Blood brain barrier transport compounds and uses thereof
阅读说明：本技术 血脑屏障迁移化合物及其用途 (Blood brain barrier transport compounds and uses thereof ) 是由 巴律·查克拉瓦蒂 D·斯坦尼米洛维克 Y·杜罗切尔 于 2018-07-31 设计创作，主要内容包括：公开了一种淀粉样蛋白-β结合肽的大脑穿透组合物。这在阿尔茨海默病的治疗中可能是有用的,例如作为双功能分子,所述双功能分子包括血脑屏障跨越抗体和通过Fc片段连接的淀粉样蛋白-β靶向肽,所述双功能分子能够跨越血脑屏障迁移至大脑,以及包括所述双功能分子的组合物。公开了使用该组合物治疗阿尔茨海默病的方法。(A brain-penetrating composition of amyloid-beta binding peptides is disclosed. This may be useful in the treatment of alzheimer's disease, for example as a bifunctional molecule comprising a blood brain barrier spanning antibody and an amyloid- β targeting peptide linked via an Fc fragment, capable of migrating across the blood brain barrier to the brain, and compositions comprising the bifunctional molecule. Methods of treating alzheimer's disease using the compositions are disclosed.)
1. An isolated peptide that binds beta-amyloid, the isolated peptide comprising the amino acid sequence:
X1TFX2TX3X4ASAQASLASKDKTPKSKSKKX5X6
wherein, X1K, G or A or X1G or A, X2K, G or V, X3R, G or A, X4K, G or A, X5R, G or V, X6N, G or V, (SEQ ID NO: 46).
2. An isolated peptide that binds beta-amyloid, the isolated peptide comprising the amino acid sequence:
X1TFX2TX3X4ASAQASLASKDKTPKSKSKKX5X6STQLX7SX8VX9NI(SEQ ID NO:31)
wherein, X1K, G or A, or X1G or A, X2K, G or V, X3R, G or A, X4K, G or A, X5R, G or V, X6N, G or V, X7K, G or V, X8R, G or A, X9Either K, G or A, respectively,
or any C-terminally cleaved amyloid-beta binding product thereof.
3. The isolated peptide of claim 1 or 2, wherein the amino acid sequence comprises a sequence selected from the group consisting of:
KTFKTRKASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI(SEQ ID NO:32)；
KTFKTRKASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI(SEQ ID NO:33)；
KTFKTRGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI(SEQ ID NO:34)；
KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI(SEQ ID NO:35)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI(SEQ ID NO:36)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVK(SEQ ID NO:37)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRV(SEQ ID NO:38)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSR(SEQ ID NO:39)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKS(SEQ ID NO:40)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLK(SEQ ID NO:41)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQL(SEQ ID NO:42)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQ(SEQ ID NO:43)；
KTFKTRKASAQASLASKDKTPKSKSKKGGSTVKNI(SEQ ID NO:44)；
KTFKTRKASAQASLASKDKTPKSKSKKRG(SEQ ID NO:45)；
GTFGTGGASAQASLASKDKTPKSKSKKGG (SEQ ID NO: 47); and
a sequence substantially equivalent thereto, or a C-terminally cleaved β -amyloid binding peptide thereof.
4. The isolated peptide of claim 3, wherein the C-terminally cleaved beta-amyloid binding peptide comprises a sequence selected from the group consisting of:
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVK(SEQ ID NO:37)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRV(SEQ ID NO:38)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSR(SEQ ID NO:39)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKS(SEQ ID NO:40)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLK(SEQ ID NO:41)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQL (SEQ ID NO: 42); and
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQ(SEQ ID NO:43)。
5. the isolated peptide of any one of claims 1 to 4, fused to an antibody or antibody fragment, said antibody and antibody fragment being capable of migrating across the blood-brain barrier.
6. The isolated peptide of claim 5, linked to an Fc fragment.
7. The isolated peptide of claim 6, wherein the isolated peptide, the antibody or fragment thereof, and the Fc fragment form a single chain polypeptide fusion protein.
8. The isolated peptide of any one of claims 6 to 7, wherein the Fc fragment comprises an Fc having reduced effector function.
9. A fusion protein comprising the isolated peptide of any one of claims 1 to 8, and an antibody or antibody fragment that migrates across the Blood Brain Barrier (BBB).
10. The fusion protein of claim 9, wherein the fusion protein is a single chain polypeptide further comprising an Fc fragment.
11. The fusion protein of claim 10, wherein the single chain polypeptide forms a dimer.
12. The fusion protein of claim 11, wherein the dimer is a homodimer or heterodimer with respect to ABP and comprises at least one ABP capable of binding β -amyloid.
13. The fusion protein of any one of claims 10 to 12, wherein the single chain polypeptide comprises:
an antibody or fragment thereof;
a peptide that binds to amyloid-beta, selected from the group consisting of SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, and SEQ ID NO 47; and
an Fc fragment selected from the group consisting of SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50.
14. The fusion protein of claim 13, wherein the antibody or fragment thereof comprises a sequence comprising
Complementarity Determining Region (CDR)1 sequence GFKITHYTMG (SEQ ID NO:1), CDR2 sequence RITWGGDNTFYSNSVKG (SEQ ID NO:2), CDR3 sequence GSTSTATPLRVDY (SEQ ID NO: 3).
15. The fusion protein of claim 14, wherein the antibody or fragment thereof comprises a sequence selected from any one of: 13, 14, 15, 16, 17 and sequences substantially identical to any of the above.
16. The fusion protein of claim 13, wherein the antibody or fragment thereof comprises a sequence selected from the group consisting of seq id no:
an antibody or fragment thereof comprising the CDR1 sequence EYPSNFYA (SEQ ID NO:4), the CDR2 sequence VSRDGLTT (SEQ ID NO:5), the CDR3 sequence AIVITGVWNKVDVNSRSYHY (SEQ ID NO: 6);
an antibody or fragment thereof comprising the CDR1 sequence GGTVSPTA (SEQ ID NO:7), the CDR2 sequence ITWSRGTT (SEQ ID NO:8), the CDR3 sequence AASTFLRILPEESAYTY (SEQ ID NO: 9); and
an antibody or fragment thereof comprising the CDR1 sequence GRTIDNYA (SEQ ID NO:10), the CDR2 sequence IDWGDGGX (SEQ ID NO: 11); wherein X is A or T, CDR3 sequence AMARQSRVNLDVARYDY (SEQ ID NO: 12).
17. The fusion protein of claim 16, wherein the antibody or fragment thereof comprises a sequence selected from any one of: 18, 21, and 24.
18. The fusion protein of claim 17, wherein the antibody or fragment thereof comprises a sequence selected from any one of: 19, 20, 22, 23, 25, 26 and sequences substantially identical to any of the above.
19. The fusion protein of claims 13 to 18, wherein a single chain polypeptide comprising the antibody or fragment thereof is linked to the amyloid-beta-binding peptide via the Fc fragment.
20. The fusion protein of claim 13 or 19, wherein the fusion protein is a single chain polypeptide comprising a sequence selected from the group consisting of seq id no:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 and 71 SEQ ID NO and sequences substantially identical thereto.
21. The fusion protein of claim 20, wherein the single chain polypeptide forms a dimer.
22. The fusion protein of claim 21, wherein the fusion protein is a single chain polypeptide comprising any suitable peptide linker.
23. The fusion protein of claim 22, wherein the peptide linker comprises an amino acid sequence that enables the linking component of the fusion protein to maintain its unrestricted desired biological function.
24. The fusion protein of claim 22 or 23, wherein the peptide linker comprises the sequence (GGGS)n、(GGGGS)nOr any suitable peptide linker sequence.
25. The fusion protein according to any one of claims 11 to 24, wherein the single chain polypeptide forms a dimeric polypeptide.
26. The fusion protein of claim 25, wherein the dimeric polypeptide comprises a homodimer or a heterodimer with respect to beta-Amyloid Binding Protein (ABP) included in the dimeric polypeptide.
27. The fusion protein of claim 26, wherein the heterodimer comprises at least one functional ABP capable of binding β -amyloid.
28. The fusion protein of any one of claims 9 to 27, wherein the antibody or fragment thereof is humanized.
29. The fusion protein of any one of claims 9 to 28, wherein the antibody or fragment thereof is a single domain antibody (sdAb).
30. The fusion protein of any one of claims 9 to 29, wherein the Fc fragment is mouse Fc2a or human Fc 1.
31. The fusion protein of claim 30, wherein the Fc fragment comprises SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 50.
32. The fusion protein of any one of claims 9 to 31, wherein the fusion protein comprises the antibody or fragment thereof linked to the N-terminus of the Fc fragment, and a polypeptide linked to the C-terminus of the Fc fragment that binds to β -amyloid.
33. The fusion protein of any one of claims 9-13 and 16-31, wherein the antibody or fragment thereof is linked to the C-terminus of the Fc fragment and the polypeptide that binds β -amyloid is linked to the N-terminus of the Fc fragment.
34. The fusion protein of claims 33 to 33, wherein the linker is independently selected to link the antibody or fragment thereof, and/or to link the polypeptide that binds β -amyloid to the Fc.
35. The fusion protein of claim 34, wherein the linker sequence is GGGGSGGGGS, GGGSGGGGS, any suitable linker or any suitable linker as provided in SEQ ID NO. 71.
36. The fusion protein of any one of claims 9 to 35, wherein the fusion protein forms a dimer comprising at least one peptide according to any one of claims 1 to 8.
37. The fusion protein of claim 38, wherein the dimer comprises two single chain polypeptides, wherein the two single chain polypeptides are: a bifunctional homodimer comprising two ABPs selected from any one of SEQ ID NOs 31 to 47; a bifunctional heterodimer comprising two different ABPs selected from any one of SEQ ID NOs 31 to 47; or a monofunctional heterodimer comprising one ABP selected from any one of SEQ ID NOs 31 to 47.
38. A pharmaceutical composition comprising the isolated peptide of any one of claims 1 to 8, or any combination thereof, and a pharmacologically acceptable carrier.
39. A pharmaceutical composition comprising the fusion protein of any one of claims 9 to 37, or any combination thereof, and a pharmacologically acceptable carrier.
40. A nucleic acid molecule encoding any of the proteins of claims 1 to 39.
41. A vector comprising the nucleic acid molecule of claim 40.
42. A kit comprising the pharmaceutical composition of claim 38 or 39.
43. The pharmaceutical composition according to claim 38 or 39, for use in treating Alzheimer's disease in a patient.
44. A method of treating Alzheimer's disease, comprising administering the fusion protein of any one of claims 9 to 37 or the pharmaceutical composition of claim 38 or 39 to a subject in need thereof.
45. A method of reducing the level of β -amyloid in a subject having elevated levels of amyloid β, comprising repeating the step of parenterally administering a sufficient amount of the pharmaceutical composition of claim 38 or 39 to the subject.
46. The method of claim 45, wherein parenteral administration is subcutaneous or intravenous.
47. The method of claim 45 or 46, wherein the level of β -amyloid is decreased in a subject with elevated levels of amyloid β following repeated parenteral administration of the composition of claim 38 or 39.
48. The method of claim 47, wherein β -amyloid is reduced for four weeks following parenteral administration of the composition of claim 38 or 39.
49. The method of claim 47, wherein the level of β -amyloid in cerebrospinal fluid (CSF) is decreased in a subject having elevated CSF following parenteral administration of the composition of claim 38 or 39.
50. The method of claim 46, wherein the level of β -amyloid in cerebrospinal fluid (CSF) is decreased in a subject having an elevated CSF within 24 hours of a single parenteral administration of the composition of claim 38 or 39.
51. A method of reducing the level of β -amyloid in a subject having elevated levels of amyloid β, comprising administering the fusion protein of any one of claims 9-37.
52. A pharmaceutical composition comprising a mixture of fusion proteins according to any one of claims 9 to 37.
53. The pharmaceutical composition of claim 52, wherein the mixture of fusion proteins comprises an ABP bifunctional homodimer fusion protein, an ABP bifunctional heterodimer fusion protein, and an ABP monofunctional heterodimer fusion protein, or any combination thereof.
54. The pharmaceutical composition of claims 52 and 53, wherein said mixture of isolated single chain polypeptide fusions is a pharmaceutically effective mixture comprising an effective yield of functional fusion protein.
55. The pharmaceutical composition of any one of claims 52 to 54, for use in alleviating a symptom of Alzheimer's disease, in combination with a pharmaceutically acceptable diluent, carrier, vehicle or excipient.
56. The pharmaceutical composition of claim 55, for use in reducing β -amyloid levels in a subject having elevated amyloid β levels.
57. A method of reducing the level of β -amyloid in a subject having elevated levels of amyloid β, comprising the step of introducing into the body of the subject the pharmaceutical composition of claim 55.
58. The pharmaceutical composition of claim 56, for use in reducing the level of β -amyloid in the brain, tissue, or body fluids (CSF and blood) of a subject having an elevated level of β -amyloid.
Technical Field
The present invention relates to compounds that migrate across the blood brain barrier, and uses thereof. More specifically, the present invention relates to compounds, which may include antibodies or fragments thereof that cross the blood brain barrier, immunoglobulin Fc domains or fragments thereof, and polypeptides that bind beta-amyloid, fusion proteins and compositions thereof, and their use in treating alzheimer's disease.
Background
Neurodegenerative diseases, such as alzheimer's disease and parkinson's disease, place an increasing burden on our aging society, as there is currently no effective treatment for these disabling (disabling) conditions. Alzheimer's Disease (AD) is an irreversible neurodegenerative disorder affecting about 15% of the population over the age of 65 and is the leading cause of progressive intellectual and cognitive failure in the aging population (Hardy et al, 2014).
In AD, cholinergic neurons are severely lost, leading to a decrease in the levels of acetylcholine (ACh), a key neurotransmitter involved in memory processing and storage. In addition, excitotoxicity caused by the neurotransmitter glutamate is also involved in the pathogenesis of AD. Thus, cholinergic enhancement and/or inhibition of glutamate toxicity may improve cognitive function in AD. Indeed, the only FDA-approved drugs for the treatment of AD are acetylcholinesterase (AChE) inhibitors (e.g., donepezil, rivastigmine, galantamine) to prevent loss of ACh, and specific glutamate receptor inhibitors (e.g., memantine) (mangialsche et al, 2010; Ji and Ha, 2010; Savonenko et al, 2012). However, the beneficial effects of these symptomatic drugs are limited and transient, providing temporary improvement of cognitive function, and do not prevent the progression of the disease. While other treatments are contemplated, including antioxidants, anti-inflammatory drugs (NSAIDS), cholesterol lowering drugs, and estrogen treatments, none of these treatments appear to have any long-term benefit, particularly in improving memory and cognitive function in AD patients (Magialasche et al, 2010; Ji and Ha, 2010).
