WO2021032966A1 - Detection of a-beta oligomers - Google Patents

Abstract

The invention is in the field of Aβ oligomers and in particular relates to binding molecules for detecting such Aβ oligomers. The binding molecules find utility in identifying Aβ oligomers both in vitro and in vivo, in screening for drugs targeting Aβ oligomers and in therapeutic methods.

Description

DETECTION OF A-BETA OLIGOMERS

Field of the Invention

The present invention relates to the detection of Ab oligomers. In particular, the present invention relates to an Ab binding molecule which can detect such oligomers. The invention also relates to methods utilising the binding molecule, including detection of Ab oligomers in a patient, screening a patient for treatment with an anti-amyloid drug and in testing of anti-amyloid drug candidates. Background of the Invention

Alzheimer’s disease (AD), which is the most prevalent cause of dementia, affects over 50 million people worldwide¹. A molecular hallmark of AD is the accumulation of insoluble protein deposits, most notably amyloid plaques and neurofibrillary tangles, in specific brain tissues^2-6. Amyloid plaques, in particular, predominantly consist of aggregated forms of the amyloid beta peptide (Ab)^2-6. The aggregation process of Ab is complex as it involves a variety of structurally different aggregated species^7,8. Among such species, increasing evidence has implicated small soluble oligomers as major agents responsible for neurotoxicity in AD^9-15. In particular, these oligomers have been shown to trigger various neurotoxic pathways, including synaptic dysregulation, membrane permeabilisation, oxidative stress, mitochondrial dysfunction and activation of pro- inflammatory response^4,9.

The conformational heterogeneity, low concentrations, and transient nature of these oligomeric species have made it extremely difficult to isolate and study them. Antibodies offer a possible way to overcome this challenge, as they represent a powerful and versatile tool owing to their high binding specificity and affinity and well-established discovery methods^16-18. These molecules have highly successful applications in diagnostics, therapeutics and targeted drug delivery systems, for infectious diseases, cancer, metabolic and hormonal disorders^16,19. In particular, many diagnostic tests routinely used in the clinic are based on antibodies. For this reason, in the last 20 years, major efforts have been made in overcoming the challenges in isolating and stabilising oligomeric species for immunisation or phage display protocols to develop antibodies that could selectively recognise such species in PET scans and biological samples from patients^9,20-29. Summary of the invention

The present inventors explored a novel route for discovering oligomer-specific antibodies, which exploits advances in computational methods that have recently opened time- and cost- effective opportunities for the rational design of protein-protein interactions.

The invention therefore provides an Ab binding molecule comprising the sequence of SEQ ID NO: 4.

The invention also provides a proteolysis targeting chimera comprising a binding molecule of the invention.

Furthermore, the invention provides an isolated polynucleotide encoding a binding molecule of the invention, a vector comprising the polynucleotide and a host cell comprising the vector.

The invention also provides use of a binding molecule of the invention in detecting and/or quantifying Ab oligomers. Similarly, the invention provides a method of detecting and/or quantifying the numbers of Ab oligomers in a patient, the method comprising contacting a sample from the patient with a binding molecule of the invention and/or administering a binding molecule of the invention to the patient.

In addition, the invention provides a method of selecting an anti-amyloid drug for a patient, the method comprising (i) contacting a sample from the patient with a binding molecule of the invention and/or (ii) and administering a binding molecule of the invention to the patient, and selecting an anti-amyloid drug for the patient based on the presence or absence of oligomers.

The invention also provides use of a binding molecule of the invention in testing the ability of an anti-amyloid drug candidate to reduce the number of Ab oligomers.

Similarly, the invention provides a method of assessing Ab oligomer inhibition by an antiamyloid drug candidate, the method comprising monitoring Ab oligomers in the presence of a binding molecule of the invention and an anti-amyloid drug candidate.

The invention further provides use of a binding molecule of the invention in delivering a drug to Ab oligomers.

The invention also provides a method of therapy comprising administration of a binding molecule of the invention to a patient in need thereof. Finally, the invention provides a method of delaying, preventing onset or progression of an amyloid disease, said method comprising administration of a binding molecule of the invention to a patient in need thereof. Brief Description of the Figures

Figure 1 shows rational design of a conformation-specific antibody for Ab42 oligomers. (A) Schematic representation of the target selection strategy used, which aims at generating an antibody with higher affinity for Ab42 oligomers than for monomers and fibrils. (B) Representation of the aggregation mechanism of Ab42⁸. Primary nucleation (k_n), secondary nucleation (kz) and elongation (k₊) rate constants are shown. The dark grey arrow indicates secondary nucleation processes, which are primarily responsible for the production of oligomers and involve the C-terminal region of Ab42³¹. (C) Sequence of Ab42³¹; the grey gradient provides a visual representation of the results of the scanning phase. The six designed CDR3 sequences (on the left) are shown together with their corresponding Ab42 epitopes (on the right). The sequence of the complementary peptide of DesAb-O, the designed antibody with highest binding affinity for Ab oligomers, is shown in a grey box on the right hand side. (D) ThT-based in vitro aggregation assay of 3 mM Ab42. The dashed line indicates the time at which samples were collected from the aggregation reaction to perform the ELISA experiment. (E) ELISA experiment performed on samples collected from aggregation reaction shown in panel (D), using the six DesAbs as primary antibodies. The bar corresponding to DesAb-0 is coloured in dark grey, whilst the one corresponding to the original DesAb29-36 is in light grey. The dashed line corresponds to the value of DesAb29-36. Error bars are representative of standard error. Statistical analysis was performed by one-way ANOVA with multiple-comparison (95% Cl, ** P-value = 0.0045).

Figure 2 shows characterisation of the conformational specificity of DesAb-0 by TIRF, AFM and STORM. (A) Coincidence of ThT and AF647-DesAb-0 signals in the TIRF single-molecule imaging of aggregates from an aggregation reaction of Ab42. Three independent experiments (dots, stars, squares) are shown. Each point is the average of 10 fields. Error bars represent one standard error. (B) TIRF images at 80 and 240 minutes of aggregation time are shown (the complete set is shown in Fig 6). ThT, AF647 and composite channel images are shown. Bars indicate 20 mm. (C) Wide (top) and detailed (bottom) high-resolution AFM 3D morphology maps of samples at 0, 20, 80 and 240 minutes. (D) Super-resolution STORM imaging of an aggregation reaction of Ab42 after 40 minutes of incubation. On the left: diffraction limited image; on the right: STORM image. Scale bars indicate 2 mm in the lower magnification images and 500 nm in the zoomed images. (E) Representation of the experimental setup for super-resolution imaging. (F) Estimation of the Kd of binding of DesAb-0 with the different aggregated species of Ab42.

Figure 3 shows development and validation of a real-time ELISA assay with DesAb-0 for detecting Ab oligomers during an aggregation reaction. (A) Graphical representation of the experimental setup of the time-course ELISA. Briefly, samples from 5 mM Ab42 aggregation reactions were collected at specific incubation times and loaded onto an ELISA plate. The amount of oligomers is determined from absorbance measurements upon incubation with DesAb-0 and a commercial HRP-conjugated anti -His tag antibody. (B) ELISA measurements taken at 0, 0.5 (50% of aggregation) and 2 h (plateau, 100% of aggregation) from a 5 mM Ab42 aggregation reaction using DesAb-0 (bars with stripes), DesAb-Ab 18-24 (dotted bars) and the commercial antibody 6E10 (grey bars). Data were normalised for smallest values measured. Statistical analysis was performed by ANOVA with multiple comparisons (C.I. 95%, ****p < 0.0001). (C) Log- scale-plot showing the concentrations of monomers (grey), oligomers (stripes) and fibrils (dots) at varying time points for a 5 mM Ab42 aggregation reaction derived from 8. (D) ThT aggregations of 3 mM Ab42 alone (black dots) or in the presence of 5-fold molar excess bexarotene (grey stars). (E) Time-course ELISA of 3 mM Ab42 alone (black dots) or in the presence of 5-fold molar excess bexarotene (grey stars).

Figure 4 shows quantification of Ab42 oligomers in C. elegans and mouse hippocampal tissue (A) From left to right: plots showing body bends per minute of

GMC101 worms, NIAD-4 fluorescence intensities of GMCIOI (dotted bars) and N2 (white bars) worms, ELISA absorbance of DesAb-0 of GMCIOI (bars with stripes) and N2 (white bars) worms at different days of adulthood. (B) NIAD-4 fluorescence of CA3 areas from J20 (grey squares) and control wild-type (black dots) mice at 4, 9, and 18 months of age. Representative fluorescence images are shown in the insets. (C) Absorbance of an ELISA using DesAb-0 on hippocampus CA3 area from J20 (grey stars) and control wild- type (black dots) mice at 4, 9, and 18 months of age. (D) Absorbance of an ELISA using the monoclonal antibody 6E10 on hippocampus CA3 area from J20 (grey triangles) and control wild-type (black dots) mice at 4, 9, and 18 months of age. (E) Bar plot showing the NIAD-4 (dotted bars) and DesAb-0 (bars with stripes) signals of J20 mice from panels B) and C) divided for the 6E10 signals of J20 mice from panel D). Error bars are representative of standard errors. Statistical analysis in (A) was performed by ANOVA with multiple-comparison. Statistical analysis in (C) was performed by t-test (C.I. 0.95;

**** P< 0.0001).

Figure 5 shows characterisation of the preparations of the DesAbs used in the present work. SDS-PAGE (A) and CD analysis (B) of the purified antibodies. In panel (A), line 1 : PMSAIVS, line 2: YHADISNE, line 3 : LEVIVRS, line 4: ESAFGRA, line 5 :

PYGSMYVHS, line 6: GAVLTAK. In panel (B) black squares: PMSAIVS, light grey triangles: YHADISNE, light grey diamonds: LEVIVRS, dark grey dots: ESAFGRA, light grey squares: PYGSMYVHS, dark grey triangles: GAVLTAK.

