lines. Through lentiviral transduction, we generated cell lines that constitutively or through induction with doxycycline express R11.1.6 or a control protein YW1 and show specific binding by R11.1.6 to endogenous Ras through microscopy and co-immunoprecipitation experiments. Genetically encoded intracellular expression of this high-affinity Ras antagonist, however, fails to measurably disrupt signaling through either the MAPK or PI3K pathway. Consistently, cellular proliferation was unaffected as well. To understand this lack of signaling inhibition, we quantified the number of molecules of R11.1.6 expressed by the inducible cell lines and developed a simple mathematical model describing the competitive binding of Ras by R11.1.6 and Raf. This model supports a potential mechanism for the lack of biological effects that we observed, suggesting stoichiometric and thermodynamic barriers that should be overcome in pharmacologic efforts to directly compete with downstream effector proteins localized to membranes at very high effective concentrations. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0645DOI Listing
August 2018

Artificial Anti-Tumor Opsonizing Proteins with Fibronectin Scaffolds Engineered for Specificity to Each of the Murine FcγR Types.

J Mol Biol 2018 06 25;430(12):1786-1798. Epub 2018 Apr 25.

Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, United States; Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, United States; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, United States. Electronic address:

We have engineered a panel of novel Fn3 scaffold-based proteins that bind with high specificity and affinity to each of the individual mouse Fcγ receptors (mFcγR). These binders were expressed as fusions to anti-tumor antigen single-chain antibodies and mouse serum albumin, creating opsonizing agents that invoke only a single mFcγR response rather than the broader activity of natural Fc isotypes, as well as all previously reported Fc mutants. This panel isolated the capability of each of the four mFcγRs to contribute to macrophage phagocytosis of opsonized tumor cells and in vivo tumor growth control with these monospecific opsonizing fusion proteins. All activating receptors (mFcγRI, mFcγRIII, and mFcγRIV) were capable of driving specific tumor cell phagocytosis to an equivalent extent, while mFcγRII, the inhibitory receptor, did not drive phagocytosis. Monospecific opsonizing fusion proteins that bound mFcγRI alone controlled tumor growth to an extent similar to the most active IgG2a murine isotype. As expected, binding to the inhibitory mFcγRII did not delay tumor growth, but unexpectedly, mFcγRIII also failed to control tumor growth. mFcγRIV exhibited detectable but lesser tumor-growth control leading to less overall survival compared to mFcγRI. Interestingly, in vivo macrophage depletion demonstrates their importance in tumor control with mFcγRIV engagement, but not with mFcγRI. This panel of monospecific mFcγR-binding proteins provides a toolkit for isolating the functional effects of each mFcγR in the context of an intact immune system.
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http://dx.doi.org/10.1016/j.jmb.2018.04.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984174PMC
June 2018

Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis.

Nat Commun 2018 04 13;9(1):1461. Epub 2018 Apr 13.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA, 02139, USA.

Chemokine receptors typically have multiple ligands. Consequently, treatment with a blocking antibody against a single chemokine is expected to be insufficient for efficacy. Here we show single-chain antibodies can be engineered for broad crossreactivity toward multiple human and mouse proinflammatory ELR CXC chemokines. The engineered molecules recognize functional epitopes of ELR CXC chemokines and inhibit neutrophil activation ex vivo. Furthermore, an albumin fusion of the most crossreactive single-chain antibody prevents and reverses inflammation in the K/BxN mouse model of arthritis. Thus, we report an approach for the molecular evolution and selection of broadly crossreactive antibodies towards a family of structurally related, yet sequence-diverse protein targets, with general implications for the development of novel therapeutics.
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http://dx.doi.org/10.1038/s41467-018-03687-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899157PMC
April 2018

Reduction of Nonspecificity Motifs in Synthetic Antibody Libraries.

J Mol Biol 2018 01 26;430(1):119-130. Epub 2017 Nov 26.

