Biparatopic abeta binding polypeptides

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The invention relates to biparatopic A-beta binding polypeptides and, more specifically, to biparatopic A-beta binding polypeptides comprising at least two immunoglobulin single variable domains binding to different epitopes of A-beta. The invention also relates to specific sequences of such polypeptides, methods of their production, and methods of using them, including methods of treatment of diseases such as Alzheimer's Disease.

As an alternative to active immunization approaches, antibodies directed against A-beta may be administered to a patient. Such passive immunization approach was shown to be successful in reducing brain A-beta burden in transgenic AD mice. The underlying mechanisms remain open for speculation since it was thought unlikely that antibodies could cross the blood-brain barrier and target the plaques present in brain. The authors therefore suggested that the antibody created an 'A-beta sink' in the plasma which titrated A-beta out of the brain. Subsequently, using gelsolin and ganglioside 1 , it was demonstrated that any A-beta-binding ligand has the potential to reduce amyloid burden in transgenic AD mice without crossing the blood-brain barrier. Short-term (24 hours) passive immunization appeared to restore cognitive deficits of transgenic AD mice even without affecting the total brain amyloid load (Dodart et al. (2002) Nature Neuroscience 5, 452-457). The result would suggest that smaller, still soluble aggregates of A-beta are targeted first by some antibodies, and also that these are the most toxic forms of A-beta. Hence, clearance of proto-fibrillar A-beta could restore memory, at least in transgenic APP-mice.

The humanized anti-A-beta monoclonal antibody bapineuzumab (an analogue of the anti-A-beta mouse antibody known as "3D6") has meanwhile entered clinical trials. However, the first data reported from Phase II trial showed mixed results: Statistically significant effects on several efficacy endpoints were observed in ApoE4 non-carriers only. Furthermore, bapineuzumab was well tolerated and safe in ApoE4 non-carriers, while in ApoE4 carriers, serious adverse events were more frequently observed in bapineuzumab-treated patients than in the placebo arm. Moreover, vasogenic edema events have been observed. The induction of cerebral microhemorrhages has also been described pre-clinically in transgenic APP mice.

Conventional antibodies (containing an Fc part) used in anti-A-beta passive immunizations are suspected to account for the induction of vasogenic edema or microhemorrhages observed in humans and animal models, which are associated with a targeting of cerebral vascular A-beta deposits (Cerebral amyloid angiopathy) leading to microbleedings via ADCC and/or CDC.

Finally, the binding affinity of about 2.5 nM of this antibody, as measured by Biacore, is assumed to be too low to induce an effective "peripheral sink effect".

Another anti-A-beta antibody, solanezumab (humanized antibody m266; LY- 2062430), has also entered clinical testings. The maximal plaque load reduction that could be achieved was published to be about 60%.In addition, specificity of this antibody is limited to soluble A-beta, so that binding of aggregates or plaques cannot be expected.

A third anti-A-beta antibody, ponezumab, only binds to A-beta(x-40) molecules, and not to A-beta(x-42) molecules, the latter being assumed to be the (more) pathogenic A-beta species. Its affinity is even lower than the affinity of bapineuzumab, and the risk of cerebral microhemorrhages can not yet be ruled out, due to its ability to bind to A-beta plaques in blood vessels, combined with a remaining ADCC/CDC activity of its Fc portion.

In summary, the above demonstrates that even if A-beta binding and clearance by (classical) antibodies appears to be an attractive mode-of-action for the development of therapeutical agents for the treatment of e.g. AD, other characteristics and effects of such immunoglobulins which have not yet been fully elucidated, such as the pharmacological implications of their property to bind to certain forms of A-beta, make it far more difficult than one might have initially assumed to find and develop safe and efficient therapeutical antibodies. Antibody fragments, such as immunoglobulin single variable domain antibodies or VHH domains (as defined below), having specificity for A-beta have also been described in the art: WO2004/44204; WO2006/40153; WO2007/35092;

Definitive diagnosis of AD still requires post-mortem pathological examination of the brain to demonstrate the presence of amyloid plaques, neurofibrillary tangles, synaptic loss and neuronal degeneration. This is essentially the same procedure as defined by Alois Alzheimer in 1906. In 1984 the National Institute of

Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) established formal criteria for the diagnosis of AD (reviewed in Petrella et al.

Radiology 226, 315-336). Patients meeting all of the following criteria are diagnosed probable AD: dementia evidenced by examination and testing (e.g. Mini-Mental Test, Blessed Dementia Scale, or similar tests), impairment of memory and at least one other cognitive function, normal consciousness, onset between 40 and 90 years of age, absence of signs of other diseases that cause dementia (exclusion criterion). A gradual progressive, cognitive impairment without an identifiable cause will be diagnosed as possible AD. Probable AD is further defined as mild (early), moderate (middle) or severe (late) dementia.

Laboratory analysis is used to objectively define or exclude alternative causes of dementia. ELISA assays of A-beta(1 -42) and phospho-tau in cerebrospinal fluid (CSF), combined with genotyping for ApoE4 (a predisposing genetic factor) appear to be sensitive and specific. The methods are, however, not widely applicable because of the invasive CSF puncture, preventing this to become routine screening. ELISA for the neural thread protein (AD7C-NTP) (developed by Nymox) demonstrated higher levels in urine from AD patients than from non- AD dementia patients or healthy controls. However, the mean levels were significantly lower in early AD cases, suggesting the test is not reliable for testing for early onset of AD.

No biochemical method is as yet suited for the firm diagnosis of early stages of AD, rather they merely help to confirm the clinical diagnosis of advanced cases. Clearly, more advanced techniques are needed to allow early diagnosis before onset of clinical symptoms that signal irreversible brain damage.

Finally, not only for diagnostic purposes but also in e.g. pre-clinical research and development, A-beta binding molecules are useful as research tools. Widely used are the antibodies already mentioned above, i.e. antibody 3D6 and antibody m266. Antibody 3D6 binds to A-beta with a relatively low affinity and may therefore not be suitable for all purposes. Antibody m266 cross-reacts with N- termially truncated versions of A-beta, such as p3, which does not allow to distinguish between disease-relevant A-beta species, such as A-beta(1 -40) and A-beta(1 -42), and other molecules such as p3.