Fusion protein comprising a blood-brain barrier (BBB)-crossing single domain antibody Fc5, an immunoglobulin Fc fragment and a beta-amyloid binding polypeptide (ABP)

Abstract

A brain-penetrating composition of amyloid-ß binding peptide is disclosed. This may be useful in the treatment of Alzheimer's disease, for example as a bifunctional molecule, comprising a blood-brain barrier crossing antibody and an amyloid-ß targeting peptide linked via an Fc fragment that is able to transmigrate across the blood-brain barrier into the brain, and compositions comprising same. Methods of using this composition for treating Alzheimer's disease are disclosed.

Claims

1. A fusion protein comprising the amino acid sequence of SEQ ID NO: 47.

2. The fusion protein of claim 1, consisting of the amino acid sequence of SEQ ID NO: 47.

3. A pharmaceutical composition comprising the fusion protein of claim 1 and a pharmaceutically acceptable diluent, carrier, vehicle or excipient.

4. A pharmaceutical composition comprising the fusion protein of claim 2 and a pharmaceutically acceptable diluent, carrier, vehicle or excipient.

5. A dimer of two fusion proteins of claim 1.

6. The dimer of claim 5, wherein each of the two fusion proteins consists of the amino acid sequence of SEQ ID NO: 47.

7. A pharmaceutical composition comprising the dimer of claim 5 and a pharmaceutically acceptable diluent, carrier, vehicle or excipient.

8. A pharmaceutical composition comprising the dimer of claim 6 and a pharmaceutically acceptable diluent, carrier, vehicle or excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

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

(2) 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 FIGS. 1A, 1B, 1C, and 1D.

(3) 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: Aß-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 Aß. Bound Aß was detected with Aß-specific antibody 6E10 or 4G8. The fusion molecules were also separated by SDS-PAGE, transferred to PVDF paper and exposed to Aß. Bound Aß was detected with specific antibody as above. Results show that ABP retained its Aß oligomer binding ability after fusion with the BBB carrier. Mo: Aß monomers; Oli: Aß 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 Aß 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 Aß deposits and not in brain sections from wild type mice that does not produce amyloid deposits.

(4) 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).

(5) 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).

(6) 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.

(7) 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 Aß-binding ability and transport across the blood brain barrier.

(8) FIG. 8: shows levels of FC5-mFc-ABP measured in various brain regions (cortex and hippocampus) after intravenous injection in mice. 15 mg/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.

(9) FIG. 9: shows the effect of ABP on Aß 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 (3×Tg-AD, sv129/C57BL6 mice harboring PS1M146V, APP.sub.Swe and tauP301L transgenes, Dr. F. M. LaFerla, University of California) and double transgenic (B6.Cg-Tg, harboring PSEN1dE9 and APP.sub.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, Aß levels in the brain were measured by ELISA. The treatment with ABP alone resulted in 25-50% reduction in brain Aß 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 Aß 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 Aß levels. CSF analysis also indicated a significant decrease in Aß.sub.1-42 levels within 24 hrs following FC5-mFc-ABP treatment (FIG. 9C). The signature peptide or epitope of Aß detected by MRM or ELISA analyses is remote/different from the Aß epitope recognized by ABP (therefore, not interfering with its quantification by either ELISA or MRM).

(10) 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.

(11) 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 30 mg/kg and subsequent four weekly doses of 15 mg/kg). CSF levels of FC5-mFc-ABP and Aß were analyzed by nanoLC MRM (FIG. 11A and FIG. 11B). Before and after four weeks of treatment, brain Aß levels were determined by PET scan using a specific Aß-binding agent [18F] NAV4694. Following tracer injection, 60 min Dynamic images were acquired, transmission scans were obtained, images were reconstructed and Binding Potential (BP.sub.ND) parametric maps were generated. FC5-mFc-ABP reduced CSF Aß level in rats within 24 hrs (FIG. 11A and FIG. 11B). An inverse relationship between the CSF levels of FC5-mFc-ABP and Aß was observed, as in Tg rats, suggesting target engagement and rapid clearance of Aß by ABP delivered to the brain and CSF by FC5 (FIG. 11B). This was further corroborated by PET scan which clearly indicated a significant reduction (30-50%) of rat brain Aß levels following four weeks of treatment with FC5-mFc-ABP (FIG. 11C).

(12) 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).

(13) FIG. 13: shows time- and dose-dependent appearance of FC5-mFc-ABP in the CSF of beagle dog and a decrease in CSF Aß levels as seen Tg mice (FIG. 9C) and Tg rats (FIGS. 11A 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 Aß 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 Aß 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 Aß burden.

(14) 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 IGF1R5 (H2). As shown, the bi-functionality of the molecule was retained, ABP's ability to bind Aß oligomer (ELISA and overlay assays) and also IGF1R5'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.

(15) FIG. 15: shows Aß oligomer binding by different single-domain antibody-Fc-ABP constructs (FIGS. 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 Aß oligomers by ELISA method.

(16) FIG. 16: shows the production of FC5-hFc1×7-ABP with specific mutations to improve stability and bio-manufacturability. FC5-hFc1×7-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, Aß-binding of ABP in the fusion molecule was also tested by overlay assay. Bound Aß was detected with Aß-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.

(17) FIG. 17: shows blood brain barrier permeability of various FC5-Fc-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 blood brain barrier were detected by nanoLC-MRM method. All ABP variants fused to humanized FC5 and IGF1R 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 “finger-print” 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.

(18) FIG. 18: shows humanized FC5(H3)-hFc1×7-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-specific antibody on ELISA plate and detected with ABP-specific antibody. This sandwich ELISA confirmed that FC5(H3)-hFc1×7-ABP remained intact after crossing rat blood brain barrier in vitro. Similar results were obtained with FC5(H3)-hFc1×7-ABP (ABP, SEQ ID NO: 36); FIG. 18(2) A, B, and C.