Department of Biological, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, 02142, MA, USA; Department of Chemical Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, 02142, MA, USA. Electronic address:

Successful antibody development requires both functional binding and desirable biophysical characteristics. In the current study, we analyze the causes of one hurdle to clinical development, off-target reactivity, or nonspecificity. We used a high-throughput nonspecificity assay to isolate panels of nonspecific antibodies from two synthetic single-chain variable fragment libraries expressed on the surface of yeast, identifying both individual amino acids and motifs within the complementarity-determining regions which contribute to the phenotype. We find enrichment of glycine, valine, and arginine as both individual amino acids and as a part of motifs, and additionally enrichment of motifs containing tryptophan. Insertion of any of these motifs into the complementarity-determining region H3 of a "clean" antibody increased its nonspecificity, with greatest increases in antibodies containing Trp or Val motifs. We next applied these rules to the creation of a synthetic diversity library based on natural frameworks with significantly decreased incorporation of such motifs and demonstrated its ability to isolate binders to a wide panel of antigens. This work both provides a greater understanding of the drivers of nonspecificity and provides design rules to increase efficiency in the isolation of antibodies with drug-like properties.
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http://dx.doi.org/10.1016/j.jmb.2017.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738262PMC
January 2018

CD38-bispecific antibody pretargeted radioimmunotherapy for multiple myeloma and other B-cell malignancies.

Blood 2018 02 20;131(6):611-620. Epub 2017 Nov 20.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA.

Pretargeted radioimmunotherapy (PRIT) has demonstrated remarkable efficacy targeting tumor antigens, but immunogenicity and endogenous biotin blocking may limit clinical translation. We describe a new PRIT approach for the treatment of multiple myeloma (MM) and other B-cell malignancies, for which we developed an anti-CD38-bispecific fusion protein that eliminates endogenous biotin interference and immunogenic elements. In murine xenograft models of MM and non-Hodgkin lymphoma (NHL), the CD38-bispecific construct demonstrated excellent blood clearance and tumor targeting. Dosimetry calculations showed a tumor-absorbed dose of 43.8 Gy per millicurie injected dose of Y, with tumor-to-normal organ dose ratios of 7:1 for liver and 15:1 for lung and kidney. In therapy studies, CD38-bispecific PRIT resulted in 100% complete remissions by day 12 in MM and NHL xenograft models, ultimately curing 80% of mice at optimal doses. In direct comparisons, efficacy of the CD38 bispecific proved equal or superior to streptavidin (SA)-biotin-based CD38-SA PRIT. Each approach cured at least 75% of mice at the highest radiation dose tested (1200 µCi), whereas at 600- and 1000-µCi doses, the bispecific outperformed the SA approach, curing 35% more mice overall ( < .004). The high efficacy of bispecific PRIT, combined with its reduced risk of immunogenicity and endogenous biotin interference, make the CD38 bispecific an attractive candidate for clinical translation. Critically, CD38 PRIT may benefit patients with unresponsive, high-risk disease because refractory disease typically retains radiation sensitivity. We posit that PRIT might not only prolong survival, but possibly cure MM and treatment-refractory NHL patients.
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http://dx.doi.org/10.1182/blood-2017-09-807610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805491PMC
February 2018

Nonspecificity in a nonimmune human scFv repertoire.

MAbs 2017 10 14;9(7):1029-1035. Epub 2017 Sep 14.

a Department of Biological Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology , Cambridge , MA , U.S.A.

Efforts to develop effective antibody therapeutics are frequently hampered by issues such as aggregation and nonspecificity, often only detected in late stages of the development process. In this study, we used a high throughput cross-reactivity assay to select nonspecific clones from a naïve human repertoire scFv library displayed on the surface of yeast. Most antibody families were de-enriched; however, the rarely expressed VH6 family was highly enriched among nonspecific clones, representing almost 90% of isolated clones. Mutational analysis of this family reveals a dominant role of CDRH2 in driving nonspecific binding. Homology modeling of a panel of VH6 antibodies shows a constrained β-sheet structure in CDRH2 that is not present in other families, potentially contributing to nonspecificity of the family. These findings confirm the common decision to exclude VH6 from synthetic antibody libraries, and support VH6 polyreactivity as a possible important role for the family in early ontogeny and cause for its overabundance in cases of some forms of autoimmunity.
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http://dx.doi.org/10.1080/19420862.2017.1356528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627588PMC
October 2017

Antitumor Antibodies Can Drive Therapeutic T Cell Responses.

Authors:
K Dane Wittrup

Trends Cancer 2017 09 29;3(9):615-620. Epub 2017 Jul 29.

