BLOOD-BRAIN BARRIER TRANSMIGRATING COMPOUNDS AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to compounds that transmigrate the blood-brain barrier, and uses thereof. More specifically, the present invention relates to compounds that may comprise an antibody or fragment thereof that crosses the blood-brain barrier, an immunoglobulin Fc domain or fragment thereof, and a polypeptide binding to beta-amyloid, fusion proteins and compositions thereof and their use in the treatment of Alzheimer's disease.

BACKGROUND OF THE INVENTION

Neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, are an increasing burden on our ageing society because there are currently no effective treatments for these disabling conditions. Alzheimer's disease (AD) is an irreversible neurodegenerative disorder affecting approximately 15% of the population over 65 years of age and is the predominant cause of progressive intellectual and cognitive failure in the ageing population (Hardy et al, 2014).

In AD, there is a severe loss of cholinergic neurons with a consequent decline in the levels of acetylcholine (ACh), a key neurotransmitter involved in memory processing and storage. In addition, excitotoxicity induced by neurotransmitter glutamate is also implicated in the pathogenesis of AD. Therefore, cholinergic augmentation and/or inhibition of glutamate toxicity might improve cognition in AD. Indeed, the only FDA approved drugs for the treatment of AD are acetylcholine esterase (AChE) inhibitors (e.g., donepezil, rivastigmine, galantamine) to prevent the loss of ACh and inhibitors of specific glutamate receptors (e.g., memantine) (Mangialasche et al, 2010; Ji and Ha, 2010; Savonenko et al, 2012). However, the beneficial effects of these symptomatic drugs are limited and transient, providing temporary improvement in cognitive functions and do not stop the progression of the disease. While other treatments including antioxidants, anti-inflammatory drugs (NSAIDS), cholesterol-lowering drugs and estrogen therapy are considered, none of these treatments appear to have any long-term beneficial effects, particularly in improving memory and cognitive function in AD patients (Magialasche et al, 2010; Ji and Ha, 2010). A major hallmark of Alzheimer's disease is the accumulation of a 39-43 amino acid peptide β- amyloid (Αβ) in the brain in the form of aggregates and plaques. A considerable body of evidence based on genetic, pathological and biochemical studies indicate that Αβ, particularly its oligomeric aggregates, plays a central role in the development of AD pathology (Hardy et al, 2014; DeLaGarza, 2003; Selkoe and Hardy, 2016). According to amyloid hypothesis, a chronic imbalance in the production and clearance of Αβ in the brain results in its accumulation and aggregation with ageing. These Αβ aggregates are believed to initiate a cascade of events leading to synaptic loss and neuronal functions, leading to a progressive loss of memory and other cognitive functions (Hardy et al, 2014; DeLaGarza, 2003; Selkoe and Hardy, 2016; Sengupta et al, 2016).

The generation of Αβ from its precursor protein APP is achieved by the sequential proteolysis of APP by proteases β and γ secretases (Barageb and Sonawane, 2015). Inhibitors of these enzymes have been shown to reduce Αβ production and are being developed as potential drugs for treating AD (Hardy et al, 2014; Mangialasche et al, 2010; Selkoe and Hardy, 2016; Ji and Ha, 2010). Similarly, agents that sequester and/or promote Αβ clearance are also being developed. Notable among these are the development of immunotherapies with AD vaccine. Both active (Αβ peptides) and passive immunization (Αβ-antibodies) have been shown to be effective in preventing amyloid deposition as well as clearing of preformed amyloid plaques in transgenic animal models of AD and in clinical trials involving AD patients (Mangialasche et al, 2010; Ji and Ha, 2010; Morrone et al, 2015; Lannfelt et al, 2014; Selkoe and Hardy, 2016; Goure et al, 2014). Inhibitors of β and γ secretases that prevent proteolytic cleavage of amyloid precursor protein (APP) and thereby reduce or suppress brain Αβ production are being developed (e.g., tarenflurbil, semagacestat, verubecestat). However, their therapeutic efficacy in reducing Αβ burden is not yet known and many of these drugs have failed in pre-clinical or clinical trials (Savonenko et al, 2012; Musiek and Holtzman, 2015). Moreover, since these enzymes are also involved in the processing of other enzymes and signaling molecules such as Notch that are linked to neuronal development (Savonenko et al, 2012; Musiek and Holtzman, 2015), these inhibitors may have serious non-specific side effects.

Immunotherapeutic approaches such as active (Αβ vaccine, AN1792) and passive immunization (e.g., Bapineuzumab, Solanezumab, Crenezumab, aducanumab etc) have been shown to be quite effective in reducing Αβ deposition and partial elimination of memory deficits in transgenic animals (Monsonego and Weiner, 2003; Bard et al, 2000, Sevigny J et al., 2016). Several clinical trials using both active and passive immunization have shown reduction in brain Αβ deposition with moderate improvement in cognition. However, clinical trials had to be abandoned due to severe inflammatory reactions (meningo-encephalitic presentation), vasogenic edema, and micro-haemorrhages in AD patients. Despite these limitations, the immunotherapy approach indicates that agents that effectively sequester Αβ, and prevent its deposition and toxicity, could potentially serve as effective drugs in arresting the progression of AD, and even prevent its development (Rafii and Aisen, 201 5, Selkoe and Hardy, 201 6).

Treatment as well as early diagnosis of AD and other diseases that originate in the brain remain challenging because the majority of suitable therapeutic molecules and diagnostics cannot penetrate the tight and highly restrictive blood-brain barrier (BBB) (Abbott, 201 3). The BBB constitutes a physical barricade that is formed by brain endothelial cells (BECs) that line the blood vessels and connect with each other through tight junctions (Abbott, 201 3). The tight junctions formed between the BECs are essential for the integrity of the BBB and prevent the paracellular transport of molecules larger than 500 daltons (Da). Because brain endothelial cells exhibit very low pinocytosis rates (Abbott, 201 3), transcellular transport of larger molecules is limited to the highly specific receptor mediated transcytosis (RMT) pathway, and the passive, charge-based adsorption mediated transcytosis (Abbott, 201 3; Pardridge, 2002). Additionally, the high density of efflux pumps, such as P-glycoprotein or the multi-drug resistance protein -1 (MDR-1 ), contribute to the removal of unwanted substances from the brain (Abbott, 201 3).

While all these characteristics protect the brain from pathogens and toxins, they equally prevent the entry of most therapeutics. In fact, less than 5% of small molecule therapeutics and virtually none of the larger therapeutics can cross the BBB in pharmacologically relevant concentrations (i.e., sufficient to engage a central nervous system (CNS) target and elicit pharmacologic/therapeutic response) unless they are specifically 'ferried', that is, coupled to a transporter molecule. Due to the lack of effective 'carriers' to transport molecules across the BBB, numerous drugs against neurodegenerative diseases have been 'shelved' or eliminated from further development as they cannot be delivered to the brain in sufficient amount. Despite considerable progress in understanding the molecular mechanism of AD pathology, there are no effective drugs or treatments currently available that can prevent the progression of or cure the disease. Furthermore, the lack of high-capacity and high-selectivity BBB carriers delays the development of new therapeutics and diagnostics for diseases originating in the brain, including brain tumors and neurodegenerative diseases. SUMMARY OF THE INVENTION

The present invention relates to compounds or compositions that transmigrate the blood-brain barrier, and uses thereof. The present invention provides polypeptides which binds beta-amyloid (β-amyloid). The polypeptides (or proteins) that bind β-amyloid may selectively bind pathologically relevant β- amyloid (Αβ^) aggregates, and may be abbreviated and referred to herein as ABP or ABP variants. The present invention provides fusion proteins (also referred to herein as compounds, compositions or constructs) comprising a beta-amyloid (β-amyloid) binding polypeptide (ABP or ABP variant) linked to an antibody or fragment thereof that crosses the blood-brain barrier (BBB), wherein BBB herein refers to an abbreviation for the carrier antibody or fragment that transmigrates the blood brain barrier. In a preferred embodiment, the fusion protein comprising an ABP or ABP variant and a BBB carrier, or fragment thereof, wherein the ABP and BBB components of the fusion protein may be linked via an Fc region or portion thereof. In a preferred embodiment, the fusion protein comprising an ABP or ABP variant and a BBB, further comprises an immunoglobumin protein effector domain, known as Fc, or fragment thereof, wherein the ABP and BBB components of the fusion protein may be linked via the Fc region or portion thereof. For example, a construct of the present invention may further comprise a linker (L), wherein L is a small linking peptide or peptide-like chain. For example, the BBB-Fc-L-ABP construct provided may be a single chain polypeptide or a dimeric polypeptide, wherein the single chain polypeptide comprising BBB-Fc-L-ABP may form a dimer by Fc dimerization. Fc dimerization may be mediated by interaction of a large tightly packed hydrophobic interface between two Fc CH3 domains. For example, a construct of the present invention may comprise BBB-Fc-ABP. The BBB-Fc-ABP construct provided may be a single chain polypeptide (monovalent) or a dimeric polypeptide (bivalent), wherein the single chain polypeptide comprising BBB-Fc-ABP may form a dimer through Fc which allows the dimerization of the fusion protein. A compound of the present invention may be referred to as a fusion protein, construct, fusion molecule, formulation or composition.

The compounds of the present invention may be used in the treatment of Alzheimer's disease (AD).

The polypeptide that binds β-amyloid may comprise a sequence selected from the group consisting of: SGKTEYMAFPKPFESSSSIGAEKPRNKKLPEEEVESSRTPWLYEQEGEVEKPFIKTGFS VSVEKSTSSNRKNQLDTNGRRRQFDEESLESFSSMPDPVDPTTVTKTFKTRKASAQAS LASKDKTPKSKSKKRNSTQLKSRVKNITHARRILQQSNRNACNEAPETGSDFSMFEA

(SEQ ID NO:27); FSSMPDPVDPTTVTKTFKTRKASAQASLASKDKTPKSKSK (SEQ ID NO:28);

KDKTPKSKSKKRNSTQLKSRVKNITHARRILQQSNRNACN (SEQ ID NO:29);

KTFKTRKASAQASLASKDKTPKSKSKKRNSTQLKSRVKNI (SEQ ID NO:30);

The polypeptide that binds β-amyloid may be a variant that comprises a sequence: X ₁ TFX ₂ TX ₃ X ₄ ASAQASLASKDKTPKSKSKKX ₅ X ₆ STQLX ₇ SX ₈ VX ₉ NI where X _! =G or A, X ₂ = G or V, X ₃ = G or A, X ₄ = G or A, X ₅ = G or V, X ₆ = G or V, X ₇ = G or V, X ₈ = G or A, X ₉ = G or A (SEQ ID NO: 31)

In specific non-limiting embodiments, the ABP, or its variants, may comprise a sequence selected from any one of: KTFKTRKASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI (SEQ ID NO:32);

KTFKTRKASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI (SEQ ID NO:33);

KTFKTRGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI (SEQ ID NO:34);

KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI (SEQ ID NO:35);

GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI (SEQ ID NO:36); KTFKTRKASAQASLASKDKTPKSKSKKGGSTVKNI (SEQ ID NO:37);

KTF KTRKAS AQ AS LAS KD KTP KS KS KKRG (SEQ ID NO:38); and a sequence substantially identical to any of the above sequences.

The polypeptide that binds β-amyloid (ABP) variant may comprise a polypeptide sequence selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38. An ABP comprising a polypeptide sequence selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38 may be referred to as an ABP or ABP variant. The present invention additionally comprises an ABP variant sequence having consensus sequence SEQ ID NO: 31 , and may be for example SEQ ID NO: 35, or SEQ ID NO: 36, or any equivalent^ stable polypeptide sequence. An equivalents stable polypeptide sequence exhibits peptide stability during expression and production of fusion protein in mammalian expression system. For example, an ABP variant of the present invention may exhibit improved peptide stability over ABP peptides of the prior art (Ref. WO2006/133566).

The present invention provides an isolated polypeptide that binds β-amyloid, the isolated polypeptide that binds β-amyloid may comprise a sequence selected from the group consisting of SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, or a sequence comprising an equivalently stable polypeptide sequence.

The present invention provides a fusion protein comprising an ABP or ABP variant selected from the group consisting of SEQ ID NO: 31 , SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38. In a non-limiting example, the ABP variant may comprise the sequence KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI (SEQ ID NO:35); GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI (SEQ ID NO:36).

A fusion protein of the present invention may comprise an antibody, or fragment thereof, that transmigrates across the blood brain barrier (BBB) (as noted above, BBB is an abbreviation of the antibody carrier that transmigrates the blood brain barrier). The antibody, or fragment thereof, (BBB) may bind surface receptor epitopes on brain endothelial cell that allow for transmigration across the blood brain barrier. For example, such surface receptor epitopes may be TMEM30A or an Insulin-Like Growth Factor 1 Receptor (IGF1 R) epitope, or isoforms, variants, portions, or fragments thereof.

The antibody, or fragment thereof, may comprise a sequence selected from the group consisting of: an antibody or fragment thereof comprising a complementarity determining region (CDR) 1 sequence of GFKITHYTMG (SEQ ID NO:1), a CDR2 sequence of RITWGGDNTFYSNSVKG (SEQ ID NO:2), a CDR3 sequence of GSTSTATPLRVDY

(SEQ ID NO:3); an antibody or fragment thereof comprising CDR1 sequence of EYPSNFYA (SEQ ID NO:4), a CDR2 sequence of VSRDGLTT (SEQ ID NO:5), a CDR3 sequence of AIVITGVWNKVDVNSRSYHY (SEQ ID NO:6); an antibody or fragment thereof comprising CDR1 sequence of GGTVSPTA (SEQ ID

NO:7), a CDR2 sequence of ITWSRGTT (SEQ ID NO:8), a CDR3 sequence of AASTFLRILPEESAYTY (SEQ ID NO:9); and an antibody or fragment thereof comprising CDR1 sequence of GRTIDNYA (SEQ ID NO:10), a CDR2 sequence of IDWGDGGX; where X is A or T (SEQ ID NO:1 1 ), a CDR3 sequence of AMARQSRVNLDVARYDY (SEQ ID NO:12).

The antibody or fragment thereof may comprise a sequence selected from the group consisting of:

X ₁ VQLVX ₂ SGGGLVQPGGSLRLSCAASGFKITHYTMGWX ₃ RQAPGKX ₄ X ₅ EX ₆ VSRITW GGDNTFYSNSVKGRFTISRDNSKNTX ₇ YLQMNSLRAEDTAVYYCAAGSTSTATPLRVD YWGQGTLVTVSS (SEQ ID NO:13), where X^D or E, X ₂ =A or E, X ₃ =F or V, X ₄ =E or G, X ₅ =R or L, X ₆ =F or W, X ₇ =L or V; X _! VX ₂ LX ₃ ESGGGLVQX ₄ GGSLRLSCX ₅ ASEYPSNFYAMSWX ₆ RQAPGKX ₇ X ₈ EX ₉ VX ₁₀ G

VSRDGLTTLYADSVKGRFTX SRDNX^KNTX^XuLQMNSX^XieAEDTAVYYCAIVITG VW KVDVNSRSYHYWGQGTX ₁₇ VTVSS, (SEQ ID NO:18), where X _! is E or Q; X ₂ is K or Q; X ₃ is V or E; X ₄ is A or P; X ₅ is V or A; X ₆ is F or V; X ₇ is E or G; X ₈ is R or L; X ₉ is F or W; X ₁₀ is A or S; X is M or I; X ₁₂ is A or S; X ₁₃ is V or L; X ₁₄ is D or Y; X ₁₅ is V or L; X ₁₆ is K or R; and X ₁₇ is Q or L;

X ₁ VX ₂ LX ₃ ESGGGLVQX ₄ GGSLRLSCX ₅ X ₆ SGGTVSPTAMGWX ₇ RQAPGKX ₈ X ₉ EX ₁ oVX ₁₁ HIT SRGTTRXi2ASSVKXi3RFTISRDXi4Xi5KNTXieYLQMNSLXi ₇ Xi8EDTAVYYCAAS TFLRILPEESAYTYWGQGT X ₁₉ VTVSS, (SEQ ID NO:21 ), where X _! is E or Q; X ₂ is K or Q; X ₃ is V or E; X ₄ is A or P; X ₅ is A or E; X ₆ is V or A; X ₇ is V or F; X ₈ is G or E; X ₉ is L or R; X ₁₀ is F or W; X is G or S; X ₁₂ is V or Y; X ₁₃ is D or G; X ₁₄ is N or S; X ₁₅ is A or S;

X ₁₆ is L or V; X ₁₇ is K or R; X ₁₈ is A or S; and X ₁₉ is L or Q; and

X ₁ VX ₂ LX ₃ ESGGGLVQX ₄ GGSLRLSCAASGRTIDNYAMAWX5RQAPGKX ₆ X ₇ EX ₈ VX ₉ T IDWGDGGX^RYANSVKGRFTISRDNX KX^TX^YLQMNXuLX^X^EDTAVYX^CA MARQSRVNLDVARYDYWGQGTX ₁₈ VTVSS (SEQ ID NO: 24), where X _! is E or Q; X ₂ is K or Q; X ₃ is V or E; X ₄ is A or P; X ₅ is V or S; X ₆ is D or G; X ₇ is L or R; X ₈ is F or W; X ₉ is A or S; X ₁₀ is A or T; X is A or S; X ₁₂ is G or N; X ₁₃ is M or L; X ₁₄ is N or R; X ₁₅ is E or R; X ₁₆ is P or A; X ₁₇ is S or Y; and X ₁₈ is Q or L.

