NL2020520B1 - Multispecific binding molecules for the prevention, treatment and diagnosis of neurodegenerative disorders - Google Patents

Abstract

The invention relates to a multispecific binding molecule, preferably an antibody, comprising at least a first binding site binding to a first target selected from the group comprising human tau protein and post-translationally modified human tau protein, such as phosphorylated, acetylated, glycosylated, glycated, prolylisomerizated, nitrated, polyaminated, ubiquitinated, sumoylated, oxidated, aggregated, cleaved human tau protein and truncated versions thereof, (1- synuclein, TDP43, mHTT, and fragments thereof and at least a second binding site binding to a second target selected from the group comprising human tau protein and post-translationally modified human tau protein, such as phosphorylated, acetylated, glycosylated, glycated, prolyl-isomerizated, nitrated, polyaminated, ubiquitinated, sumoylated, oxidated and aggregated human tau protein and cleaved and truncated versions thereof, o-synuclein, TDP43, mHTT, Clq, C5a, ILlB, 1L6, TNF-o, APOE, IL12, IL23 and fragments thereof, Wherein the first and second target are different. Further, the invention relates to a method, a diagnostic method and use of the binding molecules, specifically antibodies of the invention for the treatment and prevention of neurodegenerative diseases.

Description

FIELD OF THE INVENTION

The present invention relates to multispecific binding molecules, as well as use of these binding molecules in the diagnosis or treatment of neurodegenerative disorders, such as Alzheimer’s Disease, Lewy Body Dementia and Parkinson’s Disease.

BACKGROUND OF THE INVENTION

Neurodegenerative disorders (NDDs) are a group of conditions that is characterized by the progressive loss of structure and function of the central and peripheral nervous system. Examples of neurodegenerative disorders include Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Huntington’s Disease (HD) and Amyotrophic Lateral Sclerosis (ALS). Most NDDs are associated with the aggregation and deposition of misfolded proteins, causing toxicity to the directly affected and surrounding cells, which leads to the dysfunction and loss of synaptic connections, neuroinflammation and can ultimately result in the death of neurons. These processes lead to cognitive dysfunction and memory impairment, as well as motor dysfunction and specific clinical symptoms depending on the area in the brain where the aggregates are located.

Aggregation of misfolded proteins, resulting in the formation of insoluble filaments in the brain, is a characteristic process in neurodegeneration. Although misfolding of proteins frequently occurs in healthy subjects as well, these abnormal and aggregated proteins are usually efficiently cleared or repaired by the system. However, in diseased subjects these clearing mechanisms are impaired or overwhelmed, hence continued aggregation and ultimately deposition of these aggregates occurs. The soluble oligomers and short fibrils are thought to be the most toxic as they are highly reactive molecules that can diffuse throughout the cell and abnormally interact with cellular proteins. Furthermore, intracellular aggregated proteins can be secreted into extracellular space (e.g. via synaptic release or membrane vesicles) and are subsequently taken up by neighboring or synaptically connected neurons. These protein aggregates incorporate their physiological monomeric proteins in these new cells. This process is called ‘seeding’ and leads to a vicious cycle of protein aggregation and subsequent excretion to other cells. This process leads to the so-called propagation of protein pathology throughout, the brain and is thought to contribute to the progressive nature of the protein pathology in many NDDs. There is also substantial evidence that aggregated proteins can induce misfolding and subsequent aggregation of other proteins due to direct molecular interactions, a phenomenon known as “crossseeding”. In addition, the presence of protein aggregates can possibly lead to impairment of the clearance system of the cell and therefore other aggregates comprising different proteins are also often observed. Furthermore, since age is the biggest risk factor for most sporadic (non-familiar) NDDs, it would be predicted by chance that elderly people have diverse pathology in their brain. Hence, in many NDDs protein aggregates of multiple different protein types have been observed. Disorders that are associated with the accumulation of abnormal proteins are also frequently called “proteopathies”. Proteins commonly observed to form aggregates in NDDs are tau-protein, amyloid-6, α-synuclein, TDP-43 and mutant Huntingtin protein (mHTT). (Skovronsky, D.M., et. al., Annu. Rev. Pathol. Meeh. Dis. 2006, 1:151-70; Taylor, J.P., et al., Science, 2002, 296, 1991-1995; Rubinsztein, Nature, 2006, 443, 780; Bredesen, et al., Nature, 2006, 443, 796; Spires-Jones, et. al., Acta NeuropathoL, 2017, 134, 187-205).

All proteopathies are associated with varying degrees of neuroinflammation. Several mechanisms linking neuroinflammation and proteopathies have been described. For example, extracellular deposition or secretion of protein aggregates can activate immune cells such as microglia and astrocytes, thereby causing a chronic neuroinflammatory state in the brain. In addition, neurons with proteopathy can secrete stress factors or immune molecules which activate immune cells or downregulate immune checkpoints in the brain. Prolonged upregulation of cytokines such as IL-16, IL-6 and TNF-α can have harmful effects on the brain and were also found to be able to initiate or aggravate proteinopathy. NDDs are also associated with complement activation, which may lead to increased neuroinflammation and microglial phagocytosis of synapses and neurons. Furthermore, APOE4 - the strongest AD risk factor - is expressed in microglia and astrocytes and increases proteopathy-induced neuroinflammation. Thus, a link between proteinopathy and neuroinflammation has been identified. (Leyns and Holtzman, Molecular Neurodegeneration, 2017, 12:50)

Immunotherapy is an emerging tool in the treatment of NDDs and several antibodies against protein aggregates involved in the pathophysiology of NDDs have been developed and are being investigated in clinical trials. Furthermore, immunotherapy against immune cell surface receptors, secreted immune factors or their cognate receptors have been developed and in several cases investigated in clinical trials against NNDs. However, no cure for any of the NDDs has been found up to now and most clinical trials have failed to show a significant benefit. Hence, there is an urgent need for novel effective therapeutics.

In therapy of NDDs, the effective delivery of the therapeutic agents into the brain is a major challenge, as passage through the blood brain barrier (BBB) is required. It was found that molecules known as molecular Trojan horses (MTH) bind active transport receptors such as the insulin, transferrin or Low Density Lipoprotein Receptor-related Protein 1 receptor and can enhance entry of therapeutics into the brain by enabling active transport through the BBB.

Since it has been demonstrated that binding molecules targeted at a protein involved in NDD, such as discussed above, are of clinical value, the development of these therapies has been stimulated. Yet, there is room for improved binding molecules and therapeutic uses thereof.

SUMMARY OF THE INVENTION

The invention relates to a multispecific binding molecule, preferably an antibody, comprising at least a first binding site binding to a first target selected from the group comprising human tau protein and post-translationally modified human tau protein, such as phosphorylated, acetylated, glycosylated, glycated, prolyl-isomerizated, nitrated, polyaminated, ubiquitinated, sumoylated, oxidated and aggregated human tau protein and cleaved and truncated versions thereof, asynuclein, TDP43, mHTT, and fragments thereof and at least a second binding site binding to a second target selected from the group comprising human tau protein and post-translationally modified human fan protein, such as phosphorylated, acetylated, glycosylated, glycated, prolyl-isomerizated, nitrated, polyaminated, ubiquitinated, sumoylated, oxidated and aggregated human tan protein and cleaved and truncated versions thereof, α-synuclein, TDP43, mHTT, Clq, C5a, IL1B, IL6, TNF-α, APOE, IL12, IL23 and fragments thereof, wherein the first and second target are different.

Preferably said multispecific binding molecule of the invention, comprises at least a first binding site binding to human tan protein or posttranslationally modified human tau protein, such as phosphorylated, acetylated, glycosylated, glycated, prolyl-isomerizated, nitrated, polyaminated, ubiquitinated, sumoylated, oxidated and aggregated human tau protein and cleaved and truncated versions thereof and at least a second binding site binding to u-synuclein or a fragment thereof.

In another preferred embodiment of the invention the multispecific binding molecule comprises at least a first binding site binding to human tau protein or post-translationally modified human tau protein, such as phosphorylated, acetylated, glycosylated, glycated, prolyl-isomerizated, nitrated, polyaminated, ubiquitinated, sumoylated, oxidated and aggregated human tau protein and cleaved and truncated versions thereof and at least a second binding site binding to Clq or a fragment thereof.

Further preferred is a multispecific binding molecule of the invention, comprising at least a first binding site binding to α-synuclein or a fragment thereof and at least a second binding site binding to Clq or a fragment thereof.

In another embodiment of the invention, the multispecific binding molecule according to the invention comprises at least one binding site binding to human tau protein selected from the group comprising binding fragments of taul3 and binding fragments of 5A6 and at least one binding site binding to a-synuclein selected from the group comprising binding fragments of Syn211 and binding fragments of H3C.

In yet another embodiment of the invention, the multispecific binding molecule of the invention comprises at least one binding site binding to tau protein selected from the group comprising binding fragments of taul3 and binding fragments of 5A6 and at least one binding site binding to Clq selected from the group comprising binding fragments of JL-1 and binding fragments produced by the hybridomas 23B6C8, 5B5C22, 12A5B7 and 4A4B11.

Further, the invention relates to a multispecific binding molecule comprising at least one binding site binding to Clq selected from the group comprising binding fragments of JL-land binding fragments produced by the hybridomas 23B6C8, 5B5C22, 12A5B7 and 4A4B11 and at least one binding site binding to α-synuclein selected from the group comprising binding fragments of Syn211 and binding fragments of H3C.

In a most preferred embodiment, the multispecific binding molecule of the invention further comprises a moiety that enables shuttling of the binding molecule through the blood brain barrier, preferably wherein said moiety is a binding site.

The invention further relates to a multispecific binding molecule that is humanized.

Preferably, the multispecific binding molecule of the invention is bispecific.

More preferably, the multispecific binding molecule according to the invention is trispecific.

In another aspect, the multispecific binding molecule of the invention has a format selected from the group consisting of multispecific binding formats listed in Figure 2 of Brinkmann, et al., MAbs, 2017, 9:182-212 and Figure 1 of Spiess, et al.; Molecular Immunology, 2015, 67:95-106, and multispecific antibody conjugates, for example dual-variable-domain (DVD) antibody, trispecific IgGa and tetraspecific IgGa. triple-targeting triplebody, triabody, tribody, trispecific triple heads, trispecific triple dAb, tetraspecific dAb, multispecific dAb, circular dimeric singlechain diabody (CD-scDb), linear dimeric single-chain diabody (LD-scDb), disulfide stabilized Fv fragment, bis-scFv, tandem tri-scFv, bispecific Fab2, Fabs, chemical conjugate trimeric Fab, di-miniantibody, tetrabody, IgG-scFab, scFab-dsscFv, Fv2Fc, IgG-scFv fusions, such as BslAb, Bs2Ab, Bs3Ab, Trispecific C-terminal fusion, Tri-specific N-terminal fusion, TslAb, Ts2Ab, IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, IgM, CODV-Ig, sdAb, trispecific Zybody, tetraspecific Zybody, pentaspecific Zybody, sextaspecific Zybody, septaspecific Zybody, octaspecific Zybody, Knob-into-holes molecules and duobodies

Preferably, the multispecific binding molecule of the invention has the format selected from the group comprising trispecific triabodies, trispecific tribodies, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, 2scFv-IgG, IgG2sc,Fv, DVI-IgG, sc,Fab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, IgG-taFv, scFv4-IgG and TVD-Ig

In a further aspect, the invention relates to a multispecific antibody, wherein the Fc region of the antibody comprises a mutation at one or more of the following positions: 233, 234, 235, 236, 237, 268, 269, 270, 254, 254, 294, 297, 298, 300, 318, 320, 322, 327, 329, 331.

In another embodiment the invention relates to a multispecific binding molecule according to the invention for use in treatment or prevention of neurodegenerative disorders, selected from the group comprising Alzheimer’s Disease, Lewy Body Dementia, Parkinson’s Disease, Huntington Disease, Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia, Frontotemporal Dementia with Parkisonism-17, Multiple System Atrophy, Corticobasal Degeneration, Progressive Supranuclear Palsy, Pick’s Disease, Primary Age Related Tauopathy, Argyrophilic Grain Disease and Cerebral Amyloid Angiopathy.

Preferably, the invention relates to a multispecific binding molecule for use in treatment of Alzheimer’s Disease., which preferably can be a bispecific binding molecule for use in the treatment of Alzheimer’s Disease, a trispecific binding molecule for use in the treatment of Alzheimer’s disease, or a tetraspecific binding molecule for use in the treatment of Alzheimer’s disease.

ί

Another aspect of the invention relates to the use of a therapeutic delivery vehicle, encoding any of the multispecific binding molecules of the invention in therapy of neurodegenerative disorders.

Preferably, the invention relates to the use of an adeno-associated virus vector as therapeutic delivery vehicle encoding any of the multispecific binding molecules of the invention.

In yet another embodiment, the invention relates to a method for the treatment of neurodegenerative disorders, comprising: administration of a multispecific binding molecule according to the invention to a subject in need thereof.

Preferably, the invention relates to a method for treatment of the neurodegenerative disorders, comprising: administration of a nucleic acid construct encoding a multispecific binding molecule according to the invention in a therapeutic delivery vehicle to a subject in need thereof.

Further part of the invention is a diagnostic method for the detection of 15 neurodegenerative disorders, comprising: adding a multispecific binding molecule according to the invention to a sample obtained from a subject; determining binding of said binding molecule to any of the targets selected from human tau protein and post-translationally modified human tau protein, such as phosphorylated, acetylated, glycosylated, glycated, prolyl-isomerizated, nitrated, 20 polyaminated, ubiquitinated, sumoylated, oxidated and aggregated human tau protein and cleaved and truncated versions thereof, α-synuclein, , TDP43, mHTT, Clq, C5a, IL1B, IL6, TNF-α, APOE, TREM2, IL12, IL23 and fragments thereof in said sample; diagnosis of neurodegenerative disease if said binding is detected.

The invention also relates to a kit for the diagnosis of neurodegenerative 25 disorders, comprising a multispecific binding molecule according to the invention and means for detection of said binding mofecufe.

DETAILED DESCRIPTION

The present invention relates to multispecific binding molecules, preferably antibodies and antigen-binding fragments thereof having specified structural and functional features, and methods of use of the multispecific antibodies and antigen-binding fragments thereof in the diagnosis, prevention or treatment of NDDs.

Target proteins involved in neurodegenerative disorders

The present invention relates to multispecific binding molecules for the prevention, diagnosis or treatment of “neurodegeneration”, which refers to the progressive loss of integrity and function of neurons, often leading to cell death. Neurodegeneration, is typically associated with the misfolding of proteins that are abundant in the brain, which leads to “aggregation”, e.g. the self-assembly of abnormal proteins to form larger structures and their subsequent “deposition, e.g. precipitation of such aggregates in the brain. Oligomers, as well as insoluble aggregates exhibit neurotoxic activities to the brain tissue through several mechanisms. For example, they can cause oxidative stress, e.g. the generation of reactive oxygen species, resulting in the initiation of a signaling pathway leading to the damaging or apoptosis of cells. In addition, the aggregates contribute to the dysregulation of calcium homeostasis. Both can ultimately lead to synapse and axon dysfunction and death of neurons. Furthermore, these aggregated proteins can be secreted in the extracellular space, leading to neuroinflammation and propagation of protein pathology throughout the brain.

Several proteins were found to be prone to form such aggregates in the brain, and these are therefore suitable targets to prevent or treat neurodegeneration. Treatment of proteopathy-related proteins can be targeted towards multiple forms, including their monomeric, misfolded (conformational) or oligomeric state. Additionally, different aggregated proteins may have overlapping structural features and can be targeted simultaneously with one and the same binding molecule. (Goni, F., et al., J. Neuroinflammation, 2013, 10:914; Zha, J. et al., Scientific reports, 2016, 6:36631) Furthermore, specific post-translational modifications at particular domains can be targeted, such as phosphorylation, acetylation, glycosylation, glycation, prolyl-isomerization, nitration, polyamination, ubiquitination, sumoylation, oxidation and aggregation. Also truncated and cleaved versions of the binding molecule can be targeted. If antibodies are taken up in the cell or expressed in the cell, therapeutics can promote cellular degradation of aggregated proteins. If therapeutics work outside the cell, prevention of their propagation throughout the brain by blocking neuronal uptake or prevention of neuroinflammation could be a mechanisms of action. Additionally, depending on antibody effector function, complexes of therapeutic and aggregated protein can be degraded by immune cells such as microglia or cleared via other pathways. The present invention relates to multispecific binding molecules targeting multiple of the following proteins.

α-Synuclein is abundant in the brain and is predominantly found on presynaptic terminals. It may play a role in the dynamics of presynaptic vesicle release, but its precise function in healthy subjects is not yet clearly understood. However, upon aggregation, toxic soluble oligomers are formed which can diffuse throughout the neuron and cause cellular damage. Upon their continued aggregation, insoluble fibrils called Lewy Bodies (LB) are formed, which are distinguishable features in Parkinson’s disease (PD), Lewy Body Dementia (LBD) and Multiple System Atrophy (MSA). Lewy bodies are also often observed in combination with AD pathology and - if present - contribute to the accelerated disease progression. (Chung et al, JAMA Neurol., 2015, 72, 789-796; Walker et al., Acta NeuropathoL, 2015, 129, 729-748; Brenowitz et al., Alzheimers. Dement., 2016, doi:10.1016/j.jalz.2016.09.015; Lemstra et al., J. Neurol. Neurosurg. Psychiatry, 2017, 88, 113-118; Brenowitz et al., Neurology, 2017, doi:10.1212/WNL.0000000000004567; Blanc et al., Alzheimers. Res. Ther., 2017, 9, 47; Irwin et al., Lancet. Neurol., 2017, 16, 55-65; Toledo et al., Acta NeuropathoL, 2016, 131, 393-409.

Amyloid-β peptides have about of 36-43 amino acid residues and are derived from the amyloid precursor protein (APP), which can be cleaved by 6- and γ-secretases at the N- and C-termini respectively, to form amyloid-β species. These species are prone to oligomerization and subsequent formation and extracellular depositions of toxic plaques. These Αβ plaques have multiple adverse effects on the function of synapses and axons, including causing oxidative stress, impairing calcium homeostasis and dysregulation of the function of the endoplasmatic reticulum (ER) and mitochondria. Αβ plaques are important, for the development, of Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA). Extracellular deposition of amyloid-6 is a potent inducer of the activation of microglia and astrocytes. The continued presence of extracellular plaques may therefore create a toxic micro-environment which leads to chronic neuroinflammation in the brain.

Based on these findings, multiple antibodies and small molecules targeting amyloid-6 or 6-secretase 1 (BACE) have been investigated in clinical trials. However, all failed to show significant clinical benefits.

Tan protein plays an important role in the assembly and stabilization of microtubules in the brain. The interaction of tau protein with tubulin is regulated by dynamic phosphorylation. Under pathological conditions, hyperphosphorylation and aggregation of tau protein occurs. In AD this leads to formation of straight filaments and paired helical filaments, which subsequently form the neurofibrillary tangles and neuropil threads found in patient brains. In other tauopathies, these aggregates can have different structural features and affect also other cell types, such as astrocytes. As with α-synuclein, tau aggregation therefore leads to both loss-of-function and gain-of-toxic-function, both contributing to neurodegeneration.

Furthermore, several independent studies show that tau immunization not only inhibits tau pathology in mice, but simultaneously enhanced clearance of amyloid-6 plaques. It was hypothesized that the reduction in A6 plaques is due to a reduction in APP synthesis and/or amyloidogenic processing as a result of immunization. Moreover, antibody treatment could induce activation of microglia which facilitates A6 clearance. (Rajamohamedsait, H. et al, Scientific reports, 2017, 7:17034; Castillo-Carranza, C.M. et al, J. Neurosci, 2015, 35(12):4857- 4868; Dai, C., et al., Alzheimers Research and Therapy, 2017, 9:1)

Examples of “tauopathies”, e.g. class of NDDs associated with the hyperphosphorylation and subsequent aggregation of tau proteins in the brain, are corticobasal degeneration (CBD), frontotemporal dementia (FTD) or Pick’s disease (PiD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), and chronic traumatic encephalopathy (CTE). Alzheimer’s disease (AD) is classified as a secondary tauopathy, since amyloid-6 is also additionally present. Tau pathology is strongly linked to cellular dysfunction, synapse loss and neurodegeneration and the symptoms of tauopathies therefore correspond to the affected anatomical regions.

Additionally, tau pathology is often found in synucleinopathies, HD, ALS and other NDDs.

TAR DNA-binding protein 43 (TDP-43) is a nuclear protein that binds both RNA and DNA and is involved in regulating splicing, trafficking and stabilization of RNA, as well as miRNA production. In neurodegenerative disorders TDP-43 becomes mislocalized in the cytoplasm where it aggregates, forming stress granules and insoluble inclusion bodies. TDP-43 inclusions are observed in patients with Frontotemporal Dementia (FTD) or Amyotrophic Lateral Sclerosis (ALS). In addition, TDP-43 pathology is observed in a quarter of the patients with AD. (Montine et al., Acta Neuropathol., 2012, 123, 1-11; Takeda, T., Neuropathology, 2018; 38,72-81, Josephs et al., Neurology, 2008, 70, 1850-1857; Josephs et al., Acta Neuropathol, 2014, 127, 441-450; Behrouzi et al., Acta NeuropathoL Commun., 2016, 4, 33).

The mutant Huntingtin protein (mHTT) is a mutant of the huntingtin gene caused by an expansion of the polyglutamine repeat within exon 1 of the huntingtin gene on chromosome 4, exceeding 35 CAG repeats. The mutant has been linked to the onset and progression of Huntington’s disease (HD), an inherited disorder characterized by the neuronal dysfunction and degeneration in the striatum and cerebral cortex. Although the exact mechanism behind HD has not been fully elucidated, it is clear that mHTT afters intracellular Ca²⁺ homeostasis, disrupts intracellular trafficking and impairs gene transcription, processes that induce neurodegeneration. In contrast to for example sporadic AD and PD, HD is a genetic disease and the pathology is expected to be solely the results of mHTT dysfunction. Interestingly however, HD frequently features aggregates composed of pathological tau, α-synuclein and TDP-43 (St-Amour, I, et al., Acta Neuropathol. 2017, https://doi.org/10.10()7/8()()401 -(.) 1 7-1786-7, Fernandez-Nogales et al., Nat Med, 2014, 20, 881-885; Vuono et al., Brain, 2015, 138, 1907-1918).

Neuroinflammation”, e.g. the inflammation of nervous tissue, has been linked to neurodegeneration. Pathological protein aggregates directly and indirectly activate microglia and astrocytes and may initially lead to successful phagocytosis of these proteins. However, under chronic conditions these cells secrete proinflammatory cytokines, causing harmful neuroinflammation and neurodegeneration. These cytokines are additionally involved in the activation of neuronal intracellular pathways (e.g. kinases that phosphorylate tau proteins), which induces misfolding and aggregation of NDD related proteins. These proinflammatory cytokines may also lead to further neuroinflammation and further aggravate neurodegeneration. Microglia can for example induce a neurotoxic phenotype in astrocytes by a combination of ILla, TNF-a and Clq (Liddelow, S.A., et al., Nature, 2017, 541, 481---487). Moreover, activated microglia increase tau spreading, thereby contributing to the progression of intracellular protein aggregates throughout the brain. (Maphis, N„ et. aL Brain, 2015, 138 (6):1738-55; Asai, H„ et al.. Nature Neuroscience, 2015, 18, 1584-1593). Secreted immune factors are attractive targets for therapy as they are available in the extracellular space. Their action can be neutralized by therapeutics binding directly to these immune factors or by blocking their cognate receptors. Accordingly, complement proteins and cytokines, such as Clq, C5a, IL16, IL6, TNF-u, and IL12/IL23 play an important role in the pathobiology of NDDs.