Biological Engineering and Chemical Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address:

The classical view of therapeutic monoclonal antibodies (mAbs) against tumor-associated antigens (TAAs) is that their mechanism of action is dominated by signal blocking or the cytotoxicity of Fc-driven innate immune effector functions. We review here a mounting body of evidence that anti-TAA mAbs are capable of profoundly synergizing with T cell-directed immunotherapies such as checkpoint blockade and adoptive cell therapy. Two key components account for this synergy: (i) a self-vaccinal effect mediated by dendritic cells (DCs); and (ii) an inflammatory repolarization of the tumor microenvironment. Efficient exploitation of these mechanisms has tremendous therapeutic potential.
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http://dx.doi.org/10.1016/j.trecan.2017.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657573PMC
September 2017

Chaperone proteins as single component reagents to assess antibody nonspecificity.

MAbs 2017 10 26;9(7):1036-1040. Epub 2017 Jul 26.

a Department of Biological Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology , Cambridge , MA , USA.

Early stage assays that evaluate monoclonal antibody drug-like properties serve as valuable tools for selection of lead candidates. One liability for clinical development, off-target reactivity, is often assessed by binding to a mixture or panel of noncognate proteins. While robust, these mixes are often ill-defined, and can suffer from issues such as lot-to-lot variability. In this study, we discovered in immunoprecipitation experiments that certain chaperones are present in one of these mixtures;we then explored the use of recombinant chaperone proteins as well-characterized agents to predict antibody nonspecificity. Antibody binding to the heat shock proteins HSP70, HSP90, or trigger factor all served as predictors of cross-interaction propensity, with HSP90 providing the greatest ability to predict antibody clearance rates in mouse. Individual chaperone binding correlates surprisingly closely with binding to complex cell extracts, with the exception of a few "false negatives" (assuming a complex cell extract as the "true" value). As defined reagents, these chaperone reagents present advantages for high throughput assays of nonspecificity.
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http://dx.doi.org/10.1080/19420862.2017.1356529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627595PMC
October 2017

An engineered protein antagonist of K-Ras/B-Raf interaction.

Sci Rep 2017 07 19;7(1):5831. Epub 2017 Jul 19.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Ras is at the hub of signal transduction pathways controlling cell proliferation and survival. Its mutants, present in about 30% of human cancers, are major drivers of oncogenesis and render tumors unresponsive to standard therapies. Here we report the engineering of a protein scaffold for preferential binding to K-Ras G12D. This is the first reported inhibitor to achieve nanomolar affinity while exhibiting specificity for mutant over wild type (WT) K-Ras. Crystal structures of the protein R11.1.6 in complex with K-Ras WT and K-Ras G12D offer insight into the structural basis for specificity, highlighting differences in the switch I conformation as the major defining element in the higher affinity interaction. R11.1.6 directly blocks interaction with Raf and reduces signaling through the Raf/MEK/ERK pathway. Our results support greater consideration of the state of switch I and provide a novel tool to study Ras biology. Most importantly, this work makes an unprecedented contribution to Ras research in inhibitor development strategy by revealing details of a targetable binding surface. Unlike the polar interfaces found for Ras/effector interactions, the K-Ras/R11.1.6 complex reveals an extensive hydrophobic interface that can serve as a template to advance the development of high affinity, non-covalent inhibitors of K-Ras oncogenic mutants.
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http://dx.doi.org/10.1038/s41598-017-05889-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517481PMC
July 2017

Curative Multicycle Radioimmunotherapy Monitored by Quantitative SPECT/CT-Based Theranostics, Using Bispecific Antibody Pretargeting Strategy in Colorectal Cancer.

J Nucl Med 2017 11 13;58(11):1735-1742. Epub 2017 Jul 13.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York