(19) FIG. 19: shows humanized FC5(H3)-hFc1×7-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)-hFc1×7-ABP is transported across the BBB in vivo and delivered to the brain intact.

(20) FIG. 20: shows immunohistochemical analysis of ex-vivo binding (A) and in vivo binding (B) of FC5(H3)-hFc1×7-ABP (ABP, SEQ ID NO: 36) to endogenous Aß deposits in AD-Tg mouse brain (B6.Cg-Tg, Jackson Lab). Brain sections from AD-transgenic mice were incubated with FC5(H3)-hFc1×7-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)-hFc1×7-ABP construct but not with FC5(H3)-hFc1×7 without ABP (A), indicating ABP-dependent binding of Aß 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 Aß deposits were detected (using ABP-specific antibody) following intra-hippocampal injection (4 hrs post-injection) of FC5(H3)-hFc-ABP construct into AD transgenic mice (B).

(21) FIG. 21: shows target engagement by FC5(H3)-hFc1×7-ABP (ABP, SEQ ID NO: 32) in vivo. FIG. 21 A shows binding of Alexa 647-labeled FC5-hFc-ABP construct to natural amyloid-ß (Aß) deposits in AD transgenic mice in vivo after intra-hippocampal injection. Identity of Aß was confirmed by probing the brain sections with Aß-specific antibody 6E10 labelled with Alexa 488 and demonstrating co-localization of two signals (Merge). FIG. 21B shows demonstration of target engagement by ELISA. Following intra-hippocampal injection (4 hrs 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 Aß was detected by the same sandwich ELISA but with Aß-specific antibody as the detection antibody. It is clear from the FIGS. 21 A and 21B that the FC5-ABP construct remains intact 4 hrs post-injection in both wild type and AD transgenic mice. Most importantly, in Tg mice which expresses human Aß, injected ABP binds Aß and is pulled down as a complex (FC5(H3)-hFc-ABP*Aß) indicating Aß-target engagement by ABP in vivo.

(22) FIG. 22: shows PK, PD comparison between non-humanized and humanized FC5-Fc-ABP constructs. FC5-mFc2a-ABP or FC5(H3)-hFc1×7-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)-hFc1×7-ABP was administered intravenously into Tg mice via tail vein injection at 15 mg/kg as described for FIG. 9B. FC5-Fc-ABP and Aß 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 Aß 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

(23) 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.

(24) The present invention provides polypeptides which binds beta-amyloid (ß-amyloid). The polypeptides (or proteins) that bind β-amyloid may selectively bind pathologically relevant ß-amyloid.sub.1-42 (Aβ.sub.1-42) aggregates, and may be abbreviated and referred to herein as ABP or ABP variants (or collectively as ABP).

(25) 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.

(26) 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).

(27) There is a need for therapeutic formulations that can efficiently transmigrate ABP across the blood brain barrier, and provide the clearing of Aβ through the binding of ABP. In the prior art, a 40-amino acid Aβ-binding peptide (ABP) was identified that selectively binds Aβ.sub.1-42 oligomers implicated in AD development (WO 2006/133566). This Aβ-binding peptide inhibits Aβ binding to cellular proteins and inhibits Aβ.sub.1-42-induced cell toxicity in vitro (Chakravarthy et al, 2013).

(28) This Aβ-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 Aβ, can assist in clearing Aβ 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.

(29) Accordingly, although ABP has been shown to bind Aβ deposits when directly applied, in order to bind and clear Aβ 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 IGF1R, 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 Aβ through the binding of ABP in CSF and brain parenchyma is provided in the constructs and methods of the present invention.

(30) 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 hFc1×7 (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-hFc1×7-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.

(31) The present invention provides an isolated polypeptide which binds beta-amyloid (ß-amyloid). A polypeptide of the present invention may comprise a sequence.

(32) TABLE-US-00006 (SEQ ID NO: 31) X.sub.1TFX.sub.2TX.sub.3X.sub.4ASAQASLASKDKTPKSKSKKX.sub.5X.sub.6STQLX.sub.7SX.sub.8VX.sub.9NI where X.sub.1 = G or A, X.sub.2 = G or V, X.sub.3 = G or A, X.sub.4 = G or A, X.sub.5 = G or V, X.sub.6 = G or V, X.sub.7 = G or V, X.sub.8 = G or A, X.sub.9 = G or A

(33) 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.

(34) 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.

(35) 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.

(36) For example, the immunoglobulin light chain folds into a variable (V.sub.L) and a constant (C.sub.L) domain, while the heavy chain folds into a variable (V.sub.H) and three constant (C.sub.H, C.sub.H2, C.sub.H3) domains. Interaction of the heavy and light chain variable domains (V.sub.H and V.sub.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.

(37) 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.sub.H) and light (V.sub.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.sub.H and V.sub.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.sub.H and V.sub.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.

(38) 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.sub.L and V.sub.H connected with a peptide linker), Fab, F(ab′).sub.2, single-domain antibody (sdAb; a fragment composed of a single V.sub.L or V.sub.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.

(39) 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.sub.HH. sdAb have also been observed in shark and are termed V.sub.NAR (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.sub.H, V.sub.HH, V.sub.L, or V.sub.NAR 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.

(40) 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, 2011a,b; Kim et al, 2012), may also be brought to the sdAb.

(41) 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.

(42) 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-glycoprotein (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.

(43) 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 (IGF1R) epitope, or isoforms, variants, portions, or fragments thereof.

(44) 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 AIVITGVWNKVDVNSRSYHY (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:11), a CDR3 sequence of AMARQSRVNLDVARYDY (SEQ ID NO:12).

(45) As previously stated, the antibody or fragment thereof may be an sdAb of camelid origin or derived from a camelid V.sub.HH, and thus may be based on camelid framework regions; alternatively, the CDR described above may be grafted onto V.sub.NAR, V.sub.HH, V.sub.H or V.sub.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).