For example, Interleukin (IL)-16 was found to be consistently upregulated in AD, related tauopathies, PD, HD and various proteopathy animal models of these disorders. Cacabelos et al., Methods Find. Exp. Clin. Pharmacol., 1991, 13, 455-458; Cacabelos et al., Methods Find. Exp. Clin. Pharmacol. 1994, 16, 141-151; Gitter et al., Proc. Natl. Acad. Sci. U. S. A., 1995, 92, 10738-10741; BlumDegen et al., Neurosci. Lett., 1995, 202, 17-20; Mogi et al., Neurosci. Lett., 1994, 180, 147-150; Mogi et al., Neurosci. Lett., 1996, 211, 13-16; King et al., Alzheimer Dis. Assoc. Disord., 2017, doi: 10.1097/WAD.0000000000000211; Björkqvist et al.,

J. Exp. Med., 2008, 205, 1869-1877; Ona et al., Nature, 1999, 399, 263-267; Cook et al., Hum. Mol. Genet., 2015, 24, 6198-6212). This proinflammatory cytokine is expressed by several cell types in the brain, though primarily by microglia as a result of insult or injury. Interestingly, IL-16 was found to enhance A6 phagocytosis by microglia, thereby reducing A6 plaques in the brain, which suggests a protective role of IL-16 against neurodegeneration in early stages of AD. However, several studies also suggest induction of tau pathology by IL-16. Although the exact underlying mechanism has not yet been elucidated, there is convincing evidence that IL-16 is in fact able to mediate neuronal kinase activity, thereby stimulating phosphorylation and subsequent aggregation of tau. Indeed, several studies find opposite effects of immune pathways on Αβ on the one hand, and synapse loss and tau pathology on the other hand. (Leyns and Holtzman, Molecular Neurodegeneration, 2017, 12:50) Administration of an IL1R (the shared receptor for ILIA and IL1B) monoclonal antibody in a mouse model with combined AB and tau pathology has favorable effects on inflammation, cognition, AB and tau pathology (Kitazawa, M, et al., J Immunol, 2011; 187:6539-6549)

IL-6 is also upregulated in AD, related tauopathies, PD, HD and various proteopathy animal models of these disorders. (Luterman et al. Arch. Neurol. 2000, 57, 1153-1160; Jiang et al., Neurobiol. Aging, 2015, 36, 3176-3186; Jiang et al., Neuropharmacology, 2016, 105, 196-206; Kovac et al., J. Immunol., 2011, 187, 2732-2739; Khandelwal et al., Mol. Cell. Neurosci., 2012, 49, 44-53; Cook et al., Hum. Mol. Genet., 2015, 24, 6198-6212; Mravec et al., J. Neuroinflammation, 2016, 13, 15; Blum-Degen et al., , Neurosci. Lett., 1995, 202, 17-20; Mogi et al., Neurosci. Lett., 1994, 180, 147-150: Mogi et al., Neurosci. Lett., 1996, 211, 13-16; King et al, 2017, doi:10.1097/WAD.0000000000000211; Björkqvist et al., J. Exp. Med., 2008, 205, 1869-1877; Hull et al., Neurobiol. Aging, 1996, 17, 795-800, Cojocaru et al., Rom. J. Intern. Med. 2011, 49, 55-58; Rojanathammanee et al., J. Neuroinflammation, 2011, 8, 44; Silvestroni et al, Neuroreport, 2009, 20, 10981103. Similarly to IL-IB, overexpression of IL-6 was correlated with a reduction of AB plaques in the brain, whilst tau phosphorylation was increased through activation of the kinases p38 and cdk5. A correlation between increased serum levels of IL-6 and parkinsonism has also been established. (Leyns and Holtzman, Molecular Neurodegeneration, 2017, 12:50)

The cytokines IL-12 and IL-23 were found to play a significant role in the pathology of AD and the inhibition of IL-12/IL-23 (which share the P40 subunit) signaling pathway as well as the administration of neutralizing antibodies resulted in a reduction of AB plaques in the brain, (vom Berg, et. al., Nat. Med., 2012, 18, 1812-1819)

Tumor necrosis factor alpha (TNF-α) is another proinflammatory cytokine that was correlated to tau and AB pathologies and consequent degeneration of neurons. (Jiang et al., Neurobiol. Aging, 2015, 36, 3176-3186;

Jiang et al., Neuropharmacology, 2016, 105, 196-206; Kovac et al., J. Immunol., 2011, 187, 2732-2739; Khandelwal et al., Mol. Cell. Neurosci., 2012, 49, 44-53; Cook et al., Hum. Mol. Genet., 2015, 24, 6198-6212; Mravec et al., J. Neuroinflammation, 2016, 13, 15; Novak et al., Cell. Mol. NeurobioL, 2017, doi:10.1007/sl0571-017-0491-3: Combs et al., J. Neurosci., 2001, 21, 1179-1188; Perry et al., NeurobioL Aging, 2001, 22, 873-883; King et al, 2017, doi:10.1097/WAD.0000000000000211. Sznejder-Pacholek et al., Pharmacol. Rep. 69, 2017, 242—251; Hsiao et al, Hum. Mol. Genet., 2014, 23, 4328-4344; Björkqvist et al. J. Exp. Med. 2008, 205, 1869-1877). For example, it was found that AB can bind a receptor of TNF-α (TNFR1), eventually leading to activation of Nuclear Factor κβ (NF-kB) and neuronal apoptosis. In addition, upregulation of TNF-α was found to stimulate the expression of other local inflammatory mediators, ultimately leading to increased levels of AB and hyperphosphorylated tan. Finally, it was shown that TNF-α can eventually lead to apoptosis in neurons, amongst others through activation of caspases. (Leyns and Holtzman, Molecular Neurodegeneration, 2017, 12:50) Eikewise, TNFy and TNF type 1 were linked to the pathology of AD and PI). Several studies find favorable effects of targeting TNF-α and its receptors in mouse models of AD and some compounds are currently in clinical trials (Chang, R. et al., J Cent Nerv Syst Dis, 2017, 9: 1-5)

Complement is a potent pro-inflammatory system and one of the major pathways of the innate immune system. The complement system is crucial in the first line of defense against microbes and pathogens, but may also cause damage to self through recruitment of immune cells and formation of the membrane attack complex (MAC). It is comprised of many proteins that lead to a cascade of events, ultimately forming highly inflammatory peptides such as complement component (C) 5a. Therapies targeting C5a were shown to be beneficial in animals models of AD, PD and HD (Landlinger et al., J. Neuroinflammation, 2015, 12, 150; Gordon et al., FASEB J., 2015, 29; Woodruff et al., FASEB J., 2006, 20, 1407-1417). Interestingly, a correlation between upregulation of Clq (the first subcomponent of the Cl complex) and multiple proteinopathies has been established. Clq is often upregulated in the striatum of HD patients, substantia nigra of PD and decorates both amyloid plaques and neurofibrillary tangles in AD brains. (McGeer et al.,

Neurosci. Lett., 1989, 107, 341-346; Rogers et al., Proc. Natl. Acad. Sci., 1992, 89, 10016-10020; Afagh et al., Exp. Neurol., 1996, 138, 22-32; Shen et al., Neurosci. Lett., 2001, 305, 165-168; Schwab et al., Brain Res., 1996, 707, 196-205; Depboylu et al., J. Neuropathol. Exp. Neurol., 2011, 70, 125-132; Singhrao et al., Exp. Neurol., 1999, 159, 362-376). Multiple mouse models of Alzheimer’s disease indicated upregulation of Clq prior to plaque formation. In addition, Clq-tagged synapses can be phagocytosed by microglia in a pathway involving C3 and complement receptor 3 (CR3). This pathway plays an important role in neurodevelopment but can be induced in the adult brain under a wide range of pathological conditions such as virus infection and neurodegeneration. Early synapse loss was prevented by infusion of an anti-Clq monoclonal antibody in mouse models of AD, and an immunotherapeutic is currently in clinical trials. (Hong, S. et al., Science, 2016, 352, 712-716). Clq deposition leads to early loss of synapses via the C3-CR3 pathway. Furthermore, with increased neurodegeneration or BBB breakdown, Clq deposition also leads to induction of other downstream components of the classical complement pathway. This causes recruitment of immune cells and neuroinflammation, formation of the MAC and ultimately leads to neurodegeneration. Targeting Clq with immunotherapy may therefore diminish early synapse loss, neuroinflammation, MAC formation and ultimately neurodegeneration in AD and other tauopathies, synucleinopathies and HD. Morgan, B.P., Semin Immunopathol., 2017, 1-12. Morgan, B.P., Semin. ImmunopathoL, 2018, 40:113-124.

The gene encoding Apolipoprotein E (APOE), most notably APOE4, has been identified to be the strongest risk factor for late-onset AD and was additionally correlated to other proteopathies, such as HD, DLB and PI). (Strittmatter et al., Proc. Natl. Acad. Sci. U. S. A., 1993, 90, 1977-1981; Holtzman et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 2892-2897; Lambert et al., Nat. Genet., 2009, 41, 1094-1099; Kalman et al., Neurobiol. Aging, 2000, 21, 555-558; Wakabayashi et al., Acta Neuropathol., 1998, 95, 450-454; Tsuang et al., Neurology 2005, 64, 509-513, Pankratz et al., Mov. Disord. 2006, 21, 45-49; Tsuang et al., JAMA Neurol., 2013, 70, 223-228, Panas et al., J. Neurol. 1999, 246, 574-577; Kehoe et al., J. Med. Genet., 1999, 36, 108-111; Rita Guerreiro et al.,

Lancet Neurol., 2018, 17, 64-74). For example, it was demonstrated that APOE influences deposition of AB plaques in the brain. In addition, a mouse model using P301S tau transgenic mice, indicated both increased levels of phosphorylated tau in the brain, as well as increased cytokine levels, such as IL-IB, IL-la and TNF-a for P301S/E4 mice, compared to P301S/E2 and P301S/E3 mice, whereas these changes were largely absent in P301S/KC) mice. Thus, APOE plays a prominent role in the aggravation of neurodegeneration both through mediation of aggregation, as well as by inducing neuroinflammation through increased innate immunity. (Yang Shi et al., Nature, 2017, 549, 523). Anti-APOE immunotherapy was shown to strongly reduce plaque load and behavioral deficits in mice: (Kim, J., J. Exp. Med., 2012, 209 (12) 2149-2156; Liao, F., J

Neuroscience, 2014, 34 (21) 7281-7292). Immunotherapy specifically directed towards the APOE4 allele decreased amyloid load, hyperphosphorylated tau and behavioral deficits in transgenic animals (Luz, I., Current Alzheimer’s Research, 2016, 13 (8), 918-929).

“Neurodegenerative disorders” (NDDs), e.g. disorders that are characterized by neurodegeneration, are typically characterized by the accumulation of aggregates, formed by one or multiple proteins mentioned above, in the brain. In most cases, the underlying cause of the NDD is unknown, although certain risk factors have been identified, such as old age, (multiple) head trauma or genetic predisposition.

Arguably the most common and well-known NDD is Alzheimer’s Disease (AD). Symptoms include impaired memory, disorientation and behavioral changes. It is associated with aggregates composed of hyperphosphorylated tau protein, forming neurofibrillary tangles in the neuron, and accumulation of extracellular plaques formed from amyloid B. In addition, aggregation of TDP-43 and a-synuclein have also been observed in AD patients.

Another NDD associated with accumulation of AB is cerebral amyloid angiopathy (CAA), in which AB aggregates cause multiple strokes in the brain, which can lead to paralysis, dementia or even death.

Chronic traumatic encephalopathy (CTE) is a tauopathy that is caused by multiple traumatic injuries to the brain. Traumatic brain injury can result in amyloid B plaques and tau, TDP-43 and u-synuclein inclusions. (Kenney, K., et al., J Neuropathol Exp Neurol. 2018 Jan 1:77(1):50-63.) Most patients are former athletes (of contact sports such as boxing or ice-hockey) or military veterans.

Parkinson’s Disease (PD) is a NDD that affects the dopaminergic neurons in the substantia nigra, which mainly affects the motor system. Symptoms include tremor, slowness of moving, rigidity and difficulty of walking. Although the underlying cause of PD remains largely unclear, Lewy Bodies (e.g. aggregates of usynuclein) in the nerve cells have been observed.

Multiple System Atrophy (MSA) or Shy-Drager syndrome, is a rare NDD that shows clinical overlap with PD, but shows little response to dopamine agonists that are often used in the treatment of PD. Like PD, MSA has been linked to the formation of the Lewy Bodies in the brain.

Lewy Body Dementia (LBD) is a form of dementia that is characterized by the formation of the Lewy Bodies in the brain. It shares symptoms with PD and AD.

Corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) are two other progressive NDDs that are associated with accumulation of abnormal tau aggregates. Both disorders are primarily characterized with moving dysfunction and symptoms are observed similar to PD, but cognitive and/or behavioral abnormalities have also been observed.

Huntington Disease (HD) is a genetic disorder caused by a mutation in the gene coding for the Huntington protein. Expansion of the GAG repeats in the gene results in an abnormal protein that damages brain cells, affecting movement, behavior and cognition. In most cases, the on-set of the disease is between 30-40 years of age of the patient.

Amyotrophic Lateral Sclerosis (ALS) is a motor-neuron disease, characterized by the gradually worsening of muscle weakness, which leads to difficulties in moving, speaking, swallowing and respiratory failure. In most cases, deposition of TDP-43 in the cytoplasm is observed which impairs RNA processing.

Frontotemporal Dementia (FTD) or Pick’s disease is another form of dementia which is characterized by aggregations of tau protein and/or TDP-43 in the frontal and temporal lobes of the brain. These areas of the brain are associated with language (temporal) and behavior (frontal) and consequently symptoms are indeed primarily correlated with changes in personality, behavior and speech. This type in dementia is often observed in relatively young patients (40-60) years of age.

In approximately 20% of the cases a genetic mutation is the underlying cause of the disease. For example, Tau-positive frontotemporal dementia with parkinsonism (FTDP-17) is caused by a mutation in the MAPT gene that encodes for tau protein and is a variant of FTD, in which the patient exhibits features related to movement disorders.

Multispecific binding molecules

Many NDDs are associated with mixed pathologies of different disease targets. For example AD is characterized by both AB plaques, as well as aggregates of phosphorylated tau. In addition, aggregates of α-synuclein and TDP-43 or coaggregates thereof (for example with tau or Amyloid B) are also frequently observed in patients suffering from AD. Aggregates comprised of different proteins have also been observed. Such mixed pathology is partly the result of the ability of aggregates to interact with one another and induce aggregation of other proteins. However, in the aging brain multiple types of pathology are often observed. Aging and NDDs may therefore make the brain more susceptible to the accumulation of various aggregates. In some disorders, mixed pathologies are in fact a prerequisite for full manifestation of the disease. For example, tau pathology does not progress to the neocortex in absence of AB plaques. Additionally, the presence of Lewy bodies has been associated with worse disease outcomes in AD patients. Interestingly, with advancing age the amount of mixed pathology increases, including in pre-symptomatic people. As the world population is increasingly aging, the amount of people with mixed pathology is expected to increase substantially in the future.

In addition, synergies between proteins involved in proteopathies and neuroinflammation have also been demonstrated. For example, AB plaques in AD induce microglia activation, which subsequently initiate aggregation of tau proteins, hence enhancing disease progression.

In addition, synergies can exist in immune pathways. For example, TNFα can induce microglial activation and associated neuroinflammation. This may lead to secretion of more TNF-α and other cytokines.

Immune pathways can also modulate the relationship between proteopathy and cytokine secretion. For example, APOE genotype alters pathological AB or pathological tan-induced microglia and astrocyte activation and subsequent secretion of pro-inflammatory cytokines (e.g. IL1B and TNF-α) and their downstream effects.

Immune molecules can also mediate some of the toxic effects of proteopathy. Aggregated proteins or cells containing aggregated proteins in tauopathies, synucleinopathies and HD have been shown to induce classical complement activation. AB can for example induce microglial phagocytosis of synapses in a pathway involving complement-involved proteins Clq, C3 and CR3. (Hong, S. et al., Science, 2016, 352, 712-716).

Immune cells such as microglia can also use combinations of immune factors (ILla, TNF-α and Clq) to induce reactive neurotoxic astrocytes (Liddelow, S.A., et al., Nature, 2017, 541, 481-487).

The current invention thus relates to the use of multispecific binding molecules, specifically binding at least two different molecules of the abovementioned target for therapy or diagnosis of NDDs.

Herein, the use of multispecific binding molecules offers an additional advantage over the use of multiple monospecific binding molecules. For example, the efficacy of therapy of NDDs is drastically increased when multispecific binding molecules are used instead of a combination of multiple monospecific binding molecules. It allows the targeting of several pathological proteins simultaneously, whereas in a monospecific binding molecule approach multiple binding molecules are required to achieve a similar therapeutic effect. Hence, the total concentration of the multispecific binding molecule may remain relatively low, which is associated with a decreased risk of adverse effects, fewer clinical difficulties and lower production costs. Additionally, unknown interactions, differences in brain uptake or clearance kinetics between different molecules may be a limitation in combining multiple monospecific binding molecules.

Accordingly, the present invention relates to multispecific binding molecules and the use thereof. The present invention is directed at multispecific binding molecules, comprising at least a first binding site binding to a first target selected from the group comprising human tau protein and post-translationally modified, such as phosphorylated, acetylated, glycosylated, glycated, prolylisomerizated, nitrated, polyaminated, ubiquitinated, sumoylated, oxidated, aggregated, cleaved and truncated versions thereof, α-synuclein, 6-amyloid, TDP43, mHTT and fragments thereof, and a second binding site binding to a second target selected from the group comprising human tau protein and post-translational modificated, such as phosphorylated, acetylated, glycosylated, glycated, prolylisomerizated, nitrated, polyaminated, ubiquitinated, sumoylated, oxidated, aggregated, cleaved and truncated versions thereof, a-synuclein, β-amyloid, TDP43, mHTT, Clq, C5a, IL1 6, IL6, TNF-α, APOE, IL12/IL23 and fragments thereof, wherein the first and second target is different. In some instances, the present invention relates to multispecific binding molecules having a further binding site that binds to a third target selected from the group comprising human tau protein and post-translational modificated, such as phosphorylated, acetylated, glycosylated, glycated, prolyl-isomerizated, nitrated, polyaminated, ubiquitinated, sumoylated, oxidated, aggregated, cleaved and truncated versions thereof, asynuclein, β-amyloid, TDP43, mHTT, Clq, C5a, IL1 6, IL6, TNF-α, APOE, IL12/IL23 and fragments thereof, wherein these targets are different. Many combinations are possible and accordingly the present invention comprises a plurality of different multispecific binding molecules, including but not limited to the combinations listed below.

Table 1; First group of targets

Entry	Target
Al	tau protein targets
A2	a-synuclein
A3	6-amyloid
A4	TDP43
A5	mHTT

Table 2; Second group of targets

Entry	Target
Bl	Clq
B2	C5a
B3	IL1 β
B4	IL6
B5	TNF-α
B6	APOE
B7	IL12
B8	IL23

Multispecific binding molecules having at least one of the following binding specificities are included in the present invention, where A1-A5 and B1-B8 are indicated in the Tables 1-2 above

A1+A2, A1+A3, A1+A4, A1+A5, A2+A3, A2+A4, A3+A4, A4+A5, Al+Bl, A1+B2,

A1+B3, A1+B4, A1+B5, A1+B6, A1+B7, A1+B8, A2+B1, A2+B2, A2+B3, A2+B4,

A2+B5, A2+B6, A2+B7, A2+B8, A3+B1, A3+B2, A3+B3, A3+B4, A3+B5, A3+B6,

A3+B7, A3+B8, A4+B1, A4+B2, A4+B3, A4+B4, A4+B5, A4+B6, A4+B7, A4+B8,

A5+B1, A5+B2, A5+B3, A5+B4, A5+B5, A5+B6, A5+B7, A5+B8, A1+A2+A3, A1+A2+A4, A1+A3+A4, A1+A4+A5, A2+A3+A4

A1+A2+B1,

A1+A2+B7,

A1+A3+B5,

A1+A4+B3,

A1+A5+B1,

A1+A5+B7,

A2+A3+B5,

A2+A4+B3,

A3+A4+B1,

A3+A4+B7,

A4+A5+B5,

A1+B1+B4,

A1+B2+B4,

A1+A2+B2,

A1+A2+B8,

A1+A3+B6,

A1+A4+B4,

A1+A5+B2,

A1+A5+B8,

A2+A3+B6,

A2+A4+B4,

A3+A4+B2,

A3+A4+B8,

A4+A5+B6,

A1+B1+B5,

A1+B2+B5,

A1+A2+B3,

A1+A3+B1,

A1+A3+B7,

A1+A4+B5,

A1+A5+B3,

A2+A3+B1,

A2+A3+B7,

A2+A4+B5,

A3+A4+B3,

A4+A5+B1,

A4+A5+B7,

A1+B1+B6,

A1+B2+B6,

A1+A2+B4,

A1+A3+B2,

A1+A3+B8,

A1+A4+B6,

A1+A5+B4,

A2+A3+B2,

A2+A3+B8,

A2+A4+B6,

A3+A4+B4,

A4+A5+B2,

A4+A5+B8,

A1+B1+B7,

A1+B2+B7,

A1+A2+B5,

A1+A3+B3,

A1+A4+B1,

A1+A4+B7,

A1+A5+B5,

A2+A3+B3,

A2+A4+B1,

A2+A4+B7,

A3+A4+B5,

A4+A5+B3,

A1+B1+B2,

A1+B1+B8,

A1+B2+B8,

A1+A2+B6,

A1+A3+B4,

A1+A4+B2,

A1+A4+B8,

A1+A5+B6,

A2+A3+B4,

A2+A4+B2,

A2+A4+B8,

A3+A4+B6,

A4+A5+B4,

A1+B1+B3,

A1+B2+B3,

A1+B3+B4.

A1+B3+B5,	A1+B3+B6,	A1+B3+B7,	A1+B3+B8,	A1+B4+B5,	A1+B4+B6,
	A1+B4+B7,	A1+B4+B8,	A1+B5+B6,	A1+B5+B7,	A1+B5+B8,	A1+B6+B7,
	A1+B6+B8,	A1+B7+B8,	A2+B1+B2,	A2+B1+B3,	A2+B1+B4,	A2+B1+B5,
	A2+B1+B6,	A2+B1+B7,	A2+B1+B8,	A2+B2+B3,	A2+B2+B4,	A2+B2+B5,
5	A2+B2+B6,	A2+B2+B7,	A2+B2+B8,	A2+B3+B4,	A2+B3+B5,	A2+B3+B6,
	A2+B3+B7,	A2+B3+B8,	A2+B4+B5,	A2+B4+B6,	A2+B4+B7,	A2+B4+B8,
	A2+B5+B6,	A2+B5+B7,	A2+B5+B8,	A2+B6+B7,	A2+B6+B8,	A2+B7+B8,
	A3+B1+B2,	A3+B1+B3,	A3+B1+B4,	A3+B1+B5,	A3+B1+B6,	A3+B1+B7,
	A3+B1+B8,	A3+B2+B3,	A3+B2+B4,	A3+B2+B5,	A3+B2+B6,	A3+B2+B7,
10	A3+B2+B8,	A3+B3+B4,	A3+B3+B5,	A3+B3+B6,	A3+B3+B7,	A3+B3+B8,
	A3+B4+B5,	A3+B4+B6,	A3+B4+B7,	A3+B4+B8,	A3+B5+B6,	A3+B5+B7,
	A3+B5+B8,	A3+B6+B7,	A3+B6+B8,	A3+B7+B8,	A4+B1+B2,	A4+B1+B3,
	A4+B1+B4,	A4+B1+B5,	A4+B1+B6,	A4+B1+B7,	A4+B1+B8,	A4+B2+B3,
	A4+B2+B4,	A4+B2+B5,	A4+B2+B6,	A4+B2+B7,	A4+B2+B8,	A4+B3+B4,
15	A4+B3+B5,	A4+B3+B6,	A4+B3+B7,	A4+B3+B8,	A4+B4+B5,	A4+B4+B6,
	A4+B4+B7,	A4+B4+B8,	A4+B5+B6,	A4+B5+B7,	A4+B5+B8,	A4+B6+B7,
	A4+B6+B8,	A4+B7+B8,	A5+B1+B2,	A5+B1+B3,	A5+B1+B4,	A5+B1+B5,
	A5+B1+B6,	A5+B1+B7,	A5+B1+B8,	A5+B2+B3,	A5+B2+B4,	A5+B2+B5,
	A5+B2+B6,	A5+B2+B7,	A5+B2+B8,	A5+B3+B4,	A5+B3+B5,	A5+B3+B6,
20	A5+B3+B7,	A5+B3+B8,	A5+B4+B5,	A5+B4+B6,	A5+B4+B7,	A5+B4+B8,
	A5+B5+B6, A5+B5+B7, A5+B5+B8, A5+B6+B7, A5+B6+B8, A5+B7+B8

In a preferred embodiment the multispecific binding molecule have the following binding specificities: A1+A2, Al+Bl, A2+B1.

Additionally, binding molecules have been identified that recognize a 25 common amyloid fold that is shared by multiple toxic misfolded proteins. For example, the general amyloid interaction motif (GAIM) is a fragment of a capsid protein and was found to simultaneously bind u-synuclein, tau protein and amyloid-b. Such binding molecules can also form a binding site of a multispecific binding molecule.