One of the major hallmarks of alzheimer's disease is the accumulation of the 39-43 amino acid peptide beta-amyloid (a β) in the brain in the form of aggregates and plaques. A large body of evidence based on genetic, pathological and biochemical studies suggests that Α β, and in particular its oligomeric aggregates, play a central role in the development of AD pathology (Hardy et al 2014; DeLaGarza 2003; Selkoe and Hardy 2016). According to the amyloid hypothesis, a long-term imbalance in the production and clearance of a β in the brain leads to its accumulation and aggregation with age. These Α β aggregates are thought to initiate a series of events leading to loss of synapses and neuronal function leading to progressive loss of memory and other cognitive functions (Hardy et al 2014; DeLaGarza 2003; Selkoe and Hardy 2016; sengutta et al 2016).
The production of a β from the a β precursor protein APP is achieved by sequential proteolysis of APP by proteases β and γ secretases (Barageb and sonavine, 2015). Inhibitors of these enzymes have been shown to reduce a β production and are being developed as potential drugs for the treatment of AD (Hardy et al 2014; Mangialasche et al 2010; Selkoe and Hardy 2016; Ji and Ha 2010). Likewise, agents that sequester (sequester) and/or promote a β clearance are also under development. Of note are the development of AD vaccine immunotherapy. In transgenic AD animal models and clinical trials involving AD patients, both active (Α β peptide) and passive (Α β antibody) have been demonstrated to be effective in preventing amyloid deposits and clearing pre-formed amyloid plaques (Mangialasche et al, 2010; Ji and Ha, 2010; Morrone et al, 2015; LannFet et al, 2014; Selkoe and Hardy et al, 2016; Goure et al, 2014).
Beta and gamma secretase inhibitors (e.g., darunavir, samatet, verubechestat) are being developed to prevent proteolysis of Amyloid Precursor Protein (APP) and thereby reduce or inhibit brain a β production. However, their efficacy in reducing Α β burden is unclear and many of them have failed preclinical or clinical trials (Savonenko et al, 2012; Musiek and Holtzman, 2015). In addition, these inhibitors may have serious non-specific side effects, as these enzymes are also involved in the processing of other enzymes and signaling molecules (such as Notch associated with neuronal development) (Savonenko et al, 2012; Musiek and Holtzman, 2015).
Immunotherapeutic approaches such as active immunization (A β vaccine, AN1792) and passive immunization (e.g., Bapineuzumab, Solanezumab, Crenezumab, Aducanumab, etc.) have proven quite effective in reducing A β deposits and partially eliminating memory deficits in transgenic animals (Monsonogo and Weiner, 2003; Bard et al, 2000, Sevigny J et al, 2016). Several clinical trials using active and passive immunization have shown that brain a β deposits are reduced with modest improvement in cognition. However, clinical trials have to be abandoned due to severe inflammatory reactions (meningoencephalitis manifestations), angiogenic oedema and minor bleeding in AD patients. Despite these limitations, immunotherapy approaches suggest that agents that effectively sequester a β and prevent its deposition and toxicity may be effective drugs that prevent AD progression, even its development (Rafii and Aisen, 2015, Selkoe and Hardy, 2016).
The treatment and early diagnosis of AD and other brain-derived diseases remains challenging because most suitable therapeutic molecules and diagnoses are unable to penetrate the tight and highly restricted Blood Brain Barrier (BBB) (Abbott, 2013). The BBB constitutes a physical barrier formed by Brain Endothelial Cells (BEC) lining the blood vessels and interconnected by tight junctions (Abbott, 2013). The tight junctions formed between BECs are essential for the integrity of the BBB and to prevent the paracellular transport of molecules greater than 500 daltons (Da). Since brain endothelial cells exhibit very low phagocytic rates (Abbott, 2013), transcellular transport of macromolecules is limited to highly specific receptor-mediated endocytotic transport (RMT) pathways, as well as passive charge-based adsorption-mediated endocytotic transport (Abbott, 2013; Pardridge, 2002). In addition, high density efflux pumps, such as P-glycoprotein or multidrug resistance protein-1 (MDR-1), help to clear unwanted substances from the brain (Abbott, 2013).
While all of these features protect the brain from pathogens and toxins, they also prevent the entry of most therapeutic drugs. In fact, only less than 5% of small molecule therapeutic drugs and few larger therapeutic drugs can cross the BBB at pharmacologically relevant concentrations (i.e., sufficient to bind Central Nervous System (CNS) targets and elicit a pharmacological/therapeutic response) unless they are specifically 'transported', i.e., conjugated to a transporter molecule. Due to the lack of effective "vectors" to transport molecules across the BBB, many drugs for treating neurodegenerative diseases are "shelved" or eliminated because they cannot be delivered to the brain in sufficient quantities.
Despite the great progress in understanding the molecular mechanisms of AD, there is currently no effective drug or treatment that can prevent the progression of the disease or cure the disease. In addition, the lack of a large capacity and high selectivity of the BBB vector has hampered the development of new therapies and diagnostics for brain-derived diseases, including brain tumors and neurodegenerative diseases.
Disclosure of Invention
The present invention relates to compounds or compositions that migrate across the blood-brain barrier, and uses thereof.
The present invention provides peptides that bind beta-amyloid (beta-amyloid). The peptide (or protein) that binds to amyloid beta can be selectedSelectively binding pathologically related beta-amyloid1-42(Aβ1-42) Aggregates, and herein abbreviated ABPs, wherein ABPs herein include any ABP peptide, ABP variant, any C-terminally cleaved ABP, or an equivalent functional β -amyloid binding peptide thereof. The present invention provides specific ABP peptide species (peptides) that bind to beta-amyloid and advantageously provides a mixture of functional beta-amyloid binding peptides and thereby provides a mixture of stable and functional fusion proteins comprising said specific ABP peptides.
The invention further provides a fusion protein (also referred to herein as a compound, composition, single chain polypeptide, or construct) comprising a beta-amyloid (beta-amyloid) binding peptide (any ABP provided herein) linked to an antibody or fragment thereof capable of crossing the blood-brain barrier (BBB), wherein BBB herein refers to the abbreviation of a carrier antibody or fragment that migrates across the blood-brain barrier. In one embodiment, the fusion protein comprises an ABP and BBB vector, or a fragment thereof, wherein the ABP and BBB components of the fusion protein may be linked by an Fc region or portion thereof. In one embodiment, the fusion protein comprising ABP and BBB further comprises an immunoglobulin effector domain (referred to as Fc), or a fragment thereof, wherein the ABP and BBB components of the fusion protein can be linked by the Fc region or a portion thereof. For example, the constructs of the invention may further comprise a linker (L), wherein L is a small linker peptide or peptoid chain. Single chain polypeptides including BBB-Fc-ABP can form dimers through Fc, thereby enabling dimerization of the fusion protein. The compounds of the invention may be referred to as fusion proteins, constructs, fusion molecules, formulations or compositions.
Single chain polypeptides comprising a BBB-Fc-L-ABP fusion protein may form dimers through Fc dimerization, wherein the BBB-Fc-L-ABP may be a homodimer or heterodimer with ABP. That is, the BBB-Fc-L-ABP fusion protein provided may be a single chain polypeptide or a dimeric polypeptide thereof, the dimer may be a homodimer in which both ABP arms are functional (ABP and/or C-terminally cleaved functional ABP), or the dimer may be functional comprising a functional ABP arm (ABP or C-terminally cleaved functional ABP)ABP) and a non-functional ABP arm, or a heterodimer comprising two functional ABP arms. The term "functional" denotes ABP peptides capable of binding to beta-amyloid, such as peptides having the common sequence of SEQ ID NO:31(40aa) and its C-terminal cleavage functional product, or having the common sequence of SEQ ID NO:46(29 aa); while the term "non-functional" refers to any C-terminal cleavage product that renders ABP unable to bind to beta-amyloid. Dimerization of Fc is likely mediated by the interaction of a large, tightly packed hydrophobic interface between two Fc CH3 domains in constructs comprising BBB-Fc-ABP. It should be noted that herein BBB-Fc-ABP and BBB-Fc-L-ABP refer to equivalent abbreviations for single chain polypeptide fusion proteins comprising BBB, an Fc portion and ABP, wherein each component of the fusion protein is linked or coupled by any suitable linker to form a β -amyloid binding fusion protein capable of migrating across the blood brain barrier. The BBB, Fc, ABP and linker (L) may be any BBB, Fc, ABP or L provided herein. In one embodiment, BBB is FC5 (e.g., SEQ ID NO:17), Fc is hFc1X7 (e.g., SEQ ID NO:49), ABP is a β -amyloid binding peptide, such as any peptide having SEQ ID NO:31(40aa) (e.g., ABP (6G) SEQ ID NO:36(40aa)) or any β -amyloid binding C-terminal cleavage product thereof (e.g., SEQ ID NO:37(38aa), SEQ ID NO:38(37aa), SEQ ID NO:39(36aa), SEQ ID NO:40(35aa), SEQ ID NO:41(34aa), SEQ ID NO:42(33aa), or SEQ ID NO:43(32aa)) or any peptide comprising or consisting of SEQ ID NO:46(29aa) (e.g., SEQ ID NO:47(29 aa)); and L may be (GGGGS)n、(GGGS)nOr any suitable linker thereof, e.g. T (GGGGS)2. It is to be understood that the fusion protein provided is not limited to a particular linker and may be any linker that allows operable biological functions of each component of the fusion protein (i.e., blood brain barrier migration and β -amyloid binding). For example, the fusion protein of the invention may be the fusion protein of FC5-H3-hFc1X7-L-ABP (6G) provided in SEQ ID NOs: 56, 71 or any equivalent thereof (equivalent), such as a fusion protein comprising any of the beta-amyloid binding proteins provided herein or a C-terminal cleavage product thereof.
The equivalent fusion protein thereof may include any of the amyloid-beta binding proteins (SEQ ID NO:31), the C-terminal cleavage products of SEQ ID NO:36 (e.g., SEQ ID NO:37 through SEQ ID NO 43), or a protein comprising or consisting of SEQ ID NO:46, or an equivalent amyloid-beta binding protein thereof. For example, the C-terminally cleaved ABP may comprise the sequence of SEQ ID NO 37,
The BBB-Fc-ABP constructs provided may be single chain polypeptides (referred to as monomers of single chain fusion proteins) or dimeric polypeptides thereof. The dimeric peptide comprising two single chain polypeptides may be a homodimer-the two ABP peptides are the same (e.g., two SEQ ID NOs: 36); or the dimer peptide may be a heterodimer (e.g., SEQ ID NO:36 and SEQ ID NO:40, or any combination of SEQ ID NO:31 to 47) -the dimeric ABP peptide may be any two different sequences, wherein two or at least one of the single-chain APB peptides is selected from the group consisting of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO: 43. The ABP peptide of the dimeric single-chain polypeptide may be any biologically functional β -amyloid binding ABP peptide; specifically, any ABP selected from the group consisting of: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 44, 45, 46, 47 or a C-terminally cleaved biologically functional
A dimeric peptide comprising two BBB-Fc-L-ABP single chain polypeptides may comprise two different ABP sequences, wherein at least one of said ABP sequences is a biologically functional molecule (i.e. capable of binding to β -amyloid). Quite advantageously and unexpectedly, the dimeric peptide can accommodate a single non-functional ABP peptide and still retain equivalent functional biological activity with respect to β -amyloid binding. In a dimer comprising two different ABP sequences (i.e., a heterodimer with respect to ABP), when two functional ABP peptides are provided in the heterodimer, at least one of the ABP peptides provided in the heterodimer is a biologically functional ABP, i.e.,
The present invention provides an isolated peptide that binds to beta-amyloid, comprising or consisting of the amino acid sequence:
X1TFX2TX3X4ASAQASLASKDKTPKSKSKKX5X6
wherein, X1K, G or A, X1G or A, X2K, G or V, X3R, G or A, X4K, G or A, X5R, G or V, X6N, G or V, (SEQ ID NO: 46; 29 aa).
The present invention provides an isolated peptide that binds to beta-amyloid, comprising or consisting of the amino acid sequence:
X1TFX2TX3X4ASAQASLASKDKTPKSKSKKX5X6STQLX7SX8VX9NI(SEQ ID NO:31；40aa)
wherein, X1K, G or A, X1G or A, X2K, G or V, X3R, G or A, X4K, G or A, X5R, G or V, X6N, G or V, X7K, G or V, X8R, G or A, X9Either K, G or A, respectively,
or any C-terminally cleaved amyloid-beta binding product thereof.
The isolated peptide amino acid sequence comprises a sequence selected from the group consisting of:
KTFKTRKASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI(SEQ ID NO:32；40aa)；
KTFKTRKASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI(SEQ ID NO:33；40aa)；
KTFKTRGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI(SEQ ID NO:34；40aa)；
KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI(SEQ ID NO:35；40aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI(SEQ ID NO:36；40aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVK(SEQ ID NO:37；38aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRV(SEQ ID NO:38；37aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSR(SEQ ID NO:39；36aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKS(SEQ ID NO:40；35aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLK(SEQ ID NO:41；34aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQL(SEQ ID NO:42；33aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQ(SEQ ID NO:43；32aa)；
KTFKTRKASAQASLASKDKTPKSKSKKGGSTVKNI(SEQ ID NO:44；35aa)；
KTFKTRKASAQASLASKDKTPKSKSKKRG(SEQ ID NO:45；29aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGG (SEQ ID NO: 47; 29 aa); and
a sequence substantially equivalent thereto, or a C-terminally cleaved β -amyloid binding peptide thereof. Also provided is a C-terminal cleavage of one amino acid (i.e., a deletion of isoleucine) to produce a 39 amino acid cleavage product. In the common sequence SEQ ID NO:31,
More specifically, the C-terminally cleaved amyloid-beta binding peptide comprises a sequence selected from the group consisting of:
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVK(SEQ ID NO:37；38aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRV(SEQ ID NO:38；37aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSR(SEQ ID NO:39；36aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKS(SEQ ID NO:40；35aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLK(SEQ ID NO:41；34aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQL (SEQ ID NO: 42; 33 aa); and
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQ(SEQ ID NO:43；32aa)。
any of the isolated peptides of the invention may be fused to an antibody or antibody fragment that is capable of migrating across the blood-brain barrier. The isolated peptide may be linked to an Fc fragment, wherein the isolated peptide, the antibody or fragment thereof, forms a single chain polypeptide fusion protein with the Fc fragment. In one embodiment, the Fc fragment comprises an Fc with reduced effector function.
Accordingly, the invention provides fusion proteins comprising any of the isolated peptides provided herein, and an antibody or antibody fragment that migrates across the Blood Brain Barrier (BBB). The fusion protein may be a single chain polypeptide further comprising an Fc fragment, wherein the single chain polypeptide forms a dimer. The dimer may be a homodimer or heterodimer with respect to the ABP included in the dimer, and the heterodimer may be bifunctional (two functional ABP arms) or monofunctional (only one functional ABP arm). Advantageously, a monofunctional dimer is equivalent to a bifunctional dimer regardless of the functional ABP protein included therein. That is, for example, a heterodimer can comprise an ABP having SEQ ID NO:37 on one arm of the dimer and SEQ ID NO:41 on the other arm of the dimer, or can comprise an ABP having SEQ ID NO:43 on one arm of the dimer and a non-functional ABP on the other arm, and the two dimers advantageously exhibit equivalent β -amyloid binding functionality. This advantageously enables the production of a mixture of dimers having equivalent functionality, thereby enabling an increase in product yield.