(46) 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.sub.H, or V.sub.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 IGF1R 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.

(47) The provided antibody or fragment thereof may be a humanized version of the FC5 antibody (described in WO 2002/057445) or an IGF1R 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 IGF1R (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 2011/127580). 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.

(48) For example, and without wishing to be limiting, the antibody or fragment thereof may comprise the sequence:

(49) TABLE-US-00007 (SEQ ID NO: 13) X.sub.1VQLVX.sub.2SGGGLVQPGGSLRLSCAASGFKITHYTMGWX.sub.3RQAPGKX.sub.4X.sub.6 EX.sub.6VSRITWGGDNTFYSNSVKGRFTISRDNSKNTX.sub.7YLQMNSLRAEDTAV YYCAAGSTSTATPLRVDYWGQGTLVTVSS, where X.sub.1 = D or E, X.sub.2 = A or E, X.sub.3 = F or V, X.sub.4 = E or G, X.sub.6 = R or L, X.sub.6 = F or W, X.sub.7 = L or V, or a sequence substantially identical thereto; (SEQ ID NO: 18) X.sub.1 VX.sub.2LX.sub.3ESGGGLVQX.sub.4GGSLRLSCX.sub.5ASEYPSNFYAMSWX.sub.6RQAPGK X.sub.7X.sub.8EX.sub.9VX.sub.10GVSRDGLTTLYADSVKGRFTX.sub.11SRDNX.sub.12KNTX.sub.13X.sub.14 LQMNSX.sub.15X.sub.16AEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTX.sub.17VT VSS,, where X.sub.1 is E or Q; X.sub.2 is K or Q; X.sub.3 is V or E; X.sub.4 is A or P; X.sub.5 is V or A; X.sub.6 is F or V; X.sub.7 is E or G; X.sub.8 is R or L; X.sub.9 is F or W; X.sub.10 is A or S; X.sub.11 is M or I; X.sub.12 is A or S; X.sub.13 is V or L; X.sub.14 is D or Y; X.sub.15 is V or L; X.sub.16 is K or R; and X.sub.17 is Q or L; or a sequence substantially identical thereto; (SEQ ID NO: 21) X.sub.1VX.sub.2LX.sub.3ESGGGLVQX.sub.4GGSLRLSCX.sub.5X.sub.6SGGTVSPTAMGWX.sub.7RQAPGK X.sub.8X.sub.9EX.sub.10VX.sub.11HITWSRGTTRX.sub.12ASSVKX.sub.13RFTISRDX.sub.14X.sub.15KNT X.sub.16YLQMNSLX.sub.17X.sub.18EDTAVYYCAASTFLRILPEESAYTYWGQGT X.sub.19 VTVSS,, where X.sub.1 is E or Q; X.sub.2 is K or Q; X.sub.3 is V or E; X.sub.4 is A or P; X.sub.5 is A or E; X.sub.6 is V or A; X.sub.7 is V or F; X.sub.8 is G or E; X.sub.9 is L or R; X.sub.10 is F or W; X.sub.11 is G or S; X.sub.12 is V or Y; X.sub.13 is D or G; X.sub.14 is N or S; X.sub.15 is A or S; X.sub.16 is L or V; X.sub.17 is K or R; X.sub.18 is A or S; and X.sub.19 is L or Q; or a sequence substantially identical thereto; (SEQ ID NO: 24) X.sub.1VX.sub.2LX.sub.3ESGGGLVQX.sub.4GGSLRLSCAASGRTIDNYAMAWX.sub.5RQAPGKX.sub.6 X.sub.7EX.sub.8VX.sub.9TIDWGDGGX.sub.10RYANSVKGRFTISRDNX.sub.11KX.sub.12TX.sub.13YLQM NX.sub.14LX.sub.15X.sub.16EDTAVYX.sub.17CAMARQSRVNLDVARYDY WGQGTX.sub.18VTV SS, where X.sub.1 is E or Q; X.sub.2 is K or Q; X.sub.3 is V or E; X.sub.4 is A or P; X.sub.5 is V or S; X.sub.6 is D or G; X.sub.7 is L or R; X.sub.8 is F or W; X.sub.9 is A or S; X.sub.10 is A or T; X.sub.11 is A or S; X.sub.12 is G or N; X.sub.13 is M or L; X.sub.14 is N or R; X.sub.15 is E or R; X.sub.16 is P or A; X.sub.17 is S or Y; and X.sub.18 is Q or L; or a sequence substantially identical thereto.

(50) 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:

(51) TABLE-US-00008 (SEQ ID NO: 14) DVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSR ITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAGS TSTATPLRVDYWGKGTQVTVSS; (SEQ ID NO: 15) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWVRQAPGKGLEWVSR ITWGGDNTFYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGS TSTATPLRVDYWGQGTLVTVSS; (SEQ ID NO: 16) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWVRQAPGKGLEWVSR ITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGS TSTATPLRVDYWGQGTLVTVSS; (SEQ ID NO: 17) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEFVSR ITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGS TSTATPLRVDYWGQGTLVTVSS; (SEQ ID NO: 19) QVKLEESGGGLVQAGGSLRLSCVASEYPSNFYAMSWFRQAPGKEREFVAG VSRDGLTTLYADSVKGRFTMSRDNAKNTVDLQMNSVKAEDTAVYYCAIVI TGVWNKVDVNSRSYHYWGQGTQVTVSS; (SEQ ID NO: 20) EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKEREFVSG VSRDGLTTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIVI TGVWNKVDVNSRSYHYWGQGTLVTVSS; (SEQ ID NO: 22) QVKLEESGGGLVQAGGSLRLSCEVSGGTVSPTAMGWFRQAPGKEREFVGH ITWSRGTTRVASSVKDRFTISRDSAKNTVYLQMNSLKSEDTAVYYCAAST FLRILPEESAYTYWGQGTQVTVSS; (SEQ ID NO: 23) QVQLVESGGGLVQPGGSLRLSCAVSGGTVSPTAMGWFRQAPGKGLEFVGH ITWSRGTTRYASSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAST FLRILPEESAYTYWGQGTLVTVSS (SEQ ID NO: 25) QVKLEESGGGLVQAGGSLRLSCAASGRTIDNYAMAWSRQAPGKDREFVAT IDWGDGGARYANSVKGRFTISRDNAKGTMYLQMNNLEPEDTAVYSCAMAR QSRVNLDVARYDYWGQGTQVTVSS; (SEQ ID NO: 26) QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPGKGLEWVAT IDWGDGGTRYANSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCAMAR QSRVNLDVARYDYWGQGTLVTVSS;
and
a sequence substantially identical thereto. The antibody or fragment thereof may be a single-domain antibody.