In specific non-limiting embodiments, the antibody, or fragment thereof, may comprise a sequence selected from any one of: DVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSRITWGGD NTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAGSTSTATPLRVDYWG KGTQVTVSS (SEQ ID NO:14); EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWVRQAPGKGLEWVSRITWGGD NTFYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG QGTLVTVSS (SEQ ID NO: 15)

EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWVRQAPGKGLEWVSRITWGGD NTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG QGTLVTVSS (SEQ ID NO:16);

EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEFVSRITWGGD NTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG QGTLVTVSS (SEQ ID NO:17);

QVKLEESGGGLVQAGGSLRLSCVASEYPSNFYAMSWFRQAPGKEREFVAGVSRDGL TTLYADSVKGRFTMSRDNAKNTVDLQMNSVKAEDTAVYYCAIVITGVWNKVDVNSRS YHYWGQGTQVTVSS (SEQ ID NO: 19);

EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKEREFVSGVSRDGL TTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSY HYWGQGTLVTVSS (SEQ ID NO: 20);

QVKLEESGGGLVQAGGSLRLSCEVSGGTVSPTAMGWFRQAPGKEREFVGHITWSR GTTRVASSVKDRFTISRDSAKNTVYLQMNSLKSEDTAVYYCAASTFLRILPEESAYTY WGQGTQVTVSS (SEQ ID NO: 22);

QVQLVESGGGLVQPGGSLRLSCAVSGGTVSPTAMGWFRQAPGKGLEFVGHITWSR GTTRYASSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAASTFLRILPEESAYTY WGQGTLVTVSS (SEQ ID NO: 23);

QVKLEESGGGLVQAGGSLRLSCAASGRTIDNYAMAWSRQAPGKDREFVATIDWGDG GARYANSVKGRFTISRDNAKGTMYLQMNNLEPEDTAVYSCAMARQSRVNLDVARYD YWGQGTQVTVSS (SEQ ID NO: 25);

QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPGKGLEWVATIDWGD GGTRYANSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCAMARQSRVNLDVARY DYWGQGTLVTVSS (SEQ ID NO: 26); and a sequence substantially identical to any of the above sequences.

The BBB may be an antibody, or fragment thereof, in a preferred embodiment, the BBB may be a single-domain antibody (sdAb). The sdAb may be humanized. In a preferred embodiment, the antibody or fragment thereof (BBB) may be linked to an Fc, or fragment thereof, wherein the BBB-Fc construct may form a dimer. The invention further provides a fusion peptide comprising BBB-Fc-L-ABP, wherein the BBB-Fc portion of the fusion peptide is linked to the ABP via a short peptidic linker (for example a linker having less than 12 amino acids) and the Fc or Fc fragment allows for the dimerization of said fusion peptides to provide dimers, which accordingly protects the construct from degradation and increases its serum half-life. The Fc fragment may be any suitable Fc fragment, selected in order to impart desirable pharmacokinetics, in which the Fc or Fc fragment contributes to the long half-life of the fusion molecule. Other preferred Fc or Fc fragment embodiments may modulate, modify or suppress an immunological effector function (Shields et al., 2001). Other highly preferred Fc fragment embodiments may mediate clearance of the fusion peptide from the brain (Caram- Salas N, 201 1). In a non-limiting example Fc or Fc fragments may be Fc mouse Fc2a, or a human Fc1 , selected from any one of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 , and a sequence substantially identical thereto with attenuated effector function, which, when included in said fusion peptide, may have enhanced clearance of amyloid from the brain.

In a compound of the present invention, the BBB may be linked to the ABP via an Fc fragment, and/or any additional suitable linker L.

A compound of the present invention comprises a fusion protein; wherein the fusion protein comprises an antibody or fragment thereof, an Fc fragment, and the polypeptide that binds β- amyloid. The fusion protein may comprise an antibody or fragment thereof linked to the N- terminus of Fc fragment, and the polypeptide that binds β-amyloid is linked to the C-terminus of the Fc fragment via L, a linking peptide or a chemical linker. The antibody or fragment thereof may be linked to the C-terminus of Fc fragment and the polypeptide that binds β- amyloid linked to the N-terminus of the Fc fragment. Accordingly, the present invention provides fusion proteins comprising an antibody or fragment thereof selected from the group consisting of SEQ ID NO:13 to SEQ ID NO: 26 ; a polypeptide that binds b-amyloid selected from the group consisting of SEQ ID NO: 27 to SEQ ID NO:38 ; and an Fc fragment selected from the group consisting of SEQ ID No: 39 to SEQ ID NO:41 . The provided Fc may comprise an Fc with attenuated effector functions. For example, the fusion protein may comprise SEQ ID NOs: 42 to SEQ ID NO:52, or a sequence substantially identical thereto. The fusion protein may be a single chain polypeptide, and the single chain polypeptide of the fusion protein may for a dimeric polypeptide. It is noted that the (GGGSGGGGS or GGGGSGGGGS) linker provided in the fusion protein comprised in SEQ ID NO: 42 to SEQ ID NO: 52 may be any suitable linker sequence. For example, the linker sequence highlighted (ex. GGGSGGGGS) may be any equivalent peptide linking sequence that allows for the linking of the components of the fusion protein provided, as exemplified in SEQ ID NO: 53, wherein the linker may be any peptide or chemical linker.

In an embodiment, the BBB may be linked to an Fc fragment, thus forming a dimer. The Fc fragment may be any suitable Fc fragment, for example mouse Fc2a or human Fc1 , with attenuated effector function (Shields et al., 2001 ).

An ABP variant of the present invention may comprise SEQ ID NO: 31 , for example, may be a sequence selected from any one of SEQ ID NO: 32 to 38. An ABP variant as provided herein exhibits unexpected and significant advantages over ABP polypeptides of the prior art. More specifically, the present ABP variants exhibit increased stability and bio-manufacturability. Moreover, a compound or composition of the present invention comprising an ABP or variant, as provided herein, when coupled to BBB, via a linker and/or Fc fragment as provided herein, exhibits a synergistic and unexpected efficacy in transmigrating the blood brain barrier. Such an ABP variant, when coupled to BBB, via a selected Fc fragment as provided herein, provides an unexpected, rapid and improved clearance of Αβ from the brain. Moreover, a fusion protein of the present invention comprising an ABP or ABP variant as provided herein exhibits a synergistic and unexpected efficacy in transmigrating the BBB and improved clearance of Αβ from the brain.

Accordingly, there is provided a therapeutic composition comprising a blood brain barrier transmigrating antibody or fragment thereof (BBB), an Fc (Fc), linked to a polypeptide that binds β-amyloid (ABP or ABP variant), wherein said polypeptide confers a synergistic increase in stability and efficacy of the therapeutic composition provided. As shown, unexpectedly and most significantly, a single bolus of BBB-Fc-ABP reduced brain Αβ burden by 50% within 24 hrs of treatment (Fig. 9B) compared to three months of multiple treatments with free ABP to achieve similar results in animals, (mouse model of AD) (Fig 9A). Thus, the compound as provided herein was shown to be far more potent than free ABP in reducing brain Αβ burden. Additionally, this fusion protein comprising Fc provided a substantially longer serum half-life compared to free ABP or BBB-ABP (WO 2006/133566), thereby providing an improved therapeutic compound. (Fig 10). The ABP variants of the present invention, and fusion proteins (constructs) comprising ABP overcome the disadvantages of the prior art. In the prior art, the linking of ABP with a BBB carrier (WO 2006/133566) alone does not assure the generation of an effective molecule. Fusions comprising an Fc aimed at enhancing serum half-life do not ensure efficient transport of ABP across the blood brain barrier. Specific engineering and formulation of a fusion molecule comprising a BBB carrier-, Fc fragment- and ABP provide an efficient BBB- permeable therapeutic compound. The efficient blood brain barrier-permeable therapeutic compound provided comprises a specifically engineered formulation of a BBB-Fc-ABP, wherein the formulation exhibits a synergistic improvement in compound stability and efficacy. An unexpected increase in the stability of the fusion compositions, and a synergistic increase in efficacy of transmigrating the blood brain barrier and faster clearance (within 24 hrs) of Αβ from the brain, as shown in Fig 9, is provided in a composition comprising said fusion protein constructs.

The compound provided herein may be referred to as a compound, a fusion protein, formulation, composition or construct. The provided construct may comprise an antibody or fragment thereof (which may be abbreviated BBB), an Fc fragment (abbreviated Fc), and a polypeptide that binds β-amyloid (abbreviated ABP). The construct or composition provided (which may be abbreviated herein as BBB-Fc-ABP or BBB-Fc-L-ABP), comprises components that synergistically overcome the deficiencies encountered in the prior art with respect to blood brain barrier transmigration, efficacy and compound stability. Accordingly, compounds provided herein comprise a novel formulation having superior and unexpected efficacy in transmigrating the blood brain barrier, therapeutic efficacy and compound stability.

There are instances in the prior art where Fc fusions may increase serum half-life, although serum half-life is not necessarily increased in Fc fusions. Moreover, Fc fusions do not necessarily improve transmigration across the blood brain barrier.

The present invention provides a polypeptide that binds β-amyloid, the polypeptide sequence may be selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 38 and a sequence substantially identical in peptide stability.

The present invention provides a polypeptide that binds β-amyloid, wherein when the polypeptide sequence is referred to as an ABP or an ABP variant, ABP variant sequence may comprise SEQ ID NO: 31 , and may be selected from the group consisting of SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and sequence substantially identical in peptide stability, for example, any sequence variant of SEQ ID NO:31 . An ABP variant having SEQ ID NO: 31 of the present invention overcomes the disadvantages of the prior art with respect to polypeptide stability. Moreover, a compound comprising an ABP of the present invention exhibits improved compound stability and bio-manufacturability. Furthermore, a compound comprising an ABP or ABP variant as provided herein exhibits a synergistic and unexpected therapeutic improvement.

An ABP variant of the present invention may comprise a sequence selected from any one of SEQ ID NO: 31 to SEQ ID NO: 38, and an equivalent^ stable polypeptide sequence. An ABP variant of the present invention is superior over ABP sequences of the prior art, wherein the present ABP variant polypeptides exhibit improved stability (as shown in comparison between Fig. 2A and Fig 16).

The present invention also provides fusion proteins (also referred to as compounds, constructs, or fusion molecules) comprising an ABP, or ABP variant, of the present invention. The fusion protein may comprise an Fc fragment. The fusion protein may comprise an antibody or fragment thereof that transmigrates the blood brain barrier, an Fc fragment, and a β-amyloid binding polypeptide sequence that may be selected from the group consisting of SEQ ID NO: 31 , SEQ ID NO: 32-38. The provided BBB-Fc-ABP formulations exhibit synergistic improvement in compound stability and therapeutic efficacy in removing toxic amyloid from the brain.

The fusion protein or construct, which may be referred to herein as BBB-Fc-ABP, may vary in orientations with respect to the components comprised therein. In a compound of the present invention the fusion protein forms a dimer (as shown in Fig .1 ) wherein the fusion peptide dimerizes via the Fc region. For example, in an embodiment, the provided compound may comprise a fusion protein comprising a BBB linked to the N-terminus of the Fc fragment (Fc) and an ABP or ABP variant (ABP) linked to the C-terminus of the Fc fragment via a short peptidic linker attached to the C-terminus (Fig 1 A, wherein the compound is shown as a dimer of the fusion protein). In a further embodiment, the provided compound may comprise a BBB linked to the C-terminus of the Fc fragment (Fc) wherein the ABP or ABP variant (ABP) may be linked to the N-terminus of the Fc fragment via a suitable linker (L) (Fig 1 B). It is noted that in configuration 1 A, the ABP or ABP variant may be N-terminally (fusion) or C-terminally (chemical linking) fused/linked to Fc. In other possible configurations, the BBB may be linked to the N-terminus of the ABP, and the ABP linked to the N-terminus of the Fc fragment (Fig 1 C). In yet another possible configuration the BBB may be linked to the C-terminus of the ABP, and the ABP is linked to the C-terminus of the Fc fragment (Fig 1 D).

The compounds provided herein comprise fusion proteins comprising a sequence selected from any one of SEQ ID NO:42 SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO: 53 and a sequence substantially identical to any of these sequences. The fusion protein of the present is a fusion protein (BBB-Fc-ABP or BBB-Fc-L-ABP, wherein L may be any suitable linker) dimer (as shown in Fig 1 , and preferably, Fig 1A or 1 B)". In a non- limiting example, the compound or fusion protein provided may comprise a polypeptide comprising sequence SEQ ID NO:35 (ABP-GG-G) or SEQ ID NO:36 (ABP-6G); an antibody or fragment thereof comprising sequence SEQ ID NO:17 (FC5-H3); and an Fc fragment comprising sequence SEQ ID NO:40 (hFc1 X7).

In a specific non-limiting embodiment of the present invention, the compound may comprise sequence SEQ ID NO:46 [FC5-H3-hFc1 X7-L-ABP(GG-G)] or SEQ ID NO:47 [FC5-H3- hFc1 X7-L-ABP(6G)], wherein the L may be any suitable linker. The compounds of the present invention transmigrate the blood-brain barrier.

The present invention encompasses a nucleic acid molecule encoding any compound of the present invention as described herein. Vectors comprising the nucleic acid molecule of a fusion protein or compound of the present invention are also included in the scope of the present invention. The present invention encompasses a composition comprising a compound or fusion protein of the present invention and a pharmaceutically-acceptable carrier, diluent, or excipient.

Kits comprising a pharmaceutical composition of the present invention are also included in the scope of the present invention.

The composition of the present invention may be used for treating Alzheimer's disease in a patient.

The composition of the present invention may be used for reducing toxic β-amyloid (Αβ) levels in the brain or CSF of a subject having increased levels of brain Αβ.

The present invention provides a method of treating Alzheimer's disease, wherein a pharmaceutical composition of the present invention may be administered to a subject in need thereof.

The present invention provides a method of reducing toxic β-amyloid (Αβ) levels in the brain of a subject having increased levels of brain Αβ. The method of the present invention comprises the administration of a compound of the present invention to a patient with AD. More specifically, the present method comprises the steps of repeated parenteral administration of a sufficient amount of a pharmaceutical composition of the present invention to a subject. In the method of the present invention, parenteral administration is subcutaneous or intravenous administration.

The method of the present invention reduces toxic β-amyloid levels, after repeated parenteral administration of a composition provided herein, in the brains of subjects having increased brain levels of Αβ. More specifically, toxic β-amyloid levels are reduced within four weeks of repeated parenteral administration of the composition of the present invention.

The method of the present invention reduces toxic β-amyloid levels in the cerebrospinal fluid (CSF) of subjects after parenteral administration of the composition of the present invention. More specifically, toxic β-amyloid levels in the cerebrospinal fluid (CSF) of subjects is significantly reduced within 24 hours of a single parenteral administration of the composition of the present invention; wherein significant reduction is up to 50% within 24 hours.

The present invention provides a blood brain barrier-permeable single domain antibody (sdAb), wherein the sdAb is either camelid FC5 or a humanized version thereof. FC5 displayed in bivalent format on Fc has been shown to have improved blood brain barrier-crossing properties compared to FC5 in V _H H format (Farrington et al., 2014).

The present invention provides a compound comprising a BBB that facilitates blood brain barrier transmigration in vitro and an Fc fragment and increases serum half-life of the fusion molecule, wherein serum half-life of the Fc fusion becomes similar to that of a full IgG. Prolonged serum half-life of FC5-Fc-ABP also increases overall brain exposure, which is particularly important for treating chronic diseases such as Alzheimer's Disease.

The FC5-Fc-ABP fusion molecule as well as a humanized version FC5(H3)-hFc-ABP, and IGF1 R5-H2-ABP fusion molecules were produced in CHO cells. The ABP variants of the present invention, as provided in SEQ ID NO: 31 , are methodically re-engineered with specific point-mutations or deletions that enhance bio-manufacturability and stability of the fusion molecule (SEQ ID NO. 45, SEQ ID NO. 46; SEQ ID NO. 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53). The fusion molecules of the present invention comprising the ABP variants not only retain Αβ binding ability, the provided fusion molecules comprising the ABP variants not only penetrate the blood brain barrier, but advantageously allow for the clearance of Αβ from the brain, for example, the rapid clearance of Αβ from the brain within 24 hours of administration.

The ABP variants of the present invention, for example ABP(GG-G) (SEQ ID NO: 35) or ABP(6G) (SEQ ID NO: 36), were specifically engineered to enhance stability and manufacturability of the construct (as shown in FIG. 16). The enhanced stability and bio- manufacturability of the construct of the present invention is significant in the present art. The present invention accordingly encompasses ABP and ABP variants that enhance stability and bio-manufacturability of the fusion molecules provided, but also retain therapeutic activity (as provided in FIG. 15A). Therefore, the fusion molecules comprising different ABP variants, as provided in SEQ ID NO:31 , such as in SEQ ID NOs: 32-38 retain the Αβ oligomer binding ability of the parent ABP in vitro (Figs. 2B.2C, 12 and 15 A,B and C) as well as the BBB-penetrating property of the parent FC5 both in vitro and in vivo (Figs. 4, 5A, 6, 7, 17A, 17B, 18 and 19). The ABP is transported across the BBB by the carrier, for example FC5, in vitro and in vivo, as established by its presence in the rat and dog CSF (Figs. 5 and 6). The ABP fusion was also transported and delivered to the target regions in the brain, such as hippocampus and cortex in both wild type and AD transgenic mice (Fig. 8 and 19). The brain-delivered ABP also promoted Αβ clearance in the CSF (Figs. 9C and 1 1A and 1 1 B) and in cortical and hippocampal regions of AD transgenic mice and rat (Figs. 9B and 1 1 C). In addition, and most importantly, Αβ clearance in rat AD model correlated with enhanced hippocampal volume and neuronal connectivity (Fig. 12A and 12B) indicating desired pharmacological/physiological response to treatment.

Unexpectedly and most significantly, a single bolus of BBB-Fc-ABP reduced brain Αβ burden by 50% within 24 hrs of treatment (Fig 9B) compared to three months of multiple treatments with free ABP to achieve similar results in animals (Fig 9A). Thus, the BBB-enabled ABP was shown to be far more potent than free ABP in reducing brain Αβ burden. Additionally, this compound has a substantially longer serum half-life compared to free- or FC5- ABP, an essential characteristic of a better therapeutic, due to its fusion with Fc (Fig 10).

In the prior art, the linking of ABP with a BBB carrier (WO 2006/133566) alone does not ensure the generation of an effective molecule. Fusion with Fc to enhance serum half-life also does not ensure efficient transport of ABP across blood brain barrier. Suitable engineering and formulation of BBB carrier-, Fc fragment- and ABP fusion molecule, as provided in the fusion protein constructs of present invention, provide an efficient BBB-permeable therapeutic compound. This novel bi-functional fusion molecule has distinct advantage over conventional therapeutic antibodies currently under development. First, the BBB-fused therapeutic ABP can penetrate the brain at much higher levels and at a faster rate, which substantially improves the therapeutic efficacy. In addition, ABP and BBB (ex. FC5) have relatively lower affinity towards their respective receptors and thus are likely cleared from the brain faster, and accordingly facilitate faster clearance of ABP-bound Αβ. Unlike therapeutic antibodies that mainly employ reactive microglia/astrocytes for Αβ clearance ( e.g., aducanumab), which is a slower process, the fusion molecule of the present invention likely employs faster perivascular drainage pathway for Αβ clearance. This is supported by a nearly 50% reduction in CNS Αβ within 24 hr of treatment compared to free ABP (Fig 9B) and antibody-based therapeutics, which require months of treatment with repeated multiple doses to achieve similar results.