Accordingly, fusion proteins are provided, wherein the dimer is a homodimer or heterodimer with respect to ABP and comprises at least one ABP capable of binding β -amyloid.
The provided single chain polypeptides may include antibodies or antibody fragments thereof, peptides that bind to beta-amyloid, and Fc fragments. The single chain polypeptide may further comprise any suitable linker that enables the peptide that binds β -amyloid to be linked to the Fc fragment. The antibody or fragment thereof may be coupled or linked to an Fc, and the ABP may be linked to the Fc by any suitable linker to form a single chain polypeptide. The fusion proteins provided are not limited to inclusion of the linkers provided herein, and may include any suitable linker that enables the components (i.e., antibody, Fc, and ABP) to be linked to form a fusion protein equivalent to the fusion proteins provided herein.
The antibody or fragment thereof may be any antibody selected from the group consisting of SEQ ID NO 14,
The antibody or fragment thereof may comprise sequences comprising the Complementarity Determining Region (CDR)1 sequence GFKITHYTMG (SEQ ID NO:1), the CDR2 sequence RITWGGDNTFYSNSVKG (SEQ ID NO:2), the CDR3 sequence GSTSTATPLRVDY (SEQ ID NO: 3). Specifically, the antibody or fragment thereof comprises a sequence selected from any one of SEQ ID NO 13, SEQ ID NO 14,
The antibody or fragment thereof may comprise a sequence selected from the group consisting of:
an antibody or fragment thereof comprising the CDR1 sequence EYPSNFYA (SEQ ID NO:4), the CDR2 sequence VSRDGLTT (SEQ ID NO:5), the CDR3 sequence AIVITGVWNKVDVNSRSYHY (SEQ ID NO: 6);
an antibody or fragment thereof comprising the CDR1 sequence GGTVSPTA (SEQ ID NO:7), the CDR2 sequence ITWSRGTT (SEQ ID NO:8), the CDR3 sequence AASTFLRILPEESAYTY (SEQ ID NO: 9); and
an antibody or fragment thereof comprising the CDR1 sequence GRTIDNYA (SEQ ID NO: 10); CDR2 sequence IDWGDGGX (SEQ ID NO:11), wherein X is A or T; CDR3 sequence AMAROSRVNLDVARYDY (SEQ ID NO: 12).
Specifically, the antibody or fragment thereof may comprise a sequence selected from any one of SEQ ID NO 18,
The antibody or fragment thereof may be humanized. The antibody or fragment thereof may be a single domain antibody (SdAb).
A fusion protein single chain polypeptide comprising an antibody or a fragment thereof is linked to a peptide that binds to beta-amyloid via an Fc fragment. The single-chain polypeptide may comprise any sequence selected from the group consisting of SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55,
The fusion protein may be a single chain polypeptide comprising any suitable peptide linker. The peptide linker may comprise any amino acid sequence that enables the linking components of the fusion protein to retain their desired biological functions without restriction. For example, the peptide linker may comprise (GGGS)n、(GGGGS)nOr any suitable peptide linker sequence.
In one embodiment, provided single chain polypeptides form a dimeric polypeptide, wherein the dimeric polypeptide comprises a homodimer or a heterodimer with respect to beta-Amyloid Binding Protein (ABP) comprised in the dimeric polypeptide. The heterodimer may comprise at least one functional ABP capable of binding β -amyloid.
The Fc fragment of the fusion protein may be mouse Fc2a or
In one embodiment, the fusion protein comprises an antibody or fragment thereof linked to the N-terminus of an Fc fragment, and a β -amyloid binding polypeptide linked to the C-terminus of the Fc fragment; wherein the antibody or fragment is any antibody provided herein, e.g., comprising the CDRs of
In another embodiment, the fusion protein comprises an antibody or fragment thereof linked to the C-terminus of an Fc fragment, and a polypeptide that binds to amyloid-beta linked to the N-terminus of the Fc fragment; wherein the antibody or fragment is any antibody or fragment, including: CDRs of
The fusion protein may comprise a linker, wherein the linker is an independently selected linker sequence linking the antibody or fragment thereof and/or linking the polypeptide that binds β -amyloid to Fc. The linker may comprise a sequence (GGGS)n、(GGGGS)nGGGGSGGGGS, GGGSGGGGS, any suitable linker provided in the art, or a consensus linker as provided in SEQ ID NO:71 or any suitable linker.
The invention provides a dimer of any of the fusion proteins provided, wherein the dimer comprises at least one peptide according to SEQ ID NO:31 to SEQ ID NO:47 or any combination thereof.
More specifically, the fusion protein dimer comprises two single-chain polypeptides, wherein the two single-chain polypeptides are: a bifunctional homodimer comprising two ABPs selected from any one of
Also provided is a pharmaceutical composition comprising an isolated peptide of any one of
Nucleic acid molecules encoding any of the proteins or fusion proteins of the invention are also provided. Vectors comprising a nucleic acid molecule encoding any of the proteins or fusion proteins of the invention are provided. Kits (kits) comprising the pharmaceutical compositions are provided.
The present invention provides a method of treating alzheimer's disease comprising administering to a subject in need thereof a pharmaceutical composition comprising any fusion protein or dimer thereof.
A method of reducing toxic β -amyloid levels in the brain of a subject having elevated cerebral amyloid β levels is provided, the method comprising the step of repeating the parenteral administration of a sufficient amount of a provided pharmaceutical composition to the subject. Parenteral administration can be subcutaneous or intravenous. The reduction of β -amyloid levels is not restricted to the brain and can occur in brain, tissues or biological fluids (CSF and blood) in subjects with elevated β -amyloid levels.
Methods are provided wherein toxic β -amyloid levels are reduced following repeated parenteral administration of a composition provided in the brain of a subject having elevated cerebral amyloid β levels. Toxic beta-amyloid is reduced within four weeks of repeated parenteral administration. Toxic beta-amyloid levels in cerebrospinal fluid (CSF) of subjects with elevated Central Nervous System (CNS) beta-amyloid levels are reduced following parenteral administration of the composition. The provided methods and compositions thereof advantageously reduce toxic β -amyloid levels in the cerebrospinal fluid (CSF) of a subject with elevated CNS levels within 24 hours of a single parenteral administration of the composition. In particular, toxic β -amyloid levels are reduced in subjects with elevated cerebral amyloid β levels by a method comprising administration of a provided fusion protein.
The pharmaceutical composition may include any mixture of the fusion proteins provided; wherein the mixture of fusion proteins comprises an ABP bifunctional homodimer fusion protein, an ABP bifunctional heterodimer fusion protein and an ABP monofunctional heterodimer fusion protein or any combination thereof. The medicament comprising the mixture of single chain polypeptide fusions provides a pharmaceutically effective yield of functional fusion protein having equivalent functional activity. The pharmaceutical composition may include a pharmaceutically acceptable diluent, carrier, vehicle or excipient for ameliorating the symptoms of alzheimer's disease. The pharmaceutical composition can be used to reduce toxic β -amyloid levels in the brain of a subject having elevated cerebral amyloid β levels.
The compounds of the invention are useful for the treatment of Alzheimer's Disease (AD).
The peptide that binds to β -amyloid may comprise a sequence selected from the group consisting of:
SGKTEYMAFPKPFESSSSIGAEKPRNKKLPEEEVESSRTPWLYEQEGEVEKPFIKTGFSVSVEKSTSSNRKNQLDTNGRRRQFDEESLESFSSMPDPVDPTTVTKTFKTRKASAQASLASKDKTPKSKSKKRNSTQLKSRVKNITHARRILQQSNRNACNEAPETGSDFSMFEA(SEQ ID NO:27；174aa)；
FSSMPDPVDPTTVTKTFKTRKASAQASLASKDKTPKSKSK(SEQ ID NO:28；40aa)；
KDKTPKSKSKKRNSTQLKSRVKNITHARRILQQSNRNACN(SEQ ID NO:29；40aa)；
KTFKTRKASAQASLASKDKTPKSKSKKRNSTQLKSRVKNI(SEQ ID NO:30；40aa)；
there is provided a peptide that binds to amyloid beta comprising the sequence:
X1TFX2TX3X4ASAQASLASKDKTPKSKSKKX5X6STQLX7SX8VX9NI
wherein, X1K, G or A, X1G or A, X2K, G or V, X3R, G or A, X4K, G or A, X5R, G or V, X6N, G or V, X7K, G or V, X8R, G or A, X9K, G or A (SEQ ID NO:31, 40aa), or its C-terminal cleavage protein [ SEQ ID NO:37(38aa) to SEQ ID NO:43(22aa)]。
31, variants with substitutions at variable amino acids are also provided; wherein, X1G or a; x2G or V; x3G or A, X4G or A, X5G or V; or any combination of these amino acid substitutions in SEQ ID NO: 31.
Also provided is a peptide that binds to beta-amyloid, comprising the sequence:
X1TFX2TX3X4ASAQASLASKDKTTPKSKKX5X6
here, X1K, G or A, X1G or A, X2K, G or V, X3R, G or A, X4K, G or A, X5R, G or V, X6N, G or V, (SEQ ID NO: 46; 29 aa).
46, variants with substitutions at variable amino acids are also provided; wherein, X1G or a; x2G or V; x3G or A, X4G or A, X5G or V; or any combination of these amino acid substitutions in SEQ ID NO 46.
X in certain non-limiting embodiments, the ABP, variant thereof, or C-terminal product thereof can comprise a sequence selected from any one of the following:
X1TFX2TX3X4ASAQASLASKDKTPKSKSKKX5X6STQLX7SX8VX9NI
wherein, X1K, G or A, X1G or A, X2K, G or V, X3R, G or A, X4K, G or A, X5R, G or V, X6N, G or V, X7K, G or V, X8R, G or A, X9K, G or A, (SEQ ID NO:31, 40aa),
KTFKTRKASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI(SEQ ID NO:32；40aa)；
KTFKTRKASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI(SEQ ID NO:33；40aa)；
KTFKTRGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI(SEQ ID NO:34；40aa)；
KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI(SEQ ID NO:35；40aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI (SEQ ID NO: 36; 40 aa); or the C-terminal cleavage product of SEQ ID NO:36 in which one amino acid (isoleucine) is deleted from the C-terminal.
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVK(SEQ ID NO:37；38aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRV(SEQ ID NO:38；37aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSR(SEQ ID NO:39；36aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKS(SEQ ID NO:40；35aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLK(SEQ ID NO:41；34aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQL(SEQ ID NO:42；33aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQ(SEQ ID NO:43；32aa)；
KTFKTRKASAQASLASKDKTPKSKSKKGGSTVKNI(SEQ ID NO:44；35aa)；
KTFKTRKASAQASLASKDKTPKSKSKKRG(SEQ ID NO:45；29aa)；
X1TFX2TX3X4ASAQASLASKDKTPKSKSKKX5X6
Wherein, X1K, G or A, X1G or A, X2Is K, G or V, X3＝R、GOr A, X4K, G or A, X5R, G or V, X6N, G or V, (SEQ ID NO: 46; 29 aa); and
GTFGTGGASAQASLASKDKTPKSKSKKGG(SEQ ID NO:47；29aa)
or a sequence substantially identical to any of the sequences described above, which is capable of binding to beta-amyloid.
The ABP that binds to β -amyloid may comprise a peptide sequence selected from the group consisting of: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47. ABPs comprising or consisting of a peptide sequence selected from the group consisting of: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47. The invention additionally includes ABP sequences having the common sequence SEQ ID NO:31 or a C-terminal functional product thereof, or the common sequence SEQ ID NO: 46; for example, ABP can include the sequence SEQ ID NO:32 to SEQ ID NO:36, or any C-terminally cleaved beta-amyloid binding product of SEQ ID NO:36 (i.e., SEQ ID NO:37 to SEQ ID NO:43), or beta-amyloid binding protein including SEQ ID NO: 46. The C-terminal cleavage product can be any C-terminal cleavage of
The present invention provides an isolated polypeptide that binds beta-amyloid, which may comprise a sequence selected from the group consisting of the common sequence
The present invention provides a fusion protein comprising or consisting of an ABP selected from the group consisting of: 31, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47 or a sequence comprising an equivalent polypeptide sequence. In a non-limiting example, an ABP variant may comprise a sequence
KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI(SEQ ID NO:35；40aa)；
GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI (SEQ ID NO: 36; 40aa) or any of its beta-amyloid binding C-terminal cleavage products, such as those comprising or consisting of SEQ ID NO: 46; 29 aa.
The fusion protein of the present invention may include an antibody or a fragment thereof that migrates across the Blood Brain Barrier (BBB) (as described above, BBB is an abbreviation for antibody carrier that migrates across the blood brain barrier). The antibody or fragment thereof (BBB) may bind to a brain endothelial cell surface receptor epitope, thereby enabling migration across the blood brain barrier. For example, such a surface receptor epitope may be a TMEM30A or insulin-
The antibody or fragment thereof may comprise a sequence selected from the group consisting of seq id no:
an antibody or fragment thereof comprising Complementarity Determining Region (CDR)1 sequences including GFKITHYTMG (SEQ ID NO:1), CDR2 sequence RITWGGDNTFYSNSVKG (SEQ ID NO:2), CDR3 sequence GSTSTATPLRVDY (SEQ ID NO: 3);
an antibody or fragment thereof comprising the CDR1 sequence EYPSNFYA (SEQ ID NO:4), the CDR2 sequence VSRDGLTT (SEQ ID NO:5), the CDR3 sequence AIVITGVWNKVDVNSRSYHY (SEQ ID NO: 6);
an antibody or fragment thereof comprising the CDR1 sequence GGTVSPTA (SEQ ID NO:7), the CDR2 sequence ITWSRGTT (SEQ ID NO:8), the CDR3 sequence AASTFLRILPEESAYTY (SEQ ID NO: 9); and
an antibody or fragment thereof comprising the CDR1 sequence GRTIDNYA (SEQ ID NO:10), the CDR2 sequence IDWGDGGX (SEQ ID NO: 11); wherein X is A or T, CDR3 sequence AMARQSRVNLDVARYDY (SEQ ID NO: 12).