In a preferred embodiment the multispecific binding molecule is a multispecific antibody.

The present invention includes antibodies and methods of use thereof. As used herein, the term antibody refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies comprising two light, chains and two heavy chains), polyclonal antibodies, humanized antibodies, fully human antibodies, chimeric antibodies and camelized single domain antibodies.

The present invention includes antigen-binding fragments and methods of use thereof. As used herein, unless otherwise indicated, antibody fragment or antigen-binding fragment refers to antigen-binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions. Examples of antigen-binding fragments include, but, are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.

The present invention includes Fab fragments and methods of use thereof. A Fab fragment, is comprised of one light, chain and the Cal and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot, form a disulfide bond with another heavy chain molecule. A Fab fragment can be the product of papain cleavage of an antibody.

The present invention includes antibodies and antigen-binding fragments thereof which comprise an Fc region and methods of use thereof. An Fc region contains two heavy chain fragments comprising the Cn2 and Cn3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the Ch3 domains.

The present invention includes Fab’ fragments and methods of use thereof. A Fab' fragment contains one light, chain and a portion or fragment, of one heavy chain that contains the Vn domain and the C ill domain and also the region between the Cui and C n2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')a molecule.

The present invention includes F(ab’)2 fragments and methods of use thereof. A F(ab')2 fragment contains two light chains and two heavy chains containing a portion of the constant region between the Cm and Cif2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains. A F(ab')2 fragment can be the product of pepsin cleavage of an antibody.

The present invention includes Fv fragments and methods of use thereof. The Fv region comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

The present invention includes scFv fragments and methods of use thereof. The term single-chain Fv or scFv antibody refers to antibody fragments comprising the Vu and Vl domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the Vu and Vl domains which enables the scFv to form the desired structure for antigen-binding. For a review of scFv, see Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also, International Patent Application Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946, 778 and 5,260,203.

The present invention includes domain antibodies and methods of use thereof. A domain antibody is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more Vu regions are covalently joined with a peptide linker to create a bivalent domain antibody.

The present invention includes multivalent antibodies and methods of use thereof. A multivalent antibody comprises at least two antigen-binding sites. These binding sites can have the same antigen specificity, or can bind different specificities. In the latter case, the multivalent antibody is at least bispecific. A multivalent antibody can also be at least bivalent for one target and at least bivalent for a further target. In that case the antibody is multispecific and multivalent.

The present invention includes camelized single domain antibodies and methods of use thereof. In certain embodiments, antibodies herein also include camelized single domain antibodies. See, e.g., Muyldermans et al. Trends Biochem. Sci. 2001, 26:230; Reichmann et al. J. Immunol. Methods 1999, 231:25; WO 94/04678; WO 94/25591; U.S. Pat. No. 6,005,079).

In one embodiment, the present invention provides single domain antibodies comprising two Vu domains with modifications such that single domain antibodies are formed.

The present invention includes diabodies and methods of use thereof. As used herein, the term diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (Vn) connected to a light chain variable domain (Vl) in the same polypeptide chain (Vh-Vl or Vl-Vh). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al. Proc. Natl. Acad. Sci. USA 1993, 90: 6444-6448.

Various other forms of multispecific antibodies are known. All multispecific binding formats listed in Figure 2 of Brinkmann, et al., MAbs, 2017, 9:182-212 and Figure 1 of Spiess, et al.; Molecular Immunology, 2015, 67:95-106 are possible formats used for multispecific binding molecules of the present invention. Included herein are also multispecific antibody conjugates, for example dual-variable-domain (DVD) antibody, trispecific IgCF and tetraspecific IgGa, triple-targeting triplebody, triabody, tribody, trispecific triple heads, trispecific triple dAb, tetraspecific dAb, multispecific dAb, circular dimeric single-chain diabody (CD-scDb), linear dimeric single-chain diabody (LD-scDb), disulfidestabilized Fv fragment, bis-scFv, tandem tri-scFv, bispecific Fab2, Fabs, chemical conjugate trimeric Fab, di-miniantibody, tetrabody, IgG-scFab, scFab-dsscFv, Fv2Fc, IgG-scFv fusions, such as BslAb, Bs2Ab, Bs3Ab, Trispecific C-terminal fusion, Tri-specific N-terminal fusion, TslAb, Ts2Ab, IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, IgM, CODV-Ig, sdAb, trispecific Zybody, tetraspecific Zybody, pentaspecific Zybody, sextaspecific Zybody, septaspecific Zybody, octaspecific Zybody, Knob-into-holes and duobodies.

The skilled person will know that there are multiple ways of constructing such multispecific antibodies or fragments thereof. For details on possible multispecific antibody formats, see Brinkmann, et al., MAbs, 2017, 9:182-212, Spiess, et al.; Molecular Immunology, 2015, 67:95-106; LaFleur, et al., mAbs, 2013, 5:208-218; US20170073415; Holliger and Hudson Nat. Biotechnol. 2005, 23:1126-1136; Kontermann, R.E., 2011, Bispecific Antibodies, Springer Science & Business Media. These are incorporated by reference in the present description.

In a preferred embodiment, the multispecific antibodies have a format selected from the group comprising trispecific triabodies, trispecific tribodies, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, 2scFv-IgG, IgG-2scFv, DVI-IgG, scFabFc(kih)-scFv2, scFab-Fc(kih)-scFv, IgG-taFv, scFvMgG and TVD-Ig.

Typically, an antibody or antigen-binding fragment of the invention which is modified in some way retains at least 10% of its original binding activity (when compared to the parental antibody) when that activity is expressed on a molar basis. Preferably, an antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody. It is also intended that an antibody or antigenbinding fragment of the invention can include conservative or non-conservative amino acid substitutions (referred to as conservative variants or function conserved variants of the antibody) that do not substantially alter its biologic activity.

The present invention includes antibodies and antigen-binding fragments thereof and methods of use thereof. These antibodies or antigen-binding fragments thereof are at least partially free of other biological molecules from the cells or cell cultures in which they are produced and may be considered “isolated”. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term isolated is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.

The present invention includes chimeric antibodies (e.g., human constant domain/mouse variable domain) and methods of use thereof. As used herein, a chimeric antibody is an antibody having the variable domain from a first antibody and the constant domain from a second antibody, where the first and second antibodies are from different species. (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 1984, 81: 6851-6855). Typically, the variable domains are obtained from an antibody from an experimental animal (the parental antibody), such as a rodent, and the constant domain sequences are obtained from human antibodies, so that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human subject than the parental (e.g., mouse) antibody.

The present invention preferably includes humanized antibodies and antigen-binding fragments thereof (e.g., rat or mouse antibodies that have been humanized) and methods of use thereof. As used herein, the term humanized antibody refers to forms of antibodies that contain sequences from both human and non-human (e.g., mouse or rat) antibodies. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).

In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two different pairs of polypeptide chains, each pair having one light (about 25 kDa) and one heavy chain (about 50-70 kDa). The aminotenninal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxyterminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a J region of about 12 or more amino acids, with the heavy chain also including a D region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

The variable regions of each light/heavy chain pair form the antibody binding site.

Typically, the variable domains of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C-terminal, both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5^th ed.; NIH Publ. No. 91-3242 (1991); Kabat Adv. Prot. Chem. 1978, 32:1-75; Kabat, et al., J. Biol. Chem. 1977, 252:66096616; Chothia, et al., J Mol. Biol. 1987, 196:901-917 or Chothia, et al., Nature 1989, 342:878-883.

As used herein, the term hypervariable region refers to the amino acid residues of an antibody or antigen-binding fragment thereof that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a complementarity determining region or CDR (i.e. LCDR1, LCDR2 and LCDR3 in the light chain variable domain and HCDR1, HCDR2 and HCDR3 in the heavy chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining the CDR regions of an antibody by sequence); see also Chothia and Lesk J. Mol. Biol. 1987, 196: 901-917 (defining the CDR regions of an antibody by structure). As used herein, the term framework or FR residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

The skilled person will know that there are a number of monospecific antibodies known in the art that are able to bind to pathological proteins that are 5 involved in NDDs targeted in the present invention. The CDRs of these antibodies are non-limiting examples of binding sites that can be employed in the multispecific binding molecules and are listed in Table 4. Additionally, there are also hybridomas known in the art that can produce monospecific antibodies, which can be employed in the multispecific binding molecules of the invention. For 10 example, the Clq hybridomas 4A4B11, 23B6C8, 5B5C22 and 12A5B7 are particularly suitable for producing antibodies that can be employed in the multispecific binding molecules of the invention.

Table 4; Monospecific binding molecules against proteins involved in the pathology of NDD's

Target	name	binding SEQ.	Reference
Tau	B1IB092	KYGMS (CDR-1 (HC)) TISSSGSRTYYPDSVKG (CDR-2 (HC)) SWDGAMDY (CDR-3 (HC)) KSSQSIVHSNGNTYLE (CDR-1 (LC)) KVSNRFS (CDR-2 (LC)) FQGSLVPWA (CDR-3 (LC))	WO2015081085A2
Tau	C2N 8E12	EVKVVESGGGLVQPGGSMKL SCVVSGFTFSNYWVNWVRQA PGKGLEWVAQIRLKSDNYAT HYEESVKGRFTISRDDSKSS VYLQMNNLRAEDSGIYYCTN WEDYWGQGTTVTVSSASTKG PSVF (VH) DIVLTQSPDSLAVSLGERAT 1SCRASQSVSTSRYSYIHWY QQKPGQPPKLL1KYASNLES GVPSRFSGSGSGTDFTLN1H PLEPEDFATYYCHHSWEIPL TFGQGTKLEIK (VL)	WO2015200806 A2
Tau	RG7345

Tau	RO 7105705 (37D3-H9)	SÏGH5 (CDR-1 (HC)) TiRSGG'TYTYYêPéVKrt (CDR.2 (HC)) SYSGAMDÏ (CDR_3 (HQ) S S£< IVBSN QKTV’iX (CDR-1 (LC)) ix~° (CDR-2 (LC)) - (CDR-3 (LC)) S';'iS.-VSS'S'3& ïAKSS&SijKïi 5·5?Α3ί5ϊ<Υ:?^ SYsSMëiiVSSS ? υ-ϋυχυυ?·? ;:·)$ gycy·Υίγ pixsVKUxilx siux^aksyus y^xyaysy ;·γ~·.·ν (HC) ΫΥΧ/ΥΓΰΥγϋί X.yX':$X.i«/)»\s:· XSCiYSSQSXV Κ0?Κ·;:«·>·ΧΫ·;« K/SïKjgssssk x.xx'ixvsass^s swjim'&ss ö&m«x.gz ;:?viez?xxA· yyx'yyxxvx 8T?cm?gr>E 1« (LC)	WO2016196726
Tau	54C1-H11	x-YHy (CDR-I (HC)) TÏSSGGNTTyY^yKG (CDR-2 (HC)) ώΥ.γ^ΙΙΔ ({?DR 3 (HC)) •χΤ'ι T”i q ftj·'’'V'Tiy ï?· x» x ...^,. (CDR ] (LC)) (CDR-2 (LC)) y YM&n y or* (CDR_3 (LC)) LVK^GGSLKV SGyA.SG-’Ti’R Ί3·ϊΰί^Ϋ3?ΥΥ ?ËS:TOm T’ii-nTY’AcAY TS’-TViCi (VH) ΐ?ϊ»·:Τς:ί: rk-S ï17 ;kSf?:>NTkkAA< k?$<-<$':?DK.'*';$<·<%' ·.>·>[·%k >:>Υ< vkk JÏ.Ui.'i?'·'.' kk'C i'Xx'kv't: v A $'?k' :YkYxxkkkvrt\ .( K (VL)	WO2016196726
Tau	123E9	,b:t YBk (CDR-1 (HC)) (CDR-2 (HC)) YM;0ArtYï3^Yx (CDR_3 (HC)) RssgsiwMTYLE {CDR.im) WrtNRFS (CDR_2(LC)) X’V' c< Z-^j. ryjy; . >. (C{)R , kkïkÏ^kVKkï kk*A;k?YY?Y ikv.'k.’kki'ki :'C;k; k¥ :tö;5W&7&&Tjh S?x.\¥: ?^L$SLT$Er.; kAYYYkk-kik (HC) kV/L$?kY>¥rkk· ·;·5?ν«·?·5:^.$ ^X^TYL&S kk«k?K?kk;k-kx kk-k TkVkcxkY kkCnkk^iy? ?YSW$¥Kk.S5 Ï.K (LC)	WO2016196726

Tau	24A11	UI'x (CDR-1 (HO) L r1 KG (CDR-2 (HC)) 5 ,Y C S -ΛΥ (CDR.3 (HC)) r.S«,Nt.\L’M GNTYLS (CDR-I (LC)) -''-0 0 (GDR_2(LC)) -’^^7 L'ÏO (CDR-3 (LO) SS'S LV:d?<^?>Vi;i3 L-m;< ?ays?$ MtWA. (HC) ïlVXWötF^j oUooTVX.Etf OjQK?GQS ·;Κ LLIYiiVSNF-F i-G^PrTSG5 SSSTr-FTLKI .siKVSAK&Lc^.X: ι:κ: (LC)	WO2016196726
Tau	Hu-37D3	SYGMG (cdr-1 (HC)) TlROATYTYYHYWfO (CDR-2 (HC)) cYvO:·: (CDR.3 (HC)) (CDR_I(LGJ) KVSNRFS (CDR-2 (LC)) rQGiOvWT (CDR.3 (Lcn X s ·. X XX SiCxKKV.SiWxT SMSiLTrsTYs: .*f:SVKSt«T£ WJSiSSSiUzi 3,<WSÏ>RA£i! TAV s' Y«AS.5 Ï ;SixkK<£ÏT.ftJi:i: Tx.xTVSiS (HC) EDQLTVSPSS LSASVGDWT LTCRSSQS Y -x>K PC K 5 P K T ƒKV S NR F £G¥?&R FS SSL^PFA'FAT ïYCFQGYLVE- WT.FGQGTK' (LC)	WO2016196726 I J
Tau	HU-125B11	VG'MH (CDR-1 HC)) ex F :AO'RV;^YA.^RVW (CDR_2 HC)) te-iYx (CDR-3 (HC)) ^QWG™ (CDR-! (LC)) SAR.iKYT (CDR.2(LC)) PiORRT vte-w K.tó..Kj. X X (CDR_3 (LC)) SApLVïiJ'-.te.c; ·.*ν;·'χ,i.LL tvAA;.;'?'! ;'.·; TSKUAKSSWig SSwKS^SiW WASteVSi®;? TSSRiSSSKSi·? <..γ:λ;?»αLR··. g&?,AV?ï*-?S Wfts	WO2016196726
		(VH) i-SVAnxrS£;:.'> x.S/xSVP'SSiYT ÏTCi<AS2f5VÖ TAW?s'Ö0?? A:;te>Y-:v;p;:.·; ?:»—;?.r;'-vïp STVAÏSSLp? ί::-·Α;·ϊ·γχ<· S'kiY xï-ysap atavkyy (VL)

Tau	HU-94B2	GYT1® (CDR-! (HC)) (CDR-2 (HC)) -· (CDR-3 (HC)) Xte,>K» *(CDR_( (LC)) LV&KLDS (CDR-2 (LQ) CCDR-3 (Lcn νΚΚϊ-<?Λ??ν·ί··ν FYKYLtoYuL-I skww. χγ. m c. γ :t s ? γ y;w? : 7stay (HC) ?.vto<::Ï?<<A\,':'ï'<C· rY»:.xf'Aiï :'χΆ.·'>:Ϊ/Αο'ίΐ:?'? ίλ'ΪΎ·:1:?ζΎΐ.::?< IK (LC)	WO2016196726
Tau	DC8E8	DY VIS (CDR-1 (HC)) EIFPRSGSTYYNEKFK (CDR-2 (HC)) DYYGTSFAMDY (CDR-3 (HC)) KSSQSLLNSRTRKNYL (CDR-1 (LQ) WASTRES (CDR-2 (LQ) KQSFYLRT (CDR-3 (LC))	WO 2016079597 Al
Tau	Tau 13		Am J Pathol. 2017 Jun; 187(6): 1399- 1412.
Tau	HT7		Am J Pathol. 2017 Jun;187(6):l399- 1412.
Tau	BIIB076		CN 105324394 A
Tau	5A6		Galasko D„ 1997, J. neurochem, 68.1: 430-433.
a-synuclein	PRX002/9E4		Mov Disord. 2017 Feb; 32(2):211-218. US 8673593 B2
a-synuclein	BIIB054

α-synuclein	N1-202.3G12	DFHIH (CDR-1 (HC)) WSNPQSGNSSSAQRFQG (CDR-2 (HQ) PHDGAGNYRFDT (CDR-3 (HC)) SGDALPKHYAH (CDR-1 (LQ) KDTERPS (CDR-2 (LQ) QSADVSSTYVV (CDR-3 (LQ) EVQLVQSGAEVKKPGASVRLSCRASGYN FIDFHIHWVRQAPGEGLEWMGWSNPQSG NSSSAQRFQGRVTMTTDTSMSAAYMDLN WLTLDDTAVYYCTRPHDGAGNYRFDTW GQGTLVTVSS (HC) QSVLTQPPSVSVAPGQTARITCSGDALPKH YAHWYQQKPGQVPIVVIYKDTERPSGIPE RFSGSTSGTTVTLTISGVQAEDEAHYYCQS ADVSSTYVVFGGGTKLTVL (LC)	WO 2010069603 Al
α-synuclein	NI-202.12F4	KAWMS (CDR-1 (HQ) RIKSTADGGTTSYAAPVEG (CDR-2 (HQ) AH (CDR-3 (HC)) SGEALPMQFAH (CDR-1 (LQ) KDSERPS (CDR-2 (LQ) QSPDSTNTYEV (CDR-3 (LQ) EVQLVQSGGGLVEPGGSLRLSCAVSGFDF EKAWMSWVRQAPGQGLQWVARIKSTAD GGTTSYAAPVEGRFIISRRDSRNMLYLQM NSLKTEDTAVYYCTSAHWGQGTLVTVSS (HC) QSVLTQPPSVSVSPGQTARITCSGEALPMQ FAHWYQQRPGKAPVIVVYKDSERPSGVPE RFSGSSSGTTATLT1TGVQAEDEADYYCQS PDSTNTYEVFGGGTKLTVL (LC)	WO 2010069603 Al

α-synuclein	NI-202.3D8	TYA1S (CDR-1 (HQ) IISNDGSRKYYADSVKG (CDR-2 (HC)) KRGTYASRCKAFDF (CDR-3 (HQ) RASQSISGWLA (CDR-1 (LQ) DASNLES (CDR-2 (LQ) QQYDNYWT (CDR-3 (LQ) RASHDISNYLA (CDR-1 (LQ) AASSLQS (CDR-2 (LQ) VQYRTYPLT (CDR-3 (LQ) EVQLVESGGGVVQPGRSLRLSCAASGFTF STYAISWVRQAPGKGLEWVAIISNDGSRK YYADSVKGRFTISRDNSRDTLDLEMNSLR DEDTAVYYCAKKRGTYASRCKAFDFWG QGTLVTVSS (HC) DIQLTQSPSTLSASVGDRVTITCRASQSISG WLAWYQQKPGKAPKLL1YDASNLESGVP SRFSGSGSGTEFTLTISSLQPDDFATYYCQ QYDNYWTFGQGTKVEIK (LQ EIVMTQSPSSLSAS1GDRVTFTCRASHDISN YLAWFRQQPGKAPKSLIYAASSLQSGVPS RFSASGSGTDFTLTISSLQPEDFATYYCVQ YRTYPLTFGQGTRLGIK (LC)	WO 2010069603 Al
α-synuclein	GM285	AASGFTFSRFTMT (CDR-1 (HQ) AISGSGGGTSYADSVK (CDR-2 (HQ) AKNWAPFDY (CDR-3 (HQ) RASQSVSRSYLA (CDR-1 (LC)) GASSRAT (CDR-2 (LQ) QQYGSSPWT (CDR-3 (LQ) EVQLLESGGGLVQPGGSLRLSCAASGFTF SRFTMTWVRQAPGKGLEWVSAISGSGGG TSYADSVKGRLTVSRDNSKNTLYLQMNS LRAEDTAVYYCAAKNWAPFDYWGQGTL VTVSS (HQ EIVLTQSPGTLSLSPGERATLSCRASQSVSR SYLAWYQQKPGQAPRLLIYGASSRATGIP DRFSGSG SGTDFTLTVSRLEPEDFAV YYCE EYGSSPWTFGQGTKVEIK (LC)	WO 2017009312 Al

α-synuclein	GM37	GFTFSSYAMT (CDR-1 (HC)) AIRSNGDRTDYADSVK (CDR-2 (HC)) AKNWAPFDS (CDR-3 (HC)) ASQSVSSSYLA (CDR-1 (LC)) GASSRAT (CDR-2 (LC)) QQYGSSPWT (CDR-3 (LC)) AIRSSGDRTDYADSVK (CRD-2 (HQ) A1RSQGDRTDYADSVK (CRD-2 (HC)) A1RSHGDRTDYADSVK (CRD-2 (HC)) EVQLLESGGGLVQTGGSLRLSCAASGFTF SSYAMTWVRQAPGKGLEWVSAIRSNGDR TDYADSVKGRFTISRDNSQNTLYLQMNSL RAEDTAVYYCAKNWAPFDSWGQGTLVT VSS (HC) EIVLTQSPGTLSLSPGERATLSCRASQSVSS SYLAWYQQKPGQAPRLLIYGASSRATGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCE EYGSSPWTFGQGTKVEIK (LC)	WO 2017009312 Al
α-synuclein	BAI	GFTF'NTYAMN i CDR-1 (HCs) R1RTKSNDYATYYADSV (CDR-2 (HC)) VG'i HP's AMOY (CDR-3 iHC)) RSSQNl VHS\GS Ï YLE (CDR-1 · LC)) KVSNRFS (CDR-2 (LC)) FQGSHVPLT (CDR-3 (LC)) E VQLVETGGG L V QPKGS LKL.SC ATSG FTF NTYAMNNWVRQAPGKGLEWVAR1RTKS NDYATYYADSVRniSRDDSQSMLYLQMN NLKTLDI AMYY< VkViiYRPYAMDVWGQ GTSVTVSS (HC) DVLMTQTPLSLPVSLGDQASISCRSSQNJV HSNGNTY LEWY LQKPGQSPTIJ.1YKVSNR FSGVPDRFSGSGSGLDIHLK1SRVEAEDLV YYCFQGSHVPLTFGAGTKLELK (LC)	US 8968734 B2
α-synuclein	BA2	GFTFSNYAMS (CDR-1 (HC)1 TV PSG( 1SYTYY Pl JSV < O JR-::: i {CD QNPGSRGWYFDV iCDR-3 (HO) RSSQSiVNSNGNTYLE 1CDR-1 < l.C D KVSNRFS (CDR-2 (LC)) FQGSHVPLT (CDR-3 (LCD E VQLVESGGDl. V KPGGSl. KFSC A ASG FTF S\Y \ W'SWVRQTPDK RLEW VATVTSGGS > O \ PD$VRGRFTiSRD\ -XKNTLYl.QLSS LKJSLD1 AMY FCARQN F GSRG W YPD V W A GTTVTVSS (HO DVLMTQTPLSLPVS1 G! >QaS1S( RSSQSIV NSNGN T Y {. F U YI.QKPGQSPΜ Λ. 1Y K VSNR FSGVPDR1'SGSGS( ï DF Π. KISR\ E YED1Ό VYA CFQGSHX IVfFGAGTLELK. (L.C)	US 8968734 B2