Moreover, the present compound is less likely to elicit neuro-inflammatory response compared to antibody-based therapeutics.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will now be described by way of example, with reference to the appended drawings, wherein:

Fig 1 : shows schematic drawings of a blood brain barrier-crossing, amyloid-binding fusion protein. The fusion protein comprises a BBB-crossing single-domain antibody (BBB or BBB carrier), an Fc fragment (Fc) and an amyloid-binding peptide (ABP). Fig.1 shows the corresponding dimers of the fusion protein comprising BBB-Fc-ABP. The 3 components (i.e. BBB, Fc, and ABP) are depicted in various configurations, as illustrated in FIG 1A, 1 B, 1 C, and 1 D.

Fig 2: shows the production of FC5-mFc-ABP and humanized FC5(H3)-hFc-ABP (ABP SEQ ID NO: 32) fusion molecule in CHO cells. As shown in FIG. 2A, a Coomassie blue stained gel after separation of FC5 fusion molecules by SDS-PAGE (NR- non-reducing and R- reducing conditions) showing successful production of recombinant fusion molecule. FIG. 2B and FIG. 2C: Αβ-oligomer binding of free ABP and BBB-Fc-ABP fusion protein by ELISA and Western blot (WB) Overlay assay. Free or fused ABP was immobilized on ELISA plate and exposed to AB. Bound AB was detected with AB-specific antibody 6E10 or 4G8. The fusion molecules were also separated by SDS-PAGE, transferred to PVDF paper and exposed to AB. Bound AB was detected with specific antibody as above. Results show that ABP retained its AB oligomer binding ability after fusion with the BBB carrier. Mo: AB monomers; ON: AB Oligomers

Fig 3: shows an immunohistofluorescence assay of the binding of FC5-Fc-ABP to amyloid deposits in AD- Tg mice (B6.Cg-Tg, Jackson Lab). ABP retains the ability to bind naturally produced AB aggregates in AD-Tg mouse brain as shown by immunohistofluorescence assay. Brain sections from wild type and AD-transgenic mice were incubated with IR 800-labelled FC5-mFc-ABP and the bound fusion molecule was visualized under fluorescence microscope. Selective binding (bright spots) were seen in brain sections from AD-Tg mice that produce Αβ deposits and not in brain sections from wild type mice that does not produce amyloid deposits.

Fig 4: shows BBB permeability of FC5 is retained after fusion with ABP in vitro. Blood brain barrier crossing of FC5-ABP fusion molecules was assessed in in vitro BBB models from rat and human. Fusion molecules crossing BBB were detected by nanoLC-MRM method (FIG. 4A, FIG. 4B and FIG. 4C) and by Western blot analysis using Fc-specific antibody (FIG. 4D, done in triplicate). FC5-mFc-ABP crossed the BBB as effectively as FC5-mFc, whereas Fc- ABP without the BBB carrier moiety FC5 did not traverse across the brain endothelial cell monolayer. As expected, control single domain antibodies EG2 and A20.1 , or control full IgG (anti-HEL) did not cross the blood brain barrier. Similar results were obtained with humanized FC5-H3-hFc-ABP fusion protein (FIG. 4C and FIG. 4D).

Fig 5: shows serum and CSF pharmacokinetics of FC5-Fc-ABP in vivo. FC5-mFc-ABP was administered intravenously into rats via tail vein injection at the indicated doses (2.5, 6.25, 12.5, and 25 mg/kg). Serum and CSF were serially collected. FC5-Fc-ABP levels were quantified using nanoLC- MRM method. As shown in Fig 5, FC5-mFc-ABP appeared in the CSF in a time- and dose-dependent manner with Cmax between 12 and 24 h, indicating transport of ABP by FC5 into brain and CSF compartments in vivo. Serum PK parameters (FIG. 5 and Table 1 ) show that alpha- and beta- half-life of FC5-mFc-ABP is similar to that of a full IgG (a benchmark antibody containing rat Fc). Fig 6: shows serum and CSF PK profile of FC5-mFc-ABP in beagle dog. FC5-mFc-ABP was administered by intravenous injection to 10-12-year old beagle dogs and serum and CSF were serially collected and analyzed by nanoLC-MRM (FIG. 6A) and by Western blot using Fc- specific antibody (FIG. 6B). Asterisks indicates blood-contaminated sample (not shown in MRM analyses). As can be seen, FC5-mFc-ABP appeared in the CSF in a time-dependent manner indicating transport of ABP by FC5 across dog blood brain barrier in vivo. The PK parameters and CSF exposure were analyzed by WinNonlin software and are shown in Table 2 below.

Fig 7: shows BBB permeability and CSF appearance of FC5 fused with human Fc (hFc) and chemically linked to ABP (FC5-hFc-ABP) in vivo (rat model). FC5-hFc was linked with ABP- cystamide using a heterobifunctional cross-linker sulfo-SMCC (sulfosuccinimidyl 4-[N- maleimidomethyl] cyclohexane-1 -carboxylate) according manufacturer's instructions (ThermoFisher Scientific). Chemically conjugated molecule was administered intravenously into rats via tail vein at 6.25 mg/kg and serum and CSF samples were collected at 4 and 24 hrs and analyzed by nanoLC-MRM. FC5-hFc-ABP appears in the CSF in a time-dependent manner in contrast to Fc-ABP without the BBB carrier. It should be noted that in this chemically linked construct, C-terminus of ABP is linked to Fc fragment (random region) and N-terminus is free, unlike in the fusion construct, wherein it is N-terminus of ABP that is fused to C-terminus of Fc and C-terminus of ABP is free. This reversal in the orientation of ABP did not affect its Αβ-binding ability and transport across the blood brain barrier.

Fig. 8: shows levels of FC5-mFc-ABP measured in various brain regions (cortex and hippocampus) after intravenous injection in mice. 15mg/kg dose of FC5-mFc-ABP was injected intravenously into the tail vein of either wild type (WT) or AD-transgenic (AD-Tg, B6.Cg-Tg, Jackson Lab) mice and brains were collected at 4 and 24 hrs after intra-cardiac saline perfusion. Hippocampal and cortical tissues were dissected and analyzed by nanoLC-MRM (FIG. 8A) and by Western blot using Fc-specific antibodies (FIG. 8B). Specific peptides belonging to all the three components of the fusion molecule (BBB, in this example FC5, Fc and ABP) were detected by MRM in both cortex and hippocampus, indicating that FC5 carrier successfully delivered ABP to the target areas of the brain. Measured levels ranged between 750-1400 ng/g brain tissue at different time points, compared to ~50 ng/g tissue typically measured for control single-domain antibody A20.1 fused to Fc, or Fc fragment alone. This was further confirmed by Western blot analysis probing for Fc and ABP in tissue extracts (FIG. 8B). No protein signal of the fusion molecule was detected in animals receiving just saline by Western blot. There was a dose-dependent increase in FC5-mFc-ABP levels detected by Western blot in the target regions of the brain.

Fig 9: shows the effect of ABP on Αβ levels in transgenic (Tg) mice: Comparison between treatment with ABP alone (FIG. 9A) or ABP fused with BBB carrier FC5 (FIG. 9B and FIG. 9C). Two different AD Tg mouse models, triple transgenic (3X Tg-AD, sv129/ C57BL6 mice harboring PS1 M146V, APP _Swe and tauP301 L transgenes, Dr. F.M. LaFerla, University of California) and double transgenic (B6.Cg-Tg, harboring PSEN1 dE9 and APP _Swe transgenes, Jackson Lab) were used; mice were dosed subcutaneously (sc) with 300 nmol/kg of free ABP every second day over a 3-month or a 2-month period, respectively. At the end of the treatment period, Αβ levels in the brain were measured by ELISA. The treatment with ABP alone resulted in 25-50% reduction in brain Αβ after 2-3 months of multiple treatments (every second day) (FIG. 9A). The FC5-mFc-ABP construct was injected intravenously into double- transgenic AD mice (B6.Cg-Tg ,15 mg/kg; equivalent of 220 nmol/kg) and brain Αβ levels were measured by both ELISA and nanoLC-MRM 24 h after injection. Unexpectedly, about 50% amyloid reduction was observed within 24 hr of treatment with FC5-mFc-ABP (FIG. 9B), indicating that efficient brain delivery of ABP by FC5, suitably linked to Fc, dramatically increased the efficacy of ABP in reducing brain Αβ levels. CSF analysis also indicated a significant decrease in ^.^ levels within 24 hrs following FC5-mFc-ABP treatment (FIG. 9C). The signature peptide or epitope of Αβ detected by MRM or ELISA analyses is remote/different from the Αβ epitope recognized by ABP (therefore, not interfering with its quantification by either ELISA or MRM). Fig 10: shows an example of enhanced serum half- life of FC5-Fc-ABP construct compared to FC5-ABP (i.e. without Fc component): FC5-ABP and FC5-Fc-ABP were injected into rats via tail vein and serial serum samples were collected at various time points and analyzed by direct ELISA with FC5-specific antibody. As can be seen in Fig 10 A, FC5-ABP construct was rapidly cleared in the serum (less than 1 hr) compared to FC5-Fc-ABP (Fig 10 B), indicating substantial increase in the serum stability of the molecule comprising the Fc fragment.

Fig 11 : shows the effect of FC5-mFc-ABP on brain amyloid burden in Tg rats: AD-Tg rats were dosed with either saline or FC5-mFc-ABP via tail vein every week over a period of four weeks (loading dose of 30mg/kg and subsequent four weekly doses of 15 mg/kg). CSF levels of FC5-mFc-ABP and Αβ were analyzed by nanoLC MRM (FIG. 1 1A and FIG 1 1 B). Before and after four weeks of treatment, brain Αβ levels were determined by PET scan using a specific Αβ-binding agent [18F] NAV4694. Following tracer injection, 60 min Dynamic images were acquired, transmission scans were obtained, images were reconstructed and Binding Potential (BP _ND ) parametric maps were generated. FC5-mFc-ABP reduced CSF Αβ level in rats within 24 hrs (FIG. 1 1 A and FIG. 1 1 B). An inverse relationship between the CSF levels of FC5-mFc-ABP and Αβ was observed, as in Tg rats, suggesting target engagement and rapid clearance of Αβ by ABP delivered to the brain and CSF by FC5 (FIG. 1 1 B). This was further corroborated by PET scan which clearly indicated a significant reduction (30-50%) of rat brain AB levels following four weeks of treatment with FC5-mFc-ABP (FIG. 1 1 C).

Fig 12: shows volumetric Magnetic Resonance Imaging (MRI, using Fast Imaging with Steady- state Precession) and functional MRI (fMRI) of Tg rats before and after treatment with saline or FC5-mFc-ABP. As shown in Fig 12 A, increased hippocampal volume was observed in ABP- treated Tg rats (Tg-ABP) compared to saline-treated Tg rats (Tg-Sal) after four weeks of treatment. As shown in Fig 12B, group comparison after four weeks of treatment showed that the ABP-treated Tg rats (Tg-ABP) had greater Anterior Cingulate Cortex (ACC) connectivity compared to saline-treated Tg rats (Tg-Sal).

Fig 13: shows time- and dose -dependent appearance of FC5-mFc-ABP in the CSF of beagle dog and a decrease in CSF AB levels as seen Tg mice (Fig 9C) and Tg rats (Fig 1 1A and B). FC5-mFc-ABP was administered by intravenous injection to 10-12-year old beagle dogs at 15 mg/kg and 30 mg/kg and serum and CSF were serially collected and analyzed by nanoLC- MRM for FC5-mFc-ABP and Αβ levels. As can be seen, FC5-mFc-ABP appeared in the CSF in a time-and dose-dependent manner. Importantly, as seen in Tg mice and Tg rats, there was a significant decrease in CSF Αβ levels within 24 hrs after the FC5-mFc2a-ABP injection, suggesting translational nature of FC5 carrier in larger animals and also cross-species efficacy of ABP in reducing CNS Αβ burden.

Fig 14: shows the generation of ABP fusion molecule with a different BBB carrier. To assess the versatility of ABP fusion molecule, ABP was successfully fused with another humanized BBB carrier IGF1 R5 (H2). As shown, the bi-functionality of the molecule was retained, ABP's ability to bind Αβ oligomer (ELISA and overlay assays) and also IGF1 R5's ability deliver ABP across BBB model in vitro (data not shown). This clearly indicates that ABP can be fused to different BBB-crossing single-domain antibodies to be delivered to the brain.

Fig 15: shows Αβ oligomer binding by different single-domain antibody-Fc-ABP constructs (FIG. 15A, 15B and 15C). In some of these constructs, ABP has been modified by site-specific mutations or removal of C-terminus portion of the molecules as indicated in the SEQ ID NOs. All constructs retained similar potency in binding Αβ oligomers by ELISA method.

Fig 16: shows the production of FC5-hFc1 X7-ABP with specific mutations to improve stability and bio-manufacturability. FC5-hFc1X7-ABP carrying specific mutation (such as the ABP of SEQ ID NO: 35 or SEQ ID NO: 36) were produced in CHO cells and separated on SDS-PGE under reducing (R) and non-reducing (NR) conditions and stained with Coomassie blue as described in Fig 2. Separated protein transferred to nitrocellulose membrane and immunoblotted with either FC5-specific or hFc-specific or ABP-specific antibodies. In another set, Αβ-binding of ABP in the fusion molecule was also tested by overlay assay. Bound Αβ was detected with Αβ-specific antibody 6E10. As can be seen, the methodic modification of ABP with specific mutation substantially enhanced the stability of the molecule generated, as indicated by single protein band under reducing and non-reducing conditions.

Fig 17: shows blood brain barrier permeability of various FC5-Fc-ABP constructs and IGF1 R5- Fc-ABP construct in vitro. BBB- crossing was assessed in in vitro rat BBB model as described in Fig 4 and molecules crossing blood brain barrier were detected by nanoLC-MRM method. All ABP variants fused to humanized FC5 and IGF1 R carriers crossed the blood brain barrier effectively. As expected, A20.1 , a non-BBB permeable sdAb did not cross blood brain barrier, likewise ABP fused to A20.1 did not cross BBB (FIG. 17A). In Fig 17B, it is shown that "fingerprint" peptides for all the three components of the fusion molecule, FC5, Fc and ABP were detected by nanoLC-MRM, thereby indicating the transmigration of the intact FC5, Fc and ABP across the BBB. Fig. 18: shows humanized FC5(H3)-hFc1X7-ABP construct (ABP, SEQ ID NO: 35 and SEQ ID NO: 36) are transported across in vitro blood-brain barrier intact by FC5. Blood brain barrier - crossing was assessed in in vitro rat BBB model as described for Fig. 4 and molecule crossing BBB was detected by Western blot and ELISA assays. Fig 18 (1 ) A and B show immunoblots probed with hFc- and ABP-specific antibodies, respectively. The molecular size is exactly identical to that of the fusion molecule that was applied to in vitro blood brain barrier model. Fig 18(1) C shows sandwich ELISA in which the molecule after crossing the BBB was captured by FC5-sepcific antibody on ELISA plate and detected with ABP-specific antibody. This sandwich ELISA confirmed that FC5(H3)-hFc1X7-ABP remained intact after crossing rat blood brain barrier in vitro. Similar results were obtained with FC5(H3)-hFc1X7-ABP (ABP, SEQ ID NO: 36); Fig 18(2) A, B, and C.

Fig. 19: shows humanized FC5(H3)-hFc1X7-ABP constructs (ABP, SEQ ID NO: 35 and SEQ ID NO: 36) are transported across in vivo blood-brain barrier and delivered to the brain intact by FC5. The FC5-ABP fusion molecule was administered intravenously into wild type and AD- Tg mice via tail vein and brains were collected following intra-cardiac perfusion as described for Fig. 8. Brain cortex was homogenized and extracted in RIPA buffer and the extract was subjected to Western blot and sandwich ELISA analysis as described for Fig. 18. Fig. 19A shows immunoblot probed with ABP-specific antibody. The molecular size is exactly identical to that of the fusion molecule that was injected into the animals. Fig. 19 B shows sandwich ELISA of the same extract, molecule in the extract captured with FC5-specific antibody on the ELISA plate and detected with ABP-specific antibody. This confirms immunoblot results that FC5(H3)-hFc1X7-ABP is transported across the BBB in vivo and delivered to the brain intact.

Fig. 20: shows immunohistochemical analysis of ex-vivo binding (A) and in vivo binding (B) of FC5(H3)-hFc1X7-ABP (ABP, SEQ ID NO: 36) to endogenous Αβ deposits in AD-Tg mouse brain (B6.Cg-Tg, Jackson Lab). Brain sections from AD-transgenic mice were incubated with FC5(H3)-hFc1X7-ABP (ABP, SEQ ID NO: 36) and the bound fusion molecule was visualized with HRP-conjugated FC5-specific antibody. Selective binding (black spots) was seen with FC5(H3)-hFc1X7-ABP construct but not with FC5(H3)-hFc1X7 without ABP (A), indicating ABP-dependent binding of Αβ deposits (target engagement) in the brain. No binding was seen in brain sections from wild type mice that does not produce amyloid deposits (data not shown). Similar binding of Αβ deposits were detected (using ABP-specific antibody) following intra- hippocampal injection (4hrs post-injection) of FC5(H3)-hFc-ABP construct into AD transgenic mice (B).