Figure 6 shows single molecule analysis of aggregates from an aggregation reaction of Ab42. Fluorescence microscopy images of different time points of aggregation. ThT,

Alexa647 channels and composite images are shown. Bars indicate 20 mm.

Figure 7 shows diffusion measurements to determine the affinity of DesAb-0 for Ab42 amyloid fibrils

Figure 8 shows a plot from Figure 3E, where individual points are shown.

Figure 9 shows normalised Abs (signal DesAb-O/signal 6E10) of the aggregation time course with DesAb-0 (grey) and NIAD-4 (black) in GMC2 C. elegans worms. On the right, representative NIAD-4 fluorescence images.

Figure 10 shows (A) ANS binding assay of the six DesAbs. For each DesAb (the designed CDR3 is indicated on the top of each graph), the fluorescence spectra of ANS alone are reported as thin light grey lines, of ANS in the presence of Ab40 oligomers are indicated as thick dark grey lines, of ANS in the presence of DesAb are indicated as thin dashed grey lines, of ANS with both oligomers and DesAb are reported as thick black lines. The dashed thin black lines represent the expected spectrum for solutions with both oligomers and DesAb given by the linear sum of the spectra obtained in the presence of oligomers alone and DesAb alone. (B). ANS fluorescence spectra for characterising the binding of DesAb29-36 and DesAb-0 to Ab40 oligomers. For each experiment, the fluorescence spectrum of ANS alone is reported as a continuous light grey line, of ANS in the presence of DesAb and Ab40 oligomers in black. The dashed black lines represent the expected spectrum for solutions with both oligomers and DesAb in the case this spectrum was given by the linear sum of the spectra in the presence of oligomers alone and DesAb alone (not shown in this representation. (C) Bar plot showing the relative accessible surface in the presence of the antibodies.

Figure 11 shows ELISA experiments performed to assess the binding of the DesAbs to Ab40 oligomers. The DesAbs were loaded on the ELISA plate and then incubated in the presence of Ab40 oligomer solutions. The bar corresponding to DesAb-0 is coloured with black stripes while the one corresponding to the original DesAb- Ab29-36 in grey. (B) Correlation plot of the ANS binding data, represented as relative accessible surface, and the ELISA results. The original DesAb- Ab29-36 is represented as a black square, while DesAb-0 is indicated as a black triangle. (C) Dot-blot to determine the binding of DesAb-0 to Ab40 oligomers compared to the antibody with lowest affinity

(CDR3: PYGSMYSHV), according to ANS binding assay. Also in this case, the DesAbs were spotted on the membrane plate and then incubated in the presence of Ab40 oligomer solutions.

Figure 12 shows (A, B) ITC experiments for measuring the binding of DesAb-0 to Ab40 monomers. Solutions contain minimal amounts of DMSO to ensure that Ab40 remains monomeric. Baseline corrected raw data are shown in panel A. Dilution controls have been vertically shifted for visibility. Double subtracted integrated peaks are reported in panel B. (C, D) ITC experiments to measure the binding of DesAb-0 to stabilised Ab40 oligomers. Baseline corrected raw data are shown in panel C. Dilution controls have been vertically shifted for visibility. The double subtracted integrated peaks and fit (black line) are shown in panel D. Values from fitting: Kd of 440 ± 1.5 nM; n= 4.8 ± 0.23; DH = - 1.27E4 ±9.4E2 cal/mol; AS = -13.4 ±9.4E2 cal/mol/deg. (E, F) ITC experiments for measuring the binding of DesAb-0 to a-synuclein oligomers. Baseline corrected raw data are shown in panel E, while the double subtracted integrated peaks are reported in panel F.

Brief Description of the Sequence Listing

SEQ ID NO: 1 - sequence of Ab42 SEQ ID NO: 2 - DesAb-0 CDR1

SEQ ID NO: 3 - DesAb-0 CDR2

SEQ ID NO: 4 - DesAb-0 CDR3

SEQ ID NO: 5 - DesAb-0 complete sequence

SEQ ID NOs: 6 - 10 - designed CDR3 sequences

SEQ ID NO: 11 - residues 26-42 of Ab42

Detailed Descriotion of the Invention

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In addition as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an amino acid sequence” includes two or more such sequences, and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Ab binding molecules

The present invention provides an amyloid beta (Ab) binding molecule comprising the sequence ESAFGRA (SEQ ID NO: 4). As shown in the Examples, SEQ ID NO: 4 is the complementarity determining region (CDR) 3 sequence of the Des-Ab-0 antibody of the invention.

Ab denotes peptides of 36-43 amino acids. The peptides are derived from the amyloid precursor protein (APP), which is cleaved by beta secretase and gamma secretase to yield Ab. Ab molecules can aggregate to form oligomers (typically an isolated form of the Ab peptide where the precursor Ab monomer is non-covalently aggregated in an ordered three-dimensional structure of less than about 50 monomers). These oligomers have been shown to trigger various neurotoxic pathways, including synaptic dysregulation, membrane permeabilisation, oxidative stress, mitochondrial dysfunction and activation of pro-inflammatory responses. Ab oligomers are therefore thought to be associated with the decline in memory in Alzheimer’s disease.

The most common isoforms of Ab are Ab4o (Ab1-40) and Ab42 (Ab1-42) (SEQ ID

NO: 1).

Binding molecules of the invention may bind to (recognise) any Ab isoform (including Ab40 or Ab42). Binding molecules of the invention may bind to Ab40 or Ab42, but typically bind to both Ab40 and Ab42.

Binding molecules of the invention preferentially bind to Ab oligomers (instead of Ab in its monomeric or fibrillar form). In other words, binding molecules of the invention are specific for Ab oligomers.

A binding molecule may be specific for Ab oligomers if it binds Ab oligomers but demonstrates no detectable binding for monomers or fibrils. A binding molecule may also be specific for Ab oligomers if it exhibits stronger binding to oligomers compared with binding to monomers and fibrils. For example, a binding molecule may bind to oligomers with a higher affinity (KD) than for monomers and fibrils. A binding molecule of the invention may for example exhibit (at least) 10-fold greater binding for oligomers compared with monomers and (at least) 10-fold greater binding compared with fibrils. A binding molecule of the invention may exhibit (at least) 50-, 100- or 500-fold greater binding for oligomers compared with monomers and (at least) 50-, 100- or 500-fold greater binding compared with fibrils. Preferably, a binding molecule of the invention exhibits (at least) 1000-fold greater binding for oligomers compared with monomers and (at least) 1000-fold greater binding compared with fibrils.

Binding to Ab monomers, oligomers and fibrils may be determined as described in the examples of the application, for example using total internal reflection microscopy (TIRF) and a comparison with thioflavin T (ThT) fluorescence. A binding molecule of the invention may for example show highest binding signals at a stage in the Ab aggregation reaction where oligomers are the predominant form of Ab.

As shown in the examples section, the inventors have identified a domain antibody (Des-Ab-O), which specifically binds Ab oligomers. The full sequence of Des-Ab-0 is provided in SEQ ID NO: 5. Des-Ab-0 comprises a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 3 and a CDR3 sequence of SEQ ID NO: 4. However, in Des-Ab-0 only the CDR3 is responsible for binding to the target. CDR1 and CDR2 are not involved in direct binding to the target and are relevant only in terms of the properties of the scaffold.

Binding molecules of the invention are typically antibodies or antigen-binding fragments thereof. Binding molecules of the invention may though comprise the sequence of SEQ ID NO: 4 (and optionally SEQ ID NO: 2 and/or 3) in any appropriate scaffold.

Antibodies and antigen-binding fragments thereof

The term “antibody” as used herein encompasses both full length antibodies and antigen-binding fragments thereof. An antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR, VH or VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR, VL or VL) and a light chain constant region. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

An antigen-binding fragment thereof refers to a fragment that is able to retain (specific) binding to the antigen.

Antibodies of the invention are monoclonal antibodies.

Antibodies (and antigen-binding fragments) of the invention typically comprise the sequence of SEQ ID NO: 4. This is the CDR3 of Des-Ab-O. Preferably, this is the CDR3 sequence of the V_H region in an antibody of the invention. Antibodies of the invention may also comprise the sequence of SEQ ID NO: 2 (preferably as a CDR1 in the V_H region) or SEQ ID NO: 3 (preferably as a CDR2 in the V_H region). An antibody may comprise all three of SEQ ID NOs: 2, 3 and 4 (as CDR1, CDR2 and CDR3 in the V_H region).

An antigen-binding fragment of the invention is typically a single-domain antibody (also known as a dAb, sdAb, VHH or nanobody). A single-domain antibody is an antibody fragment consisting of a single monomeric variable domain. Single-domain antibodies may be derived from camelids, which produce functional immunoglobulins that lack light chains and the first heavy chain constant region. The variable domains form heavy chain homodimers.

A binding molecule of the invention may therefore be a camelid domain antibody (utilises a camelid domain antibody scaffold) comprising a CDR3 sequence of SEQ ID NO: 4. The binding molecule may also comprise a CDR1 sequence of SEQ ID NO: 2 and/or a CDR2 sequence of SEQ ID NO: 3.

A camelid single-domain antibody of the invention may be prepared using methods known in the art, for example by replacing one or more of the CDR sequences of the parental camelid antibody with the desired sequence by CDR grafting (CDR grafted domain antibody). Camelid domain antibody scaffolds are known in the art, including so- called “universal” nanobody scaffolds (Vincke et al (2009) J Biol Chem, 30, 3273-3284).