α-synuclein	BA3	GfTESS YAMS (CDR-1 (HC)) TkSNGGS) 'm PDSV (CDR-2 (HO) HSDY SG FA) (CDR -3 · H( ) SASSSVSYMY (CDR-1 (LC)) RTSN1 \S (CDR-2 (LC)) QQ'; HSYP) 1 (CDR-3 (LC)) EVMLVESGGG LV KPGGS LK LSCAASGFTP SSYAMSWVR Q ï PEK R1..EWV AT) SNGGS Y ΊΎΥΡΗ5 VRFHSR DNA KNTL YLQMSS L.RS EDTAMY YCAR HSDY'SG aw fay w GQGTL VTVSA (HC) QIVLTQSPATMSASPGEKVTISCSASSSVSY MYWYQQKPGSSPKPWTYRTSNLASGVPA RFSGSGSGTSYSLTISSMEAEDAATYYCQ QYHSYPYTFGGGTKLE1K (LC1	US 8968734 B2
α-synuclein	BA4	GDSFTSGYWN (CDR-1 tHO) Y1RYSGNTYYNPSL (CDR-2 (HCD SYYDYDRAWFAY (CDR-3 QIC)! RSSQSl \ HS\G\K1 H <CDR-1 ;((>) KVSNRFS (CDR-2 (LC)) SQSTHVPWT (CDR-3 (LC)) EVQLQESGPSLVKPSQTLSLTCSVTGGDSF TSGYWNWIRKFPGNKLEYMGYIRYSGNT YYNPSLRJSrrRDT.SKNQYYLQLÏSVTTEDT ATFYCARSYYDYDIGAWPAYWGQGALVT VSA (HC) DVVMTQTPLSI..PVSLGDQASECRSSQSI V HSNGNTY LHWYLQKPGQSPKLl D KVSN R FS( iV PDRFSGSGSGTDFT) KISRV F -ΧΓ DL GvYJ CSQS1 HVPA DGGGibJ LiK Q C)	US 8968734 B2
α-synuclein		GFSLTSYGVH (CDR-1 (HC;) VlWRGC.SiDASAAi (CDR-2QR. r I LRSVGOFAD iCDR-3 QU ;> RSSQT)VHNNGNT YLE (CDR i (1.0) KVSXRLS (CDR-2 (LCD IQGMB'Pi 1 (CDR 1 (1 CD	US 8968734 B2
α-synuclein		GFTFSNY-UiS ((. DR- ( QK » TISIGGSY lAYPDSY Q'DR-2 QK'D HSDYSGAWFAY K DR-3 (HCp SASSSX'SYMY (CDR 1 Q O'> RTSNLAS (CDR-2 (LCD QQHISWi .CDR 'HCp	US 8968734 B2
α-synuclein		GPTFÏ'DYYMS (CDR-1 QIC!) HRNKANGYTTEYSASV (CDR-2 QIC)) DYGNYAMDY (CDR-3 (HO) 'KSSQSLLYSSNQKNYE A (CDR-1 (LC)) WASTRES (CDR-2 (LC)) QQYYSYPYT (CDR-3 (LC))	US 8968734 B2

α-synuclein	5C1	GYTFTNYWMN (CDR-1 (IK ;? ATNPNNGYTDYNQRFKD (CDR-2 (HC)) GGH1..AY (CDR-3 (HQ) RSSQSLFHSKGNTYLH (CDR-1 (LQ) RVSNRFS (CDR-2 (LQ) SQSAHVPWT (CDR-3 (LQ) QVQ.I.QQSGAF.LAKP( 1TSVQMSCK ASG7 T 1 1 XV X1XXX IK XRPGQGL1 A IG X i XPXXG YTDYNQR FK DRA ILT A DKSSNTA YMH L.SS LTSEDSAV YFCASGGHLA YWGQGTV VT V SA (HQ DVVMTQIPIAQSVSPGDQASISCRSSQSLF HSKGN’TYI.HWYl.QKPGQSPKIJJNRVSNR FSGVPDRFSGSGSGTDFFLK1SGVEAEDLG V YFCSQS AH V PWTFGGGTKLEÏR (LC)	US 20140127131 Al
α-synuclein	9E4	NYGMS (CDR-1 (HQ) SISSGGGSTY YPDNVKG (CDR-2 (HQ) GGAG1DY (CDR-3 (HQ; kSlQH.l.YSSX’QKNYl \ (CDR-1 (IQ WASIRKS (CDR-2 (LQ) QQYYSYPLT (CDR-3 (LQ? Γ v’Ql A FSGG(' i. v QPGGS.I. .RkSCAASGFTF S\ Y( > MS V RQ A PG K G1 F W V A SISSGGG S TY YPDN V KGRFÏ 1SRDDAK.NSLYLQMNSL RAEDTAV V Y CSRGG AG 1DY WGQGTLVTV SS (HC) DIQMTQSPSSLSASVGDRVTITCKSIQTLL YSSNQKN Y (..A W FQQK PGKAPK l-L.i ¥\V ASI RKSGVPSRFSGSGSGTDFTLTISSLQPEDFA T YYCQQY YSYPDEFGGGTKLEÏK (I C)	US 20130108546 Al
α-synuclein	Syn-211		Cell Reports 2014, 7, 2054-2065
α-synuclein	H3C		Clayton D.F., Neuron, 1995, 15.2: 361-72.
Amyloid β	Gantenerumab
Amyloid β	Ponezumab
Amyloid β	Solanezumab
Amyloid β	LY-3002813
Amyloid β	GSK933776
Amyloid β	BAN2401

Amyloid β	KHK6640
Amyloid β	Crenezumab	GFTFSSYGMS (CDR-1 (HC)) SINSNGGSTYYPDSVK (CDR-2 (HC)) GDY (CDR-3 (HC)) RSSQSLVYSNGDTYLH (CDR-1 (LQ) KVSNRFS (CDR-2 (LC)) SQSTHVPWT (CDR-3 (LC)) EVQLVESGGGLVQPGGSLRLSCAASGFTF SSYGMSWVRQAPGKGLELVASINSNGGST YYPDSVKGRFTISRDNAKNSLYLQMNSLR AEDTAVYYCASGDYWGQGTTVTVSS (HC) DIVMTQSPLSLPVTPGEPASISCRSSQSLVY SNGDTYLHWYLQKPGQSPQLL1YKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCSQSTHVPWTFGQGTKVE1K (LC)	WO 2015120233 Al; WO 2015120280 Al
Amyloid β	Adacanumab	SYGMH (CDR-1 (HQ) VIWFDGTKKYYTDSVK (CDR-2 (HQ) DRG1GARRGPYYMDV (CDR-3 (HC)) RASQSISSYLN (CDR-1 (LQ) AASSLQS (CDR-2 (LQ) QQSYSTPLT (CDR-3 (LQ) QVQLVESGGGVVQPGRSLRLSCAASGFAF SSYGMHWVRQAPGKGLEWVAVIWFDGT KKYYTDSVKGRFTISRDNSKNTLYLQMNT LRAEDTAVYYCARDRGIGARRGPYYMDV WGKGTTVTVSS (HQ D1QMTQSPSSLSASVGDRVT1TCRASQSISS YLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQ SYSTPLTFGGGTKVE1KR (LQ	WO 2014089500 Al
Amyloid β	9TL	GYYTEAYYIH (CDR-1 (HQ) RIDPATGNTKYAPRLQD (CDR-2 (HQ) LYSLPVY (CDR-3 (HQ) KSSQSLLYSDAKTYLN (CDR-1 (LQ) Q1SRLDP (CDR-2 (LQ) LQGTHYPVL (CDR-3 (LQ) QVQLVQSGAEVKKPGASVKVSCKASGYY TEAYY1HWVRQAPGQGLEWMGRIDPATG NTKYAPRLQDRVTMTRDTSTSTVYMELSS LRSEDTAVYYCASLYSLPVYWGQGTTVT VSS (HC) DVVMTQSPLSLPVTLGQPASISCKSSQSLL YSDAKTYLNWFQQRPGQSPRRL1YQISRL DPGVPDRFSGSGSGTDFTLK1SRVEAEDVG VYYCLQGTHYPVLFGQGTRLEIKRT (LQ	WO 2006036291 A2

Amyloid β	3D6 (Bapineuzumab)	NYGMS (CDR-1 (HC)) S1RSGGGRTYYSDNVKG (CDR-2 (HC)) YDHYSGSSDY (CDR-3 (HC)) KSSQSLLDSDGKTYLN (CDR-1 (LC)) LVSKLDS (CDR-2 (LC)) WQGTHFPRT (CDR-3 (LC))	WO 2002088306 A2
		GFTFSSYAMS (CDR-1 (HC)) A1NASGTRTYYADSVKG (CDR-2 (HC)) GKGNTHKPYGYVRYFDV (CDR-3 (HC)) RASQSVSSSYLA (CDR-1 (LC)) GASSRAT (CDR-2 (LCD LQIYNMP1 (CDR-3 (LC))
Amyloid β		QVELVESGGGIAQPGGSLRLSC A ASG 1IF SSYAMSWVRQAPGKGLEWVSAÏNASGTR TYYADSVKGRFTISRDNSKNTLYLQMNSL· RAEDTAVYYCARGKGNTHKPYGYVRYF DVWGQGTLA'TVSS (HC)	WO 2014056816 Al
		DÏVLTQSPATLSLSPGERATLSCRASQSVSS SYLAWY QQKPGQ APR LI J YGASSRATG VP ARFSGSGSG/IOFTLTISSLEPEDFATYYCL QI YNMPITI GQGTKVEIKRT (LC)
Amyloid structure	NPT088	AETVESCLAK PHTENSFTNV WKDDKTLDRY ANYEGCLWNA TGVWCTGDE TQCYGTWVPÏ. GLAIPENEGG GSEG( iGSEGG GS EGGGTKPP E YGDTP1PGY TYINPLDGTY PPGTEQNPAN
Amyloid structures	g3p	FRNRfJGALTV ΎΊ (it VTQGTD PVKT YYQYTP VSSKAMYDA Y WNGKFRDCAF HSGI'NEDPFV CE YQGQSSDL PQPP VN AGGG SGGGSGG( >SE GGGS EGG( >SE. G< i G S EG GG S G G GS G S G D F ΟΎ EK MAN A N KG A MT EN DEN A L QSDAKGKLDS VATDYGAAID GHGDVSGL,\ NGNGATGDFA GSNSQ.MAQVG DGDN SPLMNN HiQYL.PSL.PQ S VECRPFV FS AGKPYEFSID CDKINLFRGV FAFII.YVATF MYVFSTFANI l.RN.KES

TDP-43	N1-205.3F10	SQAMS (CDR-1 (HQ) ALSRTGDYTWYADSVRG (CDR-2 (HQ) NYYSSFGYNWAAFHI (CDR-3 (HQ) RASQDVNNNYLA (CDR-1 (LQ) GASRRAT (CDR-2 (LQ) QQYGGSPPYT (CDR-3 (LC)) EVQLLESGGDLVQPGGSLRLSCAASGFTF SSQAMSWVRQAPGKGLEWVSALSRTGDY TWYADSVRGRFTVSRDDSKNIFYLEMNSL RAEDTA V YYC AKNY YS SFGYNW AAFHIW GQGTMVTVSS (HC) E1VLTQSPGTLSLSPGERATLSCRASQDVN NNYLAWYQQKPGQAPRLL1YGASRRATG VPDRFSGRGSGTDFTLT1NRLEPEDFAMYF CQQYGGSPPYTFGQGTKLEIK (LQ	WO2013061163A2
TDP-43	NI-205.51C1	DYWMH (CDR-1 (HQ) R1NLDGSDT1YADSVKG (CDR-2 (HQ) SRKSV (CDR-3 (HQ) TGSNTDVGAYDYVS (CDR-1 (LC)) DVDVRPS (CDR-2 (LQ) SSYTKSGTLV (CDR-3 (LQ) EVQLVESGGGLVQPGGSLR1SCTTSGFIFS DYWMHWVRQAPGKGLTWVSRINLDGSD TIYADSVKGRFTISRDNDKNTLYLQMNSL RVEDTAIYYCARSRKSVWGQGTMVTVSS (HC) QSALTQPASVSGSPGQSITISCTGSNTDVG AYDYVSWSQQLPGKAPKFVIFDVDVRPSG ISDRFSGSKSGNTASLTISGLQAEDEADYY CSSYTKSGTLVFGGGTKVTVV (LC)	WO2013061163A2
TDP-43	NI-205.21G2	SYTLH (CDR-1 (HQ) W1NAAFINTKYSQKFQG (CDR-2 (HQ) RASGSNGLDV (CDR-3 (HQ) QASRDITNYLN (CDR-1 (LQ) DASYLET (CDR-2 (LQ) QQYDSVPLT (CDR-3 (LQ) QVQLVQSGAEVKKPGASVKVSCKTSGYS FTSYTLHWVRQAPGHRPEWMGWINAAFI NTKYSQKFQGR1TLTRDTSAN1AYLELRSL TTEDTAVYYCARRASGSNGLDVWGQGTT VTVSS (HQ D1QMTQSPSSLS AS VGDR1T1TCQASRD1TN YLNWYQQKPGKAPKLLIYDASYLETGVPS TFSGSGSGTHFTLTISSLQPDDFATYYCQQ YDSVPLTFGGGTKVEIK (LQ	WO2013061163A2

TDP-43	NI-205.8A2	DHGMH (CDR-1 (HC)) VIWLDGSSRFYADSVEG (CDR-2 (HC)) DRVASEGTAFDV (CDR-3 (HC)) WASQNVNHYLV (CDR-1 (LC)) DTSVRAA (CDR-2 (LC)) QHRSDWT (CDR-3 (LC)) QVQLVESGGGVVQPGKSLRLSCAASGFTF RDHGMHWVRQAPGKGLEWVAVIWLDGS SRFYADSVEGRFT1SRDNSKNTLYLQLTSL RAEDTAIYYCARDRVASEGTAFDVWGQG TMVTVSS (HC) EIVLTQSPATLSLSPGERATLSCWASQNVN HYLVWYQQRPGQAPRLLLYDTS VRAAGT PARFIGSGSGTHFTLTISSLEPEDSAVYYCE HRSDWTFGQGTKVE1K (LC)	WO2013061163A2
TDP-43	NI-205.15F12	GYYMH (CDR-1 (HC)) VINPNGGSTNYAQKFKG (CDR-2 (HC)) LPVNIEVLDL (CDR-3 (HC)) RSSQTVLFSSNDKNYLA (CDR-1 (LC)) WASVRAS (CDR-2 (LC)) QQSSTAPLT (CDR-3 (LC)) QVQLVQSGTAVKKPGASVKVSCKASGFS FNGYYMHWVRQAPGQGLEWMGVINPNG GSTNYAQKFKGRITMSADTPARSVSMELG SLRSDDTAMYYCARLPVNIEVLDLWGQG TLVTVSS (HC) D1VMTQSPDSLAVSLGERATINCRSSQTVL FSSNDKNYLAWYQQKPGQPPKLLIYWAS VRASGVPDRFSGSGSGTDFSLTINGLQAED VAVYYCQQSSTAPLTFGGGTKVEIK (LC)	WO2013061163A2
TDP-43	N1-205.113C4	NYYMH (CDR-1 (HC)) 11NPSGGRTSYAQKFQG (CDR-2 (HC)) QRPSGYSGYGPSESYGNPTDDAFDV (CDR-3 (HC)) GGNN1GSRGVH (CDR-1 (LC)) DDSDRPS (CDR-2 (LC)) QVWDNSSDHLVV (CDR-3 (LC)) QVQLVQSGAEVKKPGASVKVSCKASGYT FTNYYMHWVRQAPGQGLEWMGI1NPSGG RTSYAQKFQGRASMTRDTSTSTVYMEV1S LRSEDTAV Y YC ARQRPSG YSGYGPSES YG NPTDDAFDVWGQGTTVTVSS (HC) SYVLTQPPSVSVAPGQTARITCGGNNIGSR GVHWYQQRPGQAPVLVVYDDSDRPSGIP ERFSGSNSGDTATLT1SRVEVGDEADYYC QVWDNSSDHLVVFGGGTKLTVL (LC)	W02013061163A2

TDP-43	NI-205.25F3	NYVMY (CDR-1 (HC)) FISYDGSNKYYPDSVKG (CDR-2 (HC)) DTYQYDSSTYYPYFYYYGMDV (CDR-3 (HC)) IGTSSDVGGYNYVS (CDR-1 (LC)) EVSNRPS (CDR-2 (LC)) SSFASSSTSVT (CDR-3 (LC)) QVQLVESGGGVVQPGRSLRLSCAASGFTF SNYVMYWVRQAPGKGLEWVAFISYDGS NKYYPDSVKGRFTISRDNSMNTLTLQMDS LRAEDTAVYYCARDTYQYDSSTYYPYFY YYGMDVWGQGTTVTVSS (HC) QSALTQPASVSGSPGQSITISCIGTSSDVGG YNYVSWYQQHPGKAPKLMIYEVSNRPSG VSSRFSGSKSGNTASLTISGLQSEDEADYY CSSFASSSTSVTFGGGTKLTVL (LC)	WO2013061163A2
TDP-43	NI-205.87E7	SYAMS (CDR-1 (HC)) AISGGGDRTYSADSVKG (CDR-2 (HC)) GGGGEMTAVTMDGTYYGMDV (CDR-3 (HC)) TGTSSNVGTYKFVS (CDR-1 (LC)) DVTKRPS (CDR-2 (LC)) CSYAGSYTYV (CDR-3 (LC)) EVQLLESGGGLVQPGGSLRLSCAASGFTF SSYAMSWVRQAPGKGLEWVSAISGGGDR TYSADSVKGRFTISRDNSKNTLYLQINSLR VEDTAVYYCAQGGGGEMTAVTMDGTYY GMDVWGQGTTVTVSS (HC) QSALTQPRSVSGSPGQSITISCTGTSSNVGT YKFVSWYQQHPGKAPKLMIYDVTKRPSG VPDRFSGSKSGNTASLTISGLQAEDEADY YCCSYAGSYTYVFGSGTKVTVL (LC)	WO2013061163A2
TDP-43	NI-205.21G1	SHGMH (CDR-1 (HC)) VISYDASNKSYADSVKG (CDR-2 (HC)) AFSSSASGGY (CDR-3 (HC)) RSSQSLVHSDGVTYLN (CDR-1 (LC)) KVSNRDS (CDR-2 (LC)) MQGTHWPPWT (CDR-3 (LC)) QVQLVESGGGVVQPGMSLRLSCAASGFSF SSHGMHWVRQTPGKGLEWLAV1SYDASN KSYADSVKGRFT1SRDNSKKTLYLQMDSL RVEDTALYYCANAFSSSASGGYWGQGTL VTVSS (HC) DVVMTQSPLSLPVTLGQPASISCRSSQSLV HSDGVTYLNWFQQRPGQSPRRLIYKVSNR DSGVPDRFSGSGSGTDFTLEISRVEAEDVG IYYCMQGTHWPPWTFGQGTKVE1K (LC)	W02013061163A2

TDP-43	NI-205.68G5	SYGMH (CDR-1 (HC)) IIYYDSSQRYYADSVKG (CDR-2 (HC)) DLPFHYHRSASFAPSDT (CDR-3 (HC)) RASQAVTNNYLA (CDR-1 (LQ) AASSRAT (CDR-2 (LQ) QQYGTSP1T (CDR-3 (LQ) QVQLVESGGGVVQPGRSLRLSCAASGFTF SSYGMHWVRQAPGKGLEWVAIIYYDSSQ RYYADSVKGRFTISRDNSKNALYLQMNSL RAEDTALYYCARDLPFHYHRSASFSPSDT WGQGTLVTVSS (HC) EIVLTQSPGTLSLSPGERATLSCRASQAVT NNYLAWYQQKPGQAPRLLVYAASSRATG IPDRFYGSGSGADFTLTISRLEPEDFAVYY CQQYGTSPITFGQGTRLEIK (LQ	WO2013061163A2
TDP-43	NI-205.20A1	SYRMN (CDR-1 (HQ) YISTSSSTIYYADSVKG (CDR-2 (HQ) AFDY (CDR-3 (HQ) RASQSVSSSYLA (CDR-1 (LQ) GASSRAT (CDR-2 (LQ) QQYGSSPFT (CDR-3 (LQ) EVQLVESGGGLVQPGGSLRLSCAASGFTF SSYRMNWVRQAPGKGLEWVSYISTSSSTI YYADSVKGRFTISRDNAKNSLYLQMNSLR AEDTAVYYCARAFDYWGQGTLVTVSS (HQ EIVLTQSPGTLSLSPGERATLSCRASQSVSS SYLAWYQQKPGQAPRLLIYGASSRATGIP DRFSGSGSGTDFTLTIIRLEPEDFAVYYCQ QYGSSPFTFGQGTKVEIK (LQ	WO2013061163A2
mHTT	N1-302.33C11	DFGMH (CDR-1 (HQ) LIWYDGGYKYYADSVKG (CDR-2 (HQ) HLEYCSRTTCYLGH (CDR-3 (HQ) RASQGISDYLA (CDR-1 (LQ) AASTLQT (CDR-2 (LQ) QQLKTYPYT (CDR-3 (LQ) EVQLVESGGGVVQPGNSLRLSCAASGFRF SDFGMHWVRQAPGKGLEWLALIWYDGG YKYYADSVKGRFTISRDNSKNTMFLQMN SLRAQDTAVYYCATHLEYCSRTTCYLGH WGQGTLVTVSS (HC) DIQLTQSPSFLS AS VGDTVTFTCRASQL AW FQQKPGIAPKLLIYAASTLQTGVPSRFSGS GSGTEFTLTIRSLQSEDFGTYYCQQLKTYP YTFGQGTKVEIK (LC)	WO2016016278A3

mHTT	NI-302.63F3	TRSMN (CDR-1 (HC)) WINTNTGNRTYVQAFRG (CDR-2 (HC)) GAGGGYWFDS (CDR-3 (HC)) KSNQSLFYSSNNNNYLA (CDR-1 (LC)) WGSTRES (CDR-2 (LC)) HQYYHNPYT (CDR-3 (LC)) QVQLVQSGSSVKVSCKASGYTFETRSMN WVRQAPGQGLEYMGW1NTNTGNRTYVQ AFRGRFVFSLDTSVSTAYLQISNLKTEDTA VYYCARGAGGGYWFDSWGQGTLVTVSS (HC) DIQMTQSPDSLAVSLGERAT1NCKSNQSLF YSSNNNNYLAWYQHKSGQPPKLLVYWG STRESGVPDRFSGSGSGTDFTLTISSLQAE DVA1YYCHQYYHNPYTFGQGTKLEIK (LC)	WO2016016278A3
mHTT	NI-302.35C1	IT ALS (CDR-1 (HC)) AITGNAYGTYYADSVKG (CDR-2 (HC)) GIASDSSGYSAF (CDR-3 (HC)) RASQSVDNQFA (CDR-1 (LC)) DASRRAP (CDR-2 (LC)) QHRYTWLYT (CDR-3 (LC)) EVQLVESGGNLVQPGGSLRLSCTASGFTF S1TALSWVRQAPEKGPQWVSAITGNAYGT YYADSVKGRFTISRDNAKNTLYLQMNGL RAEDTAIYYCVKGIASDSSGYSAFWGPGT LVTVSS (HC) EIVLTQSPATLSLSPGERATLSCRASQSVD NQFAWYQQKPGQAPRLLIYDASRRAPG1P DRFSGSGSGTDFTLTISSLEPEDFAIYYCQH RYTWLYTFGQGTRLEIK (LC)	WO2016016278A3
mHTT	NI-302.31F11	STYMS (CDR-1 (HC)) VIFSGADTYYADSVKG (CDR-2 (HC)) HYYGSDLPSDF (CDR-3 (HC)) RSSQSLLYSNGYNYLD (CDR-1 (LC)) LGSDRAS (CDR-2 (LC)) MQGLQSPWT (CDR-3 (LC)) EVQLVESGGGL1QPGGSLRLSCAASGFTVS STYMSWVRQAPGKGLQCVSV1FSGADTY YADSVKGRFTVSRDNSKNTLFLQMNSLR VEDTATYYCVRHYYGSDLPSDFWGQGTL VTVSS (HC) DVVMTQSPLSLPVAPGEPASISCRSSQSLL YSNGYNYLDWYLQKPGKPPQLLVYLGSD RASGVPDRFSGSGSGKDFTLNISRVEAED VGVYYCMQGLQSPWTFGQGTKLE1K (LC)	WO2016016278A3