Fig. 21 : shows target engagement by FC5(H3)-hFc1X7-ABP (ABP, SEQ ID NO: 32) in vivo. Fig 21 A shows binding of Alexa 647-labeled FC5-hFc-ABP construct to natural amyloid-β (Αβ) deposits in AD transgenic mice in vivo after intra-hippocampal injection. Identity of Αβ was confirmed by probing the brain sections with Αβ-specific antibody 6E10 labelled with Alexa 488 and demonstrating co-localization of two signals (Merge). Fig 21 B shows demonstration of target engagement by ELISA. Following intra-hippocampal injection (4hrs post-injection) of FC5(H3)-hFc-ABP construct into wild type and AD transgenic mice, hippocampal formation was dissected and homogenized. FC5-ABP fusion construct was detected by sandwich ELISA using FC5 antibody as capturing antibody and ABP antibody as the detection antibody. In vivo binding of ABP to endogenous Αβ was detected by the same sandwich ELISA but with Αβ- specific antibody as the detection antibody. It is clear from the FIG 21 A and 21 B that the FC5- ABP construct remains intact 4hrs post-injection in both wild type and AD transgenic mice. Most importantly, in Tg mice which expresses human Αβ, injected ABP binds Αβ and is pulled down as a complex (FC5(H3)-hFc-ABP*AB) indicating Αβ-target engagement by ABP in vivo.

Fig. 22: shows PK, PD comparison between non-humanized and humanized FC5-Fc-ABP constructs. FC5-mFc2a-ABP or FC5(H3)-hFc1x7-ABP was administered intravenously into rats via tail vein injection at 15 mg/kg as described for Fig 5. Serum and CSF were serially collected. FC5-Fc-ABP levels were quantified using nanoLC- MRM method. As shown in Fig 22 A, serum and CSF PK profile were very similar for non-humanized and humanized constructs. FC5-mFc2a-ABP or FC5(H3)-hFc1x7-ABP was administered intravenously into Tg mice via tail vein injection at 15 mg/kg as described for Fig 9B. FC5-Fc-ABP and Αβ levels in the CSF were measured by nanoLC-MRM as described in Fig 9B. As shown in Fig 22 B, the levels of non-humanized and humanized FC5-Fc-ABP in the CSF were similar, and most importantly, changes (decrease) in CSF Αβ levels were also very similar, indicating that humanization of FC5-Fc-ABP construct did not affect the PK and PD profile of the fusion construct. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides polypeptides, fusion proteins comprising said polypeptides, and fusion proteins comprising said polypeptides and antibodies or fragments thereof that transmigrate the blood-brain barrier.

The present invention provides polypeptides which binds beta-amyloid (β-amyloid). The polypeptides (or proteins) that bind β-amyloid may selectively bind pathologically relevant β- amyloid (Αβι- ₄₂ ) aggregates, and may be abbreviated and referred to herein as ABP or ABP variants (or collectively as ABP). The present invention provides fusion proteins comprising ABP or an ABP variant linked to an antibody or fragment thereof that crosses the blood-brain barrier. In a preferred embodiment, the fusion protein comprising ABP and a BBB additionally comprises an Fc or fragment thereof, wherein the ABP and BBB components of the fusion protein may be linked via an Fc region or portion thereof. For example, a construct of the present invention may comprise BBB-Fc-ABP or BBB-Fc-L-ABP, wherein L may be any suitable linker. The BBB-Fc-APB or BBB-Fc-L-ABP construct provided may be a single chain polypeptide or a dimeric polypeptide, wherein the single-chain polypeptide comprising BBB-Fc-ABP may form a multimer (preferably a dimer) via the component Fc region. The present invention relates to compounds that transmigrate the blood-brain barrier, and uses thereof. More specifically, the present invention relates to compounds comprising a BBB and an ABP and their use in the treatment of Alzheimer's disease (AD).

There is a need for therapeutic formulations that can efficiently transmigrate ABP across the blood brain barrier, and provide the clearing of Αβ through the binding of ABP. In the prior art, a 40-amino acid Αβ-binding peptide (ABP) was identified that selectively binds Αβ^ oligomers implicated in AD development (WO 2006/133566). This Αβ-binding peptide inhibits Αβ binding to cellular proteins and inhibits Aβ -induced cell toxicity in vitro (Chakravarthy et al, 2013). This Αβ-binding peptide binds amyloid deposits in AD transgenic mice brain, as well as binds amyloid deposits in the brains from AD patients in vitro. More importantly when directly injected into live AD transgenic mice brain (Chakravarthy et al, 2014) ABP targets natural amyloid deposits in vivo. Thus, ABP can potentially target CNS Αβ, can assist in clearing Αβ from the brain, and reduce its toxic effect. However, systemically-administered ABP has limited ability to cross BBB and access the brain parenchyma by itself.

Accordingly, although ABP has been shown to bind Αβ deposits when directly applied, in order to bind and clear Αβ from the brain, parenterally administered ABP needs to permeate the blood brain barrier. The present invention advantageously provides an ABP fused to a BBB- permeable single-domain antibody, such as FC5 or IGF1 R, via an Fc fragment to provide a bi- specific blood brain barrier-permeable therapeutic (Farrington et al, 2014). The BBB-Fc-ABP construct of the present invention may dimerize, i.e. the BBB-Fc-ABP single-chain fusion protein may form dimers of two single chain fusion proteins to yield a dimeric compound wherein each single chain of the dimer comprises a BBB, a Fc fragment and an ABP, to provide a BBB-Fc-ABP dimer. The BBB-Fc-ABP or BBB-Fc-L-ABP construct and dimers thereof, allows for the efficient transmigration of ABP across the blood brain barrier. Accordingly, the advantageous therapeutic clearing of Αβ through the binding of ABP in CSF and brain parenchyma is provided in the constructs and methods of the present invention. In order to enable brain delivery of ABP and improve its efficacy, the 40-amino acid ABP polypeptide is presently fused to the C-terminus of an Fc fragment, whereas a BBB-permeable single-domain antibody, such as FC5 (WO 2002/057445), is fused to the N-terminus of the same Fc fragment, to create a bi-specific BBB-permeable therapeutic (Farrington et al, 2014). In a non-limiting embodiment of the present invention, the Fc fragment may be mouse (SEQ ID NO. 39) or human (SEQ ID NO: 40; SEQ ID NO: 41 ). In a preferred embodiment, the Fc fragment of the present invention was engineered to reduce effector functions (Shields et al., 2001 ). For example the Fc fragment may be hFc1x7 (SEQ ID NO:40) wherein the Fc fragment in the BBB-Fc-ABP fusion protein advantageously allows for the dimerization of the fusion protein to yield a therapeutically effective fusion molecule (BBB-Fc-ABP dimer) capable of transmigrating the blood brain barrier. In an embodiment, the BBB-Fc-ABP fusion protein may be FC5-H3-hFc1x7-ABP(6G) (SEQ ID NO: 47) and dimers thereof. The fusion protein may comprise a linker sequence L that is as embodied in SEQ ID NO: 47 or SEQ ID NO:53, or any suitable linker sequence. The present invention provides an isolated polypeptide which binds beta-amyloid (β-amyloid). A polypeptide of the present invention may comprise a sequence.

X FXzTXsX _^ ASAQASLASKDKTPKSKSKKXsXeSTQLXySX _f jVXgNI (SEQ ID NO: 31) where X _! =G or A, X ₂ = G or V, X ₃ = G or A, X ₄ = G or A, X ₅ = G or V, X ₆ = G or V, X ₇ = G or V, X ₈ = G or A, X ₉ = G or A A polypeptide (ABP and ABP variant) of the present invention may be selected from the group consisting of: SEQ ID NO:27 to SEQ ID NO:38 or a sequence substantially identical thereto. In an embodiment, the polypeptide provided is an ABP variant comprising a sequence substantially equivalent to SEQ ID NO: 31 . A sequence that is substantially equivalent thereto may confer equivalent stability to the fusion molecules. The present invention provides a compound, namely a fusion protein, comprising an antibody or fragment thereof that transmigrates across the blood brain barrier (BBB) and a polypeptide that binds β-amyloid. The present invention provides fusion proteins comprising a BBB, a polypeptide that binds β-amyloid, and an Fc.

The term "antibody", also referred to in the art as "immunoglobulin" (Ig), as used herein refers to a protein constructed from paired heavy and light polypeptide chains; various Ig isotypes exist, including IgA, IgD, IgE, IgG, and IgM. When an antibody is correctly folded, each chain folds into a number of distinct globular domains joined by more linear polypeptide sequences. For example, the immunoglobulin light chain folds into a variable (V _L ) and a constant (C _L ) domain, while the heavy chain folds into a variable (V _H ) and three constant (C _H , C _H 2, C _H 3) domains. Interaction of the heavy and light chain variable domains (V _H and V _L ) results in the formation of an antigen binding region (Fv). Each domain has a well-established structure familiar to those of skill in the art. The light and heavy chain variable regions are responsible for binding the target antigen and can therefore show significant sequence diversity between antibodies. The constant regions show less sequence diversity, and are responsible for binding a number of natural proteins to elicit important biochemical events. The variable region of an antibody contains the antigen- binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen. The majority of sequence variability occurs in six hypervariable regions, three each per variable heavy (V _H ) and light (V _L ) chain; the hypervariable regions combine to form the antigen-binding site, and contribute to binding and recognition of an antigenic determinant. The specificity and affinity of an antibody for its antigen is determined by the structure of the hypervariable regions, as well as their size, shape, and chemistry of the surface they present to the antigen. Various schemes exist for identification of the regions of hypervariability, the two most common being those of Kabat and of Chothia and Lesk. Kabat et al (1991 ) define the "complementarity-determining regions" (CDR) based on sequence variability at the antigen- binding regions of the V _H and V _L domains. Chothia and Lesk (1987) define the "hypervariable loops" (H or L) based on the location of the structural loop regions in the V _H and V _L domains. These individual schemes define CDR and hypervariable loop regions that are adjacent or overlapping, those of skill in the antibody art often utilize the terms "CDR" and "hypervariable loop" interchangeably, and they may be so used herein. The CDR/loops are identified herein according to the Kabat scheme.

An "antibody fragment" as referred to herein may include any suitable antigen-binding antibody fragment known in the art. The antibody fragment may be a naturally-occurring antibody fragment, or may be obtained by manipulation of a naturally-occurring antibody or by using recombinant methods. For example, an antibody fragment may include, but is not limited to a Fv, single-chain Fv (scFv; a molecule consisting of V _L and V _H connected with a peptide linker), Fab, F(ab') ₂ , single-domain antibody (sdAb; a fragment composed of a single V _L or V _H ), and multivalent presentations of any of these. Antibody fragments such as those just described may require linker sequences, disulfide bonds, or other type of covalent bond to link different portions of the fragments; those of skill in the art will be familiar with the requirements of the different types of fragments and various approaches and various approaches for their construction. In a non-limiting example, the antibody fragment may be an sdAb derived from naturally- occurring sources. Heavy chain antibodies of camelid origin (Hamers-Casterman et al, 1993) lack light chains and thus their antigen binding sites consist of one domain, termed V _H H. sdAb have also been observed in shark and are termed V _N AR (Nuttall et al, 2003). Other sdAb may be engineered based on human Ig heavy and light chain sequences (Jespers et al, 2004; To et al, 2005). As used herein, the term "sdAb" includes those sdAb directly isolated from V _H , V _H H, VL, or VNAR reservoir of any origin through phage display or other technologies, sdAb derived from the aforementioned sdAb, recombinantly produced sdAb, as well as those sdAb generated through further modification of such sdAb by humanization, affinity maturation, stabilization, solubilization, camelization, or other methods of antibody engineering. Also encompassed by the present invention are homologues, derivatives, or fragments that retain the antigen-binding function and specificity of the sdAb.

SdAb possess desirable properties for antibody molecules, such as high thermostability, high detergent resistance, relatively high resistance to proteases (Dumoulin et al, 2002) and high production yield (Arbabi-Ghahroudi et al, 1997); they can also be engineered to have very high affinity by isolation from an immune library (Li et al, 2009) or by in vitro affinity maturation (Davies & Riechmann, 1996). Further modifications to increase stability, such as the introduction of non-canonical disulfide bonds (Hussack et al, 201 1 a, b; Kim et al, 2012), may also be brought to the sdAb. A person of skill in the art would be well-acquainted with the structure of a single-domain antibody (see, for example, 3DWT, 2P42 in Protein Data Bank). An sdAb comprises a single immunoglobulin domain that retains the immunoglobulin fold; most notably, only three CDR/hypervariable loops form the antigen-binding site. However, and as would be understood by those of skill in the art, not all CDR may be required for binding the antigen. For example, and without wishing to be limiting, one, two, or three of the CDR may contribute to binding and recognition of the antigen by the sdAb of the present invention. The CDR of the sdAb or variable domain are referred to herein as CDR1 , CDR2, and CDR3.

The antibody or fragment thereof as described herein may transmigrate the blood-brain barrier. The brain is separated from the rest of the body by a specialized endothelial tissue known as the blood-brain barrier (BBB). The endothelial cells of the BBB are connected by tight junctions and efficiently prevent many therapeutic compounds from entering the brain. In addition to low rates of vesicular transport, one specific feature of the BBB is the existence of enzymatic barrier(s) and high level(s) of expression of ATP-dependent transporters on the abluminal (brain) side of the BBB, including P-glyco protein (Gottesman and Pastani, 1993; Watanabe, 1995), which actively transport various molecules from the brain into the blood stream (Samuels, 1993). Only small (<500 Daltons) and hydrophobic (Pardridge, 1995) molecules can more readily cross the BBB. Thus, the ability of the antibody or fragment thereof as described above to specifically bind the surface receptor, internalize into brain endothelial cells, and undergo transcytosis across the blood brain barrier by evading lysosomal degradation is useful in the neurological field. The antibody or fragment thereof that crosses the blood-brain barrier may be used to carry other molecules, such as therapeutics, for delivery to the brain tissue. The antibody or fragment thereof may be any suitable antibody or fragment thereof known in the art to transmigrate the blood brain barrier.

The present invention provides a compound, or fusion protein, comprising an antibody or fragment thereof that transmigrates the blood brain barrier (BBB). An antibody or fragment of the present invention may bind to, for example, transmembrane protein 30A (TMEM30A), as described in WO 2007/036021 , or to an Insulin-Like Growth Factor 1 Receptor (IGF1 R) epitope, or isoforms, variants, portions, or fragments thereof.

The antibody or fragment thereof in the compound of the present invention may comprise a complementarity determining region (CDR) 1 sequence of HYTMG (SEQ ID NO:1); a CDR2 sequence of RITWGGDNTFYSNSVKG (SEQ ID NO:2); and a CDR3 sequence of GSTSTATPLRVDY (SEQ ID NO:3); or a CDR1 sequence of EYPSNFYA (SEQ ID NO:4), a CDR2 sequence of VSRDGLTT (SEQ ID NO:5), a CDR3 sequence of AIVITGVW KVDVNSRSYHY (SEQ ID NO:6); or a CDR1 sequence of GGTVSPTA (SEQ ID NO:7), a CDR2 sequence of ITWSRGTT

(SEQ ID NO:8), a CDR3 sequence of AASTFLRILPEESAYTY (SEQ ID NO:9); or a CDR1 sequence of GRTIDNYA (SEQ ID NO:10), a CDR2 sequence of IDWGDGGX; where X is A or T (SEQ ID NO:1 1 ), a CDR3 sequence of AMARQSRVNLDVARYDY (SEQ ID NO:12). As previously stated, the antibody or fragment thereof may be an sdAb of camelid origin or derived from a camelid V _H H, and thus may be based on camelid framework regions; alternatively, the CDR described above may be grafted onto V _NAR , V _H H, V _H or V _L framework regions. In yet another alternative, the hypervariable loops described above may be grafted onto the framework regions of other types of antibody fragments (Fv, scFv, Fab) of any source (for example, mouse or human) or proteins of similar size and nature onto which CDR can be grafted (for example, see Nicaise et al, 2004). The present invention further encompasses an antibody or fragment thereof that is chimeric (or chimerized), veneered, or humanized. Chimeric antibodies or fragments thereof are constructs in which the native variable domain (of mouse or camelid origin) is linked to human constant domain(s) (see Gonzales et al 2005). Veneering or re-surfacing of antibodies involves replacing exposed residues in the framework region of the native antibody or fragment thereof with the amino acid residues in their human counterpart (Padlan, 1991 ; Gonzales et al 2005). Humanization of an antibody or antibody fragment comprises replacing an amino acid in the sequence with its human counterpart, as found in the human consensus sequence, without loss of antigen-binding ability or specificity; this approach reduces immunogenicity of the antibody or fragment thereof when introduced into human subjects. In this process, one or more than one of the CDR defined herein may be fused or grafted to a human variable region (V _H , or V _L ), to other human antibody (IgA, IgD, IgE, IgG, and IgM), to human antibody fragment framework regions (Fv, scFv, Fab), or to human proteins of similar size and nature onto which CDR can be grafted (Nicaise et al, 2004). In such a case, the conformation of said one or more than one hypervariable loop is likely preserved, and the affinity and specificity of the sdAb for its target (i.e., an epitope on brain endothelial cells, such as TMEM30A, or an IGF1 R epitope) brain endothelial cells) is likely minimally affected. As is known by those of skill in the art, it may be necessary to incorporate certain native amino acid residues into the human framework in order to retain binding and specificity. Humanization by CDR grafting is known in the art (for example, see Tsurushita et al, 2005; Jones et al, 1986; Tempest et al, 1991 ; Riechmann et al, 1988; Queen et al, 1989; reviewed in Gonzales et al, 2005 - see also references cited therein), and thus persons of skill would be amply familiar with methods of preparing such humanized antibody or fragments thereof.

