Engineering Multivalent and Multispecific Protein Therapeutics

Liu, Cassie J.; Cochran, Jennifer R.

doi:10.1007/978-1-4471-4372-7_14

Cited by 7 publications

(12 citation statements)

References 77 publications

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“…Our previous studies have shown that 2.5F and 2.5F-Fc, which bind with high affinity to both mouse and human integrins, exhibit high selectivity for tumor versus healthy tissue in contrast to other integrin-targeting agents (11,13). It is possible that multispecific receptor targeting and the low level of avb3 and a5b1 integrins present on adult epithelial tissue compared with tumors imparts selectivity for diseased tissue, a concept that has been described in the literature for multispecific proteins (37).…”

Section: Discussionmentioning

confidence: 96%

Targeted Drug Delivery with an Integrin-Binding Knottin–Fc–MMAF Conjugate Produced by Cell-Free Protein Synthesis

Currier

Ackerman

Kintzing

et al. 2016

Molecular Cancer Therapeutics

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Antibody-drug conjugates (ADC) have generated significant interest as targeted therapeutics for cancer treatment, demonstrating improved clinical efficacy and safety compared with systemic chemotherapy. To extend this concept to other tumor-targeting proteins, we conjugated the tubulin inhibitor monomethyl-auristatin-F (MMAF) to 2.5F-Fc, a fusion protein composed of a human Fc domain and a cystine knot (knottin) miniprotein engineered to bind with high affinity to tumorassociated integrin receptors. The broad expression of integrins (including avb3, avb5, and a5b1) on tumor cells and their vasculature makes 2.5F-Fc an attractive tumor-targeting protein for drug delivery. We show that 2.5F-Fc can be expressed by cellfree protein synthesis, during which a non-natural amino acid was introduced into the Fc domain and subsequently used for site-specific conjugation of MMAF through a noncleavable linker. The resulting knottin-Fc-drug conjugate (KFDC), termed 2.5F-Fc-MMAF, had approximately 2 drugs attached per KFDC. 2.5F-Fc-MMAF inhibited proliferation in human glioblastoma (U87MG), ovarian (A2780), and breast (MB-468) cancer cells to a greater extent than 2.5F-Fc or MMAF alone or added in combination. As a single agent, 2.5F-Fc-MMAF was effective at inducing regression and prolonged survival in U87MG tumor xenograft models when administered at 10 mg/kg two times per week. In comparison, tumors treated with 2.5F-Fc or MMAF were nonresponsive, and treatment with a nontargeted control, CTRL-Fc-MMAF, showed a modest but not significant therapeutic effect. These studies provide proofof-concept for further development of KFDCs as alternatives to ADCs for tumor targeting and drug delivery applications.

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Section: Discussionmentioning

confidence: 96%

Targeted Drug Delivery with an Integrin-Binding Knottin–Fc–MMAF Conjugate Produced by Cell-Free Protein Synthesis

Currier

Ackerman

Kintzing

et al. 2016

Molecular Cancer Therapeutics

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“…Toward meeting this need, one promising concept for improved design of pharmaceuticals is engineered multivalency, which can be achieved through chemical biology and protein engineering approaches. Multivalency refers to the phenomenon by which a single molecule can be involved in multiple simultaneous molecular recognition events, which is hypothesized to enhance therapeutic efficacy by means of improved avidity, residence time, selectivity, and differential receptor trafficking. − The therapeutic potential of multivalent ligands against HER3 specifically is supported by the possibility of inducing HER3 sequestration by engaging HER3 into nonsignaling homotypic interactions. This phenomenon may be promoted by the tendency of HER3 to exist predominantly in homotypic clusters on the cell surface prior to ligand stimulation .…”

Section: Introductionmentioning

confidence: 99%

Engineered Multivalency Enhances Affibody-Based HER3 Inhibition and Downregulation in Cancer Cells

et al. 2017

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The receptor tyrosine kinase HER3 has emerged as a therapeutic target in ovarian, prostate, breast, lung, and other cancers due to its ability to potently activate the PI3K/Akt pathway, especially via dimerization with HER2, as well as for its role in mediating drug resistance. Enhanced efficacy of HER3-targeted therapeutics would therefore benefit a wide range of patients. This study evaluated the potential of multivalent presentation, through protein engineering, to enhance the effectiveness of HER3-targeted affibodies as alternatives to monoclonal antibody therapeutics. Assessment of multivalent affibodies on a variety of cancer cell lines revealed their broad ability to improve inhibition of Neuregulin (NRG)-induced HER3 and Akt phosphorylation compared to monovalent analogues. Engineered multivalency also promoted enhanced cancer cell growth inhibition by affibodies as single agents and as part of combination therapy approaches. Mechanistic * Corresponding Author: smjay@umd.edu. ORCID: Steven M. Jay: 0000-0002-3827-5988The authors declare the following competing financial interest(s): S.M.J. holds a patent related to multivalent ligand technology (US 9,029,328 B2). Supporting InformationThe Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharma-ceut. 6b00919. DNA and protein sequences for affibody constructs, purified affibody mass spectra, surface plasmon resonance data, and immunoblot quantification (PDF) Graphical Abstract HHS Public Access

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“…While we used scVEGFmut for affinity maturation studies, we could have alternatively started with our first‐generation dual‐specific variant scVEGF7I to arrive at a dual‐specific variant with high affinity against integrin and VEGFR2. Multi‐specific protein therapeutics have generated great interest in drug development as they offer opportunities for: (a) improved therapeutic efficacy, (b) lower/less frequent dosing, and (c) lower risk of systemic exposure and off‐target effects . In particular, targeting VEGFR2 and α V β 3 integrin using the high‐affinity dual‐specific angiogenesis inhibitor described in this work has the potential to reduce disease burden, and benefit patient subgroups that are either unresponsive or develop resistance to current therapies.…”

Section: Discussionmentioning

confidence: 99%

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

Kapur

Silverman

et al. 2017

Bioengineering & Transla Med

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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 αvβ3 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 αvβ3 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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Engineering Multivalent and Multispecific Protein Therapeutics

Cited by 7 publications

References 77 publications

Targeted Drug Delivery with an Integrin-Binding Knottin–Fc–MMAF Conjugate Produced by Cell-Free Protein Synthesis

Targeted Drug Delivery with an Integrin-Binding Knottin–Fc–MMAF Conjugate Produced by Cell-Free Protein Synthesis

Engineered Multivalency Enhances Affibody-Based HER3 Inhibition and Downregulation in Cancer Cells

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

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