mHTT	NI-302.2A2	TYWMN (CDR-1 (HQ) NIKPDGSDKYYVDSVKG (CDR-2 (HQ) GDGSGWNVY (CDR-3 (HQ) KSSQSLLYTSKNKDSKNYLG (CDR-1 (LQ) WASTRES (CDR-2 (LQ) QQYYTTPQ (CDR-3 (LC)) EVQLVESGGGLVQPGGSLRLSCAASGFTF STYWMNWVRQAPGKGLEWVANIKPDGS DKYYVDSVKGRFTISRDNAKNSLYLQMN SLRDEDTAVYYCARGDGSGWNVYWGQG TLVTVSS (HQ DIQMTQSPNSLAVSLGERATINCKSSQSLL YTSKNKDSKNYLGWYQQKPGQPPKLLIY WASTRESGVPDRFSGSGSGTDFTLTISSLQ AEDVAVYYCQQYYTTPQFGGGTKVE1K (LC)	WO2016016278A3
mHTT	NI-302.6N9	SYAMT (CDR-1 (HQ) TISATGGSTFYTDSVRG (CDR-2 (HQ) DLFGVDTSYYGMDV (CDR-3 (HQ) RPSQSVSGRYVA (CDR-1 (LQ) AASNRA1 (CDR-2 (LQ) QHYGASSYT (CDR-3 (LQ) EVQLVESGGDLVQPGGSLRLSCVVSGFTF SSYAMTWVRQAPGKGLAWVSTISATGGS TFYTDSVRGRFT1SRDNSKNTLYLQMNSL RTDDTAIYYCVKDLFGVDTSYYGMDVW GQGTTVTVSS (HC) E1VLTQSPGTLSLSPGERATLSCRPSQSVSG RYVAWYQQKPGQAPRLLFYAASNRAIGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQ HYGASSYTFGPGTKVDIK (LQ	WO2016016278A3
mHTT	N1-302.74C11	GYFLH (CDR-1 (HQ) WINPNSGDTNYAE (CDR-2 (HQ) EAPDPGAETDV (CDR-3 (HQ) SGDAVPKQYIY (CDR-1 (LQ) KDTQRPS (CDR-2 (LQ) QSADSSATWV (CDR-3 (LC)) EVQLVQSGTEVQKPGASVKVSCKASGYSF TGYFLHWVRQAPGQGLEWMGWINPNSG DTNYAEKFRGRI1MTRDTSVSTAHMELSS LRFDDTALYYCTREAPDPGAETDVWGQG TTVTVSS (HC) QSVLTQPPSVSVSPGQTARITCSGDAVPKQ YIYWYQQKPGQAP1LVIYKDTQRPSGIPER FSGSNSGTTVTLTITGVQADDEGDYYCQS ADSSATWVFGGGTKLTVL (LC)	WO2016016278A3

mHTT	NI-302.15F9	NSSNM (CDR-1 (HC)) SIDTSATNYKYYADSVKG (CDR-2 (HC)) GYYTPRDFDY (CDR-3 (HC)) RSSQSLLYRDNNTYLN (CDR-1 (LQ) RASDRDS (CDR-2 (LQ) MQGTHWPRT (CDR-3 (LC)) EVQLVESGGGLVTPGGSLRLSCEASGFLF KNSSMNWVRQTPGKGLEWVSSIDTSATN YKYYADSVKGRFT1SRDDATNSLYLQMN SLRADDTATYYCARGYYTPRDFDYWGQG TLVTVSS (HC) DVVMTQSPQTLSVSLGQAASISCRSSQSLL YRDNNTYLNWFHQRPGQSPRRLIYRASDR DSGVPDRFSGGGSGTDFTLKISGVEAEDV GTYYCMQGTHWPRTFGQGTKVEIK (LC)	WO2016016278A3
mHTT	NI-302.39G12	NYA1T (CDR-1 (HC)) VIYRDGRTYYGDSVRG (CDR-2 (HC)) AHGQYYYGVDV (CDR-3 (HC)) RSSQSLLHSNGYNYLD (CDR-1 (LC)) LSSNRPS (CDR-2 (LQ) MQSLQTFT (CDR-3 (LQ) EVQLVQSGGGLVHPWGSLRLSCAASGFS VSNYAITWVRRAPGKGLQYISVIYRDGRT YYGDSVRGRFTISRDDSKNTLYLQMNSLR FEDTAVYYCARAHGQYYYGVDVWGQGT TVTVSS (HQ DVVMTQSPLSLSVSPGEPASISCRSSQSLL HSNGYNYLDWYRQKPGQSPQLLIYLSSNR PSGVPDRFSASGSGTEFTLQISRVEAEDVG VYYCMQSLQTFTFGGGTKVDIK (LQ	WO2016016278A3
mHTT	NI-302.11A4	SSYMS (CDR-1 (HQ) VLYRDGDTYYADSVQG (CDR-2 (HQ) DRRSSHYYYGMDV (CDR-3 (HQ) RASQSVSSSYFA (CDR-1 (LQ) GTSRRAT (CDR-2 (LQ) QQYGSSWT (CDR-3 (LQ) EVQLVESGGGLIQPGGSLRLSCAASGFPVS SSYMSWVRQAPGEGLEWVSVLYRDGDT YYADSVQGRFTISRDNSQNTFYLQMNSLK AEDTAVYYCAGDRRSSHYYYGMDVWGQ GTTVTVSS (HC) EIVMTQSPGTLSLSPGERATLSCRASQS VS SSYFAWYQQKPGQAPRLLIYGTSRRATAI PDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSWTFGPGTKVEIK (LQ	WO2016016278A3

mHTT	NI-302.22H9	NYAIT (CDR-1 (HC)) V1YRDGRTYYGDSVRG (CDR-2 (HC)) AHGQYYYGVDV (CDR-3 (HC)) RSSQSLLHSNGYNYLD (CDR-1 (LC)) LNSNRAS (CDR-2 (LC)) MQSLQTFT (CDR-3 (LC)) EVQLVESGGGLVHPWGSLRVSCAASGFS VSNYAITWVRQAPGKGLEYISVIYRDGRT YYGDSVRGRFTISRDDSKNTIYLQMNSLR FEDTAVYYCARAHGQYYYGVDVWGQGT TVTVSS (HC) DVVMTQSPLSLSVSPGEPASISCRSSQSLL HSNGYNYLDWYRQKPGQSPQLLIYLNSN RASGVPDRFSGSGSGTEFTLTISRVEAEDV GVYYCMQSLQTFTFGGGTKVE1K (LC)	WO2016016278A3
mHTT	NI-302.44D7	SYAMS (CDR-1 (HC)) GIGYSDTSTYYADSVKG (CDR-2 (HC)) GTRDYYGMDV (CDR-3 (HC)) GLSSGSVSTSYYPS (CDR-1 (LC)) STNTRSS (CDR-2 (LC)) VLFMGSG1GV (CDR-3 (LC)) EVQLVQSGGGLVQPGGSLRLSCAASGFTF SSYAMSWVRQAPGKGLEWVSGIGYSDTS TYYADSVKGRFTVSRDISKNTLYLQMNSL RAEDTAVYYCAKGTRDYYGMDVWGQGT MVTVSS (HC) QTVVTQEPSFSVSPGGTVTLTCGLSSGSVS TSYYPSWYQQTPGRAPRTLIYSTNTRSSG VPDRFSGS1LGNKAALT1TGAQADDESDY YCVLFMGSGIGVFGGGTRLTVL (LC)	WO2016016278A3
mHTT	N1-302.37C12	NSQMT (CDR-1 (HC)) V1YTSGSAYYADSVK (CDR-2 (HC)) GPSAYYYGLDL (CDR-3 (HC)) RSSQSLLHSNG YNYLD (CDR-1 (LC)) LGSTRAS (CDR-2 (LC)) MQGLQTYT (CDR-3 (LC)) EVQLVESGGGLVQPGGSLRLSCVASALTV TNSQMTWVRRAPGRGLEWVSV1YTSGSA YYADSVKGRFTISRDNSKNTVFLQMNSLR VEDTAVYYCAKGPSAYYYGLDLWGQGT TVTVSS (HC) DIVMTQSPLSLPVTPGEPASISCRSSQSLLH SNGYNYLDWYLQKPGQSPQLL1YLGSTRA SGVPDRFSGSGSGTDFTLK1SRVEAEDVGV YYCMQGLQTYTFGQGTKLEIK (LC)	WO2016016278A3

mHTT	NI-302.55D8	DYYIH (CDR-1 (HC)) R1NPNNGGTNYAQNFQGWVTMT (CDR-2 (HC)) VGGELLREGGYHYYMDV (CDR-3 (HC)) TGNSSNIGAGYDVH (CDR-1 (LC)) DNTNRPS (CDR-2 (LC)) QSYDNSLSGSWV (CDR-3 (LC)) QVQLVQSGSEVKKPGASVKVSCKASGYT FTDYYIHWVRQAPGQGLEWMGRINPNNG GTNYAQNFQGWVTMTRDTSISTAYMELS RLRSDDTAVYYCARVGGELLREGGYHYY MDVWGKGTTVTVSS (HC) QSVLTQPPSVSGAPGQRVTISCTGNSSNIG AGYDVHWYQQLPGTAPKLLIFDNTNRPS GVPDRFSGSKSGTSASLA1TGLQAEDEAN YHCQSYDNSLSGSWVFGGGTKLTVL (LC)	WO2016016278A3
mHTT	NI-302.7A8	NSWMT (CDR-1 (HC)) NIKEDGSRTYYVDSVKG (CDR-2 (HC)) GDYNSGIYYFPGDY (CDR-3 (HC)) RSSQSLVYSDGNTYLN (CDR-1 (LC)) KVSNRDS (CDR-2 (LC)) MQGTHWPGT (CDR-3 (LC)) EVQLVESGGGSVQPGGSLRLSCVASGFIFR NSWMTWVRQDPGKGLEWVANIKEDGSR TYYVDSVKGRFTISRDNAKNSLYLQMNSL RAEDTAVYYCARGDYNSGIYYFPGDYWG QGTLVTVSS (HC) D V VMTQSPLSLPVTLGQPASISCRS SQSLV YSDGNTYLNWFQQRPGQSPRRLIYKVSNR DSGVPDRFSGSGSGTDFTLRISRVEAEDVG IYYCMQGTHWPGTFGQGTKVEIK (LC)	WO2016016278A3
mHTT	N1-302.78H12	NGYYWG (CDR-1 (HC)) S1YHNGNTYYNPSLKS (CDR-2 (HC)) PSATYYYGSGTQFHAFDV (CDR-3 (HC)) TGTSRDVGNYNYVS (CDR-1 (LC)) DVSERPS (CDR-2 (LC)) CSYAGSYTFEV (CDR-3 (LC)) QVQLQESGPGLVKPSETLSLTCLVSSYSIS NGYYWGWIRQPPGKGLEWIGS1YHNGNT YYNPSLKSRVIISVDTSKNQFSLKLRSVTA ADTAVYYCAMPSATYYYGSGTQFHAFDV WGQGTTVTVSS (HC) QSALTQPRSVSGSPGQSVTISCTGTSRDVG NYNYVSWYQQHPGEVPKLIIYDVSERPSG VPDRFSGSKSGNTASLTISGLQAEDEADY YCCSYAGSYTFEVFGGGTKLTVL (LC)	WO2016016278A3

mHTT	N1-302.71F6	GYYWS (CDR-1 (HC)) EVNHSGGTNLNSSLKS (CDR-2 (HQ) GYSYDPKYYFDS (CDR-3 (HC)) TGTSSDIGSYDFVS (CDR-1 (LQ) GVNKRPS (CDR-2 (LQ) CSYAGSTTWV (CDR-3 (LQ) QVQLQQWGAGLLKPSETLSLTCAVYGGS LSGYYWSWIRQPPGKGLEWIGEVNHSGG TNLNSSLKSRVI1SVDKSKKQFSLKLSSVT AADTAMYFCARGYSYDPKYYFDSWSQGT LVTVSS (HQ QSALTQPASVSGSPGQAITISCTGTSSDIGS YDFVSWYQQDPGKAPKVIIYGVNKRPSG VSNRFSGSKSGNTASLTISGLQADDEADY YCCSYAGSTTWVFGGGTKLTVL (LQ	WO2016016278A3
mHTT	NI-302.11H6	TYSFT (CDR-1 (HQ) W1SAYNGHTNYVDSFQG (CDR-2 (HQ) VDTTYYYYGMDV (CDR-3 (HQ) ALRFGSVSSSYYPS (CDR-1 (LQ) STNTRSS (CDR-2 (LQ) VLYMGSGIGV (CDR-3 (LQ) EVQLVQSGAVMKKPGDSVRVSCRASTYS FSTYSFTWVRQVPGQGLEWMGWISAYNG HTNYVDSFQGRLTLTTDTSASTAYMELRS LRSDDTAIYYCAAVDTTYYYYGMDVWG QGTLVTVSS (HC) QTVVTQEPTFSVSPGGTVTLTCALRFGSVS SSYYPSWFQQTPGQAPRTLIYSTNTRSSGV PARFSGS1LGNKAALTIAGAQANDEADYY CVLYMGSG1GVFGGGTKLTVL (LQ	WO2016016278A3
mHTT	NI-302.3D8	TYAMS (CDR-1 (HQ) A1SATGGSTFYAESVKG (CDR-2 (HQ) GSTAVYLFDS (CDR-3 (HQ) RASQD1RNFLAW (CDR-1 (LQ) AASTLQS (CDR-2 (LQ) QQFYNYPPT (CDR-3 (LQ) EVQLVQSGGGLVQPGGSLRLSCEASGFIF KTYAMSWVRQLPGRGLEWVSAISATGGS TFYAESVKGRLTISRDTAKNTVYLQMNNL RAEDTAMYYCAKGSTAVYLFDSWGQGT LVTVSS (HQ DIQMTQSPSSLSASVGDRVTLTCRASQDIR NFLAWIQQKPGKPPKSLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLHPEDFATYYCQQ FYNYPPTFGQGTKVEIK (LC)	WO2016016278A3

rnHTT	NI-302.18A1	TDYYY (CDR-1 (HC)) TIYFGGATYYNPSLRN (CDR-2 (HC)) VGYLDRSGLL (CDR-3 (HC)) RSSQSLLHNNGYNYLD (CDR-1 (LC)) LGSTRAS (CDR-2 (LC)) MQALQTPPT (CDR-3 (LC)) QLQLQESGPGLVKPSEALSLTCTVSGGSIT TDYYYWGWIRQSPGKGLEWVGTIYFGGA TYYNPSLRNRVSISVDTSNTRLSLRLISLSA ADTAVYYCARVGYLDRSGLLVGQGTLVT VSS (HC) ErVLTQSPLSVPVTPGEPASISCRSSQSLLH NNGYNYLDWYLKKPGQSPQLL1YLGSTR ASGVPDRFSASGSGTDFTLEISRVEAEDVG VYYCMQALQTPPTFGRGTKVEIK (LC)	WO2016016278A3
rnHTT	NI-302.8F1	NAWMN (CDR-1 (HC)) H1RTQAEGGTSDYAAPVKG (CDR-2 (HC)) PPYYYYYGLDV (CDR-3 (HC)) TGASSDVGTYDLVS (CDR-1 (LC)) EVNKRPS (CDR-2 (LC)) CSYAGYSTV (CDR-3 (LC)) EVQLVESGGGLVKPGGSLT1SCAASGFTFS NAWMNWVRQAPGKGLEWVGHIRTQAEG GTSDYAAPVKGRFTISRDDSKNTLYLQMN SLKTEDTAVYYCIPPPYYYYYGLDVWGQ GTTVTVSS (HC) QSALTQPASVSGSPGQSITISCTGASSDVG TYDLVSWYQQHPGKAPKLIIYEVNKRPSG VSYRFSASKSANTASLTISGLQAEDEAEYY CCSYAGYSTVFGGGTKLTVL (LC)	WO2016016278A3
rnHTT	NI-302.52C9	DTYMS (CDR-1 (HC)) GIHAGGETYYADSVKG (CDR-2 (HC)) HYYGNDDDTDY (CDR-3 (HC)) RSSQSLLHSNGYNYLD (CDR-1 (LC)) LGSTRAS (CDR-2 (LC)) LQAQQ1PWT (CDR-3 (LC)) EVQLVQSGGGLVQPGGSLRLSCAGSGFTV SDTYMSWVRQAPGKGLEWVSGIHAGGET YYADSVKGRFTISRDNSKNTLYLQMNRLT PEDTAVFYCARHYYGNDDDTDYWGQGT LVTVSS (HC) DVVMTQSPLSLPVTPGEPASISCRSSQSLL HSNGYNYLDWYVQKPGQSPQLLIYLGST RASGVPDRFSGSGSGTDFTLKISRVEAEDV GVYYCLQAQQIPWTFGQGTKVEIK (LC)	WO2016016278A3

mHTT	NI-302.46C9	SGAYY (CDR-1 (HQ) RVYPTWSTNYNPSLES (CDR-2 (HC)) EAPGDYDAAPLAY (CDR-3 (HC)) RASQYISHYLN (CDR-1 (LQ) AASSLQS (CDR-2 (LQ) QQSYTTPRT (CDR-3 (LQ) QVQLQESGPGLVKPSQTLSLTCTVSGASV SSGAYYWSWIRQPAGKRLEWIGRVYPTW STNYNPSLESRVTISLDTSNNQFSLKLTSLT AADTAVYYCAREAPGDYDAAPLAYWGQ GTLVTVSS (HC) DIQMTQSPSSLSASVGDRVTITCRASQYIS HYLNWYRQKPGKAPQLVIYAASSLQSEVP SRFSGSGSGPEFTLTISSLQPEDFATYYCQQ SYTTPRTFGQGTKLE1K (LC)	WO2016016278A3
mHTT	NI-302.64E5	QAWMS (CDR-1 (HC)) RIKTKTEGEATDYAAPVRG (CDR-2 (HQ) TGVLAAAVDVY (CDR-3 (HQ) KSSQSLFYSYNNENYLA (CDR-1 (LQ) WASTRES (CDR-2 (LQ) QQYYSTPQT (CDR-3 (LQ) EVQLVETGGGLVKPGGSLRLSCAASGFTF DQAWMSWVRQVPGKGLEWVGRIKTKTE GEATDYAAPVRGRFT1SRDDSEDTVFLQM NSLKTEDTALYYCTSTGVLAAAVDVYWG QGTLVTVSS (HC) DIQLTQSPDSLAVSLGERATMTCKSSQSLF YSYNNENYLAWYQQRPGQPPKLLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCQQYYSTPQTFGQGTKVDIK (LC)	WO2016016278A3
mHTT	NI-302.7D8	NYAIN (CDR-1 (HQ) W1NTITGHPTYAQGFKG (CDR-2 (HC)) TYSNYGEFDY (CDR-3 (HQ) TGTSSDVGSYNLVS (CDR-1 (LQ) EGSERPS (CDR-2 (LQ) CSYAGTTTFVL (CDR-3 (LQ) QVQLVQSGSELKKPGASVKVSCKASGYN FNNYAINWLRQAPGQGLEWMGWINTITG HPTYAQGFKGRFVFSLDTSVSTAYLQ1SSL KPEDTAVYYCARTYSNYGEFDYWGQGTL VTVSS (HC) QSALTQPASVSGSRGQSITISCTGTSSDVGS YNLVSWYQQYPGKAPKL1IHEGSERPSGV SNRFSGSKSGNTASLTISGLQAEDEADYY CCSYAGTTTFVLFGGGTKLTVL (LC)	WO2016016278A3

mHTT	NI-302.72F10	SY AMS (CDR-1 (HQ) DISG1GSNTYYADSVKG (CDR-2 (HQ) DRKRSGWYEQ (CDR-3 (HQ) RASQS1SAYLG (CDR-1 (LQ) DASIRAT (CDR-2 (LQ) HQRSKWPLT (CDR-3 (LQ) EVQLVETGGGFVQPGGSLRLSCAASGFNF GSYAMSWVRQAPGKGLEWVSD1SGIGSN TYYADSVKGRFTISRDNSDNTLYLDMSSL RAEDTARYYCAKDRKRSGWYEQWGQGT LVTVSS (HC) EIVMTQSPATLTLSPGERATLSCRASQS1SA YLGWYQQKPGQAPRLLIYDASIRATGIPD RFSGSGSGTDFTLT1SSLEPEDSAVYYCHQ RSKWPLTFGGGTKVE1K (LC)	WO2016016278A3
mHTT	NI-302.4A6	A YAMS (CDR-1 (HQ) TISGSGGSTYYADSVKG (CDR-2 (HQ) VTTELYGANSYYYYMDV (CDR-3 (HQ) RASQSVVSRYLA (CDR-1 (LQ) GASSRAT (CDR-2 (LQ) QLYGNSQT (CDR-3 (LQ) EVQLVESGGGLVQPGGSLRLSCAASGFTF SAYAMSWVRQAPGKGLEWVSTISGSGGS TYYADSVKGRFSISRDNSKNTLYLQMNSL RAEDTAVYFCAKVTTELYGANSYYYYMD VWGKGTTVTVSS (HQ EIVLTQSPGTLSLSPGERATLSCRASQSVV SRYLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAMYYC QLYGNSQTFGQGTKVE1K (LQ	WO2016016278A3
mHTT	NI-302.12H2	NYAMG (CDR-1 (HQ) VISGTGGSTYYADSVKG (CDR-2 (HQ) DLRK1SGPLYYYGMDV (CDR-3 (HQ) RASQSVSSGYLA (CDR-1 (LQ) GASTRAT (CDR-2 (LQ) QHYGASSYT (CDR-3 (LQ) EVQLVQSGGGLVQPGGSLRLSCEASGFTF SNYAMGWVRQAPGKGLEWVSVISGTGGS TYYADSVKGRFTISRDNSMNTLYLQMNSP RADDTAVYYCAKDLRK1SGPLYYYGMDV WGQGTTVTVSS (HC) EIVLTQSPGTLSLSPGERATLSCRASQSVSS GYLAWYQQKPGQAPRLLIYGASTRATGIP DRFSGSGSGTDFTLT1SRLEPEDFAVYYCQ HYGASSYTFGQGTKLE1K (LC)	WO2016016278A3

mHTT	NI-302.8M1	IYYMH (CDR-1 (HC)) GISPSGAHTMYAQNFQG (CDR-2 (HC)) GSTVTNYRPFDY (CDR-3 (HC)) RASQDISNYLA (CDR-1 (LC)) AASTLQS (CDR-2 (LC)) QNYNSGPPP (CDR-3 (LC)) EVQLVQSGAEVKKPGASVKVSCKASGYT FTIYYMHWVRQAPGQGLEWMGGISPSGA HTMYAQNFQGRVTVTRDTSTSTVYMELS SLRSEDTAVYYCARGSTVTNYRPFDYWG QGTLVTVSS (HC) DIQMTQSPSSLS AS VGDR VT1TCR ASQD1S NYLAWYQQKPGKVPKLLIFAASTLQSGVP SRFGGSGSGTDFTLTISSLQPEDVATYYCQ NYNSGPPPFGPGTKVDIK (LC)	WO2016016278A3
mHTT	N1-302.33C11- PIMC	DFGMH (CDR-1 (HC)) LIWYDGGYKYYADSVKG (CDR-2 (HC)) HLEYCSRTTCYLGH (CDR-3 (HC)) RASQGISDYLA (CDR-1 (LC)) AASTLQT (CDR-2 (LC)) QQLKTYPYT (CDR-3 (LC)) QVQLVESGGGVVQPGNSLRLSCAASGFRF SDFGMHWVRQAPGKGLEWLALIWYDGG YKYYADSVKGRFT1SRDNSKNTMFLQMN SLRAEDTAVYYCATHLEYCSRTTCYLGH WGQGTLVTVSS (HC) DIQLTQSPSFLSASVGDTVTFTCRASQG1S DYLAWFQQKPG1APKLLIYAASTLQTGVP SRFSGSGSGTEFTLTIRSLQSEDFGTYYCQ QLKTYPYTFGQGTKLEIK (LC)	WO2016016278A3
mHTT	N1-302.74C11- PIMC	GYFLH (CDR-1 (HC)) WINPNSGDTNYAE (CDR-2 (HC)) EAPDPGAETDV (CDR-3 (HC)) SGDAVPKQYIY (CDR-1 (LC)) KDTQRPS (CDR-2 (LC)) QSADSSATWV (CDR-3 (LC)) QVQLVQSGTEVQKPGASVKVSCKASGYS FTGYFLHWVRQAPGQGLEWMGWINPNS GDTNYAEKFRGRHMTRDTSVSTAHMELS SLRFDDTALYYCTREAPDPGAETDVWGQ GTTVTVSS (HC) SYELTQPPSVSVSPGQTARITCSGDAVPKQ YIYWYQQKPGQAP1LVIYKDTQRPSGIPER FSGSNSGTTVTLTITGVQADDEGDYYCQS ADSSATWVFGGGTKLTVL (LC)	WO2016016278A3