(52) 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.

(53) 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.

(54) 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 (Gln 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 (Ile 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).

(55) 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.

(56) 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.

(57) 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 IgG1, IgG2, 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).

(58) 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 2011). 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.

(59) 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 (SEQ ID NO: 58). 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 (Aβ) may bind pathologically relevant β-amyloids, such as Aβ.sub.1-42 aggregates, which are implicated in AD pathology; the polypeptide may bind Aβ with high affinity (in the nM range), inhibit Aβ binding to cellular proteins and Aβ.sub.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 Aβ.sub.42-1.

(60) 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.

(61) 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.

(62) 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.

(63) 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.

(64) 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).

(65) 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 Aß levels in brain; wherein a 50% amyloid reduction was observed within 24 hr of treatment with a construct of the present invention.

(66) 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 Aβ 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 (SEQ ID NO: 56) or (GGGS).sub.n (SEQ ID NO: 57) 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 (SEQ ID NO: 58). 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.

(67) 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.

(68) 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.

(69) 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.

(70) 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.

(71) 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.

(72) 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. 1A). 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 FIGS. 1A, 1B, 1C, and 1D. In 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.

(73) The antibody or fragment thereof may comprise the sequence

(74) TABLE-US-00009 (SEQ ID NO: 17) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEFVSR ITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGS TSTATPLRVDYWGQGTLVTVSS referred to herein as FC5-H3.

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

(76) TABLE-US-00010 (SEQ ID NO: 40) AEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG, also referred to herein as hFc1X7.

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

(78) TABLE-US-00011 (SEQ ID NO: 47) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEFVSR ITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGS TSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGSGGGGSGTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRV KNI, also referred to herein as FC5-H3-hFc1X7-ABP(6G).

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

(80) TABLE-US-00012 BBB Fc ABP FC5 mFc2a (SEQ ID NO: 39) ABP (SEQ ID NO: 14) (SEQ ID NO: 30) FC5(H3) mFc2a (SEQ ID NO: 39) ABP (SEQ ID NO: 17) (SEQ ID NO: 30) FC5(H3) hFc1x7 (SEQ ID NO: 40) ABP (SEQ ID NO: 17) (SEQ ID NO: 30) FC5(H3) hFc1x7 (SEQ ID NO: 40) ABP(G) (SEQ ID NO: 17) SEQ ID NO: 32) FC5(H3) hFc1x7 (SEQ ID NO: 40) ABP(GG-G) (SEQ ID NO: 17) SEQ ID NO: 35 FC5(H3) hFc1x7 (SEQ ID NO: 40) ABP(6G) (SEQ ID NO: 17) SEQ ID NO: 36) FC5(H3) hFc1X0 (SEQ ID NO: 41) ABP(GG-G) (SEQ ID NO: 17) SEQ ID NO: 35 FC5(H3) hFc1x7 (SEQ ID NO: 40) ABP(trc) (SEQ ID NO: 17) SEQ ID NO: 38) IGF1R-5(H2) mFc2a (SEQ ID NO: 39) ABP (SEQ ID NO: 26) (SEQ ID NO: 30)

(81) 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 (SEQ ID NO: 55) or any suitable linker, as exemplified in the consensus linker sequence in SEQ ID NO:53.

(82) The compound of the present invention, as provided in the fusion protein of SEQ ID NO:47 also referred to herein as FC5-H3-hFc1×7-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.sub.5, or His.sub.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.

(83) 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.

(84) 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.

(85) 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.

(86) A method of reducing toxic ß-amyloid levels in the cerebrospinal fluid (CSF) and brain parenchyma of subjects having increased ß-amyloid levels is provided. More specifically, toxic ß-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.

(87) 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

(88) 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 IGF1R-5 sdAb (IGF1R-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 FIG. 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

(89) 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

(90) Constructs comprising FC5 variants FC5 and FC5-H3 V.sub.HH 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.

(91) 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 μm 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 μm) and aliquoted.

(92) SDS-PAGE and Aß 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/Aß overlay assay. The immunoblots were blocked with non-fat dry milk and then exposed to Aß preparations for 45 min at room temperature (50-100 nM) and the bound Aß was detected using 6E10 antibody as described previously (Chakravarthy et al. 2013).

(93) ELISA: Aß-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 Aß.sub.1-42 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 Aß was detected by incubating HRP-conjugated Aß-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 450 nm according to manufacturer's instructions.