The provided antibody or fragment thereof may be a humanized version of the FC5 antibody (described in WO 2002/057445) or an IGF1 R antibody. FC5 (comprising a sequence of any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17), and IGF1 R (comprising a sequence of any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26) bind to the surface of receptor epitopes on brain endothelial cells and subsequently transmigrates the blood-brain barrier (BBB). FC5 has also been shown to act as a carrier to usher molecules of various sizes across the BBB (see for example, WO 201 1 /127580). The antigen mediating FC5 transmigration was tentatively identified as transmembrane domain protein 30A (TMEM30A; WO 2007/036021), which is enriched on the surface of brain endothelial cells. For example, and without wishing to be limiting, the antibody or fragment thereof may comprise the sequence:

X _! VQLVXzSGGGLVQPGGSLRLSCAASGFKITHYTMGWXaRQAPGKXiXsEXeVSR IT WGGDNTFYSNSVKGRFTISRDNSKNTX ₇ YLQMNSLRAEDTAVYYCAAGSTSTATPL RVDYWGQGTLVTVSS (SEQ ID NO:13), where X,=D or E, X ₂ =A or E, X ₃ =F or V,

X ₄ =E or G, X ₅ =R or L, X ₆ =F or W, X ₇ =L or V, or a sequence substantially identical thereto;

X ₁ VX2LX3ESGGGLVQX4GGSLRLSCX ₅ ASEYPSNFYAMSWX ₆ RQAPGKX ₇ X ₈ EX ₉ VX ₁ o GVSRDGLTTLYADSVKGRFTX ₁₁ SRDNX ₁₂ KNTX ₁₃ X ₁₄ LQMNSX ₁₅ X ₁₆ AEDTAVYYCAI VITGVWNKVDVNSRSYHYWGQGTX ₁₇ VTVSS, (SEQ ID NO:18), where X _! is E or Q;

X ₂ is K or Q; X ₃ is V or E; X ₄ is A or P; X ₅ is V or A; X ₆ is F or V; X ₇ is E or G; X ₈ is R or L; X ₉ is F or W; X ₁₀ is A or S; X is M or I; X ₁₂ is A or S; X ₁₃ is V or L; X ₁₄ is D or Y; X ₁₅ is V or L; X ₁₆ is K or R; and X ₁₇ is Q or L; or a sequence substantially identical thereto; X _! VX ₂ LX ₃ ESGGGLVQX ₄ GGSLRLSCX ₅ X ₆ SGGTVSPTAMGWX ₇ RQAPGKX ₈ X ₉ EX ₁₀ V

XiiHITWSRGTTRXi2ASSVKXi3RFTISRDXi4Xi5KNTXieYLQMNSLXi ₇ Xi8EDTAVYYC AASTFLRILPEESAYTYWGQGT X ₁₉ VTVSS, (SEQ ID NO:21), where X _! is E or Q; X ₂ is K or Q; X ₃ is V or E; X ₄ is A or P; X ₅ is A or E; X ₆ is V or A; X ₇ is V or F; X ₈ is G or E; X ₉ is L or R; X ₁₀ is F or W; X is G or S; X ₁₂ is V or Y; X ₁₃ is D or G; X ₁₄ is N or S; X ₁₅ is A or S; X ₁₆ is L or V; X ₁₇ is K or R; X ₁₈ is A or S; and X _ig is L or Q; or a sequence substantially identical thereto;

X ₁ VX ₂ LX ₃ ESGGGLVQX ₄ GGSLRLSCAASGRTIDNYAMAWX5RQAPGKX ₆ X ₇ EX ₈ VX ₉ TID WGDGGX^RYANSVKGRFTISRDNX KX^TXwYLQMNXuLXisXieEDTAVYX^CAMAR QSRVNLDVARYDYWGQGTXisVTVSS (SEQ ID NO: 24), where X _! is E or Q; X ₂ is K or Q; X ₃ is V or E; X ₄ is A or P; X ₅ is V or S; X ₆ is D or G; X ₇ is L or R; X ₈ is F or W; X ₉ is A or S; X ₁₀ is A or T; X is A or S; X ₁₂ is G or N; X ₁₃ is M or L; X ₁₄ is N or R; X ₁₅ is E or R; X ₁₆ is P or A; X ₁₇ is S or Y; and X ₁₈ is Q or L; or a sequence substantially identical thereto.

More specifically, and without wishing to be limiting in any manner, the antibody or fragment thereof may comprise a sequence selected from any one of:

DVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSRITWGGDN TFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAGSTSTATPLRVDYWG KGTQVTVSS (SEQ ID NO:14); EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWVRQAPGKGLEWVSRITWGGDN TFYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWGQG TLVTVSS (SEQ ID NO: 15);

EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWVRQAPGKGLEWVSRITWGGDN TFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG QGTLVTVSS (SEQ ID NO: 16);

EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEFVSRITWGGDN TFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG QGTLVTVSS (SEQ ID NO: 17); QVKLEESGGGLVQAGGSLRLSCVASEYPSNFYAMSWFRQAPGKEREFVAGVSRDGLT TLYADSVKGRFTMSRDNAKNTVDLQMNSVKAEDTAVYYCAIVITGVWNKVDVNSRSYH YWGQGTQVTVSS (SEQ ID NO: 19);

EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKEREFVSGVSRDGLT TLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHY WGQGTLVTVSS (SEQ ID NO: 20);

QVKLEESGGGLVQAGGSLRLSCEVSGGTVSPTAMGWFRQAPGKEREFVGHITWSRGT TRVASSVKDRFTISRDSAKNTVYLQMNSLKSEDTAVYYCAASTFLRILPEESAYTYWGQ GTQVTVSS (SEQ ID NO: 22);

QVQLVESGGGLVQPGGSLRLSCAVSGGTVSPTAMGWFRQAPGKGLEFVGHITWSRGT TRYASSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAASTFLRILPEESAYTYWGQ GTLVTVSS (SEQ ID NO: 23)

QVKLEESGGGLVQAGGSLRLSCAASGRTIDNYAMAWSRQAPGKDREFVATIDWGDGG ARYANSVKGRFTISRDNAKGTMYLQMNNLEPEDTAVYSCAMARQSRVNLDVARYDYW GQGTQVTVSS (SEQ ID NO: 25); QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPGKGLEWVATIDWGDG GTRYANSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCAMARQSRVNLDVARYDY WGQGTLVTVSS (SEQ ID NO: 26); and a sequence substantially identical thereto. The antibody or fragment thereof may be a single- domain antibody. A substantially identical sequence may comprise one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence may yield a mutant peptide with no substantial change in physiological, chemical, physico-chemical or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered "substantially identical" polypeptides. A conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g. size, charge, or polarity). These conservative amino acid mutations may be made to the framework regions of the sdAb while maintaining the CDR sequences listed above and the overall structure of the CDR of the antibody or fragment; thus the specificity and binding of the antibody are maintained.

A substantially equivalent sequence may comprise one or more conservative amino acid mutations; wherein the mutant peptide is substantially equivalent with respect to peptide stability and bio-manufacturability. Substantially equivalent may refer to equivalent with respect to fusion molecule stability; for example, the lack of a degradation product, or a low molecular weight band, as seen in SDS PAGE (reducing and non-reducing conditions). It is known in the art that one or more conservative amino acid mutations to a reference sequence may yield a mutant peptide with no substantial change in physiological, chemical, physico- chemical or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered "substantially equivalent" polypeptides.

In a non-limiting example, a conservative mutation may be an amino acid substitution. Such a conservative amino acid substitution may substitute a basic, neutral, hydrophobic, or acidic amino acid for another of the same group. By the term "basic amino acid" it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). By the term "neutral amino acid" (also "polar amino acid"), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gin or Q). The term "hydrophobic amino acid" (also "non-polar amino acid") is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (lie or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). "Acidic amino acid" refers to hydrophilic amino acids having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E), and aspartate (Asp or D).

Sequence identity is used to evaluate the similarity of two sequences; it is determined by calculating the percent of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics (and as found at ca.expasy.org/tools/blast/), BLAST-P, Blast-N, or FASTA-N, or any other appropriate software that is known in the art.

The substantially identical sequences of the present invention may be at least 90% identical; in another example, the substantially identical sequences may be at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical, or any percentage therebetween, at the amino acid level to sequences described herein. Importantly, the substantially identical sequences retain the activity and specificity of the reference sequence. In a non-limiting embodiment, the difference in sequence identity may be due to conservative amino acid mutation(s). In a non-limiting example, the present invention may be directed to an antibody or fragment thereof comprising a sequence at least 95%, 98%, or 99% identical to that of the antibodies described herein. The antibody or fragment thereof in the compound of the present invention may be linked to an Fc domain, for example, but not limited to human Fc domains. The Fc domains may be selected from various classes including, but not limited to, IgG, IgM, or various subclasses including, but not limited to lgG1 , lgG2, etc. In this approach, the Fc gene is inserted into a vector along with the sdAb gene to generate a sdAb-Fc fusion protein (Bell et al, 2010; Iqbal et al, 2010); the fusion protein is recombinantly expressed then purified. For example, and without wishing to be limiting in any manner, multivalent display formats may encompass chimeric formats of FC5-H3 and its mutational variants linked to an Fc domain. Such antibodies are easy to engineer and to produce, can greatly extend the serum half-life of sdAb (Bell et al., 2010). The Fc domain in the compound as just described may be any suitable Fc fragment known in the art. The Fc fragment may be from any suitable source; for example, the Fc may be of mouse or human origin. Other preferred Fc or Fc fragment embodiments may modulate, modify or suppress an immunological effector function (Shields 2001 ). Other highly preferred Fc fragment embodiments may mediate clearance of the fusion peptide from the brain (Caram- Salas N 201 1). In a specific, non-limiting example, the Fc may be the mouse Fc2a fragment or human Fc1 fragment (Bell et al, 2010; Iqbal et al, 2010). In a specific, non-limiting example, the multimerized construct may comprise the isolated or purified antibody or fragment as described herein and an Fc of sequence of SEQ ID NO:39; SEQ ID NO:40; SEQ ID NO:41 . Accordingly, the BBB-Fc-ABP fusion proteins provided herein may form dimers via the Fc to provide a bivalent, bifunctional BBB-Fc-ABP.

The compound of the present invention comprises an antibody or fragment thereof linked to a polypeptide that binds β-amyloid. The linker may be any polypeptide, comprising neutral or hydrophilic amino acids, of suitable length. In a non-limiting example, the length is preferably less than 12 amino acids, and the polypeptide is GGGSGGGS. Other chemical linkers may be employed using non-peptidic forms such as amide or ester linkages in a correct orientation. For example, SEQ ID NO: 53 provides a fusion protein comprising any suitable linker, wherein any fusion protein of the present invention, i.e. SEQ ID NO: 42 - 52 may comprise any suitable linker as exemplified in SEQ ID NO: 53. The polypeptide that binds β-amyloid (Αβ) may bind pathologically relevant β-amyloids, such as Αβ _! ^ aggregates, which are implicated in AD pathology; the polypeptide may bind Αβ with high affinity (in the nM range), inhibit Αβ binding to cellular proteins and Aβ ₁ _42-induced cell toxicity in vitro, and binds amyloid deposits in AD transgenic mice brain as well as in the brains from AD patients in vitro. The polypeptide in the compound of the present invention does not bind the reverse peptide Αβ ₄₂ -ι . In a compound provided herein, the polypeptide that binds β-amyloid may comprise a sequence selected from the group consisting of: (SEQ ID NO:27); (SEQ ID NO:28); (SEQ ID NO:29); (SEQ ID NO:30); (SEQ ID NO:31 ); (SEQ ID NO:32); (SEQ ID NO:33); (SEQ ID NO:34); (SEQ ID NO:35); (SEQ ID NO:36); (SEQ ID NO:37); (SEQ ID NO:38); and a sequence substantially identical thereto. A "substantially identical" sequence is as described above.

Accordingly, the present invention provides a polypeptide that binds β-amyloid and variants thereof (i.e. ABP and ABP variants). The ABP variant may comprise a sequence having SEQ ID NO: 31 , for example, the ABP variant provided may comprise a sequence selected from the group consisting of: SEQ ID NO: 31 to SEQ ID NO:38 or a sequence substantially equivalent thereto.

Accordingly, there is provided ABP polypeptide sequences comprising specific systematic and methodical modifications based on detailed biophysical characterization of the ABP. The specific and methodically directed modifications to the ABP polypeptide comprise the novel and unobvious ABP variants of the present invention, as provided in SEQ ID NO:31 . The peptide provided herein may comprise an ABP comprising a sequence that may be selected from the group consisting of SEQ ID NO: 27 - SEQ ID NO: 38, and a sequence substantially equivalent thereto.

The peptide provided herein may comprise an ABP variant, for example, an ABP comprising a sequence that may be selected from any one of SEQ ID NO: 32 to SEQ ID NO:38, or any equivalent sequence to SEQ ID NO:31 . A construct comprising an ABP or an ABP variant, as provided herein, exhibits advantageous improvement in compound stability and bio- manufacturability (as can be seen in Fig.14).

The fusion proteins and compounds provided herein, exhibit improved therapeutic efficacy. More specifically, the compounds provided comprise specifically modified ABP that allows the generation of stable BBB-Fc-ABP fusion molecule for enhanced bio-manufacturability (production in human mammalian expression system). The specific modifications to ABP provided herein were systematic and methodical modifications based on detailed biophysical characterization of the ABP. The modified ABP provided herein, may comprise, for example, a sequence selected from SEQ ID NO: 32 - SEQ ID NO: 38, and a sequence substantially equivalent thereto, such as SEQ ID NO: 31 . The compounds provided herein advantageously exhibit improved stability and bio-manufacturability, and a most significant increase in the efficacy of reducing Αβ levels in brain; wherein a 50% amyloid reduction was observed within 24 hr of treatment with a construct of the present invention. By the term "linked", also referred to herein as "conjugated", it is meant that two moieties are joined directly or indirectly (e.g., via a linker), covalently or non-covalently (e.g., adsorption, ionic interaction). A covalent linkage may be achieved through a chemical cross-linking reaction, or through fusion using recombinant DNA methodology combined with any peptide expression system, such as bacteria, yeast or mammalian cell-based systems. When conjugating the antibody or fragment thereof to the polypeptide binding Αβ or Fc, a suitable linker may be used. For example, a suitable linker may be any polypeptide, comprising neutral or hydrophilic amino acids, of suitable length that allows for the conjugation of the components of the BBB-Fc-ABP protein fusion. For example, the linker that allows the components of the fusion protein (for example, in SEQ ID NO: 42-52) to be according linked, and is not limited to the GGGGSGGGGS or (GGGS) _n linker highlighted therein and may be any suitable linker (i.e. as in the non-limited fusion protein of SEQ ID NO: 53). In a non-limiting example, the length is preferably less than 12 amino acids, and the polypeptide may be GGGSGGGS. Other chemical linkers may be employed using non-peptidic forms such as amide or ester linkages in a correct orientation. One of skill in the present art would be well aware of linkers or method of linking an antibody or fragment thereof to a polypeptide. Methods for linking an antibody or fragment thereof to a polypeptide or Fc are well-known to a person of skill in the art.

The compound provided herein comprises an antibody or fragment thereof, a polypeptide that binds β-amyloid, and an Fc fragment, linked to provide a construct (also referred to herein as a compound or fusion molecule), wherein the construct comprises a fusion protein and dimers thereof. The antibody or fragment thereof may be linked to a polypeptide that binds β-amyloid via an Fc fragment, or a suitable linker.

The antibody or fragment thereof comprises a sequence selected from any one of: SEQ ID NO:14, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO 23, SEQ ID NO:25, SEQ ID NO:26, and a sequence substantially identical thereto. The antibody or fragment thereof transmigrates the blood brain barrier. The antibody or fragment may be a sdAb; wherein the sdAb may be humanized.

The polypeptide that binds β-amyloid comprises a sequence selected from the group consisting of: SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and a sequence substantially identical thereto, and preferably a sequence comprising SEQ ID NO: 31 .

The Fc fragment comprises a sequence selected from any one of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 , and a sequence substantially identical thereto. The Fc fragment of the present invention may be any suitable Fc fragment with attenuated effector function. The Fc fragment provided in the fusion protein allows for the formation of dimeric structures; wherein for example a single-chain fusion protein comprising a sequence selected from the group consisting of SEQ ID NO: 42 to SEQ ID NO: 53 may form dimeric structures conjugated via the Fc fragment therein. Accordingly, a compound or construct of the present invention may comprise a sequence selected from the group consisting of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53 and a sequence substantially identical thereto, or dimeric structures thereof. The present invention provides fusion proteins comprising a BBB-crossing single-domain antibody (BBB), an Fc fragment (Fc) and an amyloid-binding peptide (ABP), wherein each moiety may be linked to provide fusion molecules, as illustrated in FIG. 1 . For example, in a non-limiting embodiment of the represent invention, the BBB may linked to the N-terminus of the Fc and the ABP linked to the C-terminus of the Fc fragment (FIG. 1 A). Fig.1 shows the corresponding dimers of the single-chain fusion protein comprising BBB-Fc-ABP. The 3 components (i.e. BBB, Fc, and ABP) are depicted in various configurations, as illustrated in FIG 1A, 1 B, 1 C, and 1 D.ln a specific, non-limiting example of the compound of the present invention, the polypeptide that binds β-amyloid may comprise the sequence SEQ ID NO: 31 . In an embodiment the ABP variant comprises the sequence:

GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI (SEQ ID NO:36) referred to herein as ABP(6G) and a sequence substantially equivalent thereto.

The antibody or fragment thereof may comprise the sequence EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEFVSRITWGGD NTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG QGTLVTVSS (SEQ ID NO: 17) referred to herein as FC5-H3.

The antibody or fragment thereof may further comprise the sequence of a human Fc, such as

AEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEGPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO:40), also referred to herein as hFc1 X7.

Without wishing to be limiting in any manner, the compound of the present invention may comprise the sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEFVSRITWGGD NTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG QGTLVTVSSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGGGGSGGGGSGTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKS RVKNI (SEQ ID NO:47), also referred to herein as FC5-H3-hFc1X7-ABP(6G).

Table Summary of some representative BBB-Fc-ABP fusion protein constructs. BBB Fc ABP

FC5 (SEQ ID NO: 14) mFc2a (SEQ ID NO: 39) ABP (SEQ ID NO: 30)

FC5(H3) (SEQ ID NO: 17) mFc2a (SEQ ID NO: 39) ABP (SEQ ID NO: 30)

FC5(H3) (SEQ ID NO: 17) hFd x7 (SEQ ID NO: 40) ABP (SEQ ID NO: 30)

FC5(H3) (SEQ ID NO: 17) hFd x7 (SEQ ID NO: 40) ABP(G) SEQ ID NO: 32)

FC5(H3) (SEQ ID NO: 17) hFc1 x7 (SEQ ID NO: 40) ABP(GG-G) SEQ ID NO: 35

FC5(H3) (SEQ ID NO: 17) hFc1 x7 (SEQ ID NO: 40) ABP(6G) SEQ ID NO: 36)

FC5(H3) (SEQ ID NO: 17) hFdXO (SEQ ID No: 41 ) ABP(GG-G) SEQ ID NO: 35

FC5(H3) (SEQ ID NO: 17) hFc1 x7 (SEQ ID NO: 40) ABP(trc) SEQ ID NO: 38)

IGF1 R-5(H2) (SEQ ID NO: 26) mFc2a (SEQ ID NO: 39) ABP (SEQ ID NO: 30)

It is noted that in the above-table, the BBB-Fc-ABP fusion protein constructs may also comprise a linker (L) wherein the BBB-Fc-ABP construct is a BBB-Fc-L-ABP fusion protein of the present invention, wherein L may be GGGSGGGGS or any suitable linker, as exemplified in the consensus linker sequence in SEQ ID NO:53.