Sharks also produce heavy chain only antibodies, with dAbs obtained from sharks termed VNARs. A binding molecule of the invention may therefore be a shark domain antibody (utilise a shark domain antibody scaffold) comprising a CDR3 sequence of SEQ ID NO: 4, and optionally comprising a CDR1 sequence of SEQ ID NO: 2 and/or a CDR2 sequence of SEQ ID NO: 3. Once again, appropriate shark scaffolds are known in the art and the CDR sequences may be introduced by CDR grafting (CDR grafted domain antibody).

Preferably, the binding molecule of the invention utilises a human single-domain antibody scaffold and comprises a CDR3 sequence of SEQ ID NO: 4, and optionally comprises a CDR1 sequence of SEQ ID NO: 2 and/or a CDR2 sequence of SEQ ID NO: 3.

Human antibodies are naturally composed of both heavy and light chains.

However, human domain antibody scaffolds, into which CDR sequences can be grafted, have been generated. These scaffolds are typically based on a heavy chain from a human antibody, where mutations are made to stabilise the antibody in the absence of a light chain. Such autonomous human domain antibodies are described for example in Barthelemy et al (2008) The Journal of Biological Chemisty, 283, 3639-3654 and also in Famm et al (2008) J. Mol. Biol., 376, 926-931. Human domain antibody libraries have also been described.

The domain antibody may comprise the sequence of SEQ ID NO: 5. Other antigen-binding fragments that may be used in accordance with the invention include, but are not limited to, scFv, Fab, modified Fab, Fab’, modified Fab’, F(ab’)2, Fv, dAb-Fc, Fd, dsFv, ds-scFv, scFv2 or scFv-Fc. Also included in the invention are minibodies, diabodies, triabodies and tetrabodies. The methods for creating and manufacturing these antibody fragments are well known in the art (see for example Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

The constant region domains of the antibody molecule of the present invention, if present, may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. In particular, human IgG constant region domains may be used (IgGl and IgG3 isotypes when effector functions are required or IgG2 and IgG4 isotypes when antibody effector functions are not required).

When the binding molecule is an antibody (or fragment) requiring a light chain, the light chain is typically a universal (common) light chain (or appropriate fragment thereof).

Universal light chains are able to pair with different heavy chains to form antibodies with functional binding domains (WO 2004/009618, WO 2009/157771 , Merchant et al. 1998, Nissim et al. 1994, WO 2011/097603).

Antibodies of the invention may be humanized antibodies. The term “humanized antibody” includes CDR-grafted antibody molecules in which CDR sequences have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. The term “humanized antibody” also includes humanization via a chimeric intermediate, i.e. where non-human residues are substituted with human residues.

An antibody of the invention may also be a chimeric antibody.

As used herein, the term ‘CDR-grafted antibody molecule’ refers to an antibody molecule wherein the heavy and/or light chain contains one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e.g. a human antibody). For a review, see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998. In one embodiment rather than the entire CDR being transferred, only one or more of the specificity determining residues from any one of the CDRs described herein above are transferred (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment only the specificity determining residues from one or more of the CDRs described herein above are transferred. In another embodiment only the specificity determining residues from each of the CDRs described herein above are transferred.

When the CDRs or specificity determining residues are grafted, any appropriate acceptor variable region framework sequence may be used. Suitably, the CDR-grafted antibody according to the present invention has a variable domain comprising human acceptor framework regions as well as one or more of the CDRs or specificity determining residues described above. Thus, provided in one embodiment is CDR-grafted antibody wherein the variable domain comprises human acceptor framework regions and nonhuman donor CDRs.

It will also be understood by one skilled in the art that antibodies may undergo a variety of posttranslational modifications. The type and extent of these modifications often depends on the host cell line used to express the antibody as well as the culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995).

An antibody/fragment of the invention may also be multi-specific, for example bispecific or tri-specific. A multi-specific antibody may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for epitopes of more than one target polypeptide. In other words, a multi-specific antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.

A bispecific antibody may also be a kappa(lambda) antibody or a dual variable domain immunoglobulin (DVD-Ig).

A multi-specific antibody may be constructed using standard molecular biological techniques (e.g., recombinant DNA and protein expression technology), as will be well known to a person of ordinary skill in the art. Any multi-specific antibody format may be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art The antibody may be or may comprise a variant of one of the specific sequences recited above. For example, a variant may be a substitution, deletion or addition variant of any of the above amino acid sequences.

A variant antibody may comprise 1, 2, 3, 4, 5, up to 10, up to 20 or more (typically up to a maximum of 50) amino acid substitutions and/or deletions from the specific sequences discussed above (preferably only one or two changes in CDR sequences). “Deletion” variants may comprise the deletion of individual amino acids, deletion of small groups of amino acids such as 2, 3, 4 or 5 amino acids, or deletion of larger amino acid regions. "Substitution" variants typically involve the replacement of one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid or another aliphatic amino acid. Some properties of the 20 main amino acids which can be used to select suitable substituents are as follows:

"Derivatives" or "variants" generally include those in which instead of the naturally occurring amino acid the amino acid which appears in the sequence is a structural analog thereof. Amino acids used in the sequences may also be derivatized or modified, e.g. labelled, providing the function of the antibody is not significantly adversely affected.

Derivatives and variants as described above may be prepared during synthesis of the antibody or by post- production modification, or when the antibody is in recombinant form using the known techniques of site- directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.

Variant antibodies may have an amino acid sequence which has more than about 80%, preferably more than about 85%, e.g. more than about 90 or 95% amino acid identity to the amino acid sequences disclosed herein (typically to SEQ ID NO: 5). Furthermore, the antibody may be a variant which has more than 80%, typically more than about 85%, e.g. more than about 90 or 95% amino acid identity to SEQ ID NO: 5, whilst retaining the exact CDR3. Variants may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5 (in some circumstances whilst retaining the exact CDR3). Variants may retain all CDRs.

This level of amino acid identity may be seen across the full length of the relevant SEQ ID NO sequence or over a part of the sequence, such as across about 20, 30, 50, 75, 100 or more amino acids, depending on the size of the full length polypeptide.

In connection with amino acid sequences, "sequence identity" refers to sequences which have the stated value when assessed using ClustalW (Thompson et al, 1994, supra) with the following parameters:

Pairwise alignment parameters -Method: accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10;

Multiple alignment parameters -Matrix: PAM, Gap open penalty: 10.00, % identity for delay: 30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specific gap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues: GPSNDQEKR. Sequence identity at a particular residue is intended to include identical residues which have simply been derivatized.

The present invention thus provides antibodies having specific sequences and variants which maintain the function or activity of these chains.

The present invention also provides isolated polynucleotide (DNA) sequences encoding an antibody or antigen-binding fragment of the invention. In particular, the invention provides an isolated polynucleotide encoding a VH of the invention. For example, the invention includes a polynucleotide encoding SEQ ID NO: 5, or a variant thereof as described above. DNA sequences which encode an antibody molecule of the present invention can be obtained by methods well known to those skilled in the art. The present invention also includes vectors comprising the polynucleotide(s) of the invention and host cells comprising such vectors. General methods by which the vectors may be constructed, transfection methods and culture methods are well known to those skilled in the art (see for example “Current Protocols in Molecular Biology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing).

Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the antibody molecule of the present invention. Bacterial, for example K coli, and other microbial systems may be used or eukaryotic, for example mammalian, host cell expression systems may also be used. Suitable mammalian host cells include CHO, myeloma or hybridoma cells.

The present invention also provides a process for the production of an antibody molecule according to the present invention comprising culturing a host cell containing a vector of the present invention under conditions suitable for expression of the antibody, and isolating the antibody molecule so-produced.

Non-antibody binding molecules

As set out above, the invention also encompasses non-antibody binding molecules. Such binding molecules comprise a scaffold into which one or more of the CDR sequences of the invention (preferably just the CDR3) can be inserted.

Such non-antibody binding molecules include protein scaffolds, particularly peptide aptamers/antibody mimetics. For example, the invention includes scaffolds which can readily tolerate introduction of one of more CDR sequences of the invention. Peptide aptamers are reviewed for example in Reverdatto et al (2015), Curr Top Med Chem, 15, 1082-1101.

The scaffold may be an atrimer, a protein cytotoxic T-lymphocyte associated protein-4 (CTLA4)-based molecule, an adnectin, an anticalin, a Kunitz-domain based binder, an avimer, a knottin or a fynomer. These are examples of scaffolds utilising a “loop on a frame”. The scaffold may also be a DARPin (designed Ankyrin repeat protein) or a bicyclic peptide. Furthermore, the scaffold may be an affibody, an affilin, an affimer, an affitin, an alphabody, a monobody, a nanoCLAMP or an OBody. Such scaffolds are reviewed for example in Vazquez-Lombardi et al (2015) Drug Discovery Today, 20, 1271-

1283.

One or more of the CDR sequences of the invention (or variants - see above) can also be inserted into a T cell receptor. This provides utility in chimeric antigen receptor (CAR-T) therapy.

Binding molecule conjugates

Further provided is a binding molecule of the invention conjugated to one of more additional moieties.

The binding molecule may for example be conjugated to a detectable label. Such detectable labels allow visualisation of the binding molecule and may for example be used in detection of Ab oligomers. A detectable label may for example allow detection of Ab oligomers in vitro, or from outside the body (hence allowing for diagnosis). A detectable label may be a fluorescent dye, radioactive label or an enzyme.