Radioimmunotherapy of solid tumors using antibody-targeted radionuclides has been limited by low therapeutic indices (TIs). We recently reported a novel 3-step pretargeted radioimmunotherapy (PRIT) strategy based on a glycoprotein A33 (GPA33)-targeting bispecific antibody and a small-molecule radioactive hapten, a complex of Lu and -2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (Lu-DOTA-Bn), that leads to high TIs for radiosensitive tissues such as blood (TI = 73) and kidney (TI = 12). We tested our hypothesis that a fractionated anti-GPA33 DOTA-PRIT regimen calibrated to deliver a radiation absorbed dose to tumor of more than 100 Gy would lead to a high probability of tumor cure while being well tolerated by nude mice bearing subcutaneous GPA33-positive SW1222 xenografts. We treated groups of nude mice bearing 7-d-old SW1222 xenografts with a fractionated 3-cycle anti-GPA33 DOTA-PRIT regimen (total administered Lu-DOTA-Bn activity, 167 MBq/mouse; estimated radiation absorbed dose to tumor, 110 Gy). In randomly selected mice undergoing treatment, serial SPECT/CT imaging was used to monitor treatment response and calculate radiation absorbed doses to tumor. Necropsy was done on surviving animals 100-200 d after treatment to determine frequency of cure and assess select normal tissues for treatment-related histopathologies. Rapid exponential tumor progression was observed in control treatment groups (i.e., no treatment or Lu-DOTA-Bn only), leading to euthanasia due to excessive tumor burden, whereas 10 of 10 complete responses were observed for the DOTA-PRIT-treated animals within 30 d. Treatment was well tolerated, and 100% histologic cure was achieved in 9 of 9 assessable animals without detectable radiation damage to critical organs, including bone marrow and kidney. Radiation absorbed doses to tumor derived from SPECT/CT (102 Gy) and from biodistribution (110 Gy) agreed to within 6.9%. Of the total dose of approximately 100 Gy, the first dose contributes 30%, the second dose 60%, and the third dose 10%. In a GPA33-positive human colorectal cancer xenograft mouse model, we validated a SPECT/CT-based theranostic PRIT regimen that led to 100% complete responses and 100% cures without any treatment-related toxicities, based on high TIs for radiosensitive tissues. These studies support the view that anti-GPA33 DOTA-PRIT will be a potent radioimmunotherapy regimen for GPA33-positive colorectal cancer tumors in humans.
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http://dx.doi.org/10.2967/jnumed.117.193250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666642PMC
November 2017

Engineering Aglycosylated IgG Variants with Wild-Type or Improved Binding Affinity to Human Fc Gamma RIIA and Fc Gamma RIIIAs.

J Mol Biol 2017 08 8;429(16):2528-2541. Epub 2017 Jul 8.

Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, United States; Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, United States; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, United States. Electronic address:

The binding of human IgG1 to human Fc gamma receptors (hFcγRs) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 of the Fc, with deglycosylation resulting in a complete loss of hFcγR binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human FcγRII class of the low-affinity hFcγRs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFcγR engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with FcγRIIIA, as well as the human Fc gamma RII subset. We also assess the biophysical properties and serum half-life of the aglycosylated IgG variants to measure stability. Aglycosylated constructs N297D/S298T (DTT)-K326I/A327Y/L328G (IYG) and N297D/S298A-IYG optimally drove tumor cell phagocytosis. A mathematical model of phagocytosis suggests that hFcγRI and hFcγRIIIA dimers were the main drivers of phagocytosis. In vivo tumor control of B16F10 lung metastases further confirmed the variant DTT-IYG to be the best at restoring wild-type-like properties in prevention of lung metastases. While deuterium incorporation was similar across most of the protein, several peptides within the CH2 domain of DTT-IYG showed differential deuterium uptake in the peptide region of the FG loop as compared to the aglycosylated N297Q. Thus, in this study, we have found an aglycosylated variant that may effectively substitute for wild-type Fc. These aglycosylated variants have the potential to allow therapeutic antibodies to be produced in virtually any expression system and still maintain effector function.
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http://dx.doi.org/10.1016/j.jmb.2017.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584586PMC
August 2017

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins.

Nucleic Acids Res 2017 Jul;45(13):7602-7614

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.
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http://dx.doi.org/10.1093/nar/gkx546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570165PMC
July 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

Biopolymers codelivering engineered T cells and STING agonists can eliminate heterogeneous tumors.

J Clin Invest 2017 Jun 24;127(6):2176-2191. Epub 2017 Apr 24.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Therapies using T cells that are programmed to express chimeric antigen receptors (CAR T cells) consistently produce positive results in patients with hematologic malignancies. However, CAR T cell treatments are less effective in solid tumors for several reasons. First, lymphocytes do not efficiently target CAR T cells; second, solid tumors create an immunosuppressive microenvironment that inactivates T cell responses; and third, solid cancers are typified by phenotypic diversity and thus include cells that do not express proteins targeted by the engineered receptors, enabling the formation of escape variants that elude CAR T cell targeting. Here, we have tested implantable biopolymer devices that deliver CAR T cells directly to the surfaces of solid tumors, thereby exposing them to high concentrations of immune cells for a substantial time period. In immunocompetent orthotopic mouse models of pancreatic cancer and melanoma, we found that CAR T cells can migrate from biopolymer scaffolds and eradicate tumors more effectively than does systemic delivery of the same cells. We have also demonstrated that codelivery of stimulator of IFN genes (STING) agonists stimulates immune responses to eliminate tumor cells that are not recognized by the adoptively transferred lymphocytes. Thus, these devices may improve the effectiveness of CAR T cell therapy in solid tumors and help protect against the emergence of escape variants.
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http://dx.doi.org/10.1172/JCI87624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451231PMC
June 2017

Biophysical properties of the clinical-stage antibody landscape.

