Targeting A2A receptor in autism: single domain antibodies for direct inhibition or antagonist vectorization – A2utismAb

Among adenosine receptors, the A2A receptor (A2AR) is a G-protein coupled receptor (GPCR) that receives raising attention as a therapeutic target in multiple pathologies. Brain A2AR expression is quasi exclusively striatal, and A2AR anatgonists, as istradefylline, inhibit the activity of striatal neurons expressing the dopamine D2 receptors (D2-SN, direct pathway) with beneficial effects in Parkinson Disease. Based on our previous work and preliminary data, we have hypothesized that repressing D2-SN activity would also relieve symptoms of autism. Indeed, we have verified that istradefylline alleviates autistic-like symptoms in three mouse models of autism.
Clinical use of istradefylline is impeded by its side effects, due to lack of specificity towards different subtypes of adenosine receptors and diffusion in the vascular and immune systems where A2AR is highly expressed. Such limitation exists for all small chemical molecules targeting GPCRs, but could be circumvented by using single domain antibody (SdAb) directed against the GPCR of interest. SdAbs are stable, soluble, small biomolecules (proteins) characterized by their high affinity for their target, which activity they can eventually modulate. Such SdAbs can thus be used directly for their pharmacological action at the target; they can also be conjugated to a chemical molecule. The obtained bioconjugate (antibody drug conjugate: ADC) will then ally the pharmacological properties of the chemical molecule to the high affinity and selectivity of the SdAb, either for the target or for a particular tissue, allowing a better efficacy and a reduction of side effects: these are the principles of vectorization.
We have previously identified an SdAb, DRA2-01, behaving as an antagonist of A2AR in mice. Central administration of this SdAb mimics, but in a long-lasting manner, the beneficial effects of istradefylline in a mouse model of autism. DRA2-01, however, facilitates the activity of the human version of A2AR, making it unsuitable for clinical use. The objective of our project is thus to develop novel antibody fragments or bioconjugates able to durably and specifically inhibit A2AR activity in the striatum, a key region for autism etiopathology, to treat this disease. To this aim, we will 1) identify and characterize novel SbABs inhibiting human A2AR in the brain, with expected carry-over effects, and 2) conjugate an SdAb targeting GPR88, a central GPCR highly expressed in the striatum, with istradephylline or an SdAB with A2AR antagonist activity, for a vectorized effect in the striatum.
As regards the first objective, we have previously identified 12 SdAbs binding the human version of A2AR and we will now characterize their pharmacological profile in vitro. As concerns the second part of the project, we have previously selected SbAbs targeting GPR88 and performed preliminary bioconjugation assays. These experiments allowed us to verify that the affinity of the anti-GPR88 SdAB DR88-08 for its target was maintained after conjugation with different payloads (flurophores). In a next step, we will implement original strategies to develop innovative linkers cleaved by specific striatal enzymes, to allow local release of the A2AR antagonist payload (istradéfylline ou ASC) from the bioconjugate selectively in the striatum.
In this project, we will develop innovative biomolecules allying A2AR antagonist properties with the intrinsic qualities of SdAbs, high specificity, long-lasting effects and possible vectorization to a desired site of action (brain region), to durably relieve ASD symptoms with minimal side effects. The therapeutic interest of such biomolecules will extent to other pathologies, notably Parkinson disease, and the required development of novel bioconjugation technics will pave the way for multiple unforeseen therapeutic applications, including for the treatment of CNS disorders.