The compound of the present invention, as provided in the fusion protein of SEQ ID NO:47 also referred to herein as FC5-H3-hFc1X7-L-ABP(6G), may comprise variations in each of the components comprised therein, for example, ABP may be ABP (GG-G), as provided in SEQ ID NO: 46. The linker sequence provided therein (as highlighted for example, in SEQ ID NO:42 to SEQ ID NO: 53) may be any linker that allows for the linking of the BBB-Fc-ABP fusion protein. The compound of the present invention may also comprise additional sequences to aid in expression, detection or purification of a recombinant antibody or fragment thereof. Any such sequences or tags known to those of skill in the art may be used. For example, and without wishing to be limiting, the antibody or fragment thereof may comprise a targeting or signal sequence (for example, but not limited to ompA), a detection/purification tag (for example, but not limited to c-Myc, His ₅ , or His ₆ ), or a combination thereof. In another example, the additional sequence may be a biotin recognition site such as that described by Cronan et al (WO 95/04069) or Voges et al (WO/2004/076670). As is also known to those of skill in the art, linker sequences may be used in conjunction with the additional sequences or tags, or may serve as a detection/purification tag.

The present invention also encompasses nucleic acid sequences encoding the compounds as described herein. Given the degeneracy of the genetic code, a number of nucleotide sequences would have the effect of encoding the polypeptide, as would be readily understood by a skilled artisan. The nucleic acid sequence may be codon-optimized for expression in various micro-organisms. The present invention also encompasses vectors comprising the nucleic acids as just described. Furthermore, the invention encompasses cells comprising the nucleic acid and/or vector as described. The present invention further encompasses a composition comprising one or more than one compound as described herein and a pharmaceutically acceptable diluent, excipient, or carrier. The composition may also comprise a pharmaceutically acceptable diluent, excipient, or carrier. The diluent, excipient, or carrier may be any suitable diluent, excipient, or carrier known in the art, and must be compatible with other ingredients in the composition, with the method of delivery of the composition, and is not deleterious to the recipient of the composition. The composition may be in any suitable form; for example, the composition may be provided in suspension form or powder form (for example, but limited to lyophilised or encapsulated). For example, and without wishing to be limiting, when the composition is provided in suspension form, the carrier may comprise water, saline, a suitable buffer, or additives to improve solubility and/or stability; reconstitution to produce the suspension is effected in a buffer at a suitable pH to ensure the viability of the antibody or fragment thereof. Dry powders may also include additives to improve stability and/or carriers to increase bulk/volume; for example, and without wishing to be limiting, the dry powder composition may comprise sucrose or trehalose. It would be within the competency of a person of skill in the art to prepare suitable compositions comprising the present compounds.

A method of treating Alzheimer's disease is also provided, in which a compound or composition of the present invention is administered to a subject in need thereof. Any appropriate route of administration may be utilized, including but not limited to intravenous, intraperitoneal, parenteral, intracranial, intramuscular, subcutaneous, oral, or nasal. The optimal dose for administration and route of administrations are generally determined experimentally.

A method of reducing toxic β-amyloid levels in the cerebrospinal fluid (CSF) and brain parenchyma of subjects having increased β-amyloid levels is provided. More specifically, toxic β-amyloid levels in the cerebrospinal fluid (CSF) and brain parenchyma of subjects is reduced as early as 24 hours of a single parenteral administration of the composition of the present invention.

The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.

Example 1: Construction of the BBB-Fc-L-ABP fusion molecules

Fusion molecules comprising:

a) the FC5 sdAb (SEQ ID NO:14), a murine Fc (SEQ ID NO:39) and ABP (SEQ ID NO:

30),

b) a humanized version of FC5 (FC5-H3; SEQ ID NO: 17), a human Fc (SEQ ID NO: 40) and ABP (SEQ ID NO: 30; SEQ ID NO: 31 ; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38),

c) a humanized version of IGF1 R-5 sdAb (IGF1 R-5-H2; SEQ ID NO: 26), a murine Fc (SEQ ID NO:39) and ABP (SEQ ID NO: 30) were prepared. A schematic of the fusion protein construct is shown in Figure 1 . The fusion protein comprises a BBB-crossing single-domain antibody, an Fc fragment and an amyloid- binding peptide.

Example 2: Production of BBB-Fc-L-ABP fusion molecules

The constructs FC5-mFc-ABP and humanized FC5(H3)-hFc-ABP described in Example 1 were expressed in CHO cells, and the expressed compounds were purified on MabSelect Sure affinity columns

Constructs comprising FC5 variants FC5 and FC5-H3 V _H H fused to N-terminal of mouse or human Fc antibody fragment fused to ABP variants at their C-terminus as described in example 1 were prepared, expressed, and purified. The FC5-Fc-ABP variant DNAs (DNA synthesis suppliers) were cloned into mammalian expression vector pTT5 (Durocher 2002). Polyplexes for a final concentration of 1 mg of DNA per liter of cells were pre-formed by mixing a combination of plasmid vector (80%), pTT-AKTdd (15%, activated mutant of Protein Kinase B), and pTTo-GFP (5%, to monitor transfection efficiency) with PEI MAX solution (Polysciences cat no 24765). The PEI:DNA ratio was 4:1 (W:W), both prepared in supplemented F17 medium (4 mM Glutamine, 0.1 % Kolliphor). The mixture was incubated for 5 minutes at room temperature prior to addition to the cell culture. The volume of the DNA/PEI polyplexe represent 10% of final culture volume (e.i. 100 ml per 1 L culture). Twenty four hours post-transfection, the cultures were fed with tryptone N1 at a final concentration of 1 % (with 40% w/v solution, Organotechnie) and 0.5 mM valproic acid (200 mM solution). The transfection/production were monitored for cell density and viability as well as for productivity titer (mg of Fc per L) and were harvested (supernatant) by centrifugation when the cell viability reach a minimum of 65%. Clarified cell culture medium was filtered through a 0.45 μηι membrane prior to its application on a column packed with 5 ml of protein-A MabSelect SuRe resin (GE Healthcare). After loading, the column was washed with 5 volumes of phosphate-buffered saline pH 7.1 (PBS) and the antibody was eluted with 100 mM sodium citrate buffer pH 3.0. Fractions containing the eluted antibody were pooled and a buffer exchange was performed by loading on a desalting Econo-Pac column (BioRad) equilibrated in PBS. Desalted antibody was then sterile-filtered by passing through a Millex GP (Millipore) filter unit (0.22 μηι) and aliquoted.

SDS-PAGE and AB overlay: Protein samples, prepared in Laemmli sample buffer (heated at 70°C for non-reducing and at 95°C for reducing gels, BME or DTT), were separated by SDS- PAGE on 12% Tris-Tricine gels or TGX4-15% gels (BioRad). Gels were either stained with Coomassie blue or the proteins were transferred to PVDF or nitrocellulose membranes for Western blot/AB overlay assay. The immunoblots were blocked with non-fat dry milk and then exposed to AB preparations for 45 min at room temperature (50-1 OOnM) and the bound AB was detected using 6E10 antibody as described previously (Chakravarthy et al. 2013).

ELISA: Αβ-binding assays were carried out as described by Chakravarthy et al (2013). Maxisorp 96-well ELISA plates (Nunc) were coated with (100-500 ng/well) either free ABP (synthetic) or various FC5-ABP constructs overnight at 4°C in PBS. The wells were blocked with 1 % BSA in TBS-T for 30 min and then incubated with AB^ preparations consisting predominantly of either monomer and dimers (Mo) or higher oligomers (Oli) in TBS-T at RT for 45 min with gentle agitation. Following three TBS-T washes, bound AB was detected by incubating HRP-conjugated AB-specific antibody (6E10 or 4G8) for 90 min at RT in TBS-T. The bound antibody was detected with SureBlue™ TMB reagent kit (KPL) by colorimetric measurement at 450nm according to manufacturer's instructions.

A Coomassie blue stained gel after separation of FC5 fusion molecules by SDS-PAGE (NR- non-reducing and R- reducing conditions) indicating successful production of recombinant fusion molecule is shown in Fig. 2A. AB-oligomer binding of free ABP and FC5-Fc-ABP fusion protein by ELISA and Western blot (WB) Overlay assay is shown in Fig. 2B and C. Free or fused ABP was immobilized on ELISA plate by coating samples in phosphate buffered saline (PBS) overnight at 4°C and exposed to AB preparations as described by Chakravarthy et al., 2013. Bound Αβ was detected with Αβ-specific antibody 6E10 or 4G8 (Chakravarthy et al., 2013). The fusion molecules were also separated by SDS-PAGE, transferred to PVDF paper and exposed to Αβ oligomers. Bound Αβ was detected with specific antibody as above. Results show that ABP retained its Αβ oligomer binding ability after fusion with the BBB carrier. Mo: AB monomers; Oli: Αβ Oligomers

Example 3: Binding of BBB-Fc-L-ABP fusion molecules to Αβ deposits in AD-Tg mice (B6.Cg- Tg, Jackson Lab) in vitro

The constructs produced in Example 2 were submitted to immunohistofluorescence assay to evaluate whether the FC5-Fc-ABP fusion molecules retained the ability to bind naturally- produced amyloid deposits in mouse brain as described (Chakravarthy et al., 2014). Frozen hemi-brains from wild type (Wt) and AD transgenic (AD-Tg) mice were embedded in OCT and 10-μηι sections were prepared using a Jung CM 3000 cryostat and stored at -80°C. Tissue sections were thawed and OCT peeled from sections with a razor blade and then incubated with Dako protein blocking reagent for 30 min at room temperature. Blocking agent was removed and sections were gently washed in TBS. IR 800-labelled FC5-mFc-ABP (1 :250 dilution of 5.0 μ9/μΙ solution) in antibody diluent was added and incubated for 1 h at room temperature. Sections were then washed twice with TBS, rinsed in Milli Q water, excess rinse solution removed and sections were cover-slipped with Dako Fluorescent Mounting Media. Sections visualized under fluorescence microscope (Fig. 3). Selective binding (bright spots) was seen in brain sections from AD-Tg mice that produce AB deposits and not in brain sections from Wt mice that does not produce amyloid deposits, indicating that ABP in the FC5- Fc-ABP construct retains the ability to bind naturally produced AB aggregates in AD-Tg mouse brain. In the BBB-Fc-ABP constructs provided, the BBB may be FC5 or an anti-IGF1 R antibody.

Example 4: BBB transmigration of FC5-FC-L-ABP fusion molecules in vitro

BBB- crossing of FC5-Fc-ABP fusion molecules was assessed in in vitro BBB models from rat and human (Fig. 4). Fusion molecules crossing BBB were screened in in vitro BBB permeability assay, using a single-time point for Papp determination. The quantification of variants was done by MRM-ILIS (FIG. 4A, 4B and 4C).

SV40-immortalized Adult Rat Brain (SV-ARBEC) and Human Brain Endothelial Cells (HBEC) were used to generate an in vitro blood-brain barrier (BBB) model as described (Garberg et al., 2005; Haqqani et al., 2013). Sv-ARBEC (80,000 cells/membrane) were seeded on a 0.1 mg/ml_ rat tail collagen type l-coated tissue culture inserts (pore size-1 μηι; surface area 0.9 cm ² , Falcon) in 1 ml of growth medium. The bottom chamber of the insert assembly contained 2 ml of growth medium supplemented with the immortalized neonatal rat astrocytes-conditioned medium in a 1 :1 (v/v) ratio. Equimolar amounts (5.6 μΜ) of positive (FC5 constructs) or negative controls (A20.1 , a Clostridium difficile toxin A binding V _H H; and EG2, an EGFR binding V _H H), and Fc-ABP from Example 1 were tested for their ability to cross the rat or human in vitro BBB model. Following exposure of equimolar amounts of the sdAb to the luminal side of the BBB, samples were taken after 15, 30 and 60min from the abluminal side. The sdAb content of each sample was then quantified by mass spectrometry (multiple reaction monitoring - isotype labeled internal standards; MRM - ILIS) (Fig. 4A, 4B, 4C). MRM-ILIS: The methods are all as described in Haqqani et al. (2013). Briefly, to develop the SRM (selected reaction monitoring also known as multiple reaction monitoring (MRM)) assay for VHH, each V _H H was first analyzed by nanoLC-MS/MS using data-dependent acquisition to identify all ionizible peptides. For each peptide, the 3 to 5 most intense fragment ions were chosen. An initial SRM assay was developed to monitor these fragments at attomole amounts of the digest (about 100-300 amol). Fragments that showed reproducible intensity ratios at low amounts (i.e., had Pearson r2 > 0.95 compared to higher amounts) were considered stable and were chosen for the final SRM assay. To further optimize the assay, elution times for each peptide were also included, with care taken to not choose peptides that have close m/z (mass- to-charge ratio) and elution times. A typical multiplexed SRM analysis of V _H H in cell media or body fluids (serum or cerebrospinal fluid (CSF)) involved spiking known amount of ILIS (0.1 -10 nM) followed by injecting 100-400 ng of CSF or cultured media proteins (0.3-1 μΙ_) or about 50-100 ng of serum proteins (1 -3 nanoliters) into the nanoLC-MS system. The precursor m/z of each target peptide ion was selected in the ion trap (and the remaining unrelated ions were discarded) at the specified elution time for the target, followed by collision induced dissociation (CID) fragmentation, and selection of only the desired fragment ions in the ion trap for monitoring by the detector. For quantification analysis, raw files generated by the LTQ (ThermoFisher) were converted to the standard mass spectrometry data format mzXML and intensities were extracted using an in- house software called Q-MRM (Quantitative-MRM; see Haqqani et al. 2013), which is a modified version of MatchRx software. For each V _H H, extracted-ion chromatograms were generated for each of its fragment ion that consisted of combined intensities within 0.25 Da of the fragment m/z over the entire elution time. To obtain a final intensity value for each fragment, all intensities within 0.5 min of the expected retention times were summed. A V _H H was defined as detectable in a sample if the fragments of at least one of its peptides showed the expected intensity ratios, i.e., the final intensity values showed a strong Pearson correlation r >0.95 and p<0.05 compared with the final intensities values of its corresponding pure V _H H.

Samples containing mixtures of V _H H (media, serum, CSF) were reduced, alkylated and trypsin- digested as previously described (Haqqani et al., 2012; Gergov et al., 2003). The digests (tryptic peptides) were acidified with acetic acid (5% final concentration) and analyzed on a reversed-phase nanoAcquity UPLC (Waters, Milford, MA) coupled to LTQ XL ETD or LTQ Orbitrap ETD mass spectrometer (ThermoFisher, Waltham, MA). The desired aliquot of the sample was injected and loaded onto a 300 μηι I.D. ^χ 0.5 mm 3μηι PepMaps C18 trap (ThermoFisher) then eluted onto a 100 μηι I.D. x 10 cm 1 .7 μητι BEH130C18 nanoLC column (Waters) using a gradient from 0% - 20% acetonitrile (in 0.1 % formic) in 1 minute, 20% - 46% in 16 min, and 46% - 95% in 1 min at a flow rate of 400 nL/min. The eluted peptides were ionized into the mass spectrometer by electrospray ionization (ESI) for MS/MS and SRM analysis using CID for fragmentation of the peptide ions. The CID was performed with helium as collision gas at normalized collision energy of 35% and 30 ms of activation time. Ion injection times into linear ion trap were adjusted by the instrument using an automatic gain control (AGC) target value of 6 x 10 ³ and a maximum accumulation time of 200 ms.

Determination of the apparent permeability coefficient: Quantified values can be directly plotted or the P _app (apparent permeability coefficient) values can be determined with the given formula [Qr/dt = cumulative amount in the receiver compartment versus time; A = area of the cell monolayer; CO = initial concentration of the dosing solution] and plotted. The P _app value is commonly used to determine the ability of a molecule to cross the BBB. P _app values are a measure of the specific permeability of the compound across brain endothelial monolayer.

The specific peptides used for detection and quantification of FC5 constructs are shown in Table 1 below.

TABLE 1

The samples were also analyzed by Western blot analysis using Fc-specific antibody (FIG. 4D, done in triplicate) as described in Example 2. FC5-mFc-ABP crossed the blood brain barrier as effectively as FC5-mFc, whereas Fc-ABP without the BBB carrier moiety FC5 did not traverse across the brain endothelial cell monolayer. As expected, control single domain antibodies EG2 and A20.1 , or control full IgG (anti-HEL) did not cross the blood brain barrier. Similar results were obtained with humanized FC5(H3)-hFc-ABP fusion protein (FIG. 4C and FIG. 4D). Similar results were obtained with IGF1 R5-mFc-ABP ABP (Fig 17 A and B).

Example 5: BBB transmigration and Pharmacokinetics of FC5-Fc-L-ABP fusion molecules in vivo

The ability of the constructs of Example 2 to transmigrate the blood brain barrier into the brain, specifically into the cerebrospinal fluid (CSF), was evaluated in vivo, as well as to quantify the construct presence in CSF and serum. FC5-mFc-ABP was administered intravenously into rats via tail vein at the indicated doses (2.5, 6.25, 12.5, and 25 mg/kg). Serum and CSF were serially collected. FC5-Fc-ABP levels were quantified using nanoLC- MRM method. The technique used for multiple sampling of cisterna magna CSF was developed at NRC by modification of previously described methods (Huang et al.,1995; Kornhuber et al., 1986)). All animals were purchased from Charles River Laboratories International, Inc. (Wilmington, MA, USA). Animals were housed in groups of three in a 12 h light/dark cycle at a temperature of 24°C, a relative humidity of 50 ± 5%, and were allowed free access to food and water. All animal procedures were approved by the NRC's Animal Care Committee and were in compliance with the Canadian Council of Animal Care guidelines. Male Wstar rats aged 8-10 weeks (weight range, 230-250 g) were used in all studies.