mHTT	NI-302.39G12- PIMC	NY AIT (CDR-1 (HC)) V1YRDGRTYYGDSVRG (CDR-2 (HC)) AHGQYYYGVDV (CDR-3 (HC)) RSSQSLLHSNGYNYLD (CDR-1 (LQ) LSSNRPS (CDR-2 (LC)) MQSLQTFT (CDR-3 (LC)) EVQLVESGGGLVHPWGSLRLSCAASGFSV SNYAITWVRRAPGKGLQYISVIYRDGRTY YGDSVRGRFTISRDDSKNTLYLQMNSLRF EDTAVYYCARAHGQYYYGVDVWGQGTT VTVSS (HC) DIVMTQSPLSLSVSPGEPASISCRSSQSLLH SNGYNYLDWYRQKPGQSPQLLIYLSSNRP SGVPDRFSASGSGTEFTLQ1SRVEAEDVGV YYCMQSLQTFTFGGGTKVE1K (LC)	WO2016016278A3
mHTT	NI-302.15E8	SYSMN (CDR-1 (HC)) YTSSSRSNTKKYADSVKG (CDR-2 (HC)) AGDFGELLTGEGYYGMDV (CDR-3 (HQ) SGDELGDKYVG (CDR-1 (LQ) QDAKRPS (CDR-2 (LQ) QAWDSGTMV (CDR-3 (LC))	WO2016016278A3
mHTT	NI-302.64E5- PIMC	QAWMS (CDR-1 (HC)) RIKTKTEGEATDYAAPVRG (CDR-2 (HQ) TGVLAAAVDVY (CDR-3 (HQ) KSSQSLFYSYNNENYLA (CDR-1 (LQ) WASTRES (CDR-2 (LQ) QQYYSTPQT (CDR-3 (LQ) EVQLVESGGGLVKPGGSLRLSCAASGFTF DQAWMSWVRQVPGKGLEWVGRIKTKTE GEATDYAAPVRGRFT1SRDDSEDTVFLQM NSLKTEDTALYYCTSTGVLAAAVDVYWG QGTLVTVSS (HC) DIVMTQSPDSLAVSLGERATMTCKSSQSL FYSYNNENYLAWYQQRPGQPPKLL1YWA STRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQYYSTPQTFGQGTKVEIK (LQ	WO2016016278A3

mHTT	NI-302.72F10- P1MC	SYAMS (CDR-1 (HO) DISG1GSNTYYADSVKG (.CDR-2 (IIQ) DRKRSGWYEQ (CDR-3 (IIQ) RASQS1SAYLG (CDR-1 (1.0) DAS1RAT (CDR-2 (LO) IIQRSKWPl.T (CDR-3 (1.0) EVQLVESGGGFVQPGGSLRLSCAASGFNF GSYAMSWVRQAPGKGLEWVSD1SGIGSN TYYADSVKGRFTISRDNSDNTLYLDMSSL RAEDTARYY( A K DR KRSGWYEQWGQGT LVTVSS (HC) E1VLTQSPATLTLSPGERATLSCRASQSISA YLGWYQQKPGQAPRLLIYDASIRATG1PD RFSGSGSGTDFTLTISSLEPEDSAVYYCHQ RSKWPLTFGGGTKVEIK (LC)	WO2016016278A3
IL-Ια	MABpl		The Lancet Oncology, 2017 15 (6): 656-66
IL-Ιβ	Canakinumab		Mabs, 2015, 7(6): 1151-60
IL-1		[S/K/RJYDMS (CDR-1 (HQ) Y [I/V]S[S/H |GG[G/A|GTY YPD[T/S 1 [V/AJK G (CDR-2 (HQ) GGV[T/Y]KG[Y/CJFD[V/E/L/M/Q/Y] (CDR-3 (HC)) RASGNIfH/Y/W] [N/G/T/Q/E/H/D/K ||Y/W]L[ T/A/NI (CDR-1 (LQ) [N/Q/D]K[T/N/I/E/S]L[A/M/E][D/E/S/A| (CDR-2 (LQ) Q[H/Q|FW[ S/N/T/K/R/M][I/L|P[Y/A](T/I/N) (CDR-3 (LQ)	WO2011143562A2
IL-RAP	CAN04	Q '/ a L Q □ S G P E 5. k K P G A 6 V K t ¢3 ft A s GYAFSSSWMNWVKORPGKGLEWJG R1YFGDGNVHYSGKFK3KATLTA0K S S S I A Y M a u £· £· I. T $ $ q S A V Y F 0 G £ G Y L 0 P M 0 Y W G a G T S V T V s s (HQ ÖIOMTOTTSSI. SASi.GÜRVT)SCSA SaG)NNYi.NWraQXPDGTVKl). )HY TSGLHAGVPSRFSeseSQTOYSLil 5NLEPgeVATYVCa«YSIl.PWrFG GGTKI. (LQ	WO2015132602

IL-Ιβ	hlB12.4H4	DYGVS (CDR-1 (HO) L1WGGGDTYYNSPLKS (CDR-2 (HO) QRTLWGYDLYGMDY (CDR-3 (HC)) 1TSTD1DVDMN (CDR-1 (IX?)) QGNTLRP (CDR-2 (LC)) LQSDNLPLT (CDR-3 (1.,O) (HO i VÖ®. X J\sb' PC< <A Q' L LI Q pj; 1 è IE (LC)	WO2011047266A1
IL-Ια	H3D12	NYGMN (CDR-1 (HQ) W1NTYTGESTYADDFKG (CDR-2 (HQ) GIYYYGSSYAMDY (CDR-3 (HQ) RASQD1SNCLN (CDR-1 (LQ) YTSRLHS (CDR-2 (LQ) QQGKTLPYA (CDR-3 (LQ) CKk?i cxas^yïfr KïGjwsr/FGAS’ts jMOTFK^A^tgTMBÏATLQÏlSiLitSiS) SS (HQ (LC)	WO 2010087972 A2
IL-Ιβ	Gevokizumab	TSGMGVG (CDR-1 (HO) HIWWDGDESYNPSLK (CDR-2 (HQ) [A/V/F/K/N |[R/K]YDPPWFVD (CDR-3 (HC)) RASQDISNYLS (CDR-1 (LQ) YTSKLHS (CDR-2) (LC)) LQGKMLPWT (CDR-3 (LQ) DIQMTQXTSSLSASXGDRVT1XCRASQDIS NTLSWYQQKPXXXVKLL1YYTSKLHSGVP SRFSGSGSGTDYXLTISNLEQEDIATYFCL QGKMLPWTFGGGTKLEIK (HC) QVXLXESGPGXXKPSQTLSLTCSFSGFSLS TSGMGVGWIRQPSGKGLEWLHAIWWDG DESYNPSLKXXLTISKDTSKNQVXLKITSV XXXDTAXYFCARXXYDPPWFVDWGQGT LVTVSS (LC)	WO 2007002261 A2
IL-Ιβ	LY2189102		Diabetes Care 2013 Aug; 36(8): 2239- 2246.

IL-Ιβ	CYT013
IL-Ιβ	sIL-lRII
IL 1 RAP	Rilonacept		WO 2008051496 A2
IL-1RI	Anakinra
IL-1RI	MED1-8968
11-6	Tocilizumab
11-6	Sarilumab
11-6	Siltuximab
IL-6	Vobarilizumab (ALX-0061)		Arthritis Research & Therapy 2015, 17:135
IL-6	Olokizumab
IL-6	Elsnimomab
IL-6	Ctazakizumab
IL-6	Sirikumab
IL-6	Abl	NYYVT (CDR-1 (HQ) IIYGSDETAYATSAIG (CDR-2 (HC)) DDSSDWDAKFNL (CDR-3 (HC)) QASQS1NNELS (CDR-1 (LC)) RASTLAS (CDR-2 (LC)) QQGYSLRNIDNA (CDR-3 (LC)) EVQLVESGGGLVQPGGSLRLSCAASGFSL SNYYVTWVRQAPGKGLEWVGIIYGSDET AYATSA1GRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARDDSSDWDAKFNLWGQG TLVTVSS (HC) AIQMTQSPSSLSASVGDRVTITCQASQSIN NELSWYQQKPGKAPKLL1YRASTLASGVP SRFSGSGSGTDFTLTISSLQPDDFATYYCQ QGYSLRNIDNAFGGGTKVEIKR (LC)	WO 2011066371 A2; WO 2010065079 A2
TNF-a	Etanercept

IL12 and or 23		DYY[M7L]H (CDR-1 (HC)) WIDPENGDTE[ Y/H/S]APKFQG (CDR-2 (HC)) [ C/A/N/D/E/Q/G/H/I/L/P/V] KELRYFD V (CDR-3 (HQ) RA[S/P][Q/R]SISINLH (CDR-1 (LC)) FA[S/R]QS[1/T]S (CDR-2 (LQ) QQSNS[WZF]PLT (CDR-3 (LC)) TV A APS V Fl F PPSDEQLK SGTASVVCLLNN FY PREAKV QWKV D N ALQSGNSQES VTEQ DS K DST YS1 ,SSTLT LS KA D YEK Η K V Y ACE VTHQGLSSPV IKSFNRGEC (HC) QPKAAPSVTIFPPSSEELQANKATLVCI..IS DFYPGAVTVAWKADSSPVKAGVETTTPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTV APTECS (EC)	US 8563697 B2
IL12 and or 23	Ustekinumab
IL 12 and or 23	Briakinumab
IL 12 and IL23	R41F11	GFTFNNSWMT (CDR-1 (HQ) SITNTGGSTYYPDSVKG (CDR-2 (HQ) EDNSGYDY (CDR-3 (HC)) LASEDIYSNLA (CDR-1 (LQ) YANSLND (CDR-2 (LC)) QQNYYSPPT (CDR-3 (LC)) EVQLVESGGGLVQPGRSLKLSCVASGFTF NNSWMTWIRQAPGKGLECVAS1TNTGGS TYYPDSVKGRFTISRDNAKSTLYLQMNSL RSEDTATYYCSREDNSGYDYWGQGVMV TVSS (HC) DIQMTQSPASLSASLGETVT1ECLASED1YS NLAWYQQKPGKSPQLLIYYANSLNDGVP SRFSGSGSGTQYSLKINSLQSEDVSIYFCQ QNYYSPPTFGGGTKLELKR (LC)	W02008I06134

IL12 and 1L23	R8B10/HU8B10	GFDFNSYMS (CDR-1 (HC)) DINSKSYNYATYYADSVKD (CDR-2 (HQ) HHSDYFEY (CDR-3 (HC)) LASEDIYNNLA (CDR-1 (LQ) HASSLQD (CDR-2 (LC)) LQDSEYPPT (CDR-3 (LC)) EVKLVESGGGLEQPKGSLKLSCTVSGFDF NSYGMSWVRQAPGKGLDLVADINSKSYN YATYY ADS VKDRFTISRDDSQSMV YLEM DNLKTEDTALYYCTVHHSDYFEYWGQGV MVTVSS (HC) D1QMTQSPASLSASLGETVS1ECLASED1YN NLAWYQQKPGKSPQLLIYHASSLQDGVPS RFSGSGSGTQYSLKINSLESEDAATYFCLQ DSEYPPTFGGGTKLELKR (LC)	W02008106134
IL12 and 1L23	R3C11	GFSLSTYGMGVG (CDR-1 (HQ) NIWWDDDKYYNPSLKN (CDR-2 (HQ) IDAHPMGITTPDYYVVDA (CDR-3 (HC)) KASQNVGSNVD (CDR-1 (LQ) KASNRYT (CDR-2 (LQ) MQSNSYPLT (CDR-3 (LQ) QVTLKESGPGILQPSQTLSLTCTFSGFSLST YGMGVGWIRQPSGKGLEWLANIWWDDD KYYNPSLKNRLTISKDTSNNQAFLKITNV DTADTATYYCARIDAHPMGITTPDYYVV DAWGQGASVTVSS (HC) DIVMTQSPTSMSISLGDRVTMNCKASQNV GSNVDWYQQKTGQSPKLLIYKASNRYTG VPDRFTGSGSGTDFTFTISNMQAEDLSVY YCMQSNSYPLTFGSGTKKEIKR (LC)	W02008106134
IL 12 and IL23	M20E5	GYTFTSYWIH (CDR-1 (HQ) EIDPSDSYTYYNQKFKD (CDR-2 (HQ) SLYDYDGVPD (CDR-3 (HQ) HASQGIDNNIG (CDR-1 (LQ) HGTNLED (CDR-2 (LQ) VQYAQFPFT (CDR-3 (LQ) QVPLQQPGTELVKPGASVKLSCKASGYTF TSYWIHWVLQRPEQGLEWIGEIDPSDSYT YYNQKFKDKATLTVDKSSTTAFLQLSSLT SEDSAVYYCARSLYDYDGVPDWGQGTLV TVSA (HQ DILMTQSPSSMSVSLGDTVSITCHASQGID NNIGWLQQKPGKSFKGLIYHGTNLEDGVP SRFSGSGSGTDYSLTISSLESEDFADYYCV QYAQFPFTFGGGTKLE1RR (LQ	WG2008106134

IL12 and 1L23	Hu20D7	GYTFTNYAMN (CDR-1 (HQ) WINTYTGEPTYSDDFKG (CDR-2 (HQ) GGGYDEDYFDY (CDR-3 (HQ) KSSQSLFNSINQKTYLA (CDR-1 (LQ) FASTRES (CDR-2 (LQ) QQHYDTPWT (CDR-3 (LQ) QVQLVQSGAEVKKPGASVKVSCKASGYT FTNYAMNWVRQAPGQGLEWMGWINTYT GEPTYNEKFKGRVTMTTDTSTSTAYMELR SLRSDDTAVYYCARGGGYDEDYFDYWG QGTLVTVSS (HC) DIVMTQSPDSLAVSLGERATINCKSSQSLF NSINQKTYLAQYQQKPGQPPKLLIYFASTR ESGVPDRFSGSGSGTDFTLT1SSLQAEDVA VYYCQQHYDTPWTFGQGTKVEIKRT (LC)	W02008106134
Clq	ANX005 (ANX- Ml)	GYHFTSYWMH (CDR-1 (HQ) VIHPNSGSINYNEKFES (CDR-2 (HQ) ERDSTEVLPMDY (CDR-3 (HQ) RASKSINKYLA (CDR-1 (LQ) SGSTLQS (CDR-2 (LQ) QQHNEYPLT (CDR-3 (LQ) QVQLQQPGAELVKPGASVKLSCKSSGYHF TSYWMHWVKQRPGQGLEWIGV1HPNSGS 1NYNEKFESKATLTVDKSSSTAYMQLSSLT SEDSAVYYCAGERDSTEVLPMDYWGQGT SVTVSS (HC) DVQITQSPSYLAASPGETITINCRASKS1NK YLAWYQEKPGKTNKLLIYSGSTLQSG1PSR FSGSGSGTDFTLTISSLEPEDFAMYYCQQH NEYPLTFGAGTKLELK (LC)	WO 2015006504 Al; EP 3019523 A4
Clq	JL-1		J Clin Invest. 2004; 114(5):679-688.
C5a	Eculizumab	GYIFSNYWIQ (CDR-1 (HQ) EILPGSGSTEYTENFKD (CDR-2 (HQ) YFFGSSPNWYFDV (CDR-3 (HQ) GASENIYGALN (CDR-1 (LQ) GATNLAD (CDR-2 (LQ) QNVLNTPLT (CDR-3 (LC)) QVQLVQSGAEVKKPGASVKVSCKASGYIF SNYWIQWVRQAPGQGLEWMGE1LPGSGS TEYTENFKDRVTMTRDTSTSTVYMELSSL RSEDTAVYYCARYFFGSSPNWYFDVWGQ GTLVTVSS (HC) DIQMTQSPSSLSASVGDRVTITCGASENIY GALNWYQQKPGKAPKLLIYGATNLADGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQ NVLNTPLTFGQGTKVE1K (LC)	WO 2017044811 Al

		FYAMS (CDR-1 (HQ) SLSRGGSTYYPDSVKG (CDR-2 (HQ) EGATALYAMDY (CDR-3 (HQ) RSSQSIVHSNGNTYLQ (CDR-1 (LQ) KVSNRFS (CDR-2 (LC)) FQGSHVPWT (CDR-3 (LQ)
APOE	15G8	[E/Q]V[K/Q]L[V/Q]ESGGGLVKPGGSLKLS CAASGFTFSFYAMSWVRQTP[E/D]KRLEW VASLSRGGSTYYPDSVKGRFT1SR[D/S]NA KNTLYL[G/Q1MSSL[R/K]SEDTAMYYCAR EGATALYAMDYWGQGTMVTVSS (HC)	WO2012109280 A2
		DVfL/VJMTQSPLSLPVTLGQPASISCRSSQS 1VHSNGNTYLQW[YZF]LQRPGQSPR[L/R]L LYKVSNRFSGVPDRFSGSGSGTDFTLKISR VEAEDVGVYYCFQGSHVPWTFGQGTKVE IK (LC)
APOE			US 20170218058 Al
APOE			WO 2013168174 Al
APOE			CA 2819679 Al

In a preferred embodiment, the multispecific antibody comprises one binding site binding to human tau protein selected from the group comprising binding fragments of tau 13 and binding fragments of 5A6 and one binding site 5 binding to u-synuclein selected from the group comprising binding fragments of Syn211 and binding fragments of H3C.

In another preferred embodiment, the multispecific antibody comprises one binding site binding to human tau protein selected from the group comprising binding fragments of taul3 and binding fragments of 5A6 and one binding site 10 binding to Clq selected from the group comprising binding fragments of JL-1 and binding fragments produced by the hybridomas 23B6C8, 5B5C22, 12A5B7 and 4A4B11.

Also preferred is the multispecific antibody comprising one binding site binding to Clq selected from the group comprising binding fragments of JL-1 and 15 binding fragments produced by the hybridomas 23B6C8, 5B5C22, 12A5B7 and 4A4B11 and one binding site binding to α-synuclein selected from the group comprising binding fragments of Syn211 and binding fragments of H3C.

The skilled person will also know that in the prior art a number of epitopes on the human tau protein have been identified that are suitable for targeting. The present invention includes multispecific binding molecules that specifically bind these epitopes in human (phosphorylated) tau protein.

Table 5: Epitopes of tau or phosphorylated (p)tau suitable for targeting.

Epitope	Reference
2-18	WO2014031694
9-18	WO2015081085
2-24	WO2016196726
7-9	WO2016137811
7-13	W02014008404
15-24	WO2015081085
17-28	WO2015081085
19-33	WO2016196726
19-42	WO2016196726
24-46	CA2902026
25-30	WO2014008404/ WO2015200806A2
28-44	WO2016196726
35-49	US20150252102Al
37-51	WO2016196726
P59+S61+E62+T63 +D65+K67	WO2015197820
100-114	WO2016196726
109-123	WO2016196726
118-132	WO2016196726

150-158	WO2015081085
154-168	WO2016196726
172-177	WO2016196726
197-206	WO2017005732
217-231	WO2016196726
pT231	WO2012149334A2
268-273	WO2013041962A1/ WO2016079597Al
299-304	WO2013041962A1/ WO2016079597A1
330-335	WO2013041962A1/ WO2016079597A1
362-367	WO2013041962A1/ WO2016079597Al
294-305	WO2013041962Al
306-320	WO2014008404
312-322	WO2016137811
D314+L315+K317	WO2015197820
L315+K317+P312	WO2015197820
337-343	US20150252102A1
387-397	US20130224287Al
pS396/pS404	WO2017009308A2
397-411	WO2016196726
pS413	WO2013180238A1
pS422	WO2015091656A1

Antibody Engineering

Further included are embodiments in which the antibodies of the invention and antigen-binding fragments thereof are engineered antibodies to include modifications to framework residues within the variable domains of the parental monoclonal antibody, e.g. to improve the properties of the antibody or fragment. Typically, such framework modifications are made to decrease the immunogenicity of the antibody or fragment. This is usually accomplished by replacing non-CDR residues in the variable domains (i.e. framework residues) in a parental (e.g. rodent) antibody or fragment with analogous residues from the immune repertoire of the species in which the antibody is to be used, e.g. human residues in the case of human therapeutics. Such an antibody or fragment is referred to as a humanized antibody or fragment. In some cases it is desirable to increase the affinity, or alter the specificity of an engineered (e.g. humanized) antibody. One approach is to backmutate one or more framework residues to the corresponding germline sequence. More specifically, an antibody or fragment that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody or fragment framework sequences to the germline sequences from which the antibody or fragment is derived. Another approach is to revert to the original parental (e.g., rodent) residue at one or more positions of the engineered (e.g. humanized) antibody, e.g. to restore binding affinity that may have been lost in the process of replacing the framework residues. (See, e.g., U.S. Patent No. 5,693,762, U.S. Patent No. 5,585,089 and U.S. Patent No. 5,530,101.)

In certain embodiments, the antibodies of the invention and antigenbinding fragments thereof are engineered (e.g. humanized) to include modifications to in the framework and/or CDRs to improve their properties. Such engineered changes can be based on molecular modelling. A molecular model for the variable region for the parental (non-human) antibody sequence can be constructed to understand the structural features of the antibody and used to identify potential regions on the antibody that can interact with the antigen. Conventional CDRs are based on alignment of immunoglobulin sequences and identifying variable regions.

Kabat et al., (1991) Sequences of Proteins of Immunological Interest. Kabat, et al.; National Institutes of Health, Bethesda, Md. ; 5^lh ed.; NIH Puhi. No. 91-3242; Kabat Adv. Prot. Chem. 1978, 32:1-75; Kabat, et al., J. Biol. Chem. 1977, 252:66096616. Chothia and coworkers carefully examined conformations of the loops in crystal structures of antibodies and proposed hypervariable loops. Chothia, et al., () J Mol. Biol. 1987, 196:901-917 or Chothia, et al., Nature 1989, 342:878-883. There are variations between regions classified as “CDRs” and “hypervariable loops”. Later studies (Raghunathan et al, J. Mol Recog. 2012, 25, 3, 103-113) analyzed several antibody-antigen crystal complexes and observed that the antigen binding regions in antibodies do not necessarily conform strictly to the “CDR” residues or “hypervariable” loops. The molecular model for the variable region of the nonhuman antibody can be used to guide the selection of regions that can potentially bind to the antigen. In practice the potential antigen binding regions based on model differ from the conventional “CDR”s or “hyper variable loops. Commercial scientific software such as Discovery Studio (BIOVIA, Dassault Systemes) can be used for molecular modeling. Human frameworks can be selected based on best matches with the non-human sequence both in the frameworks and in the CDRs. For FR4 (framework 4) in VH, VJ regions for the human germlines are compared with the corresponding non-human region. In the case of FR4 (framework 4) in VL, J-kappa and J-Lambda regions of human germline sequences are compared with the corresponding non-human region. Once suitable human frameworks are identified, the CDRs are grafted into the selected human frameworks. In some cases certain residues in the VI.-VII interface can be retained as in the non-human (parental) sequence. Molecular models can also be used for identifying residues that can potentially alter the CDR conformations and hence binding to antigen. In some cases, these residues are retained as in the non-human (parental) sequence. Molecular models can also be used to identify solvent exposed amino acids that can result in unwanted effects such as glycosylation, deamidation and oxidation. Develop ability filters can be introduced early on in the design stage to eliminate/minimize these potential problems.

Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as deimmunization and is described in further detail in U.S. Patent No. 7,125,689.

In particular embodiments, it will be desirable to change certain amino acids containing exposed side-chains to another amino acid residue in order to provide for greater chemical stability of the final antibody, so as to avoid deamidation or isomerization. The deamidation of asparagine may occur on NG, DG, NG, NS, NA, NT, QG or QS sequences and result in the creation of an isoaspartic acid residue that introduces a kink into the polypeptide chain and decreases its stability (isoaspartic acid effect). Isomerization can occur at DG, DS, DA or DT sequences. In certain embodiments, the antibodies of the present disclosure do not contain deamidation or asparagine isomerism sites.

For example, an asparagine (Asn) residue may be changed to Gin or Ala to reduce the potential for formation of isoaspartate at any Asn-Gly sequences, particularly within a CDR. A similar problem may occur at a Asp-Gly sequence. Reissner and Aswad Cell. Mol. Life Sci. 2003, 60:1281. Isoaspartate formation may debilitate or completely abrogate binding of an antibody to its target antigen. See, Presta J. Allergy Clin. Immunol. 2005, 116:731 at 734. In one embodiment, the asparagine is changed to glutamine (Gin). It may also be desirable to alter an amino acid adjacent to an asparagine (Asn) or glutamine (Gin) residue to reduce the likelihood of deamidation, which occurs at greater rates when small amino acids occur adjacent to asparagine or glutamine. See, Bischoff & Kolbe J. Chromatog. 1994, 662:261. In addition, any methionine residues (typically solvent exposed Met) in CDRs may be changed to Lys, Leu, Ala, or Phe or other amino acids in order to reduce the possibility that the methionine sulfur would oxidize, which could reduce antigen-binding affinity and also contribute to molecular heterogeneity in the final antibody preparation. Id. Additionally, in order to prevent or minimize potential scissile Asn-Pro peptide bonds, it may be desirable to alter any Asn-Pro combinations found in a CDR to Gin-Pro, Ala-Pro, or Asn-Ala. Antibodies with such substitutions are subsequently screened to ensure that the substitutions do not decrease the affinity or specificity of the antibody for its target, or other desired biological activity to unacceptable levels.