(94) 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. Aß-oligomer binding of free ABP and FC5-Fc-ABP fusion protein by ELISA and Western blot (WB) Overlay assay is shown in FIGS. 2B and C. Free or fused ABP was immobilized on ELISA plate by coating samples in phosphate buffered saline (PBS) overnight at 40° C. and exposed to Aß preparations as described by Chakravarthy et al., 2013. Bound Aß was detected with Aß-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 Aß oligomers. Bound Aß was detected with specific antibody as above. Results show that ABP retained its Aß oligomer binding ability after fusion with the BBB carrier. Mo: Aß monomers; Oli: Aß Oligomers

Example 3: Binding of BBB-Fc-L-ABP Fusion Molecules to Aa Deposits in AD-Tg Mice (B6.Cg-Tg, Jackson Lab) In Vitro

(95) 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-μm 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 μg/μl 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 Aß 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 Aß aggregates in AD-Tg mouse brain. In the BBB-Fc-ABP constructs provided, the BBB may be FC5 or an anti-IGF1R antibody.

Example 4: BBB Transmigration of FC5-Fc-L-ABP Fusion Molecules In Vitro

(96) 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 (FIGS. 4A, 4B and 4C).

(97) 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 I-coated tissue culture inserts (pore size-1 μm; surface area 0.9 cm.sup.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 μM) of positive (FC5 constructs) or negative controls (A20.1, a Clostridium difficile toxin A binding V.sub.HH; and EG2, an EGFR binding V.sub.HH), 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 60 min 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).

(98) 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 V.sub.HH, each V.sub.HH 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.

(99) A typical multiplexed SRM analysis of V.sub.HH 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 μL) 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.sub.HH, 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.sub.HH 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.sub.HH.

(100) Samples containing mixtures of V.sub.HH (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, Mass.) coupled to LTQ XL ETD or LTQ Orbitrap ETD mass spectrometer (ThermoFisher, Waltham, Mass.). The desired aliquot of the sample was injected and loaded onto a 300 μm I.D.×0.5 mm 3 μm PepMaps C18 trap (ThermoFisher) then eluted onto a 100 μm I.D.×10 cm 1.7 μm 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 6×10.sup.3 and a maximum accumulation time of 200 ms.

(101) Determination of the apparent permeability coefficient:Quantified values can be directly plotted or the P.sub.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; C0=initial concentration of the dosing solution] and plotted. The P.sub.app value is commonly used to determine the ability of a molecule to cross the BBB. P.sub.app values are a measure of the specific permeability of the compound across brain endothelial monolayer.

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

(103) TABLE-US-00013 TABLE 1 Tryptic MRM  SEQ ID Type peptide NO Protein FC5 peptide ITWGGDNTFYSNSVK +  59 + 66 FC5-mFc-ABP ILIS mFc peptide NTEPVLDSDGSYFMYSK 60 FC5-mFc-ABP, host IgGs ABP peptide ASAQASLASK 61 FC5-mFc-ABP IGF1R5-H2  GLEWVATIDWGDGGTR 62 IGF1R5-H2-mFc-ABP peptide IGF1R5-H2  ADETAVYYCAMAR 63 IGF1R5-H2-mFc-ABP peptide Aß LVEFAEDVGSNK 64 Host APP, Aß1-42 or Aß1-40 Alubmin APQVSTPTLVEAAR 65 Host albumin

(104) 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 IGF1R5-mFc-ABP ABP (FIGS. 17 A and B).

Example 5: BBB Transmigration and Pharmacokinetics of FC5-Fc-L-ABP Fusion Molecules In Vivo

(105) 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.

(106) 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, Mass., 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 Wistar rats aged 8-10 weeks (weight range, 230-250 g) were used in all studies.

(107) 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 μl of CSF. The CSF was then transferred into the sample glass vial (Waters, Cat #186000384c) and placed in −80° C. freezer until further analysis.

(108) 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 1100 rcf (3422 rpm) 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 WinLin 6.0 program.

(109) 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.

(110) CSF collection is a delicate procedure during which CSF can be easily contaminated with blood. Since the amounts of V.sub.HH 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 (SEQ ID NO: 65) 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

(111) A ratio of 1500 and below was considered as blood contaminated.

(112) 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.

(113) TABLE-US-00014 TABLE 2 Parameter Estimate Units CV (%) mAb V1 114.6 mL/kg 2.3 V2 74.4 mL/kg 6.5 CL 0.664 mL/(kg*hr) 1.9 CLd 2.682 mL/(kg*hr) 41.8 Alpha T.sub.1/2 11.2055 hr 38.0 Beta T.sub.1/2 205.279 hr 2.2 Vss 189.0 mL/kg 1.4 FC5mFc-ABP (FC5 domain) V1 144.5 mL/kg 2.2 V2 218.4 mL/kg 27.2 CL 1.056 mL/(kg*hr) 26.5 CLd 3.789 mL/(kg*hr) 10.6 Alpha T.sub.1/2 14.36 hr 14.3 Beta T.sub.1/2 263.8 hr 41.3 Vss 362.9 mL/kg 16.3 Dose range: 2.5 mg/kg. 6 mg/kg, 12 mg/kg, 25 mg/kg,

(114) Serum half life (both distribution phase and terminal) are similar between ‘benchmark mAb’ and FC5mFc-cargo

Example 6: Delivery of FC5-ABP Construct to the Brain in Non-Rodent Larger Animal

(115) 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.

(116) TABLE-US-00015 TABLE 3 Dog 1 Dog 2 Mean Mean Parameter Unit Estimate SD Estimate SD t½ h 96 21 89 21 AUC 0-t ng/ml * h 8.80E+06 1.99E+06 8.84E+06 2.16E+06 AUC ng/ml * h 1.01E+07 2.21E+06 1.04E+07 2.76E+06 0-inf_obs Cl_obs ml/h/kg 1.54 0.36 1.53 0.48 Vss_obs ml/h/kg 193 7 205 10 Alpha half h 19 10 13 8.5 life Beta half h 114 37 166 151 life AUC.sub.0-t,csf / % 0.88 0.09 0.52 0.04 AUC.sub.0-t,serum

Example 7: BBB Permeability

(117) 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 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

(118) 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.

