Drug development of single domain antibodies to treat dementias with synucleinopathy

Immunotherapy targeting α-synuclein (αsyn) and other amyloids is the most promising treatment for such
diseases. Most involve whole antibodies (Abs) but much less attention was paid to Ab fragments, which have
key advantages. We have generated 58 unique anti-αsyn single domain Abs (sdAbs, VHHs) from phage
display libraries derived from B-cells of a llama immunized with the αsyn protein. The sdAbs recognize various
forms of αsyn and clear αsyn to prevent its toxicity in culture and in synucleinopathy mice. Our published and
preliminary findings position us strongly to prepare our two lead sdAbs for human trials.
Four IgG αsyn Abs and two αsyn vaccines are in Phase-1 or -2 clinical trials, which strongly supports this
type of therapy. sdAbs (15 kDa) have several advantages over IgGs (150 kDa) and single chain variable
fragments (30 kDa, scFvs), including more extensive brain uptake because of their smaller size, ability to bind
to cryptic epitopes that larger antibodies cannot access, and better suitability for gene therapy because of their
single domain, which facilitates cellular folding. They also have higher affinity and solubility than scFvs. Apart
from our work, a few anti-αsyn sdAbs inhibited αsyn assembly and/or toxicity in vitro. A synthetic sdAb,
expressed as a viral construct injected into the brain, impeded αsyn spreading in mice but its efficacy on αsyn
degradation was not reported. Limited clinical development of sdAbs to date relates to broad patents that
expired in Europe in 2014 and in the US in 2017. Since then, several sdAbs entered clinical development and
trials, and four are approved for human therapy for a blood disorder, cancers and arthritis.
For clinical development of our αsyn sdAbs, we meet all six Early-Stage Entry Criteria for Biologics for this
U01 program. We propose five specific aims. Aim 1: Humanize and murinize our two lead anti-αsyn sdAbs
(hsdAb and msdAb). Two forms of each sdAb will be generated, a monovalent sdAb version, with or without a
PROTAC molecule to enhance efficacy, and a bivalent Fc-(sdAb)2 version. Their affinities for various α-syn
preparations will be examined. Aim 2: Characterize the pharmacokinetic (PK) profile of the different versions of
the anti-αsyn msdAbs in mice to facilitate the selection of the development candidate. Aim 3: Conduct in vitro
bioassays and in vivo mouse efficacy studies of the sdAb versions from Aim 1 to identify the preclinical
candidate. Aim 4: Conduct PK assessment of the lead anti-αsyn hsdAb, based on the outcome of Aims 2-3, in
rats and cynomolgus monkeys, as well as Investigational New Drug-enabling toxicology in rats. Aim 5: Develop
a stable cell line for the anti-αsyn hsdAb, formulation, and method qualification. Most of Aim 1 and Aims 4 and
5 will be conducted by contract research organizations with extensive experience in such studies.
Overall, the proposed studies are likely to identify a therapeutic clinical candidate sdAb to treat
synucleinopathies such as Alzheimer’s disease with Lewy Bodies, Lewy Body Dementia, Parkinson’s disease
and Multiple System Atrophy.