In all experiments, test antibodies (FC5 Fc-fusions) were administered intravenously into tail vein in equimolar doses (7 mg/kg). CSF sample collections were made from cisterna magna by needle puncture up to five times over 96 hours. For sample collection rats were briefly and lightly anesthetized with 3% isoflurane, placed in a stereotaxic frame with the head rotated downward at a 45° angle. A 2-cm midline incision between the ears beginning at the occipital crest was made and muscles separated to expose dura mater covering cisternae magna. A 27G butterfly needle (QiuckMedical, Cat# SV27EL) with tubing attached to 1 ml syringe was used to puncture dura and aspirate the ~20 μΙ of CSF. The CSF was then transferred into the sample glass vial (Waters, Cat#186000384c) and placed in -80°C freezer until further analysis.

Blood samples were collected from the tail vein in a commercially available tube (BD microtainer, Cat# 365956). After clotting at room temperature for 15-30 minutes, the clot was removed by centrifuging at 1 100 rcf (3422rpm) for 10 min; serum was then transferred into a clean glass vial (Waters, Cat#186000384c), frozen on dry ice and stored at -80°C until further analysis. At the end of collection, rats were sacrificed by cardiac puncture. Blood and CSF PK analyses were performed using WnLin 6.0 program.

Serum and CSF samples were analyzed by mass spectrometry and nanoLC-SRM based quantification as described in Example 4 using peptide signatures shown in Table 1 .

CSF collection is a delicate procedure during which CSF can be easily contaminated with blood. Since the amounts of V _H H s were expected to be much smaller in the CSF (<0.1 %) than blood, even a slight contamination with blood could seriously compromise the value of an individual CSF sample. It was therefore necessary to develop stringent exclusion criteria for blood-contaminated CSF samples. To evaluate blood-CSF albumin ratio, a nanoLC-SRM method was developed for quantifying albumin levels in plasma and CSF. An albumin peptide APQVSTPTLVEAAR was selected based on its unique retention time and m/z value (Mol Pharm) in order to have minimum interference with other peptide peaks in the multiplex assay. The intensity of the peptide was quantified in both CSF and plasma samples using SRM as described above. The albumin ratio was calculated as follows for each rat:

Albumin Ratio = Intensity per nl_ of plasma analyzed / Intensity per nl_ of CSF analyzed

A ratio of 1500 and below was considered as blood contaminated. As shown in Fig 5, FC5-mFc-ABP appeared in the CSF in a time- and dose-dependent manner with Cmax between 12 and 24 h, indicating transport of ABP by FC5 into brain and CSF compartments in vivo (A). Serum PK parameters (FIG 5 and Table 2 below) show that alpha- and beta- half-life of FC5-mFc-ABP is similar to that of a full IgG (a benchmark antibody containing rat Fc) and is substantially higher than that of ABP or FC5 or FC5-ABP without Fc.

TABLE 2

Dose range: 2.5 mg¾g. 6 nig kg, 1 ^' 2 _. mg kg, 25 mg kg,

'mFc-AB P ( PCS domain)

lllieillll lllS llll lll lll !!!}&■¾!!

^■ Serum half life (both distribution phase and terminal) are similar between 'benchmark mAb' and FCSmFc-cargo

Example 6: Delivery of FC5-ABP construct to the brain in non-rodent larger animal

Serum and CSF PK profile of FC5-mFc-ABP was assessed in beagle dog. FC5-mFc-ABP was administered by intravenous injection to 10-12-year old beagle dogs and serum and CSF were serially collected and analyzed by nanoLC-MRM (left panel) and by Western blot using Fc- specific antibody (FIG. 6B) as described above in Example 5. Asterisks indicates blood- contaminated sample (not shown in MRM analyses). As can be seen, FC5-mFc-ABP appeared in the CSF in a time-dependent manner indicating transport of ABP by FC5 across dog blood brain barrier in vivo, confirming the translational nature of the BBB carrier. The PK parameters and CSF exposure were analyzed by WinNonlin software and are shown in Table 3 below.

Example 7: BBB permeability

BBB permeability and CSF appearance of FC5 fused with human Fc (hFc) and chemically linked to ABP (FC5-hFc-ABP) in vivo (rat model). FC5-hFc was linked with ABP-cystamide using a heterobifunctional cross-linker sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1 -carboxylate) according manufacurer's instructions (ThermoFisher Scientific). Chemically conjugated molecule was administered intravenously into rats via tail vein at 6.25 mg/kg and serum and CSF samples were collected at 4 and 24 hrs and analyzed as described in Fig. 5. FC5-hFc-ABP appears in the CSF in a time-dependent manner, however Fc-ABP without the BBB carrier does not, confirming FC5-mediated transport of ABP across the blood brain barrier, and as illustrated in FIG. 7.

Example 8: Delivery of BBB-Fc-L-ABP construct to the brain

The ability of the FC5-Fc-ABP constructs of Example 2 to transmigrate the blood brain barrier and penetrate the brain parenchyma in vivo was assessed in mice.

FC5-mFc-L-ABP was administered by intravenous injection via tail vein at either 15 mg/kg to wild type (WT) and AD-transgenic (AD-Tg, B6.Cg-Tg, Jackson Lab) mice (Fig. 8A and 8B) or at 7.5, 15 and 30 mg/kg to AD-Tg mice, and circulated for 4 and 24h. Mice were then thoroughly perfused with 10 ml of heparinized (100U/ml) saline at a rate of 1 ml/min via the left common carotid artery to facilitate specific perfusion of the brain. Brains were then removed, and hippocampal and cortical tissues were dissected and immediately frozen and stored at - 80°C until use. Frozen tissue was homogenized in ice-cold homogenization buffer containing 50 mM Tris-HCI pH 8, 150 mM NaCI and protease inhibitor cocktail (Sigma-Aldrich, Oakville, ON) using Dounce homogenizer (10-12 stroke at 4°C). Samples were then sonicated three times for 10 s each at 4°C and insoluble material was removed (10,000 xg for 10 min at 4°C). The supernatant was analyzed for protein content, and about 0.5 μg of protein was used for SRM analysis (Fig. 8A) using methods described in Example 4 and peptide signatures shown in Table 1 . Samples were also analysed by Western blot using mFc-specific antibodies (FIG. 8B and 8C). Specific "signature" peptides belonging to all the three components of the fusion molecule (FC5, Fc and ABP) were detected by MRM in both cortex and hippocampus, indicating that FC5 carrier successfully delivered ABP to the target areas of the brain (only data from FC5 peptide is shown in Fig 8A). Measured levels ranged between 750-1400 ng/g brain tissue at different time points, compared to ~50 ng/g tissue typically measured for control single-domain antibody A20.1 fused to Fc, or Fc fragment alone. This was further confirmed by Western blot analysis probing for Fc and ABP in tissue extracts (8B and 8C). No protein signal of the fusion molecule was detected in animals receiving just saline by Western blot. There was a dose-dependent increase in FC5-mFc-ABP levels detected by Western blot in the target regions of the brain (FIG. 8C). These results clearly indicate that FC5 successfully delivers ABP to the target regions of the brain (i.e., the hippocampus and cortex) in wild type (WT) and AD-Tg mice. Example 9: Clearance of Αβ from mouse brain

To evaluate the efficacy of ABP on the amyloid burden in Tg mice, the results of treatment with ABP alone or FC5-ABP construct were compared.

Comparison between treatment with ABP alone (FIG.9A) or ABP fused with BBB carrier FC5 (FIG. 9B and 9C). Two different AD Tg mouse models, triple transgenic (3X Tg-AD, sv129/ C57BL6 mice harboring PS1 M146V, APP _Swe and tauP301 L transgenes, Dr. F.M. LaFerla, University of California) and double transgenic (B6.Cg-Tg, harboring PSEN1 dE9 and APP _Sw e transgenes, Jackson Lab) were used; mice were dosed subcutaneously (sc) with 300 nmol/kg of free ABP every second day over a 3-month or a 2-month period, respectively. At the end of the treatment period, Αβ levels in the brain were measured by ELISA using a commercial assay kit (InVitrogen, KHB3544) according to manufacturer's assay procedure. The treatment with ABP alone resulted in 25-50% reduction in brain Αβ after 2-3 months of multiple treatments (every second day) (9A). The FC5-mFc-ABP construct was injected intravenously into double-transgenic AD mice (B6.Cg-Tg ,15 mg/kg; equivalent of 220 nmol/kg) and brain Αβ levels were measured by both ELISA and nanoLC-MRM 24 h after injection as described above in Example 8. Unexpectedly, about 50% amyloid reduction was observed within 24 hr of treatment with FC5-mFc-ABP (Fig 9B), indicating that efficient brain delivery of ABP by FC5 dramatically increased the efficacy of ABP in reducing brain Αβ levels. CSF analysis also indicated a significant decrease in Αβ^ levels within 24 hrs following FC5-mFc-ABP treatment (FIG. 9C). The Αβ peptide sequence detected by MRM (SEQ: LVFFAEDVGSNK, Table 1 /ELISA analyses is remote/different from the Αβ epitope recognized by ABP (therefore, not interfering with its quantification by either ELISA or MRM).

Example 10: Introduction of Fc component into FC5-ABP construct enhances its serum half-life

FC5-ABP (FC5 SEQ ID NO 17; and ABP SEQ ID NO 36) and FC5-hFc-ABP (FC5 SEQ ID NO 17; hFc 1x7 SEQ ID NO 40 and ABP SEQ ID NO 36] constructs were produced in CHO cells as described in Example 2. Serum PK was determined as described in Example 5. FC5-ABP and FC5-Fc-ABP constructs were administered intravenously into rat tail vein at 15mg/kg. Serum was serially collected and FC5-ABP and FC5-Fc-ABP levels were quantified by direct ELISA using FC5- and ABP-specific antibodies. Serum samples were diluted (1 :5,000) in phosphate-buffered saline (PBS) and applied to Maxisorb plates and incubated overnight at 4°C. ELISA plates were washed 3 X 100 μΙ PBS and blocked with 1 % BSA in TBST for 30 min at room temperature (RT). Blocking solution was removed and the plates were incubated with HRP-conjugated FC5 monoclonal antibody (90 min). Following incubation and wash, 100μΙ SureBlue reagent was added and incubated in the dark at RT for 10-15 min. At the end of reaction, 100μΙ 1 M HCI was added and the developed colour was read at 450 nm in a plate reader. As shown in Fig 10, FC5-ABP without Fc was very rapidly cleared in the serum (within an hr) compared to FC5-ABP construct containing Fc component (FC5-Fc-ABP). This assay was also repeated with ABP-specific antibody with similar results. After sample application, ELISA plates were incubated first with ABP rabbit polyclonal antibody (90 min), followed by HRP-conjugated rabbit secondary antibody (30 min) and the bound antibody was detected as described above (data not shown).

Example 11: Clearance of Αβ from rat brain

AD-Tg rats were dosed with either saline or FC5-mFc-ABP via tail vein every week over a period of four weeks (loading dose of 30mg/kg and subsequent four weekly doses of 15 mg/kg). CSF levels of FC5-mFc-ABP and Αβ were analyzed by nanoLC MRM. Before and after four weeks of treatment, brain Αβ levels were determined by PET scan using a specific Αβ-binding agent [18F] NAV4694. FC5-mFc-ABP reduced CSF Αβ level in rats within 24 hrs. An inverse relationship between the CSF levels of FC5-mFc-ABP and Αβ was observed, as in Tg mice, suggesting target engagement and rapid clearance of Αβ by ABP delivered to the brain and CSF by FC5 (FIG. 1 1 A and FIG. 1 1 B). This was further corroborated by PET scan which clearly indicated a significant reduction (30-50%) of rat brain Αβ levels following four weeks of treatment with FC5-mFc-ABP (FIG. 1 1 C).

Example 12: Increased hippocampal volume and improved neuronal connectivity

In the experiment described in Example 1 1 , saline- and FC5-mFc-ABP- treated Tg mice were subjected volumetric and functional Magnetic Resonance Imaging (MRI) before after the treatment. Volumetric MRI (Fig. 12A) showed an increased hippocampal volume in ABP- treated Tg mice compared to saline-treated controls suggesting that ABP treatment arrested hippocampal atrophy. Functional MRI (Fig. 12B) showed improved connectivity in anterior cingulated cortex in ABP-treated Tg mice compared to saline-treated controls suggesting restoration of neuronal connectivity. This data confirms the significance, efficacy and superior therapeutic advantages now provided.

Example 13: FC5-mFc2a-ABP treatment shows decreased levels of CSF Αβ in dogs

As described in Example 6, serum and CSF PK profile of FC5-mFc-ABP was assessed in beagle dog with two dose (15 mg/kg and 30 mg/kg. In addition to measuring serum and CSF levels of FC5-mFc2a-ABP by nanoLC-MRM, CSF levels of Αβ was also measured by nanoLC- MRM as described in Example 9. As can be seen, FC5-mFc-ABP appeared in the CSF in a dose- and time-dependent manner (Fig 13B). Most importantly, as seen Tg mice and Tg rat, there was a significant decrease in CSF Αβ level that was inversely proportional to CSF FC5- mFc2a-ABP levels.

Example 14: Generation of ABP fusion molecule with a different BBB carrier

To assess the versatility of ABP fusion molecule, ABP was successfully fused with another humanized BBB carrier IGF1 R5 (H2). As shown in FIG. 12, the bi-functionality of the molecule was retained, ABP's ability to bind Αβ oligomer (ELISA and overlay assays) and also IGF1 R5's ability deliver ABP across BBB model in vitro (data not shown). This clearly indicates that ABP can be fused to different BBB-crossing single-domain antibodies to be delivered to the brain.

Example 15: Αβ oligomer binding by different BBB-crossing single-domain antibodv-Fc-ABP constructs

Various FC5-Fc-ABP constructs with modified ABPs (site-specific mutations or removal of C- terminus portion of the molecule as indicated by SEQ ID Nos shown in Tablel . and Fig 15) are provided. As shown in Fig. 15, all constructs retained similar potency in binding Αβ oligomers by ELISA method.

Example 16: Production of FC5-hFc1X7-L-ABP with specific mutations to improve stability and bio-manufacturability. FC5-hFc1X7-L-ABP carrying specific mutations (ABP, SEQ ID NO 35; ABP, SEQ ID NO 36) were produced in CHO cells and separated on SDS-PGE under reducing (R) and non-reducing (NR) conditions and stained with Coomassie, blue as described in Fig 2. Separated protein transferred to nitrocellulose membrane and immunoblotted with FC5-specific, hFc-specific and ABP-specific antibodies. In another set, Αβ-binding of ABP in the fusion molecule was also tested by overlay assay. Bound Αβ was detected with Αβ-specific antibody 6E10. As can be seen, systematic modification of ABP with specific mutations (as shown here, for example, ABP SEQ ID NO: 35 and, ABP SEQ ID NO: 36) substantially enhanced the stability of the molecule generated, as is clearly indicated herein, for example, by a single protein band under reducing and non-reducing conditions (compare with other ABP constructs of Fig 2A, wherein double protein bands can be seen). This substantial enhancement in the stability of the fusion molecule advantageously facilitates the bio-manufacturability of a homogeneous molecule.

Example 17: BBB permeability of various FC5-FC-L-ABP constructs and IGF1R5-FC-ABP construct in vitro. BBB- crossing was assessed in in vitro rat BBB model as described in Fig 4 and molecules crossing the blood brain barrier were detected by nanoLC-MRM method. All ABP variants fused to humanized FC5 and IGF1 R carriers crossed the BBB effectively. As expected, A20.1 , a non-BBB permeable sdAb did not cross BBB, and likewise, ABP fused to A20.1 did not permeate the BBB (FIG. 17A). In Fig 17B, it is shown that "finger-print" peptides for all the three components of the fusion molecule, FC5, Fc and ABP were detected by nanoLC-MRM.

The embodiments and examples described herein are illustrative and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments, including alternatives, modifications and equivalents, are intended by the inventors to be encompassed by the claims. Furthermore, the discussed combination of features might not be necessary for the inventive solution.

Example 18: Humanized FC5-FC-ABP construct [FC5(H3)-hFc-ABP (with ABP SEQ ID NO: 35 and ABP SEQ ID NO: 36)1 crosses in vitro rat blood brain barrier intact. Blood brain barrier- crossing of humanized FC5-ABP fusion molecules were assessed as described in Example 4. Fusion molecules crossing the blood brain barrier were analyzed by Western blot and ELISA methods. Immunoblots were probed with hFc-specific-, and ABP-specific antibodies. Both the antibodies recognized the molecule crossing the BBB (bottom chamber) and the molecular size was identical to that of FC5-ABP fusion construct that was applied to in vitro BBB (top chamber) indicating that the molecule that crossed the BBB remained intact. This was substantiated by sandwich ELISA assay where in the BBB-crossed molecule was captured with FC5-specific antibody and the captured molecule was detected with ABP antibody.

Example 19: Humanized FC5-FC-ABP construct [FC5(H3)-hFc-L-ABP (with ABP SEQ ID NO: 35 and ABP SEQ ID NO: 36)1 is transported across the BBB in vivo and delivered to the brain intact by FC5. Blood brain barrier-crossing and brain delivery of humanized FC5-ABP fusion molecule in mice was assessed as described in Example 8. Four hrs following intravenous administration of the molecule and intracardiac perfusion, brain was removed and the cortices were extracted in RIPA buffer and the presence of injected FC5-ABP fusion molecule was detected by Western blot probed with ABP-specific antibody and by sandwich ELISA by capturing the molecule with FC5-specific antibody and detecting with ABP-specific antibody. Western blot revealed the presence of full-length FC5-ABP construct in the cortex. This was substantiated by sandwich ELISA which revealed the intact nature of the molecule as indicated by the ability to capture the molecule by FC5-specific antibody and detect the captured molecule with ABP-specific antibody. Example 20: Ex-vivo (A) and in vivo (B) binding of humanized FC5-FC-ABP construct [FC5(H3)-hFc-ABP (ABP SEQ ID NO 361 (target engagement)

Immunohistochemistry was carried out as described in Example 3. Brain sections from AD- transgenic mice were incubated with FC5(H3)-hFc1X7-ABP (ABP, SEQ ID NO: 36) and the bound fusion molecule was visualized with HRP-conjugated FC5-specific antibody. As a negative control, sections were incubated with either no construct, or FC5-hFc construct without fused ABP. Briefly, Formalin-fixed 40μηι free-floating sections containing cortex and hippocampus from APP/PS1 transgenic mouse were subjected to antigen retrieval in 10mM sodium citrate buffer (pH 9) at 80°C for 30 minutes then cooled to room temperature. Sections were rinsed in PBS, treated with 3% H ₂ 0 ₂ in PBS for 30 minutes to block endogenous peroxidase, then rinsed again. Following a one hour incubation in Dako serum free protein block containing 0.3% triton X-100, sections were incubated with FC5(H3)-hFc1x7-ABP or the molar equivalent of FC5-hFc1 x7 in Dako diluent containing 0.3% triton X-100 for 90 minutes at room temperature. After thoroughly rinsing in PBS, the sections were incubated with anti-FC5- HRP in Dako diluent for 60 minutes at room temperature, rinsed again, and then developed using Vector Immpact DAB following the kit directions. The sections were placed on Superfrost plus slides, allowed to air dry overnight, then rehydrated, counterstained with methyl green, dipped in acetone/0.05% acetic acid (v/v) and dehydrated, cleared and coverslipped with Permount. Selective binding (black spots) was seen with FC5(H3)-hFc1X7-ABP construct but not with FC5(H3)-hFc1X7 without ABP, indicating ABP-dependent binding of Αβ deposits (target engagement) in the brain. No binding was seen in brain sections from wild type mice that does not produce amyloid deposits (data not shown).