Table 6. Exemplary stabilizing CDR variants

CDR Residue	Stabilizing Variant Sequence
Asn-Gly	Gln-Gly, Ala-Gly, or Asn-Ala
(N-G)	(Q-G), (A-G), or (N-A)
Asp-Gly	Glu-Gly, Ala-Gly or Asp-Ala
(D-G)	(E-G), (A-G), or (D-A)
Met (typically solvent exposed)	Lys, Leu, Ala, or Phe
(M)	(K), (L), (A), or (F)
Asn	Gin or Ala
(N)	(Q) or (A)
Asn-Pro	Gin-Pro, Ala-Pro, or Asn-Ala
(N-P)	(Q-P), (A-P), or (N-A)

Antibody Engineering of the Fc region

The antibodies (e.,g., humanized antibodies) and antigen-binding fragments thereof disclosed herein can also be engineered to include modifications within the Fc region, typically to alter one or more properties of the antibody, such as serum, brain ISF or CSF half-life, complement fixation, Fc receptor binding, and/or effector function antigen-dependent cellular cytotoxicity).

Furthermore, the antibodies and antigen-binding fragments thereof disclosed herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more properties of the antibody or fragment. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

The antibodies and antigen-binding fragments thereof disclosed herein also include antibodies and fragments with modified (or blocked) Fc regions to provide altered effector functions. See, e.g., U.S. Pat. No. 5,624,821;

W02003/086310; W02005/120571; W02006/0057702. Such modifications can be used to enhance or suppress various reactions of the immune system, with possible beneficial effects in diagnosis and therapy. Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions), glycosylation or de glycosylation, and adding multiple Fc regions. Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, enabling less frequent dosing and thus increased convenience and decreased use of material. See Presta J. Allergy Clin. Immunol. 2005, 116:731 at 734-35.

In one embodiment, the Fc region of the antibody or antigen-binding fragment of the invention is modified to prevent possible interactions with the neonatal Fc receptor. Such modifications can be used to decrease efflux of the antibody or antigen-binding fragment out of the brain and thus increase brain halflife. (Cooper, P.R., Brain Research, 2013, 1534, 13).

In one embodiment, the antibody or antigen-binding fragment of the invention is an IgG4 isotype antibody or fragment comprising a Serine to Proline mutation at a position corresponding to position 228 (S228P; EU index) in the hinge region of the heavy chain constant region. This mutation has been reported to abolish the heterogeneity of inter-heavy chain disulfide bridges in the hinge region (Angal S. et al., Mol Immunol. 1993, 30: 105-108; position 241 is based on the Kabat numbering system).

In one embodiment of the invention, the hinge region of CHi is modified such that the number of cysteine residues in the hinge region is increased or decreased. This approach is described further in U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of CHi is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody or antigenbinding fragment of the invention is mutated to decrease the biological half-life of the antibody or fragment. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that, the antibody or fragment has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745.

In another embodiment, the antibody or antigen-binding fragment of the invention is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375. Alternatively, to increase the biological half-life, the antibody can be altered within the CHi or Ci. region to contain a salvage receptor binding epitope taken from two loops of a C112 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022.

In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody or antigen-binding fragment. For example, one or more amino acids selected from amino acid residues 233, 234, 235, 236, 237, 268, 269, 270, 254, 254, 294, 297, 298, 300, 318, 320, 322, 327, 329, 331 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand and retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the Ci component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260. Ugurlar, I)., Science, 2018, 359, 6377, 794.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551.

In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351.

In yet another example, the Fc region is modified to decrease the ability of the antibody or antigen-binding fragment of the invention to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to decrease the affinity of the antibody or fragment for an Fey receptor by modifying one or more amino acids at the following positions: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258, 264, 265,

267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295,

296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330,331,

333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416,419,

430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication WO 00/42072. Moreover, the binding sites on human IgGl for FcyRl, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al. J. Biol. Chem. 2001, 276:6591-6604).

In one embodiment of the invention, the Fc region is modified to decrease the ability of the antibody of the invention to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243 and 264. In one embodiment, the Fc region of the antibody or fragment is modified by changing the residues at positions 243 and 264 to alanine. In one embodiment, the Fc region is modified to decrease the ability of the antibody or fragment to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243, 264, 267 and 328.

Antibody Physical Properties

The antibodies and antigen-binding fragments thereof disclosed herein may further contain one or more glycosylation sites in either the light or heavy chain immunoglobulin variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or fragment or an alteration of the pK of the antibody due to altered antigen-binding (Marshall et al. Annu Rev Biochem 1972, 41:673-702; Gala and Morrison, J. Immunol 2004, 172:5489-94; Wallick et al, J Exp Med 1988, 168:1099-109; Spiro Glycobiology, 2002, 12:43R-56R; Parekh et al, Nature 1985, 316:452-7; Mimura et al., Mol Immunol 2000, 37:697-706). Glycosylation has been known to occur at motifs containing an N-X-S/T sequence.

Each antibody or antigen-binding fragment will have a unique isoelectric point (pl), which generally falls in the pH range between 6 and 9.5. The pl for an IgGl antibody typically falls within the pH range of 7-9.5 and the pl for an IgG4 antibody typically falls within the pH range of 6-8.

Each antibody or antigen-binding fragment will have a characteristic melting temperature, with a higher melting temperature indicating greater overall stability in vivo (Krishnamurthy, R and Manning, M.C. Curr Pharm Biotechnol 2002, 3:361-71). In general, the Tmi (the temperature of initial unfolding) may be greater than 60°C, greater than 65°C, or greater than 70°C. The melting point of an antibody or fragment can be measured using differential scanning calorimetry (Chen et al., Pharm Res 2003, 20:1952-60; Ghirlando et al. Immunol Lett, 1999, 68:47-52) or circular dichroism (Murray et al., J. Chromatogr Sci 2002, 40:343-9).

In a further embodiment, antibodies and antigen-binding fragments thereof are selected that do not degrade rapidly. Degradation of an antibody or fragment can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander, A.J. and Hughes, D.E. Anal Chem, 1995, 67:3626-32).

In a further embodiment, antibodies and antigen-binding fragments thereof are selected that have minimal aggregation effects, which can lead to the triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties. Generally, antibodies and fragments are acceptable with aggregation of 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. Aggregation can be measured by several techniques, including size-exclusion column (SEC), high performance liquid chromatography (HPLC), and light scattering.

Antibody Conjugates

The antibodies and antigen-binding fragments thereof disclosed herein may also be conjugated to a chemical moiety. The chemical moiety may be, inter alia, a polymer or a compound that enables transport over the blood brain barrier (BBB). In particular embodiments, the chemical moiety is a polymer which increases the half-life of the antibody or fragment in the body of a subject. Suitable polymers include, but, are not limited to, hydrophilic polymers which include but are not limited to polyethylene glycol (PEG) (e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kl)a, 30 kDa or 40 kDa), dextran and inonomethoxypolyethylene glycol (inPEG). Antibodies or antigen-binding fragments thereof may for example be PEGylated to increase its biological (e.g. serum) halflife. To PEGylate an antibody or fragment, the antibody or fragment, typically is reacted with a reactive form of polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. In particular embodiments, the PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (ClC10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody or fragment to be PEGylated is an aglycosylated antibody or fragment. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the invention. See, e.g., EP 0 154 316 and EP 0 401 384. Lee, et al., Bioconj. Chem. 1999, 10:973-981 discloses PEG conjugated single-chain antibodies. Wen, et al., Bioconj. Chem. 2001, 12:545-553 disclose conjugating antibodies with PEG which is attached to a radiometal chelator (diethylenetriaminpentaacetic acid (DTPA)).

The antibodies and antigen-binding fragments thereof, especially when used for diagnostic purposes, disclosed herein may also be conjugated with labels such as ⁹⁹Tc,⁹⁹Y, ^mIn, ^:i2P, ¹⁴C, ^l25I, ³H, ¹³¹I, ⁿC, ¹⁵O, ¹³N, ^l8F, ⁴⁵S, ⁵¹Cr, ^δ7Το, ²²«Ra, ««Co, ⁵⁹Fe, 57_Se, i52_Eu, «’CU, ²¹⁷Ci, ^21lAt, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ²³⁴Th, and ⁴⁹K, '«⁷Gd, ⁵⁵Mn, ⁵²Tr, and ⁵«Fe.

The antibodies and antigen-binding fragments disclosed herein, especially when used for diagnostic purposes, may also be conjugated with fluorescent or chemiluminescent labels, including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde, fluorescamine, ¹⁵²Eu, dansyl, umbelliferone, luciferin, luminal label, isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridimium salt label, an oxalate ester label, an aequorin label, 2,3-dihydrophthalazinediones, bio tin/avidin, spin labels and stable free radicals.

Any method known in the art for conjugating the antibodies and antigen-binding fragments thereof of the invention to the various moieties may be employed, including those methods described by Hunter, et al., Nature 1962, 144:945; David, et al., Biochemistry 1974, 13:1014; Pain, et al., J. Immunol. Meth. 1981, 40:219; and Nygren, J., Histochem. and Cytochem. 1982, 30:407. Methods for conjugating antibodies and fragments are conventional and very well known in the art.

Antibodies or other polypeptides may be immobilized onto a variety of solid supports for use in assays. Solid phases that may be used to immobilize specific binding members include those developed and/or used as solid phases in solid phase binding assays. Examples of suitable solid phases include membrane filters, cellulose-based papers, beads (including polymeric, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and multiple-well plates. An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot. Antibodies or other polypeptides may be bound to specific zones of assay devices either by conjugating directly to an assay device surface, or by indirect binding. In an example of the later case, antibodies or other polypeptides may be immobilized on particles or other solid supports, and that solid support immobilized to the device surface.

Biological assays require methods for detection, and one of the most common methods for quantitation of results is to conjugate a detectable label to a protein or nucleic acid that has affinity for one of the components in the biological system being studied. Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, ete.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dinitrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Preparation of solid phases and detectable label conjugates often comprise the use of chemical cross-linkers. Cross-linking reagents contain at least two reactive groups, and are divided generally into homofunctional cross-linkers (containing identical reactive groups) and heterofunctional cross-linkers (containing non-identical reactive groups). Homobifunctional cross-linkers that couple through amines, sulfhydryls or react non-specifically are available from many commercial sources. Maleimides, alkyl and aryl halides, u-haloacyls and pyridyl disulfides are thiol reactive groups. Maleimides, alkyl and aryl halides, and α-haloacyls react with sulfhydryls to form thiol ether bonds, while pyridyl disulfides react with sulfhydryls to produce mixed disulfides. The pyridyl disulfide product is cleavable. Imidoesters are also very useful for protein-protein crosslinks. A variety of heterobifunctional cross-linkers, each combining different attributes for successful conjugation, are commercially available.

Intracellular delivery of binding molecules

In some cases, the multispecific binding molecule targets a pathological target that is located inside of the cell. Hence, intracellular delivery of the multispecific binding molecule is required. Multispecific binding molecules such as multispecific antibodies often lack the ability to efficiently penetrate cellular membranes due to their large size and hydrophilicity. Various formats are published in literature to improve delivery of sufficient amounts of intact binding molecules into cells.

For example, cytotransmabs are antibodies comprising a cytosol penetrating light chain variable domain paired with a heavy chain variable domain of a therapeutic antibody. Internalization of the cytotransmabs was achieved through interaction with heparin sulfate proteoglycan (HSPS), resulting in clathrin-mediated endocytic pathway. The internalized cytotransmabs did not show noticeable cytotoxicity, which indicates that initialization takes place via a physical endocytosis pathway without disrupting the membrane. Hence, cytotransmabs provide a suitable method for improving internalization of a therapeutic antibody. (Choi, D-K, mAbs, 2014, 6:6, 1402). Alternatively, a fusion protein of the IgGl-Fc domain with Cre recombinase also allowed permeation of the cell membrane. (Marschall, A.J., mAbs, 2014, 6:4, 953)

Furthermore, intracellular delivery vehicles that are able to transport antibodies into the cell have been reported. A fusion protein consisting of Protein A domain B and the self-assembling Hex domain forms a nanocarrier that tightly binds antibodies. This vehicle-antibody complex shows improved internalization compared to soluble antibodies and indeed enhances the delivery of the antibodies to the cytosol. (In Lim, S., J. Control. Release, 2017).

Another possibility is to use a therapeutic delivery vehicle comprising a replication defective virus, such as an adeno-associated virus (AAV) vector. These vectors can carry genes, for example genes encoding multispecific binding molecules or fragments thereof and enable administration of those genes into the cell. Therefore, these vehicles are particularly useful for gene-therapy. The use of AAV as delivery vehicles has several advantages. For example, AAV is a virus that infects cells but is not known to cause a disease and does not exhibit significant immunogenicity. These therapeutic delivery vehicles are also suitable for enabling expression of multispecific binding molecules of the invention directly into the targeted cell. Hence, a specific cell targeted therapy effected by providing a subject a therapeutic delivery vehicle according to the invention may be used in therapies against NDDs.

Whereas AAV packages a single strand of DNA and must wait for its second strand to be synthesized, scAAV packages two shorter strands that are complementary to each other. By avoiding second-strand synthesis, scAAV can express more quickly, although as a caveat, scAAV can only encode half of the already limited capacity of AAV. This methodology is especially suitable for expressing self-complimentary antibody fragments of the invention.

Blood brain barrier (BBB) permeation

One of the major hurdles in the development of effective treatments for disorders affecting the central nervous system, such as NDDs, is the low efficiency to deliver the therapeutics into the brain. For example, in antibody-based treatment only 0.1% of circulating antibodies end up in the brain. The main reason for this low efficiency is the presence of the BBB which tightly controls the transfer of substances from the blood to the brain tissue and vice versa. It consists of tight junctions enclosing the brain endothelial and epithelial cells, which hinders the paracellular passage of molecules.

Molecules that are allowed to passage the BBB can be utilized to transport cargo across this barrier. Such transport can be achieved either specifically, by targeting transporters that are highly expressed on the BBB, or aspecifically. The family of cell penetrating peptides (CPPs), comprised of short amphiphatic or cationic sequences, constitute a group of peptides that enable aspecific passage of cargo through the BBB.

Alternatively, BBB shuttles have been developed that are able to pass the BBB through the targeting of receptors present on the BBB (receptor-mediated transport). Ideally, such receptors should be highly expressed on the luminal side of brain vasculature with respect to other tissues, have a high capacity for transcytosis, broad substrate scope and high turnover. In addition, the physiological role of the transporter should remain unaffected. Examples of such receptors include but are not limited to transferrin receptor (TfR), insulin receptor (IR), leptin receptor, low density lipoproteins (LDLRs), lipoprotein receptor-related protein (LRP), CD98hc, n4ChR, KCa channel, GSH, GM1, AMT, FC5, IGF1 receptor, IGF2 receptor, FCGRT, Scavenger receptor class B, basigin, LRP, the melanocortin receptor, the nicotinic acetylcholine receptor, the VACM-1 receptor, IGFR, EPCR, EGFR, TNFR, M6PR, the lipoprotein receptor, NCAM, LIFR, MRP1, AchR, DTr, the glutathione transporter, SR-B1, MYOF, TFRC, ECE1, PVR, CDC50A, SCARF 1, MRC1, HLA-DRA, RAMP2, VLDLR, STAB1, TLR9, CXCL16, NTRK1, CD74, DPP4, endothelial growth factor receptors 1, 2 and 3, the glucocorticoid receptor, the ionotropic glutamate receptor, the M3 receptor, the aryl hydrocarbon receptor, GLUT-1, inositol-1, 4,5-trisphosphate (IP3) receptor, the N methyl- D-aspartate receptor, SI Pl, the P2Y receptor, TMEM30A, and RAGE. These receptors can be targeted with either peptides or proteins, such as antib o dies/antib o dy- fr a ginents.

Peptides targeting these receptors were found exploiting phage display, or can be derived from natural proteins such as apoliproteins or hormones, or from exogenous proteins like neurotoxins or viruses. Examples of peptide-based BBB shuttles include but are not limited to RDP, KC2S, , Angiopep-2, ApoB(3371-3409), ApoE(159-167), peptide-22, THR, THR retro enantio, CRT, Leptin30, RVG29, CDX, Apamin, MiniAP-4, GSH, G23, g7, TGN, TAT(47-57), SynBl, (phenylproline) 4NH2, Diketopiperazine (Table 8). These shuttle components can be easily attached to the multispecific binding molecules of the present invention by using simple chemical cross-linking reagents as described above. For a full description of peptide-based BBB shuttles, including sequences of the shuttles, active transporters, passive diffusion and the retro enantio approach see Olla-Salvia et al. Chem. Soc. Rev. 2016, 45:4690-4707.

One challenge associated with the process of intracellular delivery of macromolecules using GPP’s is the efficient endosomal release of the macromolecule into the cytoplasm. It was found that linking the macromolecule/CPP complex to a hydrophobic motif led to significant enhancement of release of the complex from the endosomes. This enhanced release is due to incorporation of the hydrophobic motif into the lipid bilayer of the endosome, leading to destabilization of the membrane and consequent release of the macromolecule into the cytoplasm. (Lönn, P., Scientific reports, 2016, 6:32301). Alternatively, proteins such as antibodies or antibody-fragments can be used to enhance passage of the BBB. Increased permeation of the BBB has been demonstrated for a number of bispecific antibodies comprising one binding site targeting the T1R, insulin receptor or glycosylated protein (Cdc50A) and these antibodies are currently being investigated in preclinical trials. (Neves, V., Trends in biotechnology). Different antibodies or antibody-fragments were found to be suitable to enhance influx from the blood to the brain tissue. These examples (Table 8) are therefore also of interest in the current invention.

When binding molecules do not bind receptors directly but through the binding of a carbohydrate-ligand, absorptive-mediated transcytosis (AMT) can take place. This form of transcytosis was exemplified by the antibody FC5 that binds a2,3-linked sialic acid groups on Cdc50A. Binding of FC5 to these groups resulted into enhanced influx of FC5 into the brain.

It was additionally demonstrated that different antibody formats allow different mechanisms of uptake in the brain. For example, antibodies with high affinity for the target receptor are less likely to be released afterwards. Careful selection of the antibody format, additionally enables the adjustment of certain physical properties, such as toxicity. Toxic side effects of an anti Tffi-Ab could be significantly reduced when the equivalent antibody, containing only one Fab arm, was administered. No detectable cytotoxicity was observed when the anti Tffi Ab was linked to the C-terminal of the anti-Ab antibody, primarily because binding to transferrin receptors in the periphery was prevented due to sterical hindrance. Moreover, it was found that when two scFv of a Tffi antibody were recombinantly fused to the C-terminal of the light chain of an AB antibody, an antibody was obtained showing increased uptake into the brain. The design, comprising a short linker connecting the scFv’s to the AB antibody, enabled monovalent binding to the Tffi. Multivalent binding was prevented due to sterical hindrance. As a consequence, the antibody was released more easily, although the likelihood of Tffibinding and thus brain uptake, was doubled compared to antibodies comprising a monovalent binding site for Tffi..(Weber, F., Cell Reports, 2017, 22, 149-162; Hultqvist, G., Theranostics 2017, 7(2), 308).

For reviews on strategies crossing the BBB using antibodies or antibodyfragments see Neves, V, Trends in biotechnology, Partridge, W., Biodrugs, 2017. All the strategies that are discussed in this reference can also be used for the present invention.

Besides improving the uptake of the multispecific binding molecules in the brain, extending the brain half life of the multispecific binding molecule is equally important. One way to achieve this goal would be to increase the influx of the multispecific binding molecules into the brain. Such an effect can be achieved by using peptide- or protein-based shuttles to enable passage of the BBB as outlined above. Alternatively, smaller multispecific binding molecules such as multispecific Fab fragments, might be more successful in passing the BBB transcellularly. (Finke, J.M., Human Antibodies, 2016).

Another way to improve the influx of multispecific binding molecules in the brain, is by giving a patient ultrasonic pulses that temporarily disrupt part of the BBB via microbubble cavitation and allows passage of substances, such as drugs or endogenous proteins. To minimize side-effects, the timing and location of the ultrasonic pulse is important. Enhanced brain and neuronal uptake of a singlechain variable fragment of an anti-tau antibody was achieved, leading to decreased tau phosphorylation in a tau transgenic mouse model.

However, apart from improving influx, reducing the efflux of the multispecific binding molecules could also contribute to an extended half life of the binding molecule. Reducing efflux from the brain can be achieved by modifying the Fc region of the antibody or antibody fragment. Moreover, it was demonstrated that sialylation of glycans expressed on the Fab fragment can also extend the serum half life of the antibody or antibody fragment. (Finke, J.M., Human Antibodies, 2016).

In a preferred embodiment of the present invention, the multispecific binding molecule additionally comprises an additional binding specificity binding a further binding target selected from the group comprising transferrin receptor (TfR), insulin receptor (IR), leptin receptor, low density lipoproteins (FDFRs), lipoprotein receptor-related protein (LRP), CD98hc, n4ChR, KCa channel, GSH, GM1, AMT, IGF1 receptor, IGF2 receptor, FCGRT, Scavenger receptor class B, basigin, LRP, the melanocortin receptor, the nicotinic acetylcholine receptor, the VACM-1 receptor, IGFR, EPCR, EGFR, TNFR, M6PR, the lipoprotein receptor, NCAM, LIFR, MRP1, AchR, DTr, the glutathione transporter, SR-B1, MYOF, TFRC, ECE1, PVR, CDC50A, SCARF 1, MRC1, HLA-DRA, RAMP2, VLDLR, STAB1, TLR9, CXCL16, NTRK1, CD74, DPP4, endothelial growth factor receptors 1, 2 and 3, the glucocorticoid receptor, the ionotropic glutamate receptor, the M3 receptor, the aryl hydrocarbon receptor, GLUT-1, inositol-1, 4,5-trisphosphate (IP3) receptor, the N-methyl- D-aspartate receptor, SI Pl, the P2Y receptor, TMEM30A, and RAGE to enable passage of the multispecific binding molecule through the BBB.

Preferably, the additional binding specificity is an antibody or antibodyfragment. In one aspect the binding site specifically targets a BBB receptor selected from the group comprising transferrin receptor, insulin receptor, CD98hc, leptin and low density lipoproteins.

All combinations are possible and accordingly the present invention comprises a plurality of different multispecific binding molecules, including but not limited to the combinations listed below, where the codes A1-A5, B1-B16 and 01C6 are described in the Tables 1-2 &7.