(119) 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 (FIGS. 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 (100 U/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-HCl pH 8, 150 mM NaCl 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×g 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 (FIGS. 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 Aß from Mouse Brain

(120) 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.

(121) Comparison between treatment with ABP alone (FIG. 9A) or ABP fused with BBB carrier FC5 (FIGS. 9B and 9C). Two different AD Tg mouse models, triple transgenic (3×Tg-AD, sv129/C57BL6 mice harboring PS1M146V, APP.sub.Swe and tauP301L transgenes, Dr. F. M. LaFerla, University of California) and double transgenic (B6.Cg-Tg, harboring PSEN1dE9 and APP.sub.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, Aß 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 Aß 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 Aß 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 Aß levels. CSF analysis also indicated a significant decrease in Aß.sub.1-42 levels within 24 hrs following FC5-mFc-ABP treatment (FIG. 9C). The Aß peptide sequence detected by MRM (SEQ: LVFFAEDVGSNK (SEQ ID NO: 64), Table 1/ELISA analyses is remote/different from the Aß 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

(122) FC5-ABP (FC5 SEQ ID NO 17; and ABP SEQ ID NO 36) and FC5-hFc-ABP (FC5 SEQ ID NO 17; hFc 1×7 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 15 mg/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 3×100 μl 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, 100 μl SureBlue reagent was added and incubated in the dark at RT for 10-15 min. At the end of reaction, 100 μl 1M HCl 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 Aß from Rat Brain

(123) 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 30 mg/kg and subsequent four weekly doses of 15 mg/kg). CSF levels of FC5-mFc-ABP and Aß were analyzed by nanoLC MRM. Before and after four weeks of treatment, brain Aß levels were determined by PET scan using a specific Aß-binding agent [18F] NAV4694. FC5-mFc-ABP reduced CSF Aß level in rats within 24 hrs. An inverse relationship between the CSF levels of FC5-mFc-ABP and Aß was observed, as in Tg mice, suggesting target engagement and rapid clearance of Aß by ABP delivered to the brain and CSF by FC5 (FIG. 11A and FIG. 11B). This was further corroborated by PET scan which clearly indicated a significant reduction (30-50%) of rat brain Aß levels following four weeks of treatment with FC5-mFc-ABP (FIG. 11C).

Example 12: Increased Hippocampal Volume and Improved Neuronal Connectivity

(124) In the experiment described in Example 11, 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 Aß in Dogs

(125) 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 Aß 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 Aß level that was inversely proportional to CSF FC5-mFc2a-ABP levels.

Example 14: Generation of ABP Fusion Molecule with a Different BBB Carrier

(126) To assess the versatility of ABP fusion molecule, ABP was successfully fused with another humanized BBB carrier IGF1R5 (H2). As shown in FIG. 12, the bi-functionality of the molecule was retained, ABP's ability to bind Aß oligomer (ELISA and overlay assays) and also IGF1R5'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: Aß Oligomer Binding by Different BBB-Crossing Single-Domain Antibody-Fc-ABP Constructs

(127) 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 Table1. and FIG. 15) are provided. As shown in FIG. 15, all constructs retained similar potency in binding Aß oligomers by ELISA method.

Example 16: Production of FC5-hFc1×7-L-ABP with Specific Mutations to Improve Stability and Bio-Manufacturability

(128) FC5-hFc1×7-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, Aß-binding of ABP in the fusion molecule was also tested by overlay assay. Bound Aß was detected with Aß-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

(129) 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 IGF1R 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.

(130) 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)] Crosses In Vitro Rat Blood Brain Barrier Intact

(131) 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)] is Transported Across the BBB In Vivo and Delivered to the Brain Intact by FC5

(132) 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)

(133) Immunohistochemistry was carried out as described in Example 3. Brain sections from AD-transgenic mice were incubated with FC5(H3)-hFc1×7-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 40 μm free-floating sections containing cortex and hippocampus from APP/PS1 transgenic mouse were subjected to antigen retrieval in 10 mM 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.sub.2O.sub.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)-hFc1×7-ABP or the molar equivalent of FC5-hFc1×7 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)-hFc1×7-ABP construct but not with FC5(H3)-hFc1×7 without ABP, indicating ABP-dependent binding of Aß 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).

(134) Similar binding of Aß 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 Aß 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

(135) Fluorophore-labelled (FIG. 18A) or naïve 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 Aß deposits as confirmed by its co-localization with Aß-specific antibody. In a parallel study, four hrs after intra-hippocampal injection of naïve 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 Aß-specific antibody as detection antibody. As shown in 21B, microinjected FC5-ABP molecule remained intact and ABP was able to engage and bind the target Aß in vivo as indicated by the presence of Aß in the pulled-down complex detected by Aß-specific antibody.

Example 22: PK/PD Comparison Between Non-Humanized and Humanized FC5-Fc-L-ABP Constructs

(136) FC5-mFc2a-ABP or FC5(H3)-hFc1×7-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)-hFc1×7-ABP was administered intravenously into Tg mice via tail vein injection at 15 mg/kg as described for FIG. 9B. FC5-Fc-ABP and Aß 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 Aß 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