The antibody or fragment thereof may comprise a sequence selected from the group consisting of seq id no:
X1VQLVX2SGGGLVQPGGSLRLSCAASGFKITHYTMGWX3RQAPGKX4X5EX6VSRITWGGDNTFYSNSVKGRFTISRDNSKNTX7YLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWGQGTLVTVSS (SEQ ID NO:13), wherein X1Either D or E, X2Is A or E, X3Is F or V, X4Either E or G, X5R or L, X6F or W, X7L or V;
X1VX2LX3ESGGGLVQX4GGSLRLSCX5ASEYPSNFYAMSWX6RQAPGKX7X8EX9VX10GVSRDGLTTLYADSVKGRFTX11SRDNX12KNTX13X14LQMNSX15X16AEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTX17VTVSS (SEQ ID NO:18), where X1Is E or Q; x2Is K or Q; x3Is V or E; x4Is A or P; x5Is V or A; x6Is F or V; x7Is E or G; x8Is R or L; x9Is F or W; x10Is A or S; x11Is M or I; x12Is A or S; x13Is V or L; x14Is D or Y; x15Is V or L; x16Is K or R; and X17Is Q or L;
X1VX2LX3ESGGGLVQX4GGSLRLSCX5X6SGGTVSPTAMGWX7RQAPGKX8X9EX10VX11HITWSRGTTRX12ASSVKX13RFTISRDX14X15KNTX16YLQMNSLX17X18EDTAVYYCAASTFLRILPEESAYTYWGQGTX19VTVSS (SEQ ID NO:21), wherein X1Is E or Q; x2Is K or Q; x3Is V or E; x4Is A or P; x5Is A or E; x6Is V or A; x7Is V or F; x8Is G or E; x9Is L or R; x10Is F or W; x11Is G or S; x12Is V or Y; x13Is D or G; x14Is N or S; x15Is A or S; x16Is L or V; x17Is K or R; x18Is A or S; and X19Is L or Q; and
X1VX2LX3ESGGGLVQX4GGSLRLSCAASGRTIDNYAMAWX5RQAPGKX6X7EX8VX9TIDWGDGGX10RYANSVKGRFTISRDNX11KX12TX13YLQMNX14LX15X16EDTAVYX17CAMARQSRVNLDVARYDYWGQGTX18VTVSS (SEQ ID NO:24), where X1Is E or Q; x2Is K or Q; x3Is V or E; x4Is A or P; x5Is V or S; x6Is D or G; x7Is L or R; x8Is F or W; x9Is A or S; x10Is A or T; x11Is A or S; x12Is G or N; x13Is M or L; x14Is N or R; x15Is E or R; x16Is P or A; x17Is S or Y; and X18Is Q or L.
In some particular non-limiting embodiments, the antibody or fragment thereof may comprise a sequence selected from any one of the following sequences:
DVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAGSTSTATPLRVDYWG KGTQVTVSS(SEQ ID NO:14)；
EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWVRQAPGKGLEWVSRITWGGDNTFYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG QGTLVTVSS(SEQ ID NO:15)；
EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWVRQAPGKGLEWVSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG QGTLVTVSS(SEQ ID NO:16)；
EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEFVSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG QGTLVTVSS(SEQ ID NO:17)；
QVKLEESGGGLVQAGGSLRLSCVASEYPSNFYAMSWFRQAPGKEREFVAGVSRDGLTTLYADSVKGRFTMSRDNAKNTVDLQMNSVKAEDTAVYYCAIVITGVWNKVDVNSRS YHYWGQGTQVTVSS(SEQ ID NO:19)；
EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKEREFVSGVSRDGLTTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSY HYWGQGTLVTVSS(SEQ IDNO:20)；
QVKLEESGGGLVQAGGSLRLSCEVSGGTVSPTAMGWFRQAPGKEREFVGHITWSRGTTRVASSVKDRFTISRDSAKNTVYLQMNSLKSEDTAVYYCAASTFLRILPEESAYTY WGQGTQVTVSS(SEQ ID NO:22)；
QVQLVESGGGLVQPGGSLRLSCAVSGGTVSPTAMGWFRQAPGKGLEFVGHITWSRGTTRYASSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAASTFLRILPEESAYTY WGQGTLVTVSS(SEQ ID NO:23)；
QVKLEESGGGLVQAGGSLRLSCAASGRTIDNYAMAWSRQAPGKDREFVATIDWGDGGARYANSVKGRFTISRDNAKGTMYLQMNNLEPEDTAVYSCAMARQSRVNLDVARYD YWGQGTQVTVSS(SEQ ID NO:25)；
QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPGKGLEWVATIDWGDGGTRYANSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCAMARQSRVNLDVARY DYWGQGTLVTVSS (SEQ ID NO: 26); and
a sequence substantially identical to any of the sequences described above.
The BBB may be an antibody or fragment thereof. In one embodiment, the BBB may be a single domain antibody (SdAb). The SdAb may be humanized.
In one embodiment, the antibody or fragment thereof (BBB) may be linked to an Fc or fragment thereof, wherein the BBB-Fc construct may form a dimer. The invention further provides a fusion peptide comprising BBB-Fc-L-ABP, wherein the BBB-Fc portion of the fusion peptide is linked to the ABP by a short peptide linker (e.g., a linker of less than 12 amino acids), and the Fc or Fc fragment enables dimerization of the fusion peptide to provide a dimer, thereby protecting the construct from degradation and increasing its serum half-life. The Fc fragment may be any suitable Fc fragment selected to impart the desired pharmacokinetics, wherein the Fc or Fc fragment contributes to the long half-life of the fusion molecule. Other embodiments of the Fc or Fc fragment can modulate, alter or inhibit immune effector function (Shields et al, 2001). Other embodiments of Fc fragments may mediate clearance of the fusion peptide from the brain (Caram-Salas N, 2011). In a non-limiting example, the Fc or Fc fragment may be Fc mouse Fc2a or human Fc1 selected from any one of
In the compounds of the invention, the BBB may be linked to the ABP via an Fc fragment and/or any additional suitable linker L.
The compounds of the invention include fusion proteins; wherein the fusion protein comprises an antibody or fragment thereof, an Fc fragment, and a peptide that binds to amyloid-beta. The fusion protein may include an antibody or fragment thereof linked to the N-terminus of the Fc fragment by L, a linker peptide, or a chemical linker, and a peptide that binds to beta-amyloid linked to the C-terminus of the Fc fragment. The antibody or fragment thereof may be linked to the C-terminus of the Fc fragment and the peptide that binds β -amyloid is linked to the N-terminus of the Fc fragment.
Accordingly, the present invention provides a fusion protein comprising a single chain polypeptide comprising an antibody or fragment thereof selected from the group consisting of SEQ ID NO 13 to SEQ ID NO 26; a beta-amyloid binding peptide selected from the group consisting of SEQ ID NO:27 to SEQ ID NO: 47; linked by an Fc fragment selected from the group consisting of SEQ ID NO 48 to
For example, the fusion protein may comprise SEQ ID NO 51 to SEQ ID NO 70, or a sequence substantially identical thereto. The fusion protein may be a single chain polypeptide, and the single chain polypeptide of the fusion protein may be a dimeric polypeptide. Note that the (GGGSGGGGS or GGGGSGGGGS) linker provided in the fusion proteins comprised by SEQ ID NO:51 to SEQ ID NO:70 may be any suitable linker sequence. For example, the highlighted linker sequence (e.g., GGGGSGGGGS) can be any equivalent peptide linking sequence that enables linking of the components of the provided fusion protein, as shown in SEQ ID NO:71, wherein the linker can be any peptide or chemical linker.
In one embodiment, the BBB may be linked to an Fc fragment, thereby forming a dimer. The Fc fragment may be any suitable Fc fragment with reduced effector function, such as mouse Fc2a or human Fc1(Shields et al, 2001).
The ABP of the invention may comprise the sequence
Accordingly, a therapeutic composition is provided comprising a blood brain barrier migrating antibody or fragment thereof (BBB), fc (fc), linked to a peptide that binds beta-amyloid (ABP or ABP variant, or C-terminal cleavage product thereof), wherein the peptides synergistically increase the stability and effectiveness of the provided therapeutic composition. As shown, unexpectedly and most significantly, single dose (single bolus) BBB-Fc-ABP reduced brain Α β burden by 50% within 24 hours post-treatment (fig. 9B) compared to multiple free ABP treatments in animals (AD mouse model) for three months (fig. 9A). Thus, the compounds provided herein are much stronger than free ABP in reducing brain Α β burden. Furthermore, this Fc-comprising fusion protein provides a longer serum half-life compared to free ABP or BBB-ABP (WO2006/133566), thereby providing an improved therapeutic compound (fig. 10).
The ABP variants and fusion proteins comprising ABP (constructs thereof) of the present invention overcome the disadvantages of the prior art. In the prior art, simply linking ABP to a BBB vector (WO2006/133566) does not ensure the production of an effective molecule. Fusions including Fc intended to enhance serum half-life do not ensure efficient transport of ABP across the blood brain barrier. The specific design and formulation (formulation) of fusion molecules comprising a BBB vector, an Fc fragment and ABP provides effective BBB-penetrating therapeutic compounds. The provided high efficiency blood brain barrier penetrating therapeutic compositions include specific design and formulation of BBB-Fc-ABP, wherein the formulation exhibits synergistic improvements in compound stability and effectiveness. As shown in fig. 9, an unexpected increase in the stability of the fusion composition was provided in the composition comprising the fusion protein construct, as well as a synergistic increase in the efficiency of blood brain barrier migration and faster (within 24 hours) clearance of a β from the brain.
The ABP C-terminal cleavage products (e.g., SEQ ID NOS: 37 to 43 and SEQ ID NOS: 46 to 47) provided herein advantageously provide a specific novel and unexpected subset of functional proteins that enable improved yields in isolatable mixtures of functional fusion proteins capable of binding beta amyloid. Advantageously, the single chain polypeptide fusion protein provided may comprise any one of
The compounds provided herein may be referred to as compounds, fusion proteins, formulations, compositions, or constructs. The provided constructs may include antibodies or fragments thereof (which may be abbreviated BBB), Fc fragments (abbreviated Fc), and polypeptides that bind beta-amyloid (abbreviated ABP). The provided constructs or compositions (which may be abbreviated herein as BBB-Fc-ABP or BBB-Fc-L-ABP) include components that synergistically overcome the deficiencies encountered in the prior art with respect to blood brain barrier migration, efficacy, and stability of compounds. Thus, the compounds provided herein include novel formulations having excellent and unexpected effects in terms of blood brain barrier migration, therapeutic effects, and compound stability.
Drawings
These and other features of the invention will now be described by way of example with reference to the accompanying drawings, in which:
FIG. 1: a schematic of amyloid-binding fusion protein dimers across the blood brain barrier is shown. A schematic of a single chain polypeptide fusion protein is provided, which includes a BBB-spanning single domain antibody (BBB or BBB vector), Fc fragment (Fc), and Amyloid Binding Peptide (ABP) fused via a linker peptide, and is shown as a dimer. FIG. 1 shows the corresponding dimers of fusion proteins comprising BBB-Fc-ABP. The dimer of the fusion protein can be a bifunctional homodimer or a bifunctional heterodimer with respect to ABP, in that both ABP arms comprise a functional ABP peptide (e.g., SEQ ID No:36, FIG. 1Ai) and/or various C-terminally cleaved functional ABPs (e.g., SEQ ID No:36 and/or SEQ ID No:37 to SEQ ID No:43, FIG. 1Aii), or a monofunctional heterodimer in which one ABP arm is functional (ABP or its C-terminally cleaved functional cleavage product) and the other arm is a non-functional peptide (FIG. 1 Aiii). The components of single chain polypeptides (i.e., BBB, Fc, and ABP) are depicted in various configurations, as shown in fig. 1A, 1B, 1C, and 1D.
FIG. 2: the generation of FC5-mFc-ABP and humanized FC5(H3) -hFc-ABP (ABP SEQ ID NO:32) fusion molecules in CHO cells is shown. As shown in FIG. 2A, Coomassie blue stained gels after separation of FC5 fusion molecules by SDS-PAGE (NR-non-reducing and R-reducing conditions) showed successful production of recombinant fusion molecules. Fig. 2B and 2C: binding of free antibody to A β -oligomers of BBB-Fc-ABP fusion protein was performed by ELISA and Western Blot (WB) overlay analysis. Free or fused ABP was immobilized on ELISA assay plates and exposed to a β. Bound a β was detected with a β -specific antibody 6E10 or
FIG. 3: shown is an immunohistological fluorescence analysis of binding of FC5-Fc-ABP to amyloid deposits in AD-Tg mice (B6.Cg-Tg, Jackson Lab). It was shown by immunohistochemical fluorescence analysis that ABP retained the ability to bind naturally occurring Α β aggregates in the brains of AD-Tg mice. Brain sections of wild type and alzheimer's disease transgenic mice were incubated with IR 800 labeled FC5-mFc-ABP and bound fusion molecules were observed under a fluorescent microscope. Selective binding (bright spots) was observed in brain sections of AD-Tg mice that produced Α β deposits, whereas no selective binding was observed in brain sections of wild type mice that did not produce amyloid deposits.
FIG. 4: it is shown that BBB penetration of FC5 was retained after in vitro fusion with ABP. The blood brain barrier crossing of FC5-ABP fusion molecules was evaluated in rat and human in vitro BBB models. Fusion molecules across the BBB were detected by the nanoLC-MRM method (fig. 4A, fig. 4B and fig. 4C) and western blot analysis using Fc-specific antibodies (fig. 4D, repeated three times). FC5-mFc-ABP crossed the BBB as efficiently as FC5-mFc, whereas the Fc-ABP without the BBB vector portion FC5 did not cross the brain endothelial cell monolayer. As expected, the control single domain antibodies EG2 and a20.1, or the control whole IgG (anti-HEL) did not cross the blood brain barrier. Similar results were obtained with the humanized FC5-H3-hFc-ABP fusion protein (FIGS. 4C and 4D).
FIG. 5: serum and CSF pharmacokinetics of FC5-Fc-ABP in vivo are shown. FC5-mFc-ABP was administered intravenously to rats by tail vein injection at the indicated doses (2.5, 6.25, 12.5, and 25 mg/kg). Serum and cerebrospinal fluid were collected continuously. FC5-Fc-ABP levels were quantified using the nanolC-MRM method. As shown in figure 5, FC5-mFc-ABP appeared in time and dose dependent manner in CSF and Cmax was between 12 and 24 hours, indicating transport of ABP to the brain and CSF compartment in vivo by FC 5. Serum PK parameters (figure 5 and table 1) show that FC5-mFc-ABP has alpha and beta half-lives similar to that of intact IgG (a benchmark antibody containing rat FC).
FIG. 6: serum and CSF PK profiles of beagle FC5-mFc-ABP are shown. FC5-mFc-ABP was administered by intravenous injection to beagle dogs 10-12 years old, and serum and CSF were collected continuously and analyzed by nanoLC-MRM (fig. 6A) and western blot using FC-specific antibodies (fig. 6B). Asterisks indicate blood-contaminated samples (not shown in the MRM analysis). It can be seen that FC5-mFc-ABP appears in time-dependent manner in CSF, suggesting transport of ABP across the canine blood brain barrier in vivo via FC 5. PK parameters and CSF exposure were analyzed by WinNonlin software and are shown in table 2 below.
