Evaluation of anti-insulin receptor antibodies as potential novel therapies for human insulin receptoropathy using cell culture models

Abstract

Aims/hypothesis Bi-allelic loss-of-function mutations in the INSR gene (encoding the insulin receptor [INSR]) commonly cause extreme insulin resistance and early mortality. Therapeutic options are limited, but anti-INSR antibodies have been shown to activate two mutant receptors, S323L and F382V. This study evaluates four well-characterised murine anti-INSR monoclonal antibodies recognising distinct epitopes (83-7, 83-14, 18-44, 18-146) as surrogate agonists for potential targeted treatment of severe insulin resistance arising from insulin receptoropathies. Methods Ten naturally occurring mutant human INSRs with defects affecting different aspects of receptor function were modelled and assessed for response to insulin and anti-INSR antibodies. A novel 3T3-L1 adipocyte model of insulin receptoropathy was generated, permitting conditional knockdown of endogenous mouse Insr by lentiviral expression of species-specific short hairpin (sh)RNAs with simultaneous expression of human mutant INSR transgenes. Results All expressed mutant INSR bound to all antibodies tested. Eight mutants showed antibody-induced autophosphorylation, while co-treatment with antibody and insulin increased maximal phosphorylation compared with insulin alone. After knockdown of mouse Insr and expression of mutant INSR in 3T3-L1 adipocytes, two antibodies (83-7 and 83-14) activated signalling via protein kinase B (Akt) preferentially over signalling via extracellular signalregulated kinase 1/2 (ERK1/2) for seven mutants. These antibodies stimulated glucose uptake via P193L, S323L, F382V and D707A mutant INSRs, with antibody response greater than insulin response for D707A. Conclusions/interpretation Anti-INSR monoclonal antibodies can activate selected naturally occurring mutant human insulin receptors, bringing closer the prospect of novel therapy for severe insulin resistance caused by recessive mutations.

Achievements, prospects and challenges in precision care for monogenic insulin-deficient and insulin-resistant diabetes

“…Although rhIGF-1 has also occasionally been used in older patients with heterozygous INSR defects [63,65], the long-term risks and benefits of these agents are unclear, and they have no place in therapy outside clinical trials in this group. Reactivation of some mutant INSR using monoclonal antibodies has been tested in models [66,67], and has promise for the future, but remains experimental.…”

Anti-Insulin Receptor Antibodies Improve Hyperglycemia in a Mouse Model of Human Insulin Receptoropathy

“…Inhibitory INSR antibodies are now in phase 1 human trials ( 10 ), while stimulatory antibodies have been shown to ameliorate diabetes in rodents ( 11 – 13 ) and primates ( 14 ). Given the high clinical need in recessive insulin receptoropathy, we previously assessed the effect of monoclonal anti-INSR antibodies ( 15 – 19 ) on a series of disease-causing mutant INSRs in cell culture models, corroborating and extending prior findings by demonstrating an action of antibodies against a panel of mutant receptors ( 20 ).…”

“…We previously demonstrated the ability of bivalent, specific anti-INSR antibodies to act as surrogate ligands on a series of mutant INSR in cell culture models ( 20 ), and we now report their evaluation in vivo in a novel mouse model of human insulin receptoropathy. The humanized mouse model of insulin receptoropathy was generated by using sequential viral infection to knockout endogenous Insr and, then, to re-express human INSR .…”

“…Two INSR mutations, D734A and S350L, were selected for study based on prior evaluation in cell signaling assays ( 20 ). Both D734A ( 24 ) and S350L ( 25 ) mutations produce receptors that are normally processed and expressed at the cell surface but demonstrate severely reduced insulin binding and autophosphorylation.…”

“…Indeed, the INSR D734A mutation lies in the αCT segment of the extracellular domain of the INSR, which is a critical structural components of insulin-binding site 1, initially identified in biochemical studies ( 26 ). Importantly, both mutant INSRs have been shown to be activatable by anti-INSR antibodies ( 20 , 27 ).…”