Proc Natl Acad Sci U S A 2017 01 17;114(5):944-949. Epub 2017 Jan 17.

Department of Computational Biology, Adimab LLC, Lebanon, NH 03766;

Antibodies are a highly successful class of biological drugs, with over 50 such molecules approved for therapeutic use and hundreds more currently in clinical development. Improvements in technology for the discovery and optimization of high-potency antibodies have greatly increased the chances for finding binding molecules with desired biological properties; however, achieving drug-like properties at the same time is an additional requirement that is receiving increased attention. In this work, we attempt to quantify the historical limits of acceptability for multiple biophysical metrics of "developability." Amino acid sequences from 137 antibodies in advanced clinical stages, including 48 approved for therapeutic use, were collected and used to construct isotype-matched IgG1 antibodies, which were then expressed in mammalian cells. The resulting material for each source antibody was evaluated in a dozen biophysical property assays. The distributions of the observed metrics are used to empirically define boundaries of drug-like behavior that can represent practical guidelines for future antibody drug candidates.
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http://dx.doi.org/10.1073/pnas.1616408114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293111PMC
January 2017

Purification of common light chain IgG-like bispecific antibodies using highly linear pH gradients.

MAbs 2017 Feb/Mar;9(2):257-268. Epub 2016 Dec 12.

a Department of High-Throughput Expression , Adimab LLC , Lebanon , NH , USA.

Monovalent bispecific antibodies (BsAbs) are projected to have broad clinical applications due to their ability to bind two different targets simultaneously. Although they can be produced using recombinant technologies, the correct pairing of heavy and light chains is a significant manufacturing problem. Various approaches exploit mutations or linkers to favor the formation of the desired BsAb, but a format using a single common light chain has the advantage that no other modification to the antibody is required. This strategy reduces the number of formed molecules to three (the BsAb and the two parent mAbs), but the separation of the BsAb from the two monovalent parent molecules still poses a potentially difficult purification challenge. Current methods employ ion exchange chromatography and linear salt gradients, but are only successful if the difference in the observed isoelectric points (pIs) of two parent molecules is relatively large. Here, we describe the use of highly linear pH gradients for the facile purification of common light chain BsAbs. The method is effective at separating molecules with differences in pI as little as 0.10, and differing in their sequence by only a single charged amino acid. We also demonstrate that purification resins validated for manufacturing are compatible with this approach.
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http://dx.doi.org/10.1080/19420862.2016.1267090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297495PMC
November 2017

Temporally Programmed CD8α DC Activation Enhances Combination Cancer Immunotherapy.

Cell Rep 2016 12;17(10):2503-2511

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address:

Numerous synergistic cancer immunotherapy combinations have been identified, but the effects of relative dose timing are rarely considered. In established syngeneic mouse tumor models, we found that staggering interferon-α (IFNα) administration after, rather than before or simultaneously with, serum-persistent interleukin-2 (IL-2) and tumor-specific antibody significantly increased long-term survival. Successful combination therapy required IFNα-induced activation of cross-presenting CD8α dendritic cells (DCs) following the release of antigenic tumor debris by the IL-2- and antibody-mediated immune response. Due to decreased phagocytic ability post-maturation, DCs activated too early captured less antigen and could not effectively prime CD8 T cells. Temporally programming DC activation to occur after tumoricidal activity enhanced tumor control by multiple distinct combination immunotherapies, highlighting dose schedule as an underappreciated factor that can profoundly affect the success of multi-component immunotherapies.
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http://dx.doi.org/10.1016/j.celrep.2016.11.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5204262PMC
December 2016

Comparative Analysis of Bispecific Antibody and Streptavidin-Targeted Radioimmunotherapy for B-cell Cancers.

Cancer Res 2016 11 2;76(22):6669-6679. Epub 2016 Sep 2.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington.