FIG. 7: BBB penetration and CSF appearance (apearance) of FC5 fused in vivo (rat model) to human FC (hFc) and chemically linked to ABP (FC5-hFc-ABP) is shown. FC5-hFc was attached to ABP-cystine using the heterobifunctional crosslinker sulfo SMCC (sulfosuccinimidyl 4- [ N-maleimidomethyl ] cyclohexane-1-carboxylate) according to the manufacturer's instructions (ThermoFisher scientific). The chemical conjugate molecules were administered intravenously to rats via the tail vein at a dose of 6.25mg/kg, and serum and CSF samples were collected at 4 and 24 hours and analyzed by nanoLC-MRM. In contrast to Fc-ABP without BBB vector, FC5-hFc-ABP appeared in CSF in a time-dependent manner. It should be noted that in this chemically linked construct, the C-terminus of ABP is linked to the Fc fragment (random region) and the N-terminus is free, unlike the fusion construct, in which the N-terminus of ABP is fused to the C-terminus of Fc and the C-terminus of ABP is free. This reversal of ABP direction does not affect its Α β binding capacity and transport across the blood brain barrier.
FIG. 8: the levels of FC5-mFc-ABP measured in various brain regions (cortex and hippocampus) after intravenous injection in mice are shown. A15 mg/kg dose of FC5-mFc-ABP was injected intravenously into the tail vein of Wild Type (WT) or AD transgenic (AD-Tg, B6.Cg-Tg, Jackson Lab) mice, and brains were collected after 4 and 24 hours intracardiac saline infusion. Hippocampus 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 three components of the fusion molecule (BBB, FC5, FC and ABP in this example) were detected by MRM in the cortex and hippocampus, indicating that FC5 vector has successfully delivered ABP to the target region of the brain. The levels measured at different time points were between 750-1400ng/g brain tissue, compared to 50ng/g tissue, typically measured for the control single domain antibody A20.1 fused to Fc or Fc fragment only. This was further confirmed by western blot analysis to detect Fc and ABP in tissue extracts (fig. 8B). No protein signal of the fusion molecule was detected by western blotting in animals receiving only physiological saline. FC5-mFc-ABP levels detected by western blotting in target areas of the brain increased dose-dependently.
FIG. 9: the effect of ABP on Α β levels in transgenic (Tg) mice is shown: comparison between treatment with ABP alone (fig. 9A) or ABP fused to BBB vector FC5 (fig. 9B and 9C). Three-fold transgene (3X Tg-AD, containing PS1M146V, APP) using two different AD Tg mouse modelsSweAnd tauP301L transgenic sv129/C57BL6 mice, Dr.F.M.LaFerla, University of California) and double transgene (B6.Cg-Tg, containing PSEN1dE9 and APPSweTransgene, Jackson Lab); mice were administered subcutaneously (sc) every other day 300nmol/kg of free ABP, respectively, over a 3 or 2 month period. At the end of the treatment, a β levels in the brain were measured by ELISA. Treatment with ABP alone resulted in a 25-50% reduction in brain Α β after 2-3 months of multiple treatments (every other day) (fig. 9A). The FC5-mFc-ABP construct was injected intravenously into double transgenic AD mice (B6.Cg-Tg, 15 mg/kg; equivalent was 220nmol/kg) and brain A β
FIG. 10: an example of the serum half-life extension of the FC5-FC-ABP construct compared to FC5-ABP (i.e. without the FC component) is shown: FC5-ABP and FC5-FC-ABP were injected into rats via the tail vein and a series of serum samples were collected at different time points and analyzed by direct ELISA with FC5 specific antibodies. As can be seen from fig. 10A, the FC5-ABP construct cleared rapidly (less than 1 hour) in serum compared to FC5-FC-ABP (fig. 10B), indicating a significant increase in serum stability of the molecules including the FC fragment.
FIG. 11: the effect of FC5-mFc-ABP on brain amyloid burden in Tg rats is shown: AD-Tg rats were injected weekly with saline or FC5-mFc-ABP (burden dose of 30mg/kg and four subsequent doses of 15mg/kg weekly) via the tail vein over a period of four weeks. CSF levels of FC5-mFc-ABP and A β were analyzed by nanolC MRM (FIG. 11A and FIG. 11B). Four weeks before and after treatment, specific Abeta binding agent [18F ] was used]NAV4694 brain a β levels were determined by PET scanning. After tracer injection, a 60 minute dynamic image is acquired, a transmission scan is acquired, an image is reconstructed and a Binding Potential (BP) is generatedND) A parameter map. FC5-mFc-ABP reduced CSF Α β levels in rats over 24 hours (fig. 11A and 11B). As in Tg rats, an inverse relationship between CSF levels of FC5-mFc-ABP and Α β was observed, indicating target binding (engagement) and rapid clearance of Α β by FC5 delivery of ABP to brain and CSF (fig. 11B). This was further confirmed by PET scans which clearly showed a significant decrease in rat brain Α β levels (30-50%) after four weeks of treatment with FC5-mFc-ABP (fig. 11C).
FIG. 12: volume magnetic resonance imaging (MRI using rapid imaging with steady state precession) and functional MRI (fmri) of Tg rats are shown before and after treatment with saline or FC 5-mFc-ABP. As shown in fig. 12A, an increase in hippocampus volume after four weeks of treatment was observed in Tg rats treated with ABP (Tg-ABP) compared to saline treated Tg rats (Tg-Sal). As shown in fig. 12B, group comparisons after four weeks of treatment showed that ABP-treated Tg rats (Tg-ABP) had greater Anterior Cingulate Corticoid (ACC) connectivity compared to saline-treated Tg rats (Tg-Sal).
FIG. 13: the time and dose dependent appearance of FC5-mFc-ABP in the CSF of beagle dogs and the reduction in CSF Α β levels in Tg mice (fig. 9C) and Tg rats (fig. 11A and B) are shown. FC5-mFc-ABP was administered by intravenous injection at doses of 15mg/kg and 30mg/kg, respectively, to 10-12 year old beagle dogs, and serum and CSF were collected continuously and analyzed by nanolC-MRM for FC5-mFc-ABP and A β levels. It can be seen that FC5-mFc-ABP appears in CSF in a time and dose dependent manner. Importantly, CSF Α β levels were significantly reduced within 24 hours after FC5-mFc2a-ABP injection as seen in Tg mice and Tg rats, indicating that FC5 vector has translational properties (translational nature) in larger animals and also suggesting an effect of the explantation of ABP in reducing CNS Α β burden.
FIG. 14: generation of ABP fusion molecules with different BBB vectors is shown. To assess the versatility of the ABP fusion molecule, ABP was successfully fused with another humanized BBB vector IGF1R5 (H2). As shown, the dual functions of the molecule (the ability of ABP to bind to a β oligomers (ELISA and overlay assay), and the ability of IGF1R5 to deliver ABP in vitro via BBB model (data not shown)) were retained. This clearly demonstrates that ABP can be fused with different BBBs across single domain antibodies for delivery to the brain.
FIG. 15: different single domain antibody-Fc-ABP constructs are shown to bind to a β oligomers (fig. 15A, 15B and 15C). In some of these constructs, the ABP has been modified by site-specific mutagenesis or removal of the C-terminal portion of the molecule, as shown in SEQ ID NOs. All constructs maintained similar potency on binding a β oligomers by ELISA.
FIG. 16: the generation of FC5-hFc1X7-ABP with specific mutations to improve stability and biological manufacturability is shown. FC5-hFc1X7-ABP with specific mutations (such as ABP of SEQ ID NO:35 or SEQ ID NO:36) was generated in CHO cells and isolated on SDS-PGE under reducing (R) and non-reducing (NR) conditions and stained with Coomassie blue as depicted in FIG. 2. The isolated proteins were transferred to nitrocellulose membranes and immunoblotted with FC 5-specific or hFc-specific or ABP-specific antibodies. In another set, the fusion molecules were also tested for a β binding of ABP by an overlay assay. Bound a β was detected with a β -
Fig. 17A, 17B: the blood brain barrier penetration of various FC5-Fc-ABP constructs and IGF1R5-Fc-ABP constructs in vitro is shown. As shown in figure 4, BBB crossing was assessed in an in vitro rat BBB model and molecules crossing the blood brain barrier were detected by the nanoLC-MRM method. All ABP variants fused to the humanized FC5 and IGF1R vectors effectively crossed the blood brain barrier. As expected, the non-BBB permeable sdAb a20.1 did not cross the blood brain barrier, and as such, the ABP fused to a20.1 did not cross the BBB (fig. 17A). In fig. 17B, "fingerprint" peptides were shown that detected all three components of the fusion molecules FC5, FC and ABP with nanoLC-MRM, indicating migration of intact FC5, FC and ABP across the BBB.
FIG. 18: the humanized FC5(H3) -hFc1X7-ABP construct (ABP, SEQ ID NO:35 and SEQ ID NO:36) is shown to be transported across the blood brain barrier in vitro intact by FC 5. As shown in figure 4, blood brain barrier crossing was assessed in an in vitro rat BBB model and molecules crossing the BBB were detected by western blot and ELISA analysis. Fig. 18(1) a and B show immunoblots detected with hFc and ABP specific antibodies, respectively. The molecular size is completely the same as the fusion molecule applied to the blood brain barrier model in vitro. Figure 18(1) C shows a sandwich ELISA in which molecules spanning the BBB are captured by FC 5-specific antibodies on ELISA plates and detected with ABP-specific antibodies. This sandwich ELISA confirmed that FC5(H3) -hFc1X7-ABP remained intact after crossing the blood brain barrier in rats in vitro. Similar results were obtained with FC5(H3) -hFc1X7-ABP (ABP, SEQ ID NO: 36); FIG. 18(2) A, B and C.
FIG. 19: the humanized FC5(H3) -hFc1X7-ABP construct (ABP, SEQ ID NO:35 and SEQ ID NO:36) is shown to be transported across the blood brain barrier in vivo and delivered intact to the brain via FC 5. As depicted in fig. 8, FC5-ABP fusion molecules were administered intravenously via tail vein to wild-type and AD-Tg mice, and brains were collected after intracardiac infusion. The cerebral cortex was homogenized and extracted in RIPA buffer, and then the extracts were subjected to western blot and sandwich ELISA analysis as described in fig. 18. Figure 19A shows an immunoblot detected with ABP-specific antibodies. The molecular size is identical to the fusion molecule injected into the animal. Figure 19B shows a sandwich ELISA of the same extract, with molecules in the extract captured with FC5 specific antibodies on the ELISA plate and detected with ABP specific antibodies. This confirms the immunoblotting result, that is FC5(H3) -hFc1X7-ABP is transported across the BBB in vivo and delivered intact to the brain.
FIG. 20: immunohistochemical analysis of ex vivo binding (A) and in vivo binding (B) of FC5(H3) -hFc1X7-ABP (ABP, SEQ ID NO:36) to endogenous A β deposits (B6.Cg-Tg, Jackson Lab) in the brains of AD-Tg mice is shown. Brain sections from AD transgenic mice were incubated with FC5(H3) -hFc1X7-ABP (ABP, SEQ ID NO:36) and bound fusion molecules were visualized with an FC5 specific antibody coupled with HRP. Selective binding was seen with the FC5(H3) -hFc1X7-ABP construct (black dots), whereas FC5(H3) -hFc1X7 without ABP was not seen (a), indicating that Α β deposits (target binding) in the brain were dependent on ABP binding. No binding was observed in brain sections of wild type mice that did not produce amyloid deposits (data not shown). Similar Α β deposit binding (B) was detected after hippocampal injection (4 hours post-injection) of FC5(H3) -hFc-ABP construct into AD transgenic mice (using ABP-specific antibodies).
FIG. 21: target binding of FC5(H3) -hFc1X7-ABP (ABP, SEQ ID NO:32) in vivo is shown. Figure 21A shows in vivo binding of Alexa 647-labeled FC5-hFc-ABP construct to native amyloid beta (Α β) deposits in AD transgenic mice following in vivo injection into hippocampus. The identity of a β was confirmed by detecting brain sections with Alexa 488-labeled a β -specific antibody 6E10 and confirming co-localization (pooling) of the two signals. Fig. 21B shows the demonstration of target binding by ELISA. After in vivo (4 hours post injection) injection of FC5(H3) -hFc-ABP construct into hippocampus of wild type and AD transgenic mice, hippocampus formation was dissected and homogenized. The FC5-ABP fusion construct was detected by a sandwich ELISA using FC5 antibody as capture antibody and ABP antibody as detection antibody. The in vivo binding of ABP to endogenous a β was detected by the same sandwich ELISA but using a β -specific antibodies as detection antibodies. As is clear from fig. 21A and 21B, the FC5-ABP construct remained intact 4 hours after injection in both wild type and AD transgenic mice. Most importantly, in Tg mice expressing human Α β, injected ABP bound to Α β and pulled down as a complex (FC5(H3) -hFc-ABP × Α β), indicating that ABP is involved in Α β -target binding in vivo.
Fig. 22A, B: a PK, PD comparison between the non-humanized and humanized FC5-FC-ABP constructs is shown. FC5-mFc2a-ABP or FC5(H3) -hFc1x7-ABP was administered intravenously into rats by tail vein injection at a dose of 15mg/kg as described in figure 5. Serum and CSF were collected continuously. FC5-Fc-ABP levels were quantified using the nanolC-MRM method. As shown in figure 22A, serum and CSF PK profiles were very similar for the non-humanized and humanized constructs. As described in fig. 9B, FC5-mFc2a-ABP or FC5(H3) -hFc1x7-ABP were administered intravenously to Tg mice by tail vein injection at 15 mg/kg. FC5-Fc-ABP and A β levels in CSF were measured by nanolC-MRM as described in FIG. 9B. As shown in fig. 22B, the levels of non-humanized and humanized FC5-FC-ABP in CSF were similar, and most importantly, the changes (decreases) in CSF Α β levels were also very similar, indicating that humanization of the FC5-FC-ABP construct did not affect the PK and PD profiles of the fusion construct.