Exemplary fluorescent labels are fluorescein isothiocyanate, or rhodamine. Exemplary enzymes are alkaline phosphatase, b-galactosidase, horseradish peroxidase and luciferase. Such labels may be incorporated using known techniques.

Suitable radioactive atoms include ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I. Also included are ^99mTc or ¹²³I for scintigraphic studies. Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRI) such as ¹²³1, ¹³¹I, ⁱⁿIn, ¹⁹F1, ¹³C, ¹⁵N,

¹⁷ O, gadolinium, manganese or iron. Sufficient of the appropriate atomic isotopes must be provided in order for the molecule to be readily detectable.

The radio- or other labels may be incorporated in known ways. For example, if the binding molecule is a polypeptide it may be biosynthesised or may be synthesised by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as ^99mTc, ¹²³1, ¹⁸⁶Rh, ¹⁸⁸Rh and ¹¹¹In can, for example, be attached via cysteine residues. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker er al (1978) Biochem. Biophys. Res. Comm. 80, 49-57) can be used to incorporate iodine-123. Reference (“Monoclonal Antibodies in Immunoscintigraphy”, J-F Chatal, CRC Press, 1989) describes other methods in detail. Binding molecules of the invention may also be conjugated to drugs, including anti-amyloid drugs, particularly anti-amyloid drugs designed to target Ab oligomers. Examples of such drugs include bexarotene, adapalene and UVD 003.

Binding molecules of the invention may also be utilised in targeting Ab oligomers to degradation pathways, including the ubiquitin-proteasome pathway and autophagic pathways. Binding molecules of the invention may therefore be modified/conjugated to appropriate molecules in order to target the oligomers for degradation.

For example, a binding molecule of the invention may form part of a proteolysis targeting chimera (PROTAC). PROTACs are bifunctional molecules that typically consist of a target protein ligand (the binding molecule of the invention), a linker and a second molecule that targets the protein for degradation. The second molecule may be an E3 ligase ligand (such as VHL ligand 1, analogues of thalidomide and analogues of hydroxyproline), which mediates polyubiqutination of the target protein and ultimately leads to degradation via the ubiquitin-proteasome pathway. PROTAC systems are known in the art and are reviewed for example in Qi and Zhang (2019) Future Medicinal Chemistry, 11.

Uses of binding molecules of the invention

The binding molecules of the invention are typically used in the detection and/or quantification of oligomeric Ab. Binding molecules may be used to detect the presence of/quantify oligomeric Ab over time; in other words to monitor oligomeric Ab. The binding molecules are typically used to detect/quantify oligomeric Ab in a patient, particularly in a patient diagnosed with or at risk from an amyloid disease (see below). The patient is typically a human, but may also be a domestic, companion (such as a dog, cat etc) or livestock animal.

The binding molecules may be used in vitro, i.e. to detect oligomeric Ab in a sample from a patient. Alternatively, the binding molecules may be used in vivo.

When used in vitro, the sample from the patient may be any appropriate sample type in which Ab may be present. The sample may be a brain, blood, serum, plasma, cerebrospinal fluid or urine sample. Typically, the sample is a cerebrospinal fluid sample, nasal wash or plasma sample. For detection in vitro, the binding molecule may be labelled using a detectable label (such as a fluorescent, radioactive or enzymatic label as described above). The binding molecule is contacted with the sample and the presence of Ab oligomers may then be detected using known techniques, such as those described in the examples section below. For example, appropriate techniques include enzyme-linked immunosorbent assay (ELISA) and microscopy, including super-resolution microscopy, as the oligomers are smaller (about 20 nm) than the diffraction limit (about 200 nm).

For detection in vivo the binding molecule is administered to a patient via an appropriate route. For example, binding molecules of the invention may be administered by intravenous infusion or subcutaneous injection. Binding molecules may also be administered directly to the CNS. Further, binding molecules of the invention may be administered to the CSF. The binding molecule may be administered intrathecally or intracerebroventricularly. The binding molecule may also be administered intraparenchymally directly to brain tissue of a subject.

Once again, it is typical for the binding molecule to be labelled using a detectable label, particularly a label appropriate for in vivo imaging techniques (such as a magnetic or radiolabel). Once the binding molecule has been administered to the patient such techniques are then used to detect binding of the binding molecule to Ab oligomers.

The invention also provides methods of identifying a patient suffering from, in the preclinical phase, or at risk of developing, an amyloid disease. The patient is once again typically a human, but may also be a domestic, companion (such as a dog, cat etc) or livestock animal.

The term “amyloid disease” includes any disease associated with aberrant Ab, in particular diseases associated with the formation of amyloid plaques/aggregates. The disease is typically Alzheimer’s disease. However, amyloid plaques have also been found in some Lewy body dementia patients, in some cerebral amyloid angiopathy (CAA) patients and in inclusion body myositis. Ab has also been implicated in the development of some cancers, particularly hepatic cancers. Adults with Down syndrome have also been shown to have accumulations of amyloid. Ophthalmological diseases, including dry age- related macular degeneration (AMD) and glaucoma, are also associated with the formation of Ab aggregates. Patients suffering from, or at risk of developing the above diseases may be identified using methods known in the art, particularly based on the patient’s clinical presentation. The methods of the invention may be used in conjunction with other techniques for diagnosing the relevant disease.

Those at risk of Alzheimer’s disease include those of advancing age, family history of the disease, mutations in APP or related genes, having heart disease risk factors, having stress or high levels of anxiety. Identification of those suffering from or at risk of Alzheimer’s can be readily accomplished by a physician. Diagnosis may also be based on mental, psychiatric and neuropsychological assessments, blood tests, brain imaging (PET, MRI, CT scan), urine tests, tests on the cerebrospinal fluid obtained through lumbar puncture, or the like. The diagnostic methods of the invention may be used in conjunction with one or more of these techniques.

Methods of the invention may for example be used to identify patients with early stage Alzheimer’s disease.

Symptoms of CAA include weakness or paralysis of the limbs, difficulty speaking, loss of sensation or balance, or even coma. If blood leaks out to the sensitive tissue around the brain, it can cause a sudden and severe headache. Other symptoms sometimes caused by irritation of the surrounding brain are seizures (convulsions) or short spells of temporary neurologic symptoms such as tingling or weakness in the limbs or face. CAA patients can be identified by, e.g., examination of an evacuated hematoma or brain biopsy specimen, the frequency of APOE e2 or e4 alleles, with clinical or radiographic (MRI and CT scans) grounds according the Boston Criteria (Knudsen et al., 2001, Neurol ogy;56:537- 539 ), or the like. Those at risk of CAA include those of advancing age, those having the APOE genotype, and those having other risk factors associated with AD. Once again, the diagnostic methods of invention may be used in conjunction with one or more of these assessments.

A newborn with Down syndrome can be identified at birth by a physician's physical exam. The diagnosis may be confirmed through kaiiotyping. Multiple screening tests may be used to test or diagnosis a patient prior to birth (biomarkers, nuchal translucency, amniocentesis, etc.).

Those suffering from or at risk ofLewy body dementia can be identified by mental, psychiatric or neuropyschological assessments, blood tests, brain imaging (PET, MRI, CT scan), urine tests, tests on the cerebrospinal fluid obtained through lumbar puncture, or the like. Those at risk ofLewy body dementia include those of advancing age.

Those suffering or at risk from glaucoma and AMD could also be identified using routine methods.

Once a patient has been identified as having Ab oligomers, the patient may be administered an appropriate drug, for example a drug targeting such oligomers.

The binding molecules of the invention may also be used in selecting an antiamyloid drug for a patient. Once again, such methods involve detecting the presence or absence of Ab oligomers in the patient, either in vivo or in vitro in a sample from the patient. An appropriate anti-amyloid drug is then selected for patient based on whether Ab oligomers are found to be present or absent.

Based on the presence or absence of Ab oligomers, the skilled person would then readily understand which drugs would be appropriate to treat the patient. For example, anti-Ab antibodies (e.g. BAN2401) or small molecules (e.g. bexarotene)

Binding molecules of the invention may also be used to monitor the efficacy of a drug in a particular patient, for example by monitoring the presence/number of oligomers following treatment with the drug.

Furthermore, binding molecules of the invention may also be used in screening candidate anti-amyloid drugs, in particular drugs thought to inhibit Ab oligomers. The method typically comprises performing an aggregation assay which involves contacting a sample comprising Ab with both a binding molecule of the invention and a candidate drug. Binding of the binding molecule to oligomers is monitored (once again, the binding molecule is typically labelled and binding may detected using known techniques, such as time course ELISA). Binding may then be compared for example with the binding molecule in the absence of the candidate compound. A compound may be identified as an inhibitor if it delays, reduces or prevents the formation of Ab oligomers.

The anti-amyloid compounds are typically small molecules.

Binding molecules of the invention may also be used in delivering a drug to Ab oligomers. The drug may be any appropriate drug for treating an amyloid disease. Amyloid diseases are discussed above. The drug is typically a compound which inhibits Ab oligomers (but can be anything which is desirable to target to Ab oligomers). The drug is conjugated to the binding molecule of the invention using any appropriate means. For example, techniques for preparing antibody-drug conjugates are well known in the art. Appropriate routes for administering a binding-molecule-drug conjugate are discussed above.

Furthermore, binding molecules of the invention may also be used in targeting Ab molecules for degradation, for example using the ubiquitin-proteasome pathway or using autophagy. As discussed above, the binding molecule of the invention may form part of a proteolysis targeting chimera.

The invention also provides a method of therapy comprising administration of a binding molecule of the invention to a patient in need thereof. The invention in particular provides a method of delaying, preventing onset or progression of an amyloid disease, said method comprising administering a binding molecule of the invention. Amyloid diseases are as described above as are patients and suitable routes of administration. The amyloid disease is typically Alzheimer’s disease.