Streptavidin (SA)-biotin pretargeted radioimmunotherapy (PRIT) that targets CD20 in non-Hodgkin lymphoma (NHL) exhibits remarkable efficacy in model systems, but SA immunogenicity and interference by endogenous biotin may complicate clinical translation of this approach. In this study, we engineered a bispecific fusion protein (FP) that evades the limitations imposed by this system. Briefly, one arm of the FP was an anti-human CD20 antibody (2H7), with the other arm of the FP an anti-chelated radiometal trap for a radiolabeled ligand (yttrium[Y]-DOTA) captured by a very high-affinity anti-Y-DOTA scFv antibody (C825). Head-to-head biodistribution experiments comparing SA-biotin and bispecific FP (2H7-Fc-C825) PRIT in murine subjects bearing human lymphoma xenografts demonstrated nearly identical tumor targeting by each modality at 24 hours. However, residual radioactivity in the blood and normal organs was consistently higher following administration of 1F5-SA compared with 2H7-Fc-C825. Consequently, tumor-to-normal tissue ratios of distribution were superior for 2H7-Fc-C825 (P < 0.0001). Therapy studies in subjects bearing either Ramos or Granta subcutaneous lymphomas demonstrated that 2H7-Fc-C825 PRIT is highly effective and significantly less myelosuppressive than 1F5-SA (P < 0.0001). All animals receiving optimal doses of 2H7-Fc-C825 followed by Y-DOTA were cured by 150 days, whereas the growth of tumors in control animals progressed rapidly with complete morbidity by 25 days. In addition to demonstrating reduced risk of immunogenicity and an absence of endogenous biotin interference, our findings offer a preclinical proof of concept for the preferred use of bispecific PRIT in future clinical trials, due to a slightly superior biodistribution profile, less myelosuppression, and superior efficacy. Cancer Res; 76(22); 6669-79. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-16-0571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5290195PMC
November 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

Target-independent variable region mediated effects on antibody clearance can be FcRn independent.

MAbs 2016 10 9;8(7):1269-1275. Epub 2016 Sep 9.

a Department of Biological Engineering , Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology , Cambridge , MA , USA.

The importance of the neonatal Fc receptor (FcRn) in extending the serum half-life of monoclonal antibodies (mAbs) is well demonstrated, and has led to the development of multiple engineering approaches designed to alter Fc interactions with FcRn. Recent reports have additionally highlighted the effect of nonspecific interactions on antibody pharmacokinetics (PK), suggesting an FcRn-independent mechanism for mAb clearance. In this report we examine a case study of 2 anti-interleukin-12/23 antibodies, ustekinumab and briakinumab, which share the same target and Fc, but differ in variable region sequences. Ustekinumab displayed near baseline signal in a wide range of early stage developability assays for undesirable protein/protein interactions, while briakinumab showed significant propensity for self- and cross-interactions. This phenotypic difference correlates with faster clearance rates for briakinumab in both human FcRn transgenic and FcRn knockout mice. These findings support a dominant contribution for FcRn-independent clearance for antibodies with high nonspecificity, and highlight a key role for early stage developability screening to eliminate clones with such high nonspecific disposition PK.
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http://dx.doi.org/10.1080/19420862.2016.1208330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5058615PMC
October 2016

Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library.

J Biol Chem 2016 Oct 30;291(43):22496-22508. Epub 2016 Aug 30.

From the Koch Institute for Integrative Cancer Research and

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (T of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation.
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http://dx.doi.org/10.1074/jbc.M116.741314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5077188PMC
October 2016

Design Principles for SuCESsFul Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions.

J Mol Biol 2016 10 21;428(20):4228-4241. Epub 2016 Jul 21.

Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, 500 Main Street, Cambridge, MA 02139, USA; Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address:

Quantifying protein location and concentration is critical for understanding function in situ. Scaffold conjugated to environment-sensitive fluorophore (SuCESsFul) biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can, in principle, detect analytes of interest with high temporal and spatial resolution. However, their adoption has been limited due to the extensive empirical screening required for their development. We sought to establish design principles for this class of biosensor by characterizing over 400 biosensors based on various protein analytes, binding proteins, and fluorophores. We found that the brightest readouts are attained when a specific binding pocket for the fluorophore is present on the analyte. Also, interaction of the fluorophore with the binding protein it is conjugated to can raise background fluorescence, considerably limiting sensor dynamic range. Exploiting these two concepts, we designed biosensors that attain a 100-fold increase in fluorescence upon binding to analyte, an order of magnitude improvement over the previously best-reported SuCESsFul biosensor. These design principles should facilitate the development of improved SuCESsFul biosensors.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5048519PMC
http://dx.doi.org/10.1016/j.jmb.2016.07.004DOI Listing
October 2016