Fig. 23A, B, C: cation Exchange (CEX) chromatograms of FC5(H3) -hFc1x7-ABP (SEQ ID NO:56) generated in a stable CHO cell line are shown, indicating that various forms of FC5(H3) -hFc1x7-ABP (ABP variant) were generated during manufacture. Detailed Western blot analysis (FIG. 23A) and mass spectrometry analysis (FIG. 23B) with anti-hFc antibodies showed that during production, different forms of fusion proteins (as shown in SEQ ID NO:37 to SEQ ID NO:43 of SEQ ID NO:36) were produced due to C-terminal cleavage of the ABP peptide at different positions, producing bifunctional dimers, i.e., bifunctional homodimers and heterodimers for ABP functionality (both ABP arms functional) and heterodimers of monofunctional fusion proteins (one functional ABP arm and the other non-functional arm), as shown in FIGS. 1Ai, 1Aii and 1 Aiii. Representative profile data of the CEX portion of the FC5(H3) -hFc1x7-L-ABP (6G) (SEQ ID NO:56) dimer produced in CHO cells showed the presence of intact and C-terminally cleaved ABP (FIG. 23B). Homodimer of SEQ ID NO:56 including ABP SEQ ID NO:36 (full chain dimer peak), heterodimer of ABP variants including C-terminal cleavage [ SEQ ID NO:57(NI, Asn-Ile peak); the SEQ ID NO:60(RVKNI peak) and the non-functional ABP (ASAQ/ASLA peak) are shown (FIG. 23B). The precise nature of the cleavage product of the fusion protein (SEQ ID NO:56) was unknown and unobvious until a detailed analysis was performed. Further shown is the β -amyloid binding activity of the CEX fraction containing a mixture of homodimers (bifunctional) and heterodimers (mono-and bifunctional) (peak 2) (western blot a β overlay and ELISA analysis, EC 50), fig. 23A, (representing approximately 25% of total protein), very similar to the bifunctional homodimers and heterodimers (peak 3) (representing approximately 65% of total protein). This strongly suggests that heterodimeric and homodimeric moieties (the central and main peaks of CEX) can be used in combination without significantly reducing the binding activity of β -amyloid. This advantageously will substantially increase the yield of FC5(H3) -hFc1x7-ABP fusion protein during large scale biological manufacturing, particularly during downstream processing of the fusion protein product. This is demonstrated in fig. 23C, which shows that modified CEX conditions produce fractions consisting of homodimers and heterodimers (monofunctional and bifunctional) with high yields (more than 85% of total protein applied on the column). The beta-amyloid binding activity (EC 50) of this combinatorial library (sections B3 to B6) is very similar to other CEX moieties comprising mainly homodimeric (C10-D2, E12) or heterodimeric forms (C6-C8).
FIG. 24: brain delivery of FC5(H3) -hFc1x7-ABP fusion protein (a mixture of homodimers and heterodimers) in vivo following intravenous injection (tail vein) of wild type (wt) and Tg mice as assessed by sandwich ELISA (24A) and western blot analysis (24B) is shown (see detailed information in fig. 8 and 19), which is very similar to brain delivery of the major homodimer form of FC5(H3) -hFc1x7-ABP (SEQ ID NO:56, see fig. 19), confirming that the heterodimer form of FC5(H3) -hFc1x7-ABP in the mixture does not affect brain delivery of the fusion protein across the blood brain barrier in vivo.
FIG. 25: CSF exposure of FC5(H3) -hFc1x7-ABP fusion protein (mixture of homodimers and heterodimers) and its effect on CSF beta-amyloid following intravenous injection into Tg mice is shown (see fig. 22 and 24). As shown in FIG. 25, the CSF exposure and change (decrease) in CSF Abeta levels of FC5(H3) -hFc1x7-ABP were very similar to those seen with the main homodimeric form of FC5(H3) -hFc1x7-ABP (SEQ ID NO:56 see FIG. 22), further confirming that FC5(H3) -hFc1x7-ABP in the mixture did not affect the functional effects of the FC5(H3) -hFc1x7-ABP fusion protein in vivo.
Detailed Description
The present invention provides polypeptides, fusion proteins comprising the polypeptides, and fusion proteins comprising the polypeptides and antibodies or fragments thereof that migrate across the blood-brain barrier.
The present invention provides peptides that bind beta-amyloid (beta-amyloid). Peptides (or proteins) that bind beta-amyloid protein can selectively bind pathologically related beta-amyloid protein1-42(Aβ1-42) Aggregates, and may be abbreviated and referred to herein as ABP or ABP variants (or collectively ABP), or C-terminally cleaved ABP products.
The present invention provides fusion proteins comprising ABP (ABP variants and C-terminal cleavage products thereof) attached to an antibody or fragment thereof that crosses the blood-brain barrier. In one embodiment, the fusion protein comprising ABP and BBB further comprises Fc or a fragment thereof, wherein the ABP and BBB components of the fusion protein can be linked through the Fc region or a portion thereof to form a single chain polypeptide (herein abbreviated as BBB-Fc-ABP or BBB-Fc-L-ABP). For example, the constructs of the invention may comprise BBB-Fc-ABP or BBB-Fc-L-ABP, wherein L may be any suitable linker. The provided BBB-Fc-ABP (also abbreviated BBB-Fc-L-ABP) constructs may be single chain polypeptides or dimeric polypeptides thereof, wherein the single chain polypeptides comprising BBB-Fc-ABP may form multimers (preferably dimers) by the component Fc regions, which may be homodimers or heterodimers with respect to ABP in the fusion protein, and may be monofunctional or bifunctional with respect to heterodimers having one functional ABP arm or two different functional ABP arms, respectively.
The present invention relates to compounds that migrate across the blood brain barrier, and uses thereof. More specifically, the present invention relates to compounds comprising BBB and ABP and their use in the treatment of Alzheimer's Disease (AD).
There is a need for therapeutic agents that can effectively transport ABP across the blood brain barrier and clear a β through binding of ABP. In the prior art, a 40 amino acid a β binding peptide (ABP) was identified that selectively binds to a β involved in the development of AD1-42Oligomers (WO 2006/133566). The A beta binding peptide inhibits the binding of A beta to cell protein in vitro and inhibits the A beta1-42Induced cytotoxicity (Chakravarthy et al, 2013). The Abeta binding peptide can bind to amyloid deposits in the brain of AD transgenic mice, and can also bind to amyloid deposits in the brain of AD patients in vitro. More importantly, ABPs target native amyloid deposits in vivo when injected directly into the brain of live AD transgenic mice (Chakravarthy et al, 2014). Thus, human ABP can potentially target CNS a β, helping to clear a β from the brain and reduce its toxic effects. However, systemically administered ABP has a limited ability to cross the BBB itself and enter the brain parenchyma.
Thus, although ABP has been shown to bind to a β deposits when administered directly, parenteral injection of ABP requires penetration of the blood brain barrier in order to bind and clear a β from the brain. The present invention advantageously provides ABPs fused via Fc fragments to BBB-penetrating single domain antibodies such as Fc5 or IGF1R to provide bispecific blood brain barrier penetrating therapeutics (Farrington et al, 2014). The BBB-Fc-ABP constructs of the invention can dimerize, i.e., the BBB-Fc-ABP single chain fusion protein can form a dimer of two single chain fusion proteins to produce a dimeric compound, wherein each single chain of the dimer comprises a BBB, an Fc fragment, and an ABP to provide a BBB-Fc-ABP dimer. The BBB-Fc-ABP or BBB-Fc-L-ABP constructs and their dimers enable efficient ABP migration across the blood brain barrier. Thus, in the constructs and methods of the invention, therapeutic clearance of a β via binding of CSF and ABP in brain parenchyma is provided to be advantageous.
To enable brain delivery of ABP and improve its effect, ABP peptides are currently fused to the C-terminus of an Fc fragment, while blood brain barrier penetrating single domain antibodies such as Fc5(WO2002/057445) are fused to the N-terminus of the same Fc fragment to produce a bispecific blood brain barrier penetrating therapeutic (Farrington et al 2014). In non-limiting embodiments of the invention, the Fc fragment can be mouse (SEQ ID NO:48) or human (SEQ ID NO: 49; SEQ ID NO: 50). In one embodiment, the Fc fragment of the invention is designed to reduce effector function (Shields et al, 2001). For example, the Fc fragment can be hFc1x7(SEQ ID NO:49), wherein the Fc fragment in the BBB-Fc-ABP fusion protein advantageously enables dimerization of the fusion protein to produce a therapeutically effective fusion molecule (BBB-Fc-ABP dimer) that is capable of migrating across the blood-brain barrier. In one embodiment, the BBB-Fc-ABP fusion protein may be FC5-H3-hFc1x7-ABP (6G) (SEQ ID NO:56) and its dimer, or a functionally equivalent fusion protein of SEQ ID NO:56, i.e., SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 that comprises an ABP product capable of binding the C-terminal cleavage of beta-amyloid. The fusion protein may comprise the linker sequence L as embodied in SEQ ID NO:56 or SEQ ID NO:71, or any suitable linker sequence.
The present invention provides isolated peptides that bind beta-amyloid (beta-amyloid). The peptides of the invention may comprise or consist of a sequence.
X1TFX2TX3X4ASAQASLASKDKTPKSKSKKX5X6STQLX7SX8VX9NI(SEQ ID NO:31；40aa)
Wherein, X1K, G or A, X1G or A, X2K, G or V, X3＝R、G or A, X4K, G or A, X5R, G or V, X6N, G or V, X7K, G or V, X8R, G or A, X9Either K, G or a, or any C-terminally cleaved β -amyloid binding product thereof.
The present invention provides C-terminal cleavage products of SEQ ID NO:36(40aa), i.e., SEQ ID NO:37(38aa) to SEQ ID NO:43(32 aa). The present invention provides specific C-terminal deletions and mixtures comprising these C-terminal deletions. Specifically,
The present invention provides a beta-amyloid binding peptide comprising or consisting of SEQ ID NO 46(29 aa).
The ABP peptide (ABP, ABP variant or C-terminal cleavage product thereof) of the invention may be selected from the group consisting of: 27 to 36 or a sequence substantially identical thereto, or a C-terminally cleaved ABP peptide capable of binding beta-amyloid, such as
The present invention provides a compound, a fusion protein, comprising an antibody or fragment thereof that migrates across the Blood Brain Barrier (BBB) and a polypeptide that binds beta-amyloid. The present invention provides fusion proteins comprising a BBB, a polypeptide that binds to beta-amyloid, and an Fc.
The term "antibody" is also known in the art as an "immunoglobulin" (Ig), and as used herein refers to a protein composed of paired heavy and light polypeptide chains; there are various Ig isotypes, including IgA, IgD, IgE, IgG, and IgM. When the antibody is correctly folded, each chain folds into many different globular domains, and is linked together by a more linear polypeptide sequence. For example, immunoglobulin light chain folding is variable (V)L) And constant (C)L) Domain, and heavy chain folded variable (V)H) And three constants (C)H、CH2、CH3) A domain. Heavy chain variable region and light chain variable region (V)HAnd VL) The interaction of (a) results in the formation of an antigen binding region (Fv). Each domain has an established structure familiar to those skilled in the art.
The light chain variable region and the heavy chain variable region are responsible for binding to the target antigen and thus may show significant sequence diversity between antibodies. The constant region displays less sequence diversity and is responsible for binding to many native proteins to trigger important biochemical events. The variable region of an antibody comprises the antigen binding determinants of the molecule and thus determines the antibody's targetSpecificity of the target antigen. Most of the sequence variability occurs in the 6 hypervariable regions, the variable heavy chain (V)H) And light chain (V)L) 3 of each; the hypervariable regions combine to form an antigen-binding site and facilitate the binding and recognition of antigenic determinants. The specificity and affinity of an antibody for its antigen depends on the structure of the hypervariable regions, as well as the size, shape and chemical composition of the surface on which they are presented to the antigen. There are various approaches to identifying regions of high variability, the two most common being Kabat, Chothia and Lesk. Kabat et al (1991) according to VHAnd VLThe sequence variability of the antigen-binding regions of the domains defines the "complementarity determining regions" (CDRs). Chothia and Lesk (1987) according to VHAnd VLThe position of the structural loop region in the structure defines the "hypervariable loop" (H or L). These separate schemes define adjacent or overlapping CDR and hypervariable loop regions, which terms are often used interchangeably by those skilled in the art of antibodies and may be used as such herein. Herein CDRs/loops are identified according to the Kabat scheme.
The "antibody fragment" referred to herein may include any suitable antigen-binding antibody fragment known in the art. Antibody fragments may be naturally occurring antibody fragments, or may be obtained by manipulation of naturally occurring antibodies or using recombinant methods. For example, antibody fragments may include, but are not limited to, Fv, single chain Fv (scFv; V linked by a peptide linker)LAnd VHComposed molecule), Fab, F (ab')2Single domain antibodies (sdAb; consisting of a single V)LOr VHFragments of composition), as well as multivalent presentations of any of these. Antibody fragments such as those just described may require linker sequences, disulfide bonds, or other types of covalent bonds to join the different portions of the fragment; those skilled in the art will be familiar with the requirements of different types of fragments and various methods and the various methods for constructing them.
In a non-limiting example, an antibody fragment can be an sdAb derived from a naturally-occurring source. Camel-derived heavy chain antibodies (Hamers-Casterman et al, 1993) lack a light chain and therefore their antigen binding site consists of one domain, designated VHH. In thatThe sdAb was also observed in sharks and designated as VNAR(Nutall et al, 2003). Other sdabs can be designed based on human Ig heavy and light chain sequences (jesperss et al, 2004; To et al, 2005). As used herein, the term "sdAb" includes V from any source by phage display or other techniquesH、VHH、VLOr VNARSdabs isolated directly from libraries, sdabs derived from the aforementioned sdabs, recombinantly produced sdabs, and sdabs produced by further modifying such sdabs by humanization, affinity maturation, stabilization, solubilization, camelization, or other antibody engineering methods. The invention also includes homologs, derivatives or fragments that retain the antigen binding function and specificity of the sdabs.
SdAb has desirable antibody molecular properties, such as high thermal stability, high lotion resistance, relatively high protease resistance (Dumoulin et al, 2002) and high yield (Arbabi-Ghahroudi et al, 1997); it can also be made to have very high affinity by isolation from immune pools (Li et al, 2009) or by in vitro affinity maturation (Davies & Riechmann, 1996). Further modifications to sdabs, such as the introduction of atypical disulfide bonds, may also increase stability (Hussack et al, 2011a, b; Kim et al, 2012).
The structure of single domain antibodies will be well known to those skilled in the art (see, e.g., 3DWT, 2P42 in protein databases). The sdAb comprises a single immunoglobulin domain that retains the immunoglobulin fold; most notably, only three CDRs/hypervariable loops form the antigen binding site. However, it will be appreciated by those skilled in the art that not all CDRs need to bind antigen. For example, and without intending to be limiting, one, two, or three of the CDRs can contribute to binding and recognition of an antigen by an sdAb of the invention. The CDRs of the SdAb or variable domain are referred to herein as
The antibodies or fragments thereof described herein can migrate across the blood brain barrier. The brain is separated from other parts of the body by a special endothelial tissue called the Blood Brain Barrier (BBB). The endothelial cells of the BBB are linked together by tight junctions and effectively prevent many therapeutic compounds from entering the brain. In addition to the low rate of vesicle trafficking, a particular feature of the BBB is the presence of an enzymatic barrier on the open (brain) side of the blood brain barrier and high levels of ATP-dependent transporter expression, including P-glycoprotein (Gottesman and Pastani, 1993; Watanabe, 1995), which actively transports various molecules from the brain into the bloodstream (Samuels, 1993). Only small molecules (<500 daltons) and hydrophobic molecules (Pardridge, 1995) are able to cross the blood-brain barrier more easily. Thus, the ability of an antibody or fragment thereof as described above to specifically bind to a surface receptor, internalize into brain endothelial cells, and undergo transcellular action across the blood brain barrier by avoiding lysosomal degradation is useful in the neurological field. Antibodies or fragments thereof that cross the blood brain barrier can be used to carry other molecules, such as therapeutic drugs, for delivery to brain tissue. The antibody or fragment thereof may be any suitable antibody or fragment thereof known in the art to migrate across the blood brain barrier.