The method may involve administering a binding molecule-drug conjugate to the patient. Once again, the binding molecule may be modified so that it target the oligomers for degradation (for example the binding molecule of the invention may form part of a proteolysis targeting chimera).

Examoles

Example 1 : Antibody scanning to find an epitope on Ab42 for oligomer-specific binding and rational design of a panel of oligomer-specific antibody candidates

The object was to generate an antibody able to selectively target oligomeric species of Ab, rather than its monomeric and fibrillar forms (Fig. la). An antibody scanning method based on the use of rationally designed single-domain antibodies (DesAbs) for sequence-activity studies has been developed³¹. Using this strategy, it was found that the antibody DesAb-Ab29-36, targeting the epitope 29-36 at the C-terminus of 42-residue form of Ab (Ab42), was able to inhibit at sub-stoichiometric concentrations the secondary nucleation step during the aggregation of Ab42 (Fig. lb). Therefore DesAb-Ab29-36 is likely to bind to aggregated species with higher affinity than to Ab42 monomers. However, recent structural characterisations of Ab42 amyloid fibrils have shown that the C-terminal region of this peptide is buried within the core of the cross-b structures 2-34, and accordingly DesAb- Ab₂9-36 showed only negligible binding towards mature fibrils³¹. Taken together, these results indicated that the 29-36 region of Ab42 is likely solvent-exposed when the peptide is an oligomeric conformation, before becoming buried in the amyloid fibrils, and that targeting epitopes within this region may be a viable strategy for generating oligomer-specific antibodies

Starting from this consideration, the rational design of a number of antibodies binding epitopes close to or including region 29-36 was performed. Specifically, a panel of six additional antibodies was designed, with the aim of selecting the antibody with highest preferential binding to Ab42 oligomers with respect to monomers and fibrils (Fig. 1). All the DesAb variants were expressed in E. coli and purified as previously reported³¹ (Fig. 5). Circular dichroism (CD) spectroscopy revealed that all the six DesAbs have a secondary structure content compatible with the native conformation of a V_H domain (Fig. 5).

In order to identify the DesAbs with the strongest binding to Ab42 oligomers, an ELISA-based screening assay was developed (Fig. lc-e). The analysis was performed on samples collected directly from aggregation reactions at the half time of aggregation (Fig. Id), when the oligomers are present at their highest concentration³⁵. These samples were immobilised onto an ELISA plate and the various DesAbs used as primary antibodies in an indirect ELISA set up (Fig. ld,e). All the antibodies in the panel were able to bind to the oligomeric mixtures. In particular, the antibody with the amino acid sequence ESAFGRA in its CDR3 (DesAb-O), showed the strongest binding (Fig. le).

Example 2: Characterisation of the conformational specificity of DesAb-O.

In order to determine whether DesAb-0 is specific for oligomers over fibrils, total internal reflection fluorescence (TIRF) microscopy was used to visualise individual aggregates in samples taken from aggregation reactions of 3 mM Ab42 at specific incubation times (Figs. 2 and 6). These protein samples were deposited on glass slides and probed with 5 mM thioflavin T (ThT) and 1 nM AF647-labelled DesAb-O. The aggregates contained in the samples were then imaged and how many of them were recognised by ThT and AF647-DesAb-0 and how many by both were measured. In agreement with expectations, many aggregates were probed by both ThT and AF647-DesAb-0 at early aggregation times, with a percentage of coincidence of the two signals up to 50% between 60 and 100 min (Fig. 2a). By contrast, at later incubation times (beyond 140 minutes), when mature amyloid fibrils are formed, the percentage of coincidence dramatically drops down to less that 10%.

The content and heterogeneity of samples was further investigated at 0, 20, 80, and 240 minutes at the single aggregate nanoscale acquiring high-resolution, 3D morphology maps by phase-controlled atomic force microscopy (AFM)³⁶. Samples at 20 minutes contained predominantly spheroidal oligomeric species and few elongated pre-fibrillar species. The sample after 80 and 240 minutes contained an abundant population of elongated pre-fibrillar and fibrillar aggregates³⁷ (Fig. 2c). Starting from 80 minutes, the presence of ring-shape toroidal and prefibrillar oligomers was observed ^37,38, whose size matches those recognised by DesAb-0 and observed by TIRF. Taken together, these data indicate that DesAb-0 is able to preferentially bind oligomers over monomers and fibrils of Ab42.

Example 3: ELISA-based real-time oligomer quantification using DesAb-O.

Given the high oligomer specificity of DesAb-O, an assay was developed to monitor the formation and subsequent conversion into larger aggregates of oligomeric populations during the aggregation of Ab42 in the presence and absence of anti-amyloid compounds. First, Ab42 oligomers formed during an in vitro aggregation assay were investigated³⁵. Aggregation reactions of Ab42 were used at 3 mM of peptide concentration, from which 20 pi samples were taken at specific incubation times. These samples were then analysed in an indirect ELISA set up using DesAb-0 as primary antibody (Fig. 3a).

In order to assess whether this antibody was able to specifically detect Ab42 oligomers under our experimental conditions, its performance was compared to that of a commercial antibody (6E10), which targets the N-terminal region of Ab42, and of a previously described DesAb

which binds to all aggregated species, oligomers and fibrils with no particular preference, at the beginning (0 hours), at the half time (1 hour) and at the plateau (2 hours) of aggregation (Fig. 3a, b). As expected, 6E10 leads to similar signals at the three incubation times (Fig. 3b). As this antibody binds the N-terminus of Ab42 and this portion of the peptide is exposed in most conformations, this result proves that the total amount of Ab42 in the wells is similar for all three time points and the presence of aggregates does not affect the absorption of the samples on the ELISA wells. By contrast, the DesAb- Ab18-₂₄ signal increased with the aggregation time, proving that this antibody binds preferentially aggregated species, but cannot distinguish between oligomers and fibrils (Fig. 3b). Finally, DesAb-0 showed the highest signal at the halftime of the aggregation reaction (Fig. 3b), indicating that this antibody binds specifically oligomers, which transiently form during the aggregation of Ab42.

A time course experiment was then performed (Fig. 3ed), where more aggregation time points were analysed in order to verify whether the evolution over time of the Ab42 oligomer population during the aggregation process could be characterised. It was found that absorbance progressively increased, reaching the highest value at 1 hour of incubation. This value was approximately 3 -fold higher than that one at the initial time of aggregation.

This difference is highly significant. At a comparable initial concentration of monomer (5 mM), it has been shown that the concentration of monomers is 35-fold higher than the concentration of oligomers (2.4 mM vs 70 nM) at this time point⁸ (Fig. 3c). After 1 hour of incubation, the absorbance progressively decreased to values even lower than the initial ones, indicating that DesAb-0 does not bind fibrillar species (Fig. 3e).

Example 4: Using DesAb-0 to assess Ab42 oligomer inhibition by drug candidates

Next, the ability of the DesAb-0 assay to capture changes in the oligomeric population in the presence of anti-amyloid compounds was evaluated. To do so, an aggregation assay was performed in the presence of the small molecule bexarotene (Fig. 3d, e), which has been shown to inhibit primary nucleation and thus to delay the formation of Ab42 oligomers³⁹. In this case, the DesAb-O-based ELISA assay detected a shift of 30 minutes in the peak of the oligomers (Fig. 3d), which matches the shift in half time of aggregation observed by ThT assay (Fig. 3d). These results indicate that the antibody- based assay is able to detect oligomeric populations formed during in vitro aggregation of Ab42 and that this technique can be used to probe the effects of anti-amyloid compounds on these populations.

Example 5: DesAb-0 detects Ab42 oligomers in a C. elegans model of AD

In order to verify whether DesAb-0 could specifically detect Ab42 oligomers formed in vivo, a C. elegans model of Ab42-mediated dysfunction was used, denoted GMC101, in which human Ab42 is expressed in body wall muscle cells where it forms aggregates and results in severe age-progressive paralysis⁴⁰. The analysis was performed on protein extracts from 500 GMC101 worms at day 0, 3, 5, 7, 10 of adulthood (Fig. 4a). The fitness of the worms was first assessed staring from after 24 h of induction of aggregation for 10 days. It was found that the GMC101 worms had the most dramatic drop of mobility between day 5 and 7, which indicates that the toxic species likely reach their highest concentrations at that time. Then the formation of the amyloid aggregates was monitored using the amyloid-specific compound NIAD-4, whose fluorescence increases with the concentration of amyloid aggregates. No significant NIAD-4 fluorescence change able to capture the pathological behaviours observed between day 5 and 7 was found, suggesting that this compound is not selective for toxic aggregates. Then, it was verified whether these species could be specifically detected using DesAb-O. An ELISA protocol similar was performed to that previously described for the in vitro aggregation and compared the result of this experiment with a quantification of the aggregates using the amyloid-specific compound NIAD-4. In particular, the ELISA wells were coated with the protein extracts and DesAb-0 used as a primary antibody. As a control, the same procedure was performed on protein extracts from the wild type C. elegans model N2 and these signals used as a reference (Figs. 4a). DesAb-0 was able to specifically recognise NIAD-4 negative, toxic Ab42 species in the GMC101 protein extracts formed between day 5 and 7.

