Cytokine Activation by Antibody Fragments Targeted to Cytokine-Receptor Signaling Complexes

Kuruganti, Srilalitha; Miersch, Shane; Deshpande, Ashlesha; Speir, Jeffrey A.; Harris, Bethany; Schriewer, Jill; Buller, R. Mark; Sidhu, Sachdev S.; Walter, Mark R.

doi:10.1074/jbc.m115.665943

Cited by 9 publications

(4 citation statements)

References 53 publications

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“…Recently, Kuruganti et al (32) selected antibody fragments that stabilize the IFN/interferon receptor 2 (IFNAR2) binary complex to potentiate IFN signaling. Following a selection scheme similar to those used for IL-4 stapler discovery (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1 A , center ) (31). Antibodies that potentiate cytokine action by enhancing cytokine–receptor binding have also been reported (32, 33). Here, we sought to develop an evolutionary strategy for the discovery of antibody-based constructs that simultaneously engage two interacting subunits within dimerized receptors, as they are disposed in their native signaling conformations induced by endogenous cytokines (Fig.…”

Section: Introductionmentioning

confidence: 99%

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A strategy for the selection of monovalent antibodies that span protein dimer interfaces

Spangler

Moraga

Jude

et al. 2019

Journal of Biological Chemistry

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Ligand-induced dimerization is the predominant mechanism through which secreted proteins activate cell surface receptors to transmit essential biological signals. Cytokines are a large class of soluble proteins that dimerize transmembrane receptors into precise signaling topologies, but there is a need for alternative, engineerable ligand scaffolds that specifically recognize and stabilize these protein interactions. Recombinant antibodies can potentially serve as robust and versatile platforms for cytokine complex stabilization, and their specificity allows for tunable modulation of dimerization equilibrium. Here, we devised an evolutionary strategy to isolate monovalent antibody fragments that bridge together two different receptor subunits in a cytokine–receptor complex, precisely as the receptors are disposed in their natural signaling orientations. To do this, we screened a naive antibody library against a stabilized ligand–receptor ternary complex that acted as a “molecular cast” of the natural receptor dimer conformation. Our selections elicited “stapler” single-chain variable fragments (scFvs) of antibodies that specifically engage the interleukin-4 receptor heterodimer. The 3.1 Å resolution crystal structure of one such stapler revealed that, as intended, this scFv recognizes a composite epitope between the two receptors as they are positioned in the complex. Extending our approach, we evolved a stapler scFv that specifically binds to and stabilizes the interface between the interleukin-2 cytokine and one of its receptor subunits, leading to a 15-fold enhancement in interaction affinity. This demonstration that scFvs can be selected to recognize epitopes that span protein interfaces presents new opportunities to engineer structurally defined antibodies for a broad range of research and therapeutic applications.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A strategy for the selection of monovalent antibodies that span protein dimer interfaces

Spangler

Moraga

Jude

et al. 2019

Journal of Biological Chemistry

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“…These computational tools have been used for large scale structural comparisons of naive and antigen‐experienced repertoires . Often, structure modeling tools are also used to design antibodies that target a specific epitope or to improve antigen binding of known antibodies . Moreover, several studies combined computational structure modeling and crystallographic approaches .…”

Section: Structure and Function Of Antibodiesmentioning

confidence: 99%

Assessing human B cell repertoire diversity and convergence

Imkeller

Wardemann

2018

Immunological Reviews

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A hallmark of the adaptive immune system is the specificity of B cell and T cell responses. Mechanistically, this feature relies on the fact that the two genes that encode B cell and T cell antigen receptors are not germline encoded and instead are assembled from a large number of small gene segments during lymphocyte development. The underlying somatic gene recombination process can generate a quasi-unlimited repertoire of antigen receptors. The high degree of diversity is essential to guarantee recognition of nearly any antigenic structure to protect from the large variety of potential invading pathogens and to keep the balance with commensals. Due to the enormous complexity of the antigen receptor repertoire, our understanding of its actual size and functional convergence at the level of the individual and the population is still limited. A better understanding of the actual degree of diversity could help to predict adaptive immune responses and would have wide implications for the development of preventive and therapeutic measures against infectious and autoimmune diseases as well as cancer. Here, we discuss the recent advances in the field with a specific focus on B cells and the function of antibodies.

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“…For binding selections against each of the 12 IFNα subtypes, we used library F, a phage-displayed library of synthetic antigenbinding fragments (Fabs) that has been described previously (Persson et al, 2013), and has yielded tight and specific Fabs for many antigens (Hornsby et al, 2015;Huang et al, 2015;Kuruganti et al, 2016;Na et al, 2016). Library F was built using a highly stable human framework by incorporating optimized diversity within the three heavy chain complementarity determining regions (CDR-H1, -H2, and -H3) and the third light chain CDR (CDR-L3).…”

Section: Isolation and Characterization Of Phage-displayed Anti-ifnα Antibodiesmentioning

confidence: 99%

A panel of synthetic antibodies that selectively recognize and antagonize members of the interferon alpha family

Miersch

Kuruganti

Walter

et al. 2017

Protein Engineering, Design and Selection

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The 12 distinct subtypes that comprise the interferon alpha (IFNα) family of cytokines possess anti-viral, anti-proliferative and immunomodulatory activities. They are implicated in the etiology and progression of many diseases, and also used as therapeutic agents for viral and oncologic disorders. However, a deeper understanding of their role in disease is limited by a lack of tools to evaluate single subtypes at the protein level. Antibodies that selectively inhibit single IFNα subtypes could enable interrogation of each protein in biological samples and could be used for characterization and treatment of disease. Using phage-displayed synthetic antibody libraries, we have conducted selections against 12 human IFNα subtypes to explore our ability to obtain fine-specificity antibodies that recognize and antagonize the biological signals induced by a single IFNα subtype. For the first time, we have isolated antibodies that specifically recognize individual IFNα subtypes (IFNα2a/b, IFNα6, IFNα8b and IFNα16) with high affinity that antagonize signaling. Our results show that highly specific antibodies capable of distinguishing between closely related cytokines can be isolated from synthetic libraries and can be used to characterize cytokine abundance and function.

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Cytokine Activation by Antibody Fragments Targeted to Cytokine-Receptor Signaling Complexes

Cited by 9 publications

References 53 publications

A strategy for the selection of monovalent antibodies that span protein dimer interfaces

A strategy for the selection of monovalent antibodies that span protein dimer interfaces

Assessing human B cell repertoire diversity and convergence

A panel of synthetic antibodies that selectively recognize and antagonize members of the interferon alpha family

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