(137) TABLE-US-00016 SEQ ID NO: Sequence Description 1 GFKITHYTMG CDR1 FC5 2 RITWGGDNTFYSNSVKG CDR2 FC5 3 GSTSTATPLRVDY CDR3 FC5 4 EYPSNFYA CDR1 IGF1R-3 5 VSRDGLTT CDR2 IGF1R-3 6 AIVITGVWNKVDVNSRSYHY CDR3 IGF1R-3 7 GGTVSPTA CDR1 IGF1R-4 8 ITWSRGTT CDR2 IGF1R-4 9 AASTFLRILPEESAYTY CDR3 IGF1R-4 10 GRTIDNYA CDR1 IGF1R-5 11 IDWGDGGX; where X is A or T CDR2 IGF1R-5 12 AMARQSRVNLDVARYDY CDR3 IGF1R-5 13 X.sub.1VQLVX.sub.2SGGGLVQPGGSLRLSCAASGFKITHYTMGWX.sub.3RQAPGKX.sub.4 Humanized FC5 X.sub.5EX.sub.6VSRITWGGDNTFYSNSVKGRFTISRDNSKNTX.sub.7YLQMNSLRAED consensus TAVYYCAAGSTSTATPLRVDYWGQGTLVTVSS, where X.sub.1 = D or E, X.sub.2 = A or E, X.sub.3 = F or V, X.sub.4 = E or G, X.sub.5 = R or L, X.sub.6 = F or W, X.sub.7 = L or V; 14 DVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKERE FC5 FVSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADY YCAAGSTSTATPLRVDYWGKGTQVTVSS 15 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWVRQAPGKGLE FC5-H1 WVSRITWGGDNTFYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCAAGSTSTATPLRVDYWGQGTLVTVSS 16 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGVVVRQAPGKGLE FC5-H2 WVSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAV YYCAAGSTSTATPLRVDYWGQGTLVTVSS 17 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3 VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY CAAGSTSTATPLRVDYWGQGTLVTVSS 18 X.sub.1VX.sub.2LX.sub.3ESGGGLVQX.sub.4GGSLRLSCX.sub.5ASEYPSNFYAMSWX.sub.6RQAPGK IGF1R-3 X.sub.7X.sub.8EX.sub.9VX.sub.10GVSRDGLTTLYADSVKGRFTX.sub.11SRDNX.sub.12KNTX.sub.13X.sub.14LQM Consensus NSX.sub.15X.sub.16AEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTX.sub.17VTVSS, where X.sub.1 is E or Q; X.sub.2 is K or Q; X.sub.3 is V or E; X.sub.4 is A or P; X.sub.5 is V or A; X.sub.6 is F or V; X.sub.7 is E or G; X.sub.8 is R or L; X.sub.9 is F or W; X.sub.10 is A or S; X.sub.11 is M or I; X.sub.12 is A or S; X.sub.13 is V or L; X.sub.14 is D or Y; X.sub.15 is V or L; X.sub.16 is K or R; and X17 is Q or L, 19 QVKLEESGGGLVQAGGSLRLSCVASEYPSNFYAMSWFRQAPGKERE IGF1R-3 FVAGVSRDGLTTLYADSVKGRFTMSRDNAKNTVDLQMNSVKAEDTAV YYCAIVITGVWNKVDVNSRSYHYWGQGTQVTVSS 20 EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKERE IGF1R-3-H5 FVSGVSRDGLTTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS 21 X.sub.1VX.sub.2LX.sub.3ESGGGLVQX.sub.4GGSLRLSCX.sub.5X.sub.6SGGTVSPTAMGWX.sub.7RQAPG IGF1R-4 KX.sub.8X.sub.9EX.sub.10VX.sub.11HITWSRGTTRX.sub.12ASSVKX.sub.13RFTISRDX.sub.14X.sub.15KNTX.sub.16YLQ Consensus MNSLX.sub.17X.sub.18EDTAVYYCAASTFLRILPEESAYTYWGQGT X.sub.19VTVSS, where X.sub.1 is E or Q; X.sub.2 is K or Q; X.sub.3 is V or E; X.sub.4 is A or P; X.sub.5 is A or E; X.sub.6 is V or A; X.sub.7 is V or F; X.sub.8 is G or E; X.sub.9 is L or R; X.sub.10 is F or W; X.sub.11 is G or S; X.sub.12 is V or Y; X.sub.13 is D or G; X.sub.14 is N or S; X.sub.15 is A or S; X.sub.16 is L or V; X.sub.17 is K or R; X.sub.18 is A or S; and X.sub.19 is L or Q, 22 QVKLEESGGGLVQAGGSLRLSCEVSGGTVSPTAMGWFRQAPGKERE IGF1R-4 FVGHITWSRGTTRVASSVKDRFTISRDSAKNTVYLQMNSLKSEDTAVY YCAASTFLRILPEESAYTYWGQGTQVTVSS 23 QVQLVESGGGLVQPGGSLRLSCAVSGGTVSPTAMGWFRQAPGKGLE IGF1R-4-H3 FVGHITWSRGTTRYASSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVY YCAASTFLRILPEESAYTYWGQGTLVTVSS 24 X.sub.1VX.sub.2LX.sub.3ESGGGLVQX.sub.4GGSLRLSCAASGRTIDNYAMAWX.sub.5RQAPGKX.sub.6 IGF1R-5 X.sub.7EX.sub.8VX.sub.9TIDWGDGGX.sub.10RYANSVKGRFTISRDNX.sub.11KX.sub.12TX.sub.13YLQMNX.sub.14 Consensus LX.sub.15X.sub.16EDTAVYX.sub.17CAMARQSRVNLDVARYDYWGQGTX.sub.18VTVSS, where X.sub.1 is E or Q; X.sub.2 is K or Q; X.sub.3 is V or E; X.sub.4 is A or P; X.sub.5 is V or S; X.sub.6 is D or G; X.sub.7 is L or R; X.sub.8 is F or W; X.sub.9 is A or S; X.sub.10 is A or T; X.sub.11 is A or S; X.sub.12 is G or N; X.sub.13 is M or L; X.sub.14 is N or R; X.sub.15 is E or R; X.sub.16 is P or A; X.sub.17 is S or Y; and X.sub.18 is Q or L, 25 QVKLEESGGGLVQAGGSLRLSCAASGRTIDNYAMAWSRQAPG IGF1R-5 KDREFVATIDWGDGGARYANSVKGRFTISRDNAKGTMYLQMN NLEPEDTAVYSCAMARQSRVNLDVARYDYWGQGTQVTVSS 26 QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPG IGF1R-5-H2 KGLEWVATIDWGDGGTRYANSVKGRFTISRDNSKNTMYLQMN SLRAEDTAVYYCAMARQSRVNLDVARYDYWGQGTLVTVSS 27 SGKTEYMAFPKPFESSSSIGAEKPRNKKLPEEEVESSRTPWLYEQEG PK-4 EVEKPFIKTGFSVSVEKSTSSNRKNQLDTNGRRRQFDEESLESFSSMP DPVDPTTVTKTFKTRKASAQASLASKDKTPKSKSKKRNSTQLKSRVKN ITHARRILQQSNRNACNEAPETGSDFSMFEA 28 FSSMPDPVDPTTVTKTFKTRKASAQASLASKDKTPKSKSK P4 peptide 29 KDKTPKSKSKKRNSTQLKSRVKNITHARRILQQSNRNACN P5 peptide 30 KTFKTRKASAQASLASKDKTPKSKSKKRNSTQLKSRVKNI P4-5 peptide (ABP) 31 X.sub.1TFX.sub.2TX.sub.3X.sub.4ASAQASLASKDKTPKSKSKKX.sub.5X.sub.6STQLX.sub.7SX.sub.8VX.sub.9NI ABP consensus here X.sub.1 = G or A, X.sub.2 = G or V, X.sub.3 = G or A, X.sub.4 = G or A, X.sub.5 = G or V, X.sub.6 = G or V, X.sub.7 = G or V, X.sub.8 = G or A, X.sub.9 = G or A (SEQ ID NO: 31) 32 KTFKTRKASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI [ABP(G)] 33 KTFKTRKASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI [ABP(GG)] 34 KTFKTRGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI [ABP(RG-G)] 35 KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI [ABP(GG-G)] 36 GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI [ABP(6G)] 37 KTFKTRKASAQASLASKDKTPKSKSKKGGSTVKNI [ABP(GGspv)] 38 KTFKTRKASAQASLASKDKTPKSKSKKRG [ABP(trc)] 39 ASEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCV Mouse Fc2a VVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQH QDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEE MTKKQVTLTCMVTDFMPEDIYVEVVTNNGKTELNYKNTEPVLDSDGSY FMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG 40 AEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV Human Fc1x7 VVDVSHEGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 41 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV Human Fc1X0 VVDVSHEGPEVKFNWHVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQK SLSLSPG 42 EVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKERE FC5-mFc2a- FVSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADY ABP YCAAGSTSTATPLRVDYWGKGTQVTVSSASEPRGPTIKPCPPCKCPA PNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVN NVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKD LPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPE DIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNS YSCSVVHEGLHNHHTTKSFSRTPGTGGGGSGGGGSKTFKTRKASAQ ASLASKDKTPKSKSKKRNSTQLKSRVKNI 43 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3-mFc2a- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY L-ABP CAAGSTSTATPLRVDYWGQGTLVTVSSASEPRGPTIKPCPPCKCPAP NLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLP APIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIY VEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYS CSVVHEGLHNHHTTKSFSRTPGTGGGGSGGGGSKTFKTRKASAQAS LASKDKTPKSKSKKRNSTQLKSRVKNI 44 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L-ABP CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGSGGGGSKTFKTRKASAQASLA SKDKTPKSKSKKRNSTQLKSRVKNI 45 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FCS-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(G) LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGSGGGGSKTFKTRKASAQASLA SKDKTPKSKSKKRGSTQLKSRVKNI 46 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(GG-G) LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGTGGGSGGGGSKTFKTGG ASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI 47 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(6G) LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGSGGGGSGTFGTGGASAQASL ASKDKTPKSKSKKGGSTQLKSRVKNI 48 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5(H3)-L- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY ABP(GG-G)-L CAAGSTSTATPLRVDYWGQGTLVTVSSGGGGSGGGGSKTFKTGGAS hFc1X7 AQASLASKDKTPKSKSKKRGSTQLKSRVKNIGGGGSGGGGSAEPKSS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 49 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X0-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSCDKTHTCPPCPAPEL ABP(GG-G) LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWHVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEGLHNHYTQKSLSLSPGTGGGSGGGGSKTFKTGGASAQAS LASKDKTPKSKSKKRGSTQLKSRVKNI 50 QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPGKGLE IGF1R5-H2- WVATIDWGDGGTRYANSVKGRFTISRDNSKNTMYLQMNSLRAEDTAV hFc1X7-L- YYCAMARQSRVNLDVARYDYWGQGTLVTVSSAEPKSSDKTHTCPPC ABP(GG-G) PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGTGGGSGGGGSKTFKTGGASA QASLASKDKTPKSKSKKRGSTQLKSRVKNI 51 KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNIGGGSGGG ABP(GG-G)-L- GSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT hFc1X7- CVVVDVSHEGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT IGF1R5-H2 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGQVQL VESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPGKGLEWVATI DWGDGGTRYANSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCA MARQSRVNLDVARYDYWGQGTLVTVSS 52 AEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV hFc1X7-L- VVDVSHEGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL ASP(GG-G)-L- HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE IGF1R5-H2 LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSG GGGSKTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNIGGG SGGGGSQVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQA PGKGLEWVATIDWGDGGTRYANSVKGRFTISRDNSKNTMYLQMNSLR AEDTAVYYCAMARQSRVNLDVARYDYWGQGTLVTVSS 53 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL L(consensus)- LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV ABP(6G) EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTX.sub.1GX.sub.2X.sub.3GX.sub.4X.sub.5GX.sub.6,GTFGTGGASAQ ASLASKDKTPKSKSKKGGSTQLKSRVKNI where X.sub.1 = A or G., X.sub.2 = A or G, X.sub.3 = S or T, X.sub.4 = G or V, X.sub.5 = A or V, X.sub.6 = S or T

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