Example 6: DesAb-0 detects Ab42 oligomers in a mouse model of AD

To further validate the ability of DesAb-0 to detect Ab42 oligomers formed in vivo, similar analyses were performed using a J20 mouse model of Alzheimer’s disease (PDGF-APPSw,Ind), which overexpresses a variant of human APP carrying both the

Swedish and Indiana familiar mutations⁴¹. ELISA assays were performed on hippocampus protein extracts using DesAb-0 and 6E10, and NIAD-4 fluorescence quantified from tissue slices from mice at 4, 9, 18 months of age. These signals were then normalised using a control wild-type mouse. In agreement with the C. elegans experiments, a DesAb-0 positive signal was detected at 4 months of age, when amyloid plaques cannot be significantly detected with NIAD-4 yet, but the mice show the initial signs of memory deficit⁴². Furthermore, the DesAb-0 signal increases between 4 and 9 months and then decreases at 18 months, while both 6E10 and NIAD-4 signals progressively increase (Fig. 4b-e). The decrease of DesAb-0 signal observed at 18 months suggests that oligomers are less accessible at this age, in agreement with observations that plaques appear starting from 9 months.

Example 7: DesAb-0 detects Ab40 oligomers

Given that an heterogeneous population of Ab oligomers⁴³ is likely to exist upon in vivo aggregation, the ability of DesAb-0 to detect also other types of Ab oligomers was tested. For this purpose, a fluorescence-based screening assay in combination with Zn²⁺- stabilised Ab40 oligomers was developed⁴³. This assays uses the fluorescent probe 8- anilinonaphthalene-1 -sulfonic acid (ANS), whose fluorescence emission increases upon binding the oligomers. The change in ANS fluorescence was monitored in the presence of a 1 : 1 ratio DesAb: Ab40 (Fig. 10). The addition of the DesAbs produced a decrease of ANS fluorescence, which suggests a displacement of ANS molecules from the surface of the oligomers as a result of the antibody binding. Among all the antibodies the antibody DesAb-0 showed the greatest binding also in this set up, producing a 55% decrease of the ANS fluorescence of the oligomers (Fig 10), almost 30% higher than that of DesAb- Ab29- 36. As a complementary assay, an ELISA was performed (Fig. 11) monitoring the binding of immobilised DesAbs in the presence of Ab40 oligomer solutions and these results correlated with the ANS binding analysis. Comparing these two data sets, it was found that, while the majority of the antibodies had a binding similar to the original antibody DesAb-Ab29-36 , DesAb-0 showed a significantly stronger binding to Ab40 oligomers (Fig.

11).

These results were further confirmed with a dot blot analysis. The antibodies DesAb-0 and PYGSMYVHS were immobilised, this last being the least reactive antibody in the ANS quenching experiments (Fig. 11), on a nitrocellulose membrane. The membrane was then incubated in the presence of a solution of stabilised Ab40 oligomers and the bound oligomers revealed using 6E10, a commercial primary antibody targeting Ab. In agreement with the previous experiments, also in this set up, DesAb-0 was the antibody showing the strongest binding to Ab40 oligomers.

Oligomer-specific binding was then quantified. It was first determined whether this antibody was selective for stabilised oligomers over monomers of Ab40. Isothermal calorimetry experiments were performed (Fig. 12), where the heat change was measured overtime upon injections of DesAb into solutions containing Ab40 oligomers or monomers. It was found that DesAb-0 was capable of binding these Zn²⁺-stabilised oligomers with a K_d of approximately 500 nM (Fig. 12), while showed only a weak binding for monomers (Fig. 12). Notably, while the binding to monomers is mainly endothermic and, thus, probably entropically driven, the binding to oligomers is exothermic.

Methods

Rational design of the antibodies.

A detailed description of the method is provided in Ref. ³⁰. The complementary peptide design procedure consists of two steps. First, given a target linear epitope, from the Protein Data Bank (PDB) all protein fragments that face in a b-strand any sub-sequence of at least three residues in which the target epitope can be fragmented were collected.

Second, complementary peptides predicted to bind the target epitope were built by merging together these fragments using a ‘cascade method’. In essence, this cascade method starts from one of these fragments and extend it to the length of the target epitope by linking it to some of the others. Fragments are linked using three rules: (i) fragments can be joined together only if found in b-strands of the same type (i.e. parallel or antiparallel), (ii) all fragments making up a complementary peptide must partly overlap with their neighbouring fragments and (iii) the overlapping regions must be identical both in the sequence and in the backbone hydrogen-bond pattern that is extracted from the b-strand where each fragment is found. Since the identification of the complementary peptides is based on the analysis of amino acid sequences facing each other in b-strands in the PDB, the interaction with the target sequence is already shown to be viable in a biological context. In addition, given this design strategy, the resulting complementary peptides are expected to bind the target epitope by enforcing a b-strand-like conformation. Therefore, such complementary peptides will be particularly effective in binding to solvent-exposed regions of protein sequences that do not form persistent hydrogen bonds with other parts of the protein molecule, such in the case of disordered regions³⁰. Protein expression and purification.

The various complementary peptides were grafted into the CDR3 of the DesAb scaffold by means of mutagenic PCR with phosphorylated oligonucleotides³¹. The different DesAb constructs were then expressed and purified using Ni²⁺ chromatography, as previously described³¹. Imidazole was finally removed using size exclusion chromatography with a HiLoad 16/600 Superdex 75 pg column (GE Healthcare, Chicago, IL, USA). Protein concentration was determined by absorbance measurement at 280 nm using theoretical extinction coefficients calculated with ExPASy ProtParam⁴⁴. Ab40 (Ml- 40) and Ab42 (Ml- 42) peptides were expressed in E. coli BL21 (DE3) Gold Strain (Agilent Technologies, Santa Clara, CA, USA) and purified as described previously⁸. Aliquots of purified Ab42 and Ab40 were lyophilized and stored at -80 °C.

Circular dickroism (CD)

Far-UV CD spectra of the DesAbs were recorded using a Jasco J-810 spectropolarimeter equipped with a Peltier holder (Jasco UK Ltd, Great Dunmow, UK), using a 0.1 -cm-pathlength cuvette. Samples contained 10 mM protein in 20 mM Tris pH 7.4, 100 mM NaCl. The far-UV CD spectra of all DesAbs were recorded from 200 to 250 nm at 20 °C, and the spectrum of the buffer was systematically subtracted from the spectra of all DesAbs.

Aggregation conditions for Ab42

The lyophilized Ab42 peptide was dissolved in 6 M urea pH 8 and incubated for 3 h at room temperature. This protein solution was then subjected to an additional gel filtration chromatograpy using a Superdex 75 10/300 GL column (GE Healthcare, Chicago, IL, USA), and the peak corresponding to the monomeric Ab42 peptide was collected in low-binding test tubes (Coming, New York, NY, USA) on ice⁸. Monomeric Ab42 peptides were aggregated at a protein concentration of 3 mM in 20 mM sodium phosphate buffer (pH 8), 200 mM EDTA under quiescent conditions. ELISA-based binding screening of the antibodies

20 ml Aliquots were taken at the tso (i.e. half-time) of aggregation from aggregation reactions prepared as described above (see Aggregation conditions for Ab42). Samples were then immobilised on a 96 or 384 well Maxisorp ELISA plate (Nunc, Roskilde, Denmark) with no shaking for 1 h at room temperature. The plate was then washed three times with 20 mM Tris pH 7.4, 100 mM NaCl and incubated in 20 mM Tris pH 7.4, 100 mM NaCl, 5% BSA under constant shaking overnight at 4 °C. The day after the plate was washed six times with 20 mM Tris pH 7.4, 100 mM NaCl and then incubated with 30 ml solutions of 5 mM DesAb-0 under constant shaking either for 1 hour or overnight at room temperature. At the end of this incubation, the plate was washed six times with 20 mM Tris pH 7.4, 100 mM NaCl and incubated with 30 ml solutions of Rabbit polyclonal to 6X His tag® HRP conjugated (Abeam, Cambridge, UK) at a dilution of 1 :4000 in 20 mM Tris pH 7.4, 100 mM NaCl, 5% BSA under shaking for 1 hour at room temperature. Finally the plate was washed 3 times with 20 mM Tris pH 7.4, 100 mM NaCl, then twice with 20 mM Tris pH 7.4, 100 mM NaCl, 0.02% Tween-20 and again three times with 20 mM Tris pH 7.4, 100 mM NaCl. Finally, the amount of bound DesAb-0 was quantified by using 1- Step™ Ultra TMB-ELISA Substrate Solution (ThermoFisher Scientific, Waltham, MA, United States), according to manufacturer instructions, and measuring the absorbance at 450 nm by means of a CLARIOstar plate reader (BMG Labtech, Aylesbury, UK).

Preparation for ThT/AF647- DesAb-0 fluorescence imaging

Aliquots were taken from aggregation reactions at 20 min intervals and imaged immediately. Borosilicate coverslips (22x22mm, 630-2186, VWR, Radnor, PA, USA) were cleaned for 1 hour using argon plasma (PDC-002, Harrick Plasma). Frame-Seal slide chambers (9 x 9 mm, Bio-Rad, Hercules, SLF-0201) were affixed to the glass, and poly-L- lysine (50 ml, 70-150 kDa, Sigma-Aldrich, P4707-50 ML) was added to the coverslip and incubated for 30 minutes and then washed three times with filtered (0.2 mm syringe filter, 6780-1302, Whatman, Maidstone, UK) PBS buffer. A working solution containing

AF647-DesAb-0 (1 nM) and ThT (5 mM) in filtered (0.2 mm syringe filter, 6780-1302, Whatman, Maidstone, UK) PBS (pH 7.4) was prepared 5 minutes prior to imaging. Ab aliquots were diluted (500 nM), incubated on the poly-l-lysine coated coverslip for 5 minutes and then washed twice with filtered PBS. The DesAb-ThT working solution (50 mL) was added to the cover-slide and incubated for 2 minutes prior to imaging.