The present invention provides a compound, or fusion protein, comprising an antibody or fragment thereof that migrates across the Blood Brain Barrier (BBB). The antibodies or fragments of the invention may bind to, for example, transmembrane protein 30A (TMEM30A) (as described in WO 2007/036021), or to an insulin-
The antibody or fragment thereof of the composition of the invention may include Complementarity Determining Region (CDR)1 sequence GFKITHYTMG (SEQ ID NO:1), CDR2 sequence RITWGGDNTFYSNSVKG (SEQ ID NO:2), and CDR3 sequence GSTSTATPLRVDY (SEQ ID NO: 3); or
CDR1 sequence EYPSNFYA (SEQ ID NO:4), CDR2 sequence VSRDGLTT (SEQ ID NO:5), CDR3 sequence AIVITGVWNKVDVNSRSYHY (SEQ ID NO: 6); or
CDR1 sequence GGTVSPTA (SEQ ID NO:7), CDR2 sequence ITWSRGTT (SEQ ID NO:8), CDR3 sequence AASTFLRILPEESAYTY (SEQ ID NO: 9); or
CDR1 sequence GRTIDNYA (SEQ ID NO:10), CDR2 sequence IDWGDGGX (SEQ ID NO: 11); wherein X is A or T, CDR3 sequence AMARQSRVNLDVARYDY (SEQ ID NO: 12).
As mentioned previously, the antibody or fragment thereof may be a camelid-derived sdAbOr from camel VHSdabs of H, and thus can be based on camelid framework regions; alternatively, the above CDRs may be grafted to VNAR、VHH、VHOr VLOn the frame area. In yet another alternative, the hypervariable loops described above can be grafted onto the framework regions of other types of antibody fragments (Fv, ScFv, Fab) of any origin (e.g., mouse or human), or the CDRs can be grafted onto proteins of similar size and nature (see, e.g., Nicaise et al, 2004).
The invention further includes chimeric (or chimerized), veneered (veneered) or humanized antibodies or fragments thereof. Chimeric antibodies or fragments thereof are constructs of naturally variable domains (of mouse or camelid origin) linked to human constant domains (see Gonzales et al 2005). Antibody veneering or refinishing involves the replacement of exposed residues in the framework regions of the native antibody or fragment thereof with amino acid residues in its human counterpart (Padlan, 1991; Gonzales et al, 2005). Humanization of an antibody or antibody fragment involves the replacement of amino acids in the sequence with human counterparts found in human common sequences without loss of antigen binding ability or specificity; this method reduces the immunogenicity of the antibody or fragment thereof when introduced into a human subject. In this process, one or more of the CDRs defined herein may be fused or grafted to a human variable region (V)HOr VL) Other human antibodies (IgA, IgD, IgE, IgG and IgM), human antibody fragment framework regions (Fv, scFv, Fab), or CDRs can be grafted onto human proteins of similar size and nature (Nicaise et al, 2004). In this case, the conformation of the one or more hypervariable loops may be preserved and the affinity and specificity of the sdAb for its target (i.e., an epitope on brain endothelial cells such as TMEM30A, or IGF1R epitope) may be minimally affected. As is well known to those skilled in the art, certain natural amino acid residues may need to be incorporated into the human framework to maintain binding and specificity. Humanization by CDR grafting is well known in the art (see, e.g., Tsurushita et al, 2005; Jones et al, 1986; Tempest et al, 1991; Riechm)ann et al, 1988; queen et al, 1989; gonzales et al, for review in 2005-see also the references cited therein) and thus the person skilled in the art will be very familiar with the methods for preparing such humanized antibodies or fragments thereof.
The antibody or fragment thereof provided may be a humanized version of the FC5 antibody (described in WO2002/057445) or the IGF1R antibody. FC5 (comprising 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 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 epitopes on the receptor surface on brain endothelial cells and subsequently migrate across the Blood Brain Barrier (BBB). FC5 has also been shown to act as a vector to introduce molecules of various sizes across the BBB (see, e.g., WO 2011/127580). Antigens mediating FC5 migration were transiently identified as transmembrane domain protein 30A (TMEM 30A; WO 2007/036021), which is enriched on the surface of brain endothelial cells.
For example, and without intending to be limiting, an antibody or fragment thereof may comprise the sequence:
X1VQLVX2SGGGLVQPGGSLRLSCAASGFKITHYTMGWX3RQAPGKX4X5EX6VSRITWGGDNTFYSNSVKGRFTISRDNSKNTX7YLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWGQGTLVTVSS (SEQ ID NO:13), wherein X1Either D or E, X2Is A or E, X3Is F or V, X4Either E or G, X5R or L, X6F or W, X7L or V, or a sequence substantially identical thereto;
X1VX2LX3ESGGGLVQX4GGSLRLSCX5ASEYPSNFYAMSWX6RQAPGKX7X8EX9VX10GVSRDGLTTLYADSVKGRFTX11SRDNX12KNTX13X14LQMNSX15X16AEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTX17VTVSS (SEQ ID NO:18), where X1Is E or Q; x2Is K or Q; x3Is V or E; x4Is A or P; x5Is V or A; x6Is F or V; x7Is E or G; x8Is R or L; x9Is F or W; x10Is A or S; x11Is M or I; x12Is A or S; x13Is V or L; x14Is D or Y; x15Is V or L; x16Is K or R; and X17Is Q or L, or a sequence substantially identical thereto;
X1VX2LX3ESGGGLVQX4GGSLRLSCX5X6SGGTVSPTAMGWX7RQAPGKX8X9EX10VX11HITWSRGTTRX12ASSVKX13RFTISRDX14X15KNTX16YLQMNSLX17X18EDTAVYYCAASTFLRILPEESAYTYWGQGTX19VTVSS (SEQ ID NO:21), wherein X1Is E or Q; x2Is K or Q; x3Is V or E; x4Is A or P; x5Is A or E; x6Is V or A; x7Is V or F; x8Is G or E; x9Is L or R; x10Is F or W; x11Is G or S; x12Is V or Y; x13Is D or G; x14Is N or S; x15Is A or S; x16Is L or V; x17Is K or R; x18Is A or S; and X19Is L or Q, or a sequence substantially identical thereto;
X1VX2LX3ESGGGLVQX4GGSLRLSCAASGRTIDNYAMAWX5RQAPGKX6X7EX8VX9TIDWGDGGX10RYANSVKGRFTISRDNX11KX12TX13YLQMNX14LX15X16EDTAVYX17CAMARQSRVNLDVARYDYWGQGTX18VTVSS (SEQ ID NO:24), where X1Is E or Q; x2Is K or Q; x3Is V or E; x4Is A or P; x5Is V or S; x6Is D or G; x7Is L or R; x8Is F or W; x9Is A or S; x10Is A or T; x11Is A or S; x12Is G or N; x13Is M or L; x14Is N or R; x15Is E or R; x16Is P or A; x17Is S or Y; and X18Is Q or L, or a sequence substantially identical thereto.
More specifically, without wishing to be limited in any way, the antibody or fragment thereof may comprise a sequence selected from any one of the following sequences:
DVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAGSTSTATPLRVDYWG KGTQVTVSS(SEQ ID NO:14)；
EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWVRQAPGKGLEWVSRITWGGDNTFYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG QGTLVTVSS(SEQ ID NO:15)；
EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWVRQAPGKGLEWVSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG QGTLVTVSS(SEQ ID NO:16)；
EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEFVSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG QGTLVTVSS(SEQ ID NO:17)；
QVKLEESGGGLVQAGGSLRLSCVASEYPSNFYAMSWFRQAPGKEREFVAGVSRDGLTTLYADSVKGRFTMSRDNAKNTVDLQMNSVKAEDTAVYYCAIVITGVWNKVDVNSRS YHYWGQGTQVTVSS(SEQ ID NO:19)；
EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKEREFVSGVSRDGLTTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSY HYWGQGTLVTVSS(SEQ ID NO:20)；
QVKLEESGGGLVQAGGSLRLSCEVSGGTVSPTAMGWFRQAPGKEREFVGHITWSRGTTRVASSVKDRFTISRDSAKNTVYLQMNSLKSEDTAVYYCAASTFLRILPEESAYTY WGQGTQVTVSS(SEQ ID NO:22)；
QVQLVESGGGLVQPGGSLRLSCAVSGGTVSPTAMGWFRQAPGKGLEFVGHITWSRGTTRYASSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAASTFLRILPEESAYTY WGQGTLVTVSS(SEQ ID NO:23)；
QVKLEESGGGLVQAGGSLRLSCAASGRTIDNYAMAWSRQAPGKDREFVATIDWGDGGARYANSVKGRFTISRDNAKGTMYLQMNNLEPEDTAVYSCAMARQSRVNLDVARYD YWGQGTQVTVSS(SEQ ID NO:25)；
QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPGKGLEWVATIDWGDGGTRYANSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCAMARQSRVNLDVARY DYWGQGTLVTVSS (SEQ ID NO: 26); and
a sequence substantially identical thereto. The antibody or fragment thereof may be a single domain antibody.
With respect to any constituent peptide in the fusion protein of the invention, a "substantially identical" sequence may include one or more conservative amino acid mutations, or amino acid deletions, to enable retention of biological functional activity (such as in the C-terminally cleaved ABP peptides provided herein). It is known in the art that one or more conservative amino acid mutations to a reference sequence can result in a mutant peptide that has no substantial change in a physiological, chemical, physicochemical, or functional property as compared to the reference sequence; in this case, the reference sequence and the mutant sequence will be considered to be "substantially identical" polypeptides. A conservative amino acid substitution is defined herein as the replacement of one amino acid residue with another amino acid residue having similar chemical properties (e.g., size, charge, or polarity). These conservative amino acid mutations can be made to the framework regions of the sdabs while maintaining the CDR sequences listed above and the overall structure of the CDRs of the antibody or fragment, thereby maintaining the specificity and binding of the antibody.
With respect to any constituent peptide in the fusion protein of the invention, a "substantially equivalent" sequence may include one or more conservative amino acid mutations or amino acid deletions to enable retention of a biologically functional activity (e.g., in a C-terminally cleaved ABP peptide provided herein); wherein the mutant peptide is substantially equivalent with respect to peptide stability and biological manufacturability. Substantial equivalence may refer to equivalence with respect to the stability of the fusion molecule; for example, degradation products or low molecular weight bands are absent 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 can result in a mutant peptide that has no substantial change in a physiological, chemical, physicochemical, or functional property as compared to the reference sequence; in this case, the reference sequence and the mutant sequence will be considered to be "substantially equivalent" polypeptides.
In a non-limiting example, a conservative mutation may be an amino acid substitution or deletion that maintains functionality. Such conservative amino acid substitutions may replace another amino acid of the same group with a basic, neutral, hydrophobic, or acidic amino acid. The term "basic amino acid" refers to a hydrophilic amino acid with a side chain pk value greater than 7, which is generally positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). The term "neutral amino acid" (also referred to as "polar amino acid") refers to a hydrophilic amino acid with a side chain that is uncharged at physiological pH, but has at least one bond in which the electron pair common to two atoms is held closer 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 referred to as "non-polar amino acid") refers to an amino acid that exhibits a hydrophobicity greater than zero according to the standardized common hydrophobicity scale (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). The term "acidic amino acid" refers to a hydrophilic amino acid having a side chain pk value of less than 7, which is generally negatively charged at physiological pH. Acidic amino acids include glutamic acid (Glu or E), and aspartic acid (Asp or D).
Sequence identity is used to assess the similarity of two sequences; it is determined by calculating the percentage of residues that are identical when the two sequences for maximum correspondence between residue positions are aligned. Sequence identity can be calculated using any known method; for example, computer software can be used to calculate sequence identity. Without wishing to be limiting, sequence identity may be calculated by software such as the NCBI BLAST2 service maintained by Swiss bioinformatics research (as found in ca. expasy. org/tools/BLAST /), BLAST-P, BLAST-N or FASTA-N, or any other suitable software known in the art.
Substantially identical sequences of the invention may be at least 74% identical; in another example, a substantially identical sequence may be at least 74, 75, 76, 77, 78, 79, 80-90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical, or any percentage therebetween, at the amino acid level to a sequence described herein. Importantly, substantially identical sequences retain the activity and specificity of the reference sequence. In non-limiting embodiments, the difference in sequence identity may be due to conservative amino acid mutations. In non-limiting examples, the invention can be directed to an antibody or fragment thereof comprising a sequence at least 95%, 98%, or 99% identical to the sequence of an antibody described herein. In another non-limiting example, as provided herein, an ABP peptide of the invention can include a sequence at least 74% identical to SEQ ID NO:36(40 aa); that is, the invention encompasses ABP peptides that are C-terminal cleavage products of SEQ ID NO:36, and can be at least 74%, 75%, 76%, 77%, 78% -100% identical thereto, or any percentage therebetween. It should be noted that the percent homology is calculated based on each constituent peptide with respect to the reference peptide sequence (i.e., comparing ABP constituent peptide sequences). The ABP of the invention comprises the sequence of SEQ ID NO:46(29aa), or its equivalent amyloid-beta binding protein.
The antibody or fragment thereof in the compounds of the invention may be linked to, for example, an Fc domain, but is not limited to a human Fc domain. The Fc domain may be selected from various classes, including but not limited to IgG, IgM, or various subclasses, including but not limited to IgG1, IgG2, and the like. In this approach, the Fc gene was inserted into a vector along with the sdAb gene, producing an sdAb-Fc fusion protein (Bell et al, 2010; Iqbal et al, 2010); the fusion protein is expressed recombinantly and then purified. For example, and without wishing to be limited in any way, multivalent display formats may include chimeric versions of FC5-H3 and mutant variants thereof linked to an FC domain. Such antibodies are easy to design and produce and can greatly extend the serum half-life of sdabs (Bell et al, 2010).
The Fc domain in the compounds 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 murine or human origin. Other embodiments of the Fc or Fc fragment may modulate, modify or inhibit immune effector function (Shields, 2001). Other embodiments of Fc fragments may mediate clearance of the fusion peptide from the brain (Caram-Salas N, 2011). In a specific non-limiting example, the Fc can be a mouse Fc2a fragment or a human Fc1 fragment (Bell et al, 2010; Iqbal et al, 2010). In particular non-limiting examples, multimeric constructs may include isolated or purified antibodies or fragments as described herein and antibodies having the sequence of SEQ ID No. 48; 49 of SEQ ID NO; fc of
The compounds of the invention include antibodies or fragments thereof linked to a polypeptide that binds to beta-amyloid. The linker may be any polypeptide of suitable length including neutral or hydrophilic amino acids. In a non-limiting example, preferably less than 12 amino acids in length, and the polypeptide is GGGGSGGGGS or TGGGGSGGGGS or any suitable linker (e.g., (GGGSGGGGS)nOr (GGGGSGGG)nOr any suitable combination). Other chemical linkers may take the form of properly oriented non-peptides, such as amide or ester linkages. For example, SEQ ID NO:71 provides a fusion protein comprising any suitable linker, wherein any of the fusion proteins of the present invention (i.e., SEQ ID NOS: 51-70) can comprise any suitable linker as shown in SEQ ID NO: 71. Polypeptides that bind beta-amyloid (A β) may be associated with pathologically relevant beta-amyloid (such as A β associated with AD pathology1-42Aggregate) bonding; the polypeptides may bind to A.beta.with high affinity (in the nM range), inhibit binding of A.beta.to cellular proteins and A.beta.in vitro1-42Induced cytotoxicity and binding of amyloid deposits in AD transgenic mouse brains and AD patients' brains in vitro. The polypeptides in the compounds of the invention do not interact with the inverse peptide Abeta42-1And (4) combining.