Similar binding of Αβ deposits were detected following intra-hippocampal injection of FC5(H3)- hFc-ABP construct into AD transgenic mice (B). Four hours after intra-hippocampal injection, Tg mice were perfused and the brains were removed and sectioned. FC5(H3)-hFc-ABP binding to Αβ deposits (shiny white dots) were visualised with ABP-specific polyclonal antibody. Brain sections were first incubated with ABP-specific monoclonal antibody followed by Alexa-647 conjugated anti rabbit-Fc secondary antibody

Example 21: Target engagement by humanized FC5-Fc-ABP construct (FC5(H3)-hFc-ABP [SEQ ID NO: 46) in vivo. Fluorophore-labelled (FIG.18A) or naive FC5-ABP (FIG 18B) fusion molecule was microinjected into the hippocampal region of wild-type (Wt) and AD-Tg (Tg) mice. 30 min after microinjection of fluorophore-labelled FC5-ABP fusion molecule, brains were removed, sectioned and observed under fluorescence microscope. As shown in 21A, injected molecule bound to Αβ deposits as confirmed by its co-localization with AB-specific antibody. In a parallel study, four hrs after intra-hippocampal injection of naive FC5-ABP fusion molecule, hippocampal formation from injected (ips) and non-injected (con) regions were collected and homogenized in Tris-buffered saline. Hippocampal extracts were subjected to sandwich ELISA with FC5 antibody as capturing antibody and either ABP or AB-specific antibody as detection antibody. As shown in 21 B, microinjected FC5-ABP molecule remained intact and ABP was able to engage and bind the target Αβ in vivo as indicated by the presence of AB in the pulled-down complex detected by AB-specific antibody.

Example 22: PK/PD comparison between non-humanized and humanized FC5-Fc-L-ABP constructs

FC5-mFc2a-ABP or FC5(H3)-hFc1 x7-ABP was administered intravenously into rats via tail vein injection at 15 mg/kg as described for Fig 5. Serum and CSF were serially collected. FC5- Fc-ABP levels were quantified using nanoLC- MRM method. As shown in Fig 22 A, serum and CSF PK profile were very similar for non-humanized and humanized constructs. FC5-mFc2a- ABP or FC5(H3)-hFc1 x7-ABP was administered intravenously into Tg mice via tail vein injection at 15 mg/kg as described for Fig 9B. FC5-Fc-ABP and AB levels in the CSF were measured by nanoLC-MRM as described in Fig 9B. As shown in Fig 22 B, the levels of non- humanized and humanized FC5-Fc-ABP in the CSF were similar, and most importantly, changes (decrease) in CSF AB levels were also very similar, indicating that humanization of FC5-Fc-ABP construct did not affect the PK and PD profile of the fusion construct.

SEQUENCES

SEQ Sequence Description ID NO:

1 GFKITHYT G CDR1 FC5

2 RITWGGDNTFYSNSVKG CDR2 FC5

3 GSTSTATPLRVDY CDR3 FC5

4 EYPSNFYA CDR1 IGF1 R-3

5 VSRDGLTT CDR2 IGF1 R-3

6 AIVITGVWNKVDVNSRSYHY CDR3 IGF1 R-3

7 GGTVSPTA CDR1 IGF1 R-4

8 ITWSRGTT CDR2 IGF1 R-4

9 AASTFLRILPEESAYTY CDR3 IGF1 R-4

10 GRTIDNYA CDR1 IGF1 R-5

1 1 IDWGDGGX; where X is A or T CDR2 IGF1 R-5

12 A ARQSRVN LDVARYDY CDR3 IGF1 R-5

13 X ₁ VQLVX ₂ SGGGLVQPGGSLRLSCAASGFKITHYT GWX ₃ RQAPGKX ₄ Humanized FC5 X ₅ EX ₆ VSRITWGGDNTFYSNSVKGRFTISRDNSKNTX ₇ YLQIV1NSLRAED consensus TAVYYCAAGSTSTATPLRVDYWGQGTLVTVSS, where X-,=D or E,

X ₂ =A or E, X ₃ =F or V, X ₄ =E or G, X ₅ =R or L, X ₆ =F or W, X ₇ =L or V;

14 DVQLQASGGGLVQAGGSLRLSCAASGFKITHYT GWFRQAPGKERE FC5

FVSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQ NSLKPEDTADY

YCAAGSTSTATPLRVDYWGKGTQVTVSS

15 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYT GWVRQAPGKGLE FC5-H1 WVSRITWGGDNTFYSNSVKGRFTISRDNSKNTLYLQ NSLRAEDTAV

YYCAAGSTSTATPLRVDYWGQGTLVTVSS

EVQLVESGGGLVQPGGSLRLSCAASGFKITHYT GWVRQAPGKGLE FC5-H2 WVSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQ NSLRAEDTAV YYCAAGSTSTATPLRVDYWGQGTLVTVSS

EVQLVESGGGLVQPGGSLRLSCAASGFKITHYT GWFRQAPGKGLEF FC5-H3 VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQ NSLRAEDTAVYY CAAGSTSTATPLRVDYWGQGTLVTVSS

X ₁ VX ₂ LX3ESGGGLVQX4GGSLRLSCX ₅ ASEYPSNFYA SWX ₆ RQAPGK IGF1 R-3 X7X8EX9VX10GVSRDGLTTLYADSVKGRFTX11SRDNX12KNTX13X14LQ Consensus NSX15X16AEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTX17VTVSS

, where X-, is E or Q; X ₂ is K or Q; X ₃ is V or E; X is A or P; X ₅ is V or A;

X ₆ is F or V; X ₇ is E or G; X ₈ is R or L; X _g is F or W; Χ·, ₀ is A or S; X·,·, is

or I ; Χ·, ₂ is A or S; Χ·, ₃ is V or L; X is D or Y; Χ·, ₅ is V or L; Χ·, ₆ is K or

R; and X-| ₇ is Q or L,

QVKLEESGGGLVQAGGSLRLSCVASEYPSNFYA SWFRQAPGKERE IGF1 R-3 FVAGVSRDGLTTLYADSVKGRFT SRDNAKNTVDLQ NSVKAEDTAV YYCAIVITGVWNKVDVNSRSYHYWGQGTQVTVSS

EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYA SWFRQAPGKERE IGF1 R-3-H5 FVSGVSRDGLTTLYADSVKGRFTISRDNSKNTLYLQ NSLRAEDTAVY YCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS

X1VX2LX3ESGGGLVQX4GGSLRLSCX5X6SGGTVSPTA GWX7RQAPG IGF1 R-4 Consensus NSLX ₁₇ X ₁₈ EDTAVYYCAASTFLRILPEESAYTYWGQGT X ₁₉ VTVSS,

where X-i is E or Q; X ₂ is K or Q; X ₃ is V or E; X is A or P; X ₅ is A or E;

X ₆ is V or A; X ₇ is V or F; X ₈ is G or E; X _g is L or R; Χ·, ₀ is F or W; X·,·, is

G or S; Χ·, ₂ is V or Y; Χ·, ₃ is D or G; X is N or S; Χ·, ₅ is A or S; X ₆ is L or

V; Xi7 is K or R; X-| ₈ is A or S; and X-ig is L or Q,

QVKLEESGGGLVQAGGSLRLSCEVSGGTVSPTA GWFRQAPGKERE IGF1 R-4 FVGHITWSRGTTRVASSVKDRFTISRDSAKNTVYLQ NSLKSEDTAVY YCAASTFLRILPEESAYTYWGQGTQVTVSS

QVQLVESGGGLVQPGGSLRLSCAVSGGTVSPTA GWFRQAPGKGLE IGF1 R-4-H3 FVGHITWSRGTTRYASSVKGRFTISRDNSKNTVYLQ NSLRAEDTAVY YCAASTFLRILPEESAYTYWGQGTLVTVSS

X1VX2LX3ESGGGLVQX4GGSLRLSCAASGRTIDNYA AWX5RQAPGKX IGF1 R-5 ₆ X7EX ₈ VX ₉ TIDWGDGGXioRYANSVKGRFTISRDNXii KXi2TXi3YLQIVlNX Consensus i4LXi5Xi ₆ EDTAVYXi ₇ CAIVlARQSRVNLDVARYDYWGQGTXi ₈ VTVSS,

where X-i is E or Q; X ₂ is K or Q; X ₃ is V or E; X ₄ is A or P; X ₅ is V or S;

X ₆ is D or G; X ₇ is L or R; X ₈ is F or W; X _g is A or S; Χ·, ₀ is A or T; X·,·, is A

or S; X12 is G or N; X-| ₃ is or L; X-| ₄ is N or R; X-| ₅ is E or R; X-| ₆ is P or

A; Xi7 is S or Y; and X-| ₈ is Q or L,

QVKLEESGGGLVQAGGSLRLSCAASGRTIDNYAMAWSRQAPG IGF1 R-5

KDREFVATIDWGDGGARYANSVKGRFTISRDNAKGTMYLQMN

NLEPEDTAVYSCAMARQSRVNLDVARYDYWGQGTQVTVSS

QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPG IGF1 R-5-H2

KGLEWVATIDWGDGGTRYANSVKGRFTISRDNSKNTMYLQMN

SLRAEDTAVYYCAMARQSRVNLDVARYDYWGQGTLVTVSS

SGKTEY AFPKPFESSSSIGAEKPRNKKLPEEEVESSRTPWLYEQEG PK-4 EVEKPFIKTGFSVSVEKSTSSNRKNQLDTNGRRRQFDEESLESFSS P DPVDPTTVTKTFKTRKASAQASLASKDKTPKSKSKKRNSTQLKSRVKN ITHARRILQQSNRNACNEAPETGSDFS FEA

FSS PDPVDPTTVTKTFKTRKASAQASLASKDKTPKSKSK P4 peptide

KDKTPKSKSKKRNSTQLKSRVKNITHARRILQQSNRNACN P5 peptide KTFKTRKASAQASLASKDKTPKSKSKKRNSTQLKSRVKNI P4-5 peptide

(A BP)

X ₁ TFX ₂ TX ₃ X ₄ ASAQASLASKDKTPKSKSKKX ₅ X ₆ STQLX ₇ SX ₈ VXgNI ABP consensus here X-, =G or A, X ₂ = G or V, X ₃ = G or A, X = G or A, X ₅ = G or V, X ₆ =

G or V, X ₇ = G or V, X ₈ = G or A, X _g = G or A (SEQ ID NO: 31)

KTFKTRKASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI [ABP(G)]

KTFKTRKASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI [ABP(GG)]

KTFKTRGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI [ABP(RG-G)l

KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI [ABP(GG-G)]

GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI [ABP(6G)]

KTFKTR KAS AQ AS LAS KD KTP KS KSKKG G STVKN I fABP(GGspv)l

KTFKTRKASAQASLASKDKTPKSKSKKRG [ABP(trc)]

ASEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVL ISLSPIVTCV Mouse Fc2a

VVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQH

QDW SGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEE TKKQVTLTC VTDF PEDIYVEWTNNGKTELNYKNTEPVLDSDGSY

F YSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG

AEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL ISRTPEVTCV Human Fc1x7

VVDVSHEGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE

LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

LYSKLTVDKSRWQQGNVFSCSV HEALHNHYTQKSLSLSPG

AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL ISRTPEVTCV Human FdXO VVDVSHEGPEVKFNWHVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSV HEGLHNHYTQK SLSLSPG

EVQLQASGGGLVQAGGSLRLSCAASGFKITHYT GWFRQAPGKERE FC5-mFc2a-

FVSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQ NSLKPEDTADY ABP

YCAAGSTSTATPLRVDYWGKGTQVTVSSASEPRGPTIKPCPPCKCPA

PNLLGGPSVFIFPPKIKDVL ISLSPIVTCVVVDVSEDDPDVQISWFVN

NVEVHTAQTQTHREDYNSTLRVVSALPIQHQDW SGKEFKCKVNNKD

LPAPIERTISKPKGSVRAPQVYVLPPPEEE TKKQVTLTC VTDF PE

DIYVEWTNNGKTELNYKNTEPVLDSDGSYF YSKLRVEKKNWVERNS

YSCSVVHEGLHNHHTTKSFSRTPGTGGGGSGGGGSKTFKTRKASAQ

ASLASKDKTPKSKSKKRNSTQLKSRVKN I

EVQLVESGGGLVQPGGSLRLSCAASGFKITHYT GWFRQAPGKGLEF FC5-H3-mFc2a-

VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQ NSLRAEDTAVYY L-ABP

CAAGSTSTATPLRVDYWGQGTLVTVSSASEPRGPTIKPCPPCKCPAP

NLLGGPSVFIFPPKIKDVL ISLSPIVTCVVVDVSEDDPDVQISWFVNNV

EVHTAQTQTHREDYNSTLRVVSALPIQHQDW SGKEFKCKVNNKDLP

APIERTISKPKGSVRAPQVYVLPPPEEE TKKQVTLTC VTDF PEDIY

VEWTNNGKTELNYKNTEPVLDSDGSYF YSKLRVEKKNWVERNSYS

CSVVHEGLHNHHTTKSFSRTPGTGGGGSGGGGSKTFKTRKASAQAS

LASKDKTPKSKSKKRNSTQLKSRVKNI

EVQLVESGGGLVQPGGSLRLSCAASGFKITHYT GWFRQAPGKGLEF FC5-H3-

VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQ NSLRAEDTAVYY hFc1X7-L-ABP

CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL

LGGPSVFLFPPKPKDTL ISRTPEVTCVVVDVSHEGPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSV HEALHNHYTQKSLSLSPGTGGGSGGGGSKTFKTRKASAQASLA

SKDKTPKSKSKKRNSTQLKSRVKNI

EVQLVESGGGLVQPGGSLRLSCAASGFKITHYT GWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L-

CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(G)

LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPGTGGGSGGGGSKTFKTRKASAQASLA

SKDKTPKSKSKKRGSTQLKSRVKNI

EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3-

VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L-

CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(GG-G)

LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGTGGGSGGGGSKTFKTGG

ASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI

EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3-

VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L-

CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(6G)

LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPGTGGGSGGGGSGTFGTGGASAQASL

ASKDKTPKSKSKKGGSTQLKSRVKNI

EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5(H3)-L-

VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY ABP(GG-G)-L

CAAGSTSTATPLRVDYWGQGTLVTVSSGGGGSGGGGSKTFKTGGAS hFc1X7

AQASLASKDKTPKSKSKKRGSTQLKSRVKNIGGGGSGGGGSAEPKSS

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH

EGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL

NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ

VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL

TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3-

VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X0-L-

CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSCDKTHTCPPCPAPEL ABP(GG-G)

LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWHVDG

VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI

AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF

SCSVMHEGLHNHYTQKSLSLSPGTGGGSGGGGSKTFKTGGASAQAS

LASKDKTPKSKSKKRGSTQLKSRVKNI

QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPGKGLE IGF1 R5-H2-

WVATIDWGDGGTRYANSVKGRFTISRDNSKNTMYLQMNSLRAEDTAV hFc1X7-L-

YYCAMARQSRVNLDVARYDYWGQGTLVTVSSAEPKSSDKTHTCPPC ABP(GG-G)

PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNW

YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG

NVFSCSVMHEALHNHYTQKSLSLSPGTGGGSGGGGSKTFKTGGASA

QASLASKDKTPKSKSKKRGSTQLKSRVKNI

KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNIGGGSGGG ABP(GG-G)-L-

GSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT hFc1X7-

CVVVDVSHEGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT IGF1 R5-H2

VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSV HEALHNHYTQKSLSLSPGQVQL VESGGGLVQPGGSLRLSCAASGRTIDNYA AWVRQAPGKGLEWVATI DWGDGGTRYANSVKGRFTISRDNSKNT YLQ NSLRAEDTAVYYCA ARQSRVNLDVARYDYWGQGTLVTVSS

52 AEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL ISRTPEVTCV hFc1X7- L-

VVDVSHEGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL ABP(GG-G)-L-

HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE IGF1 R5-H2

LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

LYSKLTVDKSRWQQGNVFSCSV HEALHNHYTQKSLSLSPGGGGSG

GGGSKTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNIGGG

SGGGGSQVQLVESGGGLVQPGGSLRLSCAASGRTIDNYA AWVRQA

PGKGLEWVATIDWGDGGTRYANSVKGRFTISRDNSKNT YLQ NSLR

AEDTAVYYCA ARQSRVNLDVARYDYWGQGTLVTVSS

53 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYT GWFRQAPGKGLEF FC5-H3-

VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQ NSLRAEDTAVYY hFc1X7-

CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL L(consensus)- ABP(6G)

LGGPSVFLFPPKPKDTL ISRTPEVTCVVVDVSHEGPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSV HEALHNHYTQKSLSLSPGTXiGXzXsGXiXsGXs.GTFGTGGASAQ

ASLASKDKTPKSKSKKGGSTQLKSRVKNI

where X-,= A or G. , X ₂ = A or G, X ₃ = S or T, X ₄ = G or V, X ₅ = A or V, X ₆

= S or T

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WO 201 1/127580

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