DesAb-ThT co-localization imaging Imaging experiments were carried out with bespoke total internal reflection fluorescence (TIRF) inverted microscope (Eclipse TE2000-U, Nikon) fitted with a Perfect Focus unit. Excitation of ThT and AF647 was achieved with either a 405 nm laser (LBX- 405-50-CIR-PP, Oxxius, Lannion, France) or 641 nm laser (Cube, 1150205, Coherent) respectively. The beams were aligned parallel to the optical axis and directed into an oil immersion objective lens (1.49 NA, 60x, Plan Apo, TIRF, Nikon, Tokyo, Japan) above the critical angle to ensure TIR at the coverslip-sample (glass/water) interface. Fluorescence emission was also collected by the same objective and selected by the presence of a dichroic (Di01-R405/488/561/635, Semrock, Rochester, NY, USA) and subsequently passed through appropriate emission filters (BLP01 -488R-25, FF01 -480/40-25, FF01 -

676/37-25, Semrock, Rochester, NY, USA). Image stacks of the AF647 and ThT emission channels were collected by sequential excitation of AF647 followed by ThT. Images were recorded by an electron multiplying charged coupled device (Evolve delta 512, Photometries, Tucson, A Z, USA) with an electron multiplication gain of 250 ADU/photon running in frame transfer, clear pre-sequence mode. Each pixel on the image was 237 nm. Images from 27 different fields of view were recorded at 50 ms for 200 frames in each emission channel using a custom beanshell script through Micro-manager software (v. 1.4)

⁴⁵ Fluorescence image analysis

Co-localization data was analyzed with a bespoke Image! ⁴⁶ macro. Separate average intensity z-projections of ThT and AF647 channels were created which results in single frame images representing the mean pixel intensities calculated for the total image stack. Following this, points of intensity above a background threshold were located, counted and binarized. Pixels with a value of 1 in both the AF647 and ThT images were identified as coincident points. Chance coincident spots were extracted by performing a 90° rotation of the AF647 binary image and subtracted from the total coincidence value. Percentage coincidence was calculated with the equation below:

Atomic force microscopy (AFM)

AFM measurements were performed in air of the sample deposited on glass, where TIRF measurements have been acquired. High-resolution images (1024x1024 pixels) and phase controlled³⁶ were collected using an NX10 Atomic Force Microscope (Park Systems, South Korea) under ambient conditions and in amplitude modulation non-contact (NC- AM) mode. Square areas of 2x2 mm² and 4x4 mm² were imaged. All the measurements using sharp cantilevers (PPP-NCHR, Park Systems, South Korea) with resonance frequency of 330 kHz and a typical apical radius of 8 nm. The raw images were flattened using the built-in software (XEI, Park System, South Korea). To maintain consistency in the subsequent statistical analysis, all images were processed using the same parameters. The images were first flattened by a plane and then line-by-line in 1st regression order.

This second step was repeated until a flat baseline was obtained in line profile of the image was reached. During the process of flattening of the images, the aggregates were masked from the calculation to avoid modification and underestimation of their heights.

ThT aggregation assay of Ab42

Aggregation experiments were performed in the presence of 20 mM ThT under quiescent conditions at 37 °C using a CLARIOstar plate reader (BMG Labtech, Aylesbury, UK). ThT fluorescence was measured through the bottom of the plate every minute with an excitation filter of 440 nm and an emission filter of 480 nm.

ELISA with aliquots from aggregation reactions

20 ml Aliquots were taken at specific incubation times and immobilised on a 96 or 384 well Maxisorp ELISA plate (Nunc, Roskilde, Denmark) with no shaking for 1 h at room temperature. Experiments were then performed as described above ( ELISA-based screening of the antibodies). Experiments in the presence of the compound bexarotene were performed using the same protocol on aggregation reactions supplemented with 5 molar excess bexarotene.

Strains of C elegans The following strains of C. elegans were used: dvIs100 [unc-54p::A-beta-l- 42::unc-54 3'-UTR + mtl-2p::GFP] (GMC101), which produces constitutive expression of GFP in intestinal cells; unc-54p::A-beta-l-42 which expresses full-length human Ab42 peptide in body-wall muscle cells that aggregates in vivo ; shifting L4 or young adult animals from 20 °C to 25 °C causes paralysis⁴⁰. C. elegans var Bristol (N2) was used as a control strain. Generation time is about 3 days. Isolated from mushroom compost near Bristol, England⁴⁷.

Media for C. elegans

Standard conditions were used for the propagation of C. elegans⁴⁷. Briefly, the animals were synchronized by hypochlorite bleaching, hatched overnight in M9 (3 g/l KH2PO₄, 6 g/l Na₂HPO₄, 5 g/l NaCl, 1 mM MgSO₄) buffer, and subsequently cultured at 20 °C on nematode growth medium (NGM) (CaCl2 ImM, MgSO₄ 1 mM, cholesterol 5 mg/ml, 250 mM KH2PO₄ pH 6, Agar 17 g/L, NaCl 3 g/l, casein 7.5g/l) plates seeded with the E. coli strain OP50. Saturated cultures of OP50 were grown by inoculating 50 mL of LB medium (tryptone 10 g/l, NaCl 10 g/l, yeast extract 5 g/l) with OP50 and incubating the culture for 16 h at 37 °C. NGM plates were seeded with bacteria by adding 350 ml of saturated OP50 to each plate and leaving the plates at 20 °C for 2-3 days. On day 3 after synchronization, the animals were placed on NGM plates containing 5-fluoro-2'deoxy-uridine (FUDR)

(6.83 nM, unless stated otherwise) to inhibit the growth of offspring. FUDR plates were seeded with bacteria by adding 350 ml of lOx concentrated OP50 solution to ensure starvation did not occur for the lifespan of the worm. Concentrated OP50 solution was obtained by centrifuging 1 L of saturated OP50 culture at 5000 RPM for 15 minutes and suspending the resultant pellet in 100 ml sterile water.

Staining of the aggregates using the fluorescent probe NIAD-4

To visualize the amount of aggregates in the worms, live transgenic were incubated with 1 mM NIAD-4 (0.1% dimethyl sulfoxide in M9 buffer) for 4 hours at room temperature. After staining, animals were allowed to recover on NGM plates for about 24 hours to allow destaining via normal metabolism. Stained animals were mounted on 2% agarose pads containing 40 mM NaN₃ as anesthetic on glass microscope slides for imaging. Images were captured using a Zeiss Axio Observer D1 fluorescence microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) with a 20* objective and a 49004 ET-CY3/TRITC filter (Chroma Technology Corp., Rockingham, VT, USA). Fluorescence intensity was calculated using ImageJ software (National Institutes of Health) and then normalized as the corrected total cell fluorescence. Only the head region was considered because of the high background signal in the guts. All experiments were carried out in triplicate, and the data from one representative experiment are shown.

Automated motility assay on agar plates and imaging of the aggregates

All C. elegans populations were cultured at 20 °C and developmental^ synchronized from a 4-hour egg lay. At 64 to 72 hours after egg lay (time zero), individuals were shifted to 24 °C and transferred to FUDR plates and body movements were assessed over the times indicated. Videos were analysed using a custom-made tracking code. ELISA with C elegans protein extracts

GMC101 and N2 worms were incubated on FUDR plates until DO-1-3-5-7-10 of adulthood, according to what reported in the text for each experiment. At a specific time, the worms were washed out of the plates and snap frozen using liquid nitrogen. For sample preparation, around 1000 animals were thaw and resuspended in 500 ml PBS supplemented with EDTA-free protease inhibitor tablets IX (Roche, Basel, Switzerland) after which they were sonicated (5 x 45 sec, 50% cycles, 50% maximum power on ice) using a Bandelin Sonopuls HD 2070 (Bandelin, Berlin, Germany). After sonication, the lysates were centrifuged at maximal speed using a bench centrifuge and 20 ml of supernatant (5 mg) were loaded on a 96 Maxisorp ELISA plate (Nunc, Roskilde, Denmark) with no shaking overnight at 4°C. ELISAs were conducted as described above (see ELISA- based screening of the antibodies). Absorbance values obtained for GMC101 worms we normalised over those obtained for the N2 control mice.

Use and care of the mice

Experiments were performed according to the Animals Scientific Procedures Act UK (1986). T g(PDGFB-APPSwInd)20Lms/2Mmj ax (J20) heterozygous mice were housed at University College London, maintained on a 12-hour light/dark cycle and provided with food and water ad libitum. 16-18 months old female mice transgenic or non-transgenic littermates were used for this experiment (n=3 per group).

Immunofluorescence staining

J20 mice brains were collected, immediately fixed overnight at 4 °C with 4% paraformaldehyde in PBS, then washed twice with PBS and immersed in 30% sucrose/PBS. Subsequently, brain samples were frozen in isopentane on dry ice, 50 mm sections were cut on a cryostat and stored in ethylene glycol based cryoprotectant (30% glycerol, 30% ethylene glycol in 0.1 M sodium/potassium phosphate buffer, pH 7.4). Brain sections were incubated with 70% and 80% Ethanol for 1 minute, and incubated in NIAD-4 solution (1% in 80% of Ethanol) for 15 minutes. The slices were then incubated with 80% and 70% Ethanol for 1 minute and washed twice with distilled water. Finally, brain slices were mounted in Fluoromount-G (SouthemBiotech, Birmingham, AL, USA) on a glass slide for imaging. Images of the hippocampus area were captured using Leica SP8 confocal microscope. Images were taken using a 20x (NA 0.72) dry objective, with 1024 x 1024 image resolution and 15 z-steps of 0.5 mm.