In the compounds provided herein, the polypeptide that binds to β -amyloid may comprise a sequence selected from the group consisting of: 27, SEQ ID NO; 28 in SEQ ID NO; 29 in SEQ ID NO; 30 in SEQ ID NO; 31, SEQ ID NO; 32 is SEQ ID NO; 33, SEQ ID NO; 34 in SEQ ID NO; 35 in SEQ ID NO; SEQ ID NO:36 or its beta-amyloid binding C-terminal cleavage product (i.e., SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO: 43; or SEQ ID NO: 44; SEQ ID NO: 45; SEQ ID NO: 46; SEQ ID NO:47 and sequences substantially identical thereto). Sequences that are "substantially identical" are as described above.
Accordingly, the present invention provides a polypeptide that binds to beta-amyloid and variants thereof (i.e., ABP and ABP variants). The ABP variant may comprise, for example, a sequence having SEQ ID No. 31, and the ABP variants provided may comprise a sequence selected from the group consisting of the sequences of SEQ ID nos. 31 to 47 or substantially equivalent sequences thereof.
Thus, ABP polypeptide sequences that include specific system and method modifications based on detailed biophysical characteristics of ABP are provided. Specific and methodical targeted modifications to ABP polypeptides include the novel and nonobvious ABP variants of the invention, or C-terminal cleavage products with beta-amyloid binding activity, as provided in SEQ ID NO: 31.
The peptides provided herein can include ABP comprising a sequence selected from the group consisting of SEQ ID NO:27-SEQ ID NO:47, and a sequence substantially equivalent thereto or a C-terminal cleavage product having beta-amyloid binding activity.
The fusion proteins provided herein can include an ABP comprising a sequence that can be selected from any one of
More specifically, the particular ABP cleavage species provided herein (i.e., SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43) cannot be predicted, and the particular cleavage site that is now cleaved from SEQ ID NO:36 is unknown and cannot be predicted either. These specific C-terminal cleavage products are unpredictable during the biological manufacturing of fusion peptides. The stability and biological manufacturability of ABP proteins and their C-terminal cleavage products are unpredictable.
Furthermore, it is quite advantageous and unexpected that the fusion proteins provided herein can be bifunctional dimers or monofunctional dimers, wherein the monofunctional and bifunctional dimers provide equivalent fusion proteins with respect to β -amyloid binding functionality and activity. The advantageous stability, manufacturability and equivalent beta-amyloid binding functionality of the provided C-terminal cleavage products are unknown and unobvious. This enables the production of mixtures of ABP peptides with equivalent β -amyloid binding activity, as well as the production of mixtures of fusion proteins and their dimers with equivalent β -amyloid binding activity. Furthermore, the advantageous equivalent functionalities of monofunctional and bifunctional dimers are unknown and unpredictable. Advantageously, the present invention provides a mixture of fusion proteins and dimers (mono-functional and bi-functional dimers comprising the minimal ABP sequence (which includes SEQ ID NO: 46)) with equivalent activity, thereby enabling increased yields of functional ABP protein and increased yields of functional fusion proteins and functional fusion protein dimers in biological manufacturing.
The fusion proteins and compounds provided herein exhibit improved therapeutic efficacy. More specifically, the compounds provided include specifically modified ABPs, including, for example, mutations at specific amino acids K (lysine), R (arginine), and N (aspartic acid) to G (glycine) at
The term "linked", also referred to herein as "coupled", refers to the linkage of two moieties, either directly or indirectly (e.g., through a linker), covalently or noncovalently (e.g., adsorption, ionic interaction). Covalent bonds can be achieved by chemical cross-linking reactions or by fusion using recombinant DNA methods in conjunction with any peptide expression system, such as bacterial, yeast or mammalian cell based systems. When the antibody or fragment thereof is conjugated to a polypeptide that binds to a β or Fc, a suitable linker may be used. For example, a suitable linker may be any polypeptide of suitable length comprising neutral or hydrophilic amino acids that enables coupling of the components of the BBB-Fc-ABP protein fusion. For example, the linker enables the components of the fusion protein (e.g., in SEQ ID NOS: 51-69) to be linked and is not limited to GGGGSGGGGS or (GGGS) highlighted thereinnLinker, and can be any suitable linker (i.e., as in the non-limiting fusion protein of SEQ ID NO: 71). In a non-limiting example, the length is preferably less than 12 amino acids, and the polypeptide may be GGGGSGGGS, or GGGSGGGGS, or TGGGGGSGGGS, or any suitable linker in the art. Other chemical linkers may take the form of properly oriented non-peptides, such as amide or ester linkages. Linkers or methods for attaching antibodies or fragments thereof to polypeptides will be apparent to those skilled in the art. Methods for attaching antibodies or fragments thereof to polypeptides or Fc are well known to those skilled in the art.
The compounds provided herein include linked antibodies or fragments thereof, polypeptides that bind beta-amyloid, and Fc fragments to provide constructs (also referred to herein as compounds or fusion molecules), wherein the constructs include fusion proteins and dimers thereof. The antibody or fragment thereof may be linked to the polypeptide that binds to beta-amyloid via an Fc fragment, or a suitable linker.
The antibody or fragment thereof comprises a sequence selected from any one of the following sequences: 14, 15, 16, 17, 19, 20, 22, 23, 25, 26 and sequences substantially equivalent thereto. The antibody or fragment thereof migrates across the blood brain barrier. The antibody or fragment can be an sdAb; wherein the sdAb can be humanized.
The polypeptide that binds to beta-amyloid protein comprises a sequence selected from the group consisting of:
The Fc fragment comprises a sequence selected from any one of seq id nos: SEQ ID NO 48, SEQ ID NO 49,
Thus, the compound or construct of the invention may comprise a sequence selected from the group consisting of: SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71 and sequences substantially identical thereto or dimeric structures thereof, wherein the dimeric peptide comprises two single chain polypeptides selected from the group comprising: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 and substantially the same sequence, deletion variants, such as 47 or any sequence or non-sequence C-terminally cut ABP variants from 31, consisting of a functional product comprising 46; specifically, there is provided a C-terminal deletion of 31 consecutive amino acids, or a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 amino acids (as provided in SEQ ID NOs: 37 to 43). The dimeric single chain polypeptide may be a bifunctional homodimer (fig. 1Ai), a bifunctional heterodimer (fig. 1Aii) or a monofunctional heterodimer (fig. 1 Aiii). For example, the ABP moiety of each dimer may be the same or different; when the ABP moieties in the dimer differ, at least one ABP in the single-chain polypeptide is a biologically functional peptide (i.e., capable of binding β -amyloid).
As depicted in fig. 23, 24 and 25, a dimeric peptide comprising two BBB-Fc-L-ABP single chain polypeptides may comprise two different ABP sequences (heterodimers), wherein at least one of said ABP sequences in the heterodimers is a biologically functional ABP molecule (i.e., can bind to β -amyloid, as depicted in fig. 1Aii or 1 Aiii). Very advantageously, the dimeric peptide can accommodate a single non-functional ABP peptide (i.e., a monofunctional heterodimer as shown in fig. 1Aiii) and retain equivalent functional biological activity with respect to binding β -amyloid (fig. 23B, C). When two functional ABP peptides are provided in a dimer (bifunctional heterodimer), in a dimer comprising two different ABP sequences (i.e. heterodimers with respect to ABP), at least one ABP peptide provided in the heterodimer is a biologically functional ABP, i.e. SEQ ID NO:27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 or any combination thereof. With respect to manufacturability, isolatability, and functional product yield, it is highly advantageous that the ABP peptides in the heterodimer need not all be biologically functional β -amyloid binding proteins, and that the heterodimer with a single function (or dual function) retains the overall biological activity of the fusion protein with respect to binding β -amyloid in vitro (fig. 23) and brain uptake and efficacy in vivo (fig. 24 and 25). The amyloid binding activity (EC 50) of the portion comprising the mixture of mono-and bifunctional homodimers and heterodimers (
As shown in fig. 1, the present invention provides a fusion protein comprising a BBB-spanning single domain antibody (BBB), an Fc fragment (Fc), and an Amyloid Binding Peptide (ABP), wherein each moiety can be linked to provide a fusion molecule. For example, in a non-limiting example of the invention, the BBB may be 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 single chain fusion proteins comprising BBB-Fc-ABP. The dimer of the fusion protein may be a homodimer with respect to ABPs in that both ABP arms comprise a functional full-length (e.g., a bifunctional homodimer, e.g., SEQ ID NO:36, fig. 1Ai) and/or various C-terminally cleaved ABPs (e.g., a bifunctional heterodimer, SEQ ID NO:36 and/or SEQ ID NO:37 to SEQ ID NO:43, fig. 1Aii) or heterodimers, wherein one ABP arm is functional (ABP or C-terminally cleaved ABP) and the other arm is non-functional (i.e., a monofunctional heterodimer). As shown in fig. 1A, 1B, 1C, and 1D, 3 components (i.e., BBB, FC, and ABP) are depicted in various configurations. In a specific, non-limiting example of a compound of the invention, the polypeptide that binds to amyloid beta may include the sequence
In one embodiment, the ABP variant comprises the sequence:
GTFGTGGASAQASLASKDKTPKSKKGGSTQLKSRVKNI (SEQ ID NO:36) (referred to herein as ABP (6G)) and sequences substantially equivalent thereto, or any C-terminal cleavage product having beta-amyloid binding activity, such as SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO: 43. In one embodiment, the ABP comprises the sequence of SEQ ID NO 46.
The antibody or fragment thereof may comprise a sequence
EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEFVSRITWGGDNTFYSNSVK GRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG QGTLVTVSS (SEQ ID NO:17), referred to herein as FC 5-H3.
The antibody or fragment thereof may further include sequences of human Fc, such as
AEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:49), also referred to herein as hFC1X 7.
Without wishing to be limited in any way, the compounds of the invention may comprise the sequence:
EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEFVSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSGTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI [ SEQ ID NO:56, also referred to herein as FC5-H3-hFc1X7-ABP (6G)]. The invention also provides a fusion protein of SEQ ID NO:56 wherein the linker connecting the Fc and ABP components of the fusion protein is (GGGGS)2、(GGGS)2、T(GGGGS)2Or any suitable linker known in the art, and wherein the ABP peptide of the fusion protein of SEQ ID NO:56 may be SEQ ID NO:36, a sequence substantially equivalent thereto, or any C-terminal cleavage product with functional beta-amyloid binding activity (such as SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:47) (SEQ ID NOS: 57-63, SEQ ID NO: 65).
The table summarizes some representative BBB-Fc-ABP fusion protein constructs
Note that in the above table, the BBB-Fc-ABP fusion protein construct may also include 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 GGGGSGGGGS, GGGSGGGGS, TGGGGSGGGGS or any multiple thereof, or any suitable linker, or any linker as shown by the common linker sequence in SEQ ID NO: 53.
The compounds of the invention as provided in the fusion protein of SEQ ID NO:56, also referred to herein as FC5-H3-hFc1X7-L-ABP (6G), may include variants that include variants of each component, e.g., the ABP may be an ABP as provided in SEQ ID NO:55 (GG-G) or an ABP as provided in SEQ ID NO:36 (6G), or any C-terminal cleavage product thereof. Linker sequences provided therein (e.g., as inHighlighted in SEQ ID NO:51 to SEQ ID NO:71) may be any linker that enables the connection of the BBB-Fc-ABP fusion protein. The compounds of the invention may also include additional sequences that aid in the expression, detection, or purification of the recombinant antibody or fragment thereof. Any such sequence or tag known to those skilled in the art may be used. For example, without wishing to be limiting, the antibody or fragment thereof may include a targeting or signal sequence (e.g., without limitation, OmpA), a detection/purification tag (e.g., without limitation, c-Myc, His5Or His6) Or a combination thereof. In another example, the additional sequence may be a biotin recognition site, such as the sites described by Cronan et al (WO95/04069) or Voges et al (WO/2004/076670). It is also known to those skilled in the art that linker sequences can be used in conjunction with additional sequences or tags, or can be used as detection/purification tags.
The invention also includes nucleic acid sequences encoding the compounds herein. Considering the degeneracy of the genetic code, many nucleotide sequences will have the effect of encoding a polypeptide, as will be readily understood by the skilled artisan. The nucleic acid sequence may be codon optimized for expression in various microorganisms. The invention also includes vectors comprising the nucleic acids just described. Furthermore, the invention also encompasses cells comprising said nucleic acids and/or vectors.
The invention further includes compositions comprising one or more compounds described herein and a pharmaceutically acceptable diluent, excipient, or carrier. The composition may also include a pharmaceutically acceptable diluent, excipient or carrier. The diluent, excipient or carrier can be any suitable diluent, excipient or carrier known in the art and must be compatible with the other ingredients in the composition, the method of delivery of the composition, and not deleterious to the recipient of the composition. The composition may be in any suitable form; for example, the composition may be provided in the form of a suspension or powder (e.g., but not limited to, lyophilization or encapsulation). For example, and without intending to be limiting, when the composition is provided in the form of a suspension, the carrier may include water, physiological saline, suitable buffers, or additives to improve solubility and/or stability; the recombination is performed in a buffer of appropriate pH to produce a suspension to ensure the activity of the antibody or fragment thereof. The dry powder may also include additives to improve stability and/or carriers to increase capacity/volume; for example, but not intended to be limiting, the dry powder composition may include sucrose or trehalose. It is within the ability of those skilled in the art to prepare suitable compositions comprising the present compounds.
Also provided is a method of treating alzheimer's disease, wherein a compound or composition of the invention is administered to a subject in need thereof. Any suitable route of administration may be used, including but not limited to intravenous, intraperitoneal, parenteral, intracranial, intramuscular, subcutaneous, oral, or nasal. The optimal dosage and route of administration is generally determined experimentally.
A method of reducing toxic β -amyloid levels in cerebrospinal fluid (CSF) and cerebral parenchyma of a subject having elevated β -amyloid levels is provided. More specifically, toxic β -amyloid levels in cerebrospinal fluid (CSF) and cerebral parenchyma of a subject are reduced as early as within 24 hours of a single parenteral administration of a composition of the invention.
The invention will be further illustrated in the following examples. It should be understood, however, that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any way.
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