ELISA with mouse protein extracts

30 ml Aliquots (5 mg) were immobilised on a 96 Maxisorp ELISA plate (Nunc, Roskilde, Denmark) with no shaking overnight at 4 °C. Experiments were then performed as described above ( ELISA-based screening of the cmtibodies). Absorbance values obtained for J20 mice we normalised over those obtained for the wild-type control mice.

Ab40 oligomer preparation

Lyophilized Ab40 (1 mg/ml) was solubilized overnight in 300 ml HFIP. Solvent was gently evaporated off with nitrogen, and protein resuspended in 100% DMSO. Two sonication steps of 10 minutes were preformed, after which the protein was resuspended at 100 mM in 20 mM sodium phosphate buffer, 200 mM ZnCl2, pH 6.9 for 20 hours at 20 °C. Samples were centrifuged (15,000 ref, 20 °C, 15 minutes) and the supernatant was removed. Oligomers were resuspended in buffer (20 mM tris, 100 mM NaCl) with thorough mixing. ANS binding measurements

10 mM oligomers (in monomer equivalents) were incubated for two hours in the absence and presence of an equimolar concentration of the different DesAbs at 20 °C. Subsequently, ANS was added to a final concentration of 30 mM (i.e. 3-fold excess dye). Emission spectra were recorded using a plate reader (BMG Labtech, Aylesbury, UK) with excitation at 380 nm. Duplicate samples are shown representative of three independent experiments that gave consistent results.

Isothermal titration calorimetry (ITC)

Measurements were performed on a MicroCal iTC200 (Malvern Panalytical Ltd, Malvern, UK) at 25 °C. The DesAb-0 (90 mM) was injected 16 times into a solution containing 10 mM of either monomeric Ab40 or oligomers formed from a stock solution of 10 mM monomeric Ab40. All solutions were 20 mM Tris pH 7.4, lOOmMNaCl. Solutions for the experiments involving monomeric Ab40 contained 0.05% DMSO to ensure that the peptide was indeed in it monomeric form. Each injection was 2 ml and occurred on 3- minute intervals. Heats of dilution, obtained by separately injecting the antibody into buffer (both with and without DMSO) and buffer into either the monomer or oligomer solution, were subtracted from the final data. The corrected heats were divided by the number of moles injected and analysed using Origin 7.0 software (OriginLab, Northampton, MA, USA).

ELISA with Ab40 oligomers

40 ml of 5 mM DesAb solutions were used to coat the wells of a 96 well Maxisorp ELISA plate (Nunc, Roskilde, Denmark) with no shaking for 1 h at room temperature. The plate was then washed three times with PBS and incubate in PBS, 5% BSA under constant shaking over night at 4 °C. The day after the plate was washed six times with PBS and then incubated with 40 ml solutions of 5 mM oligomers in PBS under constant shaking either for 1 hour at room temperature or over night at 4 °C. At the end of this incubation, the plate was washed six times with PBS and incubated with 40 ml solutions of the mouse monoclonal anti-amyloid b Antibody [6E10] (1 :2000 dilution; Absolute Antibody Ltd, Redcar, UK). After 6 additional washes with PBS, 0.02% Tween-20, the plate was incubated with 40 ml of goat anti-mouse IgG (H+L) secondary antibody and HRP conjugate (1:2,000 dilution; Life Technologies, Carlsbad, CA, USA) in PBS, 5% BSA for 1 h at room temperature. Finally, the plate was washes 6 times with PBS, 0.02% Tween- 20 and bound oligomers quantified by using 1-Step™ Ultra TMB-ELISA Substrate Solution (ThermoFisher Scientific, Waltham, MA, United States), according to manufacturer instructions, and measuring the absorbance at 450 nm by means of a

CLARIOstar plate reader (BMG Labtech, Aylesbury, UK).

Dot-blot with Ab40 oligomers

4 and 2 mM solutions of DesAb-0 and were spotted (3.5 ml) on a 0.2 mm pore size nitrocellulose membrane (GE Healthcare, Chicago, IL, USA). As negative control, samples with same amounts of Des Ab-P Y GSMYVHS, which showed the weakest binding to Ab40 oligomers in in the ANS binding measurements, were used. The blots were blocked in PBS, 5% BSA over night at 4 °C. Then, they were incubated in solutions containing 5 mM Ab40 oligomers in PBS over night at 4 °C. Blots with bound oligomers were then probed with the mouse monoclonal anti-amyloid b Antibody [6E10] (1 :2000 dilution; Absolute Antibody Ltd, Redcar, UK) and with goat anti-mouse IgG (H+L) secondary antibody and Alexa Fluor 488 conjugate (1:5,000 dilution; Life Technologies, Carlsbad, CA, USA). References

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Sequence listing

Claims

1. An Ab binding molecule comprising the sequence of SEQ ID NO: 4.

2. The binding molecule of claim 1, which is an antibody.

3. The binding molecule of claim 2, which is a human, humanised or chimeric antibody.

4. The binding molecule of claim 2 or 3, wherein the antibody is a single-domain antibody.

5. The binding molecule of claim 4, which is a human single-domain antibody, a camelid single-domain antibody or a shark single-domain antibody.

6. The binding molecule of claim 2 or 3, wherein the antibody is a scFv, Fab, modified Fab, Fab’, modified Fab’, F(ab’)2, Fv, dAb-Fc, Fd, dsFv, ds-scFv, scFv2 or scFv-

Fc.

7. The binding molecule of claim 2 or 3, wherein the antibody is a minibody, diabody, triabody or tetrabody.

8. The binding molecule of claim 2 or 3, wherein the antibody is multi-specific.

9. The binding molecule of any one of the preceding claims, which comprises a sequence with at least 90% identity to SEQ ID NO: 5.

10. The binding molecule of any one of the preceding claims, which comprises the sequence of SEQ ID NO: 5.

11. The binding molecule of claim 1, which is an antibody mimetic.

12. The binding molecule of claim 11, wherein the antibody mimetic is a DARPin, a peptide aptamer, an affibody, an affimer, an affitin, an alphabody, an adnectin, an anticalin, a kunitz domain inhibitor scaffold, an avimer, a knottin, a fynomer, an atrimer, a bicyclic peptide, an affilin, a monobody, a nanoCLAMP an OBody or a cytotoxic T-lymphocyte associated protein-4 (CTLA4) based binder.

13. A proteolysis targeting chimera comprising the binding molecule of any one of claims 1-12.

14. An isolated polynucleotide encoding a binding molecule of any one of claims 1-12.

15. The isolated polynucleotide of claim 14, which encodes a V_H region of an antibody of any one of claims 2-10.

16. A vector comprising the polynucleotide of claim 15, or a host cell comprising the vector.

17. Use of a binding molecule of any one of claims 1-12 in detecting and/or quantifying Ab oligomers.

18. A method of detecting and/or quantifying the numbers of Ab oligomers in a patient, the method comprising contacting a sample from the patient with a binding molecule of any one of claims 1-12 and/or administering a binding molecule of any one of claims 1-12 to the patient.

19. The method of claim 18, wherein the presence and/or number of Ab oligomers is used to identify a patient at risk of, in the preclinical phase of, or suffering from, an amyloid disease.

20. The method of claim 19, wherein the amyloid disease is Alzheimer’s disease, Lewy body dementia, cerebral amyloid angiopathy, inclusion body myositis, Down syndrome, a cancer associated with Ab or an ophthalmological disease associated with Ab, such as dry age-related macular degeneration (AMD) or glaucoma.

21. A method of selecting an anti-amyloid drug for a patient, the method comprising (i) contacting a sample from the patient with a binding molecule of any one of claims 1-12 and/or (ii) and administering a binding molecule of any one of claims 1-12 to the patient, and selecting an anti-amyloid drug for the patient based on the presence or absence of oligomers.

22. The method of claim 21, wherein patient has been diagnosed with, or is at risk of, Alzheimer’s disease, Lewy body dementia, cerebral amyloid angiopathy, inclusion body myositis, Down syndrome, an ophthalmological disease associated with Ab such as dry age-related macular degeneration (AMD) or glaucoma, or a cancer associated with Ab.

23. The method of any one of claims 18-22, wherein the binding molecule is labelled with a detectable label.

24. The method of any one of claims 18-23, wherein the sample is a cerebrospinal fluid sample, a nasal wash or a plasma sample.

25. Use of a binding molecule of any of claims 1-12 in testing the ability of an antiamyloid drug candidate to reduce the number of Ab oligomers.

26. A method of assessing Ab oligomer inhibition by an anti-amyloid drug candidate, the method comprising monitoring Ab oligomers in the presence of a binding molecule of any one of claims 1-12 and an anti-amyloid drug candidate.

27. The method of claim 26, wherein a drug candidate is identified as an inhibitor if the drug delays, reduces or prevents the formation of oligomers.

28. Use of a binding molecule of any one of claims 1-12 in delivering a drug to Ab oligomers.

29. A method of therapy comprising administration of a binding molecule of any one of claims 1-12 to a patient in need thereof.

30. A method of delaying, preventing onset or progression of an amyloid disease, said method comprising administration of a binding molecule of any one of claims 1-12 to a patient in need thereof.

31. The method of claim 29 or 30, wherein the binding molecule is conjugated to an anti-amyloid drug.

32. The method of claim 30 or 31, wherein the amyloid disease is Alzheimer’s disease, Lewy body dementia, cerebral amyloid angiopathy, inclusion body myositis, Down syndrome, a cancer associated with Ab or an ophthalmological disease associated with Ab such as dry age-related macular degeneration (AMD) or glaucoma.

33. The use of claim 28 or the method of any one of claims 29-32, wherein the binding molecule forms part of a proteolysis targeting chimera.