Table 7: Transporters used to passage the BBB

Entry	Target
Cl	transferrin receptor
C2	insulin receptor
03	leptin receptor
C4	low density lipoproteins
C5	CD98hc
06	ApoE(159-167)

A1+A2+C1,

A3+A4+C1,

A1+B5+C1,

A2+B3+C1,

A3+B1+C1,

A3+B7+C1,

A4+B5+C1,

A5+B3+C1,

A1+A2+C2,

A3+A4+C2,

A1+B5+C2,

A2+B3+C2,

A3+B1+C2,

A1+A3+C1,

A4+A5+C1,

A1+B6+C1,

A2+B4+C1,

A3+B2+C1,

A3+B8+C1,

A4+B6+C1,

A5+B4+C1,

A1+A3+C2,

A4+A5+C2,

A1+B6+C2,

A2+B4+C2,

A3+B2+C2,

A1+A4+C1,

A1+B1+C1,

A1+B7+C1,

A2+B5+C1,

A3+B3+C1,

A4+B1+C1,

A4+B7+C1,

A5+B5+C1,

A1+A4+C2,

A1+B1+C2,

A1+B7+C2,

A2+B5+C2,

A3+B3+C2,

A1+A5+C1,

A1+B2+C1,

A1+B8+C1,

A2+B6+C1,

A3+B4+C1,

A4+B2+C1,

A4+B8+C1,

A5+B6+C1,

A1+A5+C2,

A1+B2+C2,

A1+B8+C2,

A2+B6+C2,

A3+B4+C2,

A2+A3+C1,

A1+B3+C1,

A2+B1+C1,

A2+B7+C1,

A3+B5+C1,

A4+B3+C1,

A5+B1+C1,

A5+B7+C1,

A2+A3+C2,

A1+B3+C2,

A2+B1+C2,

A2+B7+C2,

A3+B5+C2,

A2+A4+C1,

A1+B4+C1,

A2+B2+C1,

A2+B8+C1,

A3+B6+C1,

A4+B4+C1,

A5+B2+C1,

A5+B8+C1,

A2+A4+C2,

A1+B4+C2,

A2+B2+C2,

A2+B8+C2,

A3+B6+C2,

A3+B7+C2,	A3+B8+C2,	A4+B1+C2,	A4+B2+C2,	A4+B3+C2,	A4+B4+C2,
	A4+B5+C2,	A4+B6+C2,	A4+B7+C2,	A4+B8+C2,	A5+B1+C2,	A5+B2+C2,
	A5+B3+C2,	A5+B4+C2,	A5+B5+C2,	A5+B6+C2,	A5+B7+C2,	A5+B8+C2,
	A1+A2+C3,	A1+A3+C3,	A1+A4+C3,	A1+A5+C3,	A2+A3+C3,	A2+A4+C3,
5	A3+A4+C3,	A4+A5+C3,	A1+B1+C3,	A1+B2+C3,	A1+B3+C3,	A1+B4+C3,
	A1+B5+C3,	A1+B6+C3,	A1+B7+C3,	A1+B8+C3,	A2+B1+C3,	A2+B2+C3,
	A2+B3+C3,	A2+B4+C3,	A2+B5+C3,	A2+B6+C3,	A2+B7+C3,	A2+B8+C3,
	A3+B1+C3,	A3+B2+C3,	A3+B3+C3,	A3+B4+C3,	A3+B5+C3,	A3+B6+C3,
	A3+B7+C3,	A3+B8+C3,	A4+B1+C3,	A4+B2+C3,	A4+B3+C3,	A4+B4+C3,
10	A4+B5+C3,	A4+B6+C3,	A4+B7+C3,	A4+B8+C3,	A5+B1+C3,	A5+B2+C3,
	A5+B3+C3,	A5+B4+C3,	A5+B5+C3,	A5+B6+C3,	A5+B7+C3,	A5+B8+C3,
	A1+A2+C4,	A1+A3+C4,	A1+A4+C4,	A1+A5+C4,	A2+A3+C4,	A2+A4+C4,
	A3+A4+C4,	A4+A5+C4,	A1+B1+C4,	A1+B2+C4,	A1+B3+C4,	A1+B4+C4,
	A1+B5+C4,	A1+B6+C4,	A1+B7+C4,	A1+B8+C4,	A2+B1+C4,	A2+B2+C4,
15	A2+B3+C4,	A2+B4+C4,	A2+B5+C4,	A2+B6+C4,	A2+B7+C4,	A2+B8+C4,
	A3+B1+C4,	A3+B2+C4,	A3+B3+C4,	A3+B4+C4,	A3+B5+C4,	A3+B6+C4,
	A3+B7+C4,	A3+B8+C4,	A4+B1+C4,	A4+B2+C4,	A4+B3+C4,	A4+B4+C4,
	A4+B5+C4,	A4+B6+C4,	A4+B7+C4,	A4+B8+C4,	A5+B1+C4,	A5+B2+C4,
	A5+B3+C4,	A5+B4+C4,	A5+B5+C4,	A5+B6+C4,	A5+B7+C4,	A5+B8+C4,
20	A1+A2+C5,	A1+A3+C5,	A1+A4+C5,	A1+A5+C5,	A2+A3+C5,	A2+A4+C5,
	A3+A4+C5,	A4+A5+C5,	A1+B1+C5,	A1+B2+C5,	A1+B3+C5,	A1+B4+C5,
	A1+B5+C5,	A1+B6+C5,	A1+B7+C5,	A1+B8+C5,	A2+B1+C5,	A2+B2+C5,
	A2+B3+C5,	A2+B4+C5,	A2+B5+C5,	A2+B6+C5,	A2+B7+C5,	A2+B8+C5,
	A3+B1+C5,	A3+B2+C5,	A3+B3+C5,	A3+B4+C5,	A3+B5+C5,	A3+B6+C5,
25	A3+B7+C5,	A3+B8+C5,	A4+B1+C5,	A4+B2+C5,	A4+B3+C5,	A4+B4+C5,
	A4+B5+C5,	A4+B6+C5,	A4+B7+C5,	A4+B8+C5,	A5+B1+C5,	A5+B2+C5,
	A5+B3+C5,	A5+B4+C5,	A5+B5+C5,	A5+B6+C5,	A5+B7+C5,	A5+B8+C5,
	A1+A2+C6,	A1+A3+C6,	A1+A4+C6,	A1+A5+C6,	A2+A3+C6,	A2+A4+C6,
	A3+A4+C6,	A4+A5+C6,	A1+B1+C6,	A1+B2+C6,	A1+B3+C6,	A1+B4+C6,
30	A1+B5+C6,	A1+B6+C6,	A1+B7+C6,	A1+B8+C6,	A2+B1+C6,	A2+B2+C6,
	A2+B3+C6,	A2+B4+C6,	A2+B5+C6,	A2+B6+C6,	A2+B7+C6,	A2+B8+C6,
	A3+B1+C6,	A3+B2+C6,	A3+B3+C6,	A3+B4+C6,	A3+B5+C6,	A3+B6+C6,
	A3+B7+C6,	A3+B8+C6,	A4+B1+C6,	A4+B2+C6,	A4+B3+C6,	A4+B4+C6,

A4+B5+C6, A4+B6+C6, A4+B7+C6, A4+B8+C6, A5+B1+C6, A5+B2+C6

A5+B3+C6. A5+B4+C6. A5+B5+C6. A5+B6+C6. A5+B7+C6. A5+B8+C6

A1+A2+A3+C1,

A1+A4+A5+C1,

A1+A2+B5+C1,

A1+A3+B2+C1,

A1+A3+B7+C1,

A1+A4+B4+C1,

A1+A5+B1+C1,

A1+A5+B6+C1,

A1+B1+B4+C1,

A1+B2+B3+C1,

A1+B2+B8+C1,

A1+B3+B8+C1,

A1+B5+B6+C1,

A1+B7+B8+C1,

A2+B1+B6+C1,

A2+B2+B5+C1,

A2+B3+B5+C1,

A2+B4+B6+C1,

A2+B5+B8+C1,

A3+B1+B3+C1,

A3+B1+B8+C1,

A3+B2+B7+C1,

A3+B3+B7+C1,

A3+B4+B8+C1,

A3+B6+B8+C1,

A4+B1+B5+C1,

A4+B2+B4+C1,

A4+B3+B4+C1,

A4+B4+B5+C1,

A4+B5+B7+C1,

A5+B1+B2+C1,

A1+A2+A4+C1,

A1+A2+B1+C1,

A1+A2+B6+C1,

A1+A3+B3+C1,

A1+A3+B8+C1,

A1+A4+B5+C1,

A1+A5+B2+C1,

A1+A5+B7+C1,

A1+B1+B5+C1,

A1+B2+B4+C1,

A1+B3+B4+C1,

A1+B4+B5+C1,

A1+B5+B7+C1,

A2+B1+B2+C1,

A2+B1+B7+C1,

A2+B2+B6+C1,

A2+B3+B6+C1,

A2+B4+B7+C1,

A2+B6+B7+C1,

A3+B1+B4+C1,

A3+B2+B3+C1,

A3+B2+B8+C1,

A3+B3+B8+C1,

A3+B5+B6+C1,

A3+B7+B8+C1,

A4+B1+B6+C1,

A4+B2+B5+C1,

A4+B3+B5+C1,

A4+B4+B6+C1,

A4+B5+B8+C1,

A5+B1+B3+C1,

A1+A2+A5+C1,

A1+A2+B2+C1,

A1+A2+B7+C1,

A1+A3+B4+C1,

A1+A4+B1+C1,

A1+A4+B6+C1,

A1+A5+B3+C1,

A1+A5+B8+C1,

A1+B1+B6+C1,

A1+B2+B5+C1,

A1+B3+B5+C1,

A1+B4+B6+C1,

A1+B5+B8+C1,

A2+B1+B3+C1,

A2+B1+B8+C1,

A2+B2+B7+C1,

A2+B3+B7+C1,

A2+B4+B8+C1,

A2+B6+B8+C1,

A3+B1+B5+C1,

A3+B2+B4+C1,

A3+B3+B4+C1,

A3+B4+B5+C1,

A3+B5+B7+C1,

A4+B1+B2+C1,

A4+B1+B7+C1,

A4+B2+B6+C1,

A4+B3+B6+C1,

A4+B4+B7+C1,

A4+B6+B7+C1,

A5+B1+B4+C1,

A1+A3+A4+C1,

A1+A2+B3+C1,

A1+A2+B8+C1,

A1+A3+B5+C1,

A1+A4+B2+C1,

A1+A4+B7+C1,

A1+A5+B4+C1,

A1+B1+B2+C1,

A1+B1+B7+C1,

A1+B2+B6+C1,

A1+B3+B6+C1,

A1+B4+B7+C1,

A1+B6+B7+C1,

A2+B1+B4+C1,

A2+B2+B3+C1,

A2+B2+B8+C1,

A2+B3+B8+C1,

A2+B5+B6+C1,

A2+B7+B8+C1,

A3+B1+B6+C1,

A3+B2+B5+C1,

A3+B3+B5+C1,

A3+B4+B6+C1,

A3+B5+B8+C1,

A4+B1+B3+C1,

A4+B1+B8+C1,

A4+B2+B7+C1,

A4+B3+B7+C1,

A4+B4+B8+C1,

A4+B6+B8+C1,

A5+B1+B5+C1,

A1+A3+A5+C1,

A1+A2+B4+C1,

A1+A3+B1+C1,

A1+A3+B6+C1,

A1+A4+B3+C1,

A1+A4+B8+C1,

A1+A5+B5+C1,

A1+B1+B3+C1,

A1+B1+B8+C1,

A1+B2+B7+C1,

A1+B3+B7+C1,

A1+B4+B8+C1,

A1+B6+B8+C1,

A2+B1+B5+C1,

A2+B2+B4+C1,

A2+B3+B4+C1,

A2+B4+B5+C1,

A2+B5+B7+C1,

A3+B1+B2+C1,

A3+B1+B7+C1,

A3+B2+B6+C1,

A3+B3+B6+C1,

A3+B4+B7+C1,

A3+B6+B7+C1,

A4+B1+B4+C1,

A4+B2+B3+C1,

A4+B2+B8+C1,

A4+B3+B8+C1,

A4+B5+B6+C1,

A4+B7+B8+C1,

A5+B1+B6+C1,

A5+B1+B7+C1,

A5+B2+B6+C1,

A5+B3+B6+C1,

A5+B4+B7+C1,

A5+B6+B7+C1,

A2+A3+B3+C1,

A2+A3+B8+C1,

A2+A4+B5+C1,

A3+A4+B2+C1,

A3+A4+B7+C1,

A5+B1+B8+C1,

A5+B2+B7+C1,

A5+B3+B7+C1,

A5+B4+B8+C1,

A5+B2+B3+C1,

A5+B2+B8+C1,

A5+B3+B8+C1,

A5+B5+B6+C1,

A5+B2+B4+C1,

A5+B3+B4+C1,

A5+B4+B5+C1,

A5+B5+B7+C1,

A5+B6+B8+C1, A5+B7+B8+C1, A2+A3+B1+C1,

A2+A3+B4+C1, A2+A3+B5+C1, A2+A3+B6+C1,

A2+A4+B1+C1, A2+A4+B2+C1, A2+A4+B3+C1,

A2+A4+B6+C1, A2+A4+B7+C1, A2+A4+B8+C1,

A3+A4+B3+C1, A3+A4+B4+C1, A3+A4+B5+C1,

A3+A4+B8+C1, A4+A5+B1+C1, A4+A5+B2+C1,

A5+B2+B5+C1

A5+B3+B5+C1

A5+B4+B6+C1

A5+B5+B8+C1

A2+A3+B2+C1

A2+A3+B7+C1

A2+A4+B4+C1

A3+A4+B1+C1

A3+A4+B6+C1

A4+A5+B3+C1

A4+A5+B4+C1, A4+A5+B5+C1, A4+A5+B6+C1, A4+A5+B7+C1, A4+A5+B8+C1,

A1+A2+A3+C2,

A1+A4+A5+C2,

A1+A2+B5+C2,

A1+A3+B2+C2,

A1+A3+B7+C2,

A1+A4+B4+C2,

A1+A5+B1+C2,

A1+A5+B6+C2,

A1+B1+B4+C2,

A1+B2+B3+C2,

A1+B2+B8+C2,

A1+B3+B8+C2,

A1+B5+B6+C2,

A1+B7+B8+C2,

A2+B1+B6+C2,

A2+B2+B5+C2,

A2+B3+B5+C2,

A2+B4+B6+C2,

A2+B5+B8+C2,

A3+B1+B3+C2,

A3+B1+B8+C2,

A1+A2+A4+C2,

A1+A2+B1+C2,

A1+A2+B6+C2,

A1+A3+B3+C2,

A1+A3+B8+C2,

A1+A4+B5+C2,

A1+A5+B2+C2,

A1+A5+B7+C2,

A1+B1+B5+C2,

A1+B2+B4+C2,

A1+B3+B4+C2,

A1+B4+B5+C2,

A1+B5+B7+C2,

A2+B1+B2+C2,

A2+B1+B7+C2,

A2+B2+B6+C2,

A2+B3+B6+C2,

A2+B4+B7+C2,

A2+B6+B7+C2,

A3+B1+B4+C2,

A3+B2+B3+C2,

A1+A2+A5+C2,

A1+A2+B2+C2,

A1+A2+B7+C2,

A1+A3+B4+C2,

A1+A4+B1+C2,

A1+A4+B6+C2,

A1+A5+B3+C2,

A1+A5+B8+C2,

A1+B1+B6+C2,

A1+B2+B5+C2,

A1+B3+B5+C2,

A1+B4+B6+C2,

A1+B5+B8+C2,

A2+B1+B3+C2,

A2+B1+B8+C2,

A2+B2+B7+C2,

A2+B3+B7+C2,

A2+B4+B8+C2,

A2+B6+B8+C2,

A3+B1+B5+C2,

A3+B2+B4+C2,

A1+A3+A4+C2,

A1+A2+B3+C2,

A1+A2+B8+C2,

A1+A3+B5+C2,

A1+A4+B2+C2,

A1+A4+B7+C2,

A1+A5+B4+C2,

A1+B1+B2+C2,

A1+B1+B7+C2,

A1+B2+B6+C2,

A1+B3+B6+C2,

A1+B4+B7+C2,

A1+B6+B7+C2,

A2+B1+B4+C2,

A2+B2+B3+C2,

A2+B2+B8+C2,

A2+B3+B8+C2,

A2+B5+B6+C2,

A2+B7+B8+C2,

A3+B1+B6+C2,

A3+B2+B5+C2,

A1+A3+A5+C2,

A1+A2+B4+C2,

A1+A3+B1+C2,

A1+A3+B6+C2,

A1+A4+B3+C2,

A1+A4+B8+C2,

A1+A5+B5+C2,

Al+B 1+B3+C2,

A1+B1+B8+C2,

A1+B2+B7+C2,

A1+B3+B7+C2,

A1+B4+B8+C2,

A1+B6+B8+C2,

A2+B1+B5+C2,

A2+B2+B4+C2,

A2+B3+B4+C2,

A2+B4+B5+C2,

A2+B5+B7+C2,

A3+B1+B2+C2,

A3+B1+B7+C2,

A3+B2+B6+C2,

A3+B2+B7+C2,

A3+B3+B7+C2,

A3+B4+B8+C2,

A3+B6+B8+C2,

A4+B1+B5+C2,

A4+B2+B4+C2,

A4+B3+B4+C2,

A4+B4+B5+C2,

A4+B5+B7+C2,

A5+B1+B2+C2,

A5+B1+B7+C2,

A5+B2+B6+C2,

A5+B3+B6+C2,

A5+B4+B7+C2,

A5+B6+B7+C2,

A2+A3+B3+C2,

A2+A3+B8+C2,

A2+A4+B5+C2,

A3+A4+B2+C2,

A3+A4+B7+C2,

A4+A5+B4+C2,

A1+A2+A3+C3,

A1+A4+A5+C3,

A1+A2+B5+C3,

A1+A3+B2+C3,

A1+A3+B7+C3,

A1+A4+B4+C3,

A1+A5+B1+C3,

A1+A5+B6+C3,

A1+B1+B4+C3,

A1+B2+B3+C3,

A1+B2+B8+C3,

A1+B3+B8+C3,

A3+B2+B8+C2,

A3+B3+B8+C2,

A3+B5+B6+C2,

A3+B7+B8+C2,

A4+B1+B6+C2,

A4+B2+B5+C2,

A4+B3+B5+C2,

A4+B4+B6+C2,

A4+B5+B8+C2,

A5+B1+B3+C2,

A5+B1+B8+C2,

A5+B2+B7+C2,

A5+B3+B7+C2,

A5+B4+B8+C2,

A5+B6+B8+C2,

A2+A3+B4+C2,

A2+A4+B1+C2,

A2+A4+B6+C2,

A3+A4+B3+C2,

A3+A4+B8+C2,

A4+A5+B5+C2,

A1+A2+A4+C3,

A1+A2+B1+C3,

A1+A2+B6+C3,

A1+A3+B3+C3,

A1+A3+B8+C3,

A1+A4+B5+C3,

A1+A5+B2+C3,

A1+A5+B7+C3,

A1+B1+B5+C3,

A1+B2+B4+C3,

A1+B3+B4+C3,

A1+B4+B5+C3,

A3+B3+B4+C2,

A3+B4+B5+C2,

A3+B5+B7+C2,

A4+B1+B2+C2,

A4+B1+B7+C2,

A4+B2+B6+C2,

A4+B3+B6+C2,

A4+B4+B7+C2,

A4+B6+B7+C2,

A5+B1+B4+C2,

A5+B2+B3+C2,

A5+B2+B8+C2,

A5+B3+B8+C2,

A5+B5+B6+C2,

A5+B7+B8+C2,

A2+A3+B5+C2,

A2+A4+B2+C2,

A2+A4+B7+C2,

A3+A4+B4+C2

A4+A5+B1+C2

A4+A5+B6+C2,

A1+A2+A5+C3

A1+A2+B2+C3,

A1+A2+B7+C3,

A1+A3+B4+C3,

A1+A4+B1+C3

A1+A4+B6+C3,

A1+A5+B3+C3,

A1+A5+B8+C3,

Al+B 1+B6+C3,

A1+B2+B5+C3,

A1+B3+B5+C3,

A1+B4+B6+C3,

A3+B3+B5+C2,

A3+B4+B6+C2,

A3+B5+B8+C2,

A4+B1+B3+C2,

A4+B1+B8+C2,

A4+B2+B7+C2,

A4+B3+B7+C2,

A4+B4+B8+C2,

A4+B6+B8+C2,

A5+B1+B5+C2,

A5+B2+B4+C2,

A5+B3+B4+C2,

A5+B4+B5+C2,

A5+B5+B7+C2,

A2+A3+B1+C2,

A2+A3+B6+C2,

A2+A4+B3+C2,

A2+A4+B8+C2,

A3+A4+B5+C2,

A4+A5+B2+C2,

A4+A5+B7+C2,

A1+A3+A4+C3,

A1+A2+B3+C3,

A1+A2+B8+C3,

A1+A3+B5+C3,

A1+A4+B2+C3,

A1+A4+B7+C3,

A1+A5+B4+C3,

Al+B 1+B2+C3,

A1+B1+B7+C3,

A1+B2+B6+C3,

A1+B3+B6+C3,

A1+B4+B7+C3,

A3+B3+B6+C2,

A3+B4+B7+C2,

A3+B6+B7+C2,

A4+B1+B4+C2,

A4+B2+B3+C2,

A4+B2+B8+C2,

A4+B3+B8+C2,

A4+B5+B6+C2,

A4+B7+B8+C2,

A5+B1+B6+C2,

A5+B2+B5+C2,

A5+B3+B5+C2,

A5+B4+B6+C2,

A5+B5+B8+C2,

A2+A3+B2+C2,

A2+A3+B7+C2,

A2+A4+B4+C2,

A3+A4+B1+C2,

A3+A4+B6+C2,

A4+A5+B3+C2,

A4+A5+B8+C2,

A1+A3+A5+C3,

A1+A2+B4+C3,

A1+A3+B1+C3,

A1+A3+B6+C3,

A1+A4+B3+C3,

A1+A4+B8+C3,

A1+A5+B5+C3,

Al+B 1+B3+C3,

A1+B1+B8+C3,

A1+B2+B7+C3,

A1+B3+B7+C3,

A1+B4+B8+C3,

A1+B5+B6+C3,

A1+B7+B8+C3,

A2+B1+B6+C3,

A2+B2+B5+C3,

A2+B3+B5+C3,

A2+B4+B6+C3,

A2+B5+B8+C3,

A3+B1+B3+C3,

A3+B1+B8+C3,

A3+B2+B7+C3,

A3+B3+B7+C3,

A3+B4+B8+C3,

A3+B6+B8+C3,

A4+B1+B5+C3,

A4+B2+B4+C3,

A4+B3+B4+C3,

A4+B4+B5+C3,

A4+B5+B7+C3,

A5+B1+B2+C3,

A5+B1+B7+C3,

A5+B2+B6+C3,

A5+B3+B6+C3,

A5+B4+B7+C3,

A5+B6+B7+C3,

A2+A3+B3+C3,

A2+A3+B8+C3,

A2+A4+B5+C3,

A3+A4+B2+C3,

A3+A4+B7+C3,

A4+A5+B4+C3,

A1+A2+A3+C4,

A1+A4+A5+C4,

A1+A2+B5+C4,

A1+B5+B7+C3,

A2+B1+B2+C3,

A2+B1+B7+C3,

A2+B2+B6+C3,

A2+B3+B6+C3,

A2+B4+B7+C3,

A2+B6+B7+C3,

A3+B1+B4+C3,

A3+B2+B3+C3,

A3+B2+B8+C3,

A3+B3+B8+C3,

A3+B5+B6+C3,

A3+B7+B8+C3,

A4+B1+B6+C3,

A4+B2+B5+C3,

A4+B3+B5+C3,

A4+B4+B6+C3,

A4+B5+B8+C3,

A5+B1+B3+C3,

A5+B1+B8+C3,

A5+B2+B7+C3,

A5+B3+B7+C3,

A5+B4+B8+C3,

A5+B6+B8+C3,

A2+A3+B4+C3,

A2+A4+B1+C3,

A2+A4+B6+C3,

A3+A4+B3+C3

A3+A4+B8+C3,

A4+A5+B5+C3,

A1+A2+A4+C4

A1+A2+B1+C4,

A1+A2+B6+C4,

A1+B5+B8+C3,

A2+B1+B3+C3,

A2+B1+B8+C3,

A2+B2+B7+C3,

A2+B3+B7+C3,

A2+B4+B8+C3,

A2+B6+B8+C3,

A3+B1+B5+C3,

A3+B2+B4+C3,

A3+B3+B4+C3,

A3+B4+B5+C3,

A3+B5+B7+C3,

A4+B1+B2+C3,

A4+B1+B7+C3,

A4+B2+B6+C3,

A4+B3+B6+C3,

A4+B4+B7+C3,

A4+B6+B7+C3,

A5+B1+B4+C3,

A5+B2+B3+C3,

A5+B2+B8+C3,

A5+B3+B8+C3,

A5+B5+B6+C3,

A5+B7+B8+C3,

A2+A3+B5+C3,

A2+A4+B2+C3,

A2+A4+B7+C3,

A3+A4+B4+C3

A4+A5+B1+C3,

A4+A5+B6+C3,

A1+A2+A5+C4

A1+A2+B2+C4,

A1+A2+B7+C4,

A1+B6+B7+C3,

A2+B1+B4+C3,

A2+B2+B3+C3,

A2+B2+B8+C3,

A2+B3+B8+C3,

A2+B5+B6+C3,

A2+B7+B8+C3,

A3+B1+B6+C3,

A3+B2+B5+C3,

A3+B3+B5+C3,

A3+B4+B6+C3,

A3+B5+B8+C3,

A4+B1+B3+C3,

A4+B1+B8+C3,

A4+B2+B7+C3,

A4+B3+B7+C3,

A4+B4+B8+C3,

A4+B6+B8+C3,

A5+B1+B5+C3,

A5+B2+B4+C3,

A5+B3+B4+C3,

A5+B4+B5+C3,

A5+B5+B7+C3,

A2+A3+B1+C3,

A2+A3+B6+C3,

A2+A4+B3+C3,

A2+A4+B8+C3,

A3+A4+B5+C3,

A4+A5+B2+C3,

A4+A5+B7+C1,

A1+A3+A4+C4,

A1+A2+B3+C4,

A1+A2+B8+C4,

A1+B6+B8+C3,

A2+B1+B5+C3,

A2+B2+B4+C3,

A2+B3+B4+C3,

A2+B4+B5+C3,

A2+B5+B7+C3,

A3+B1+B2+C3,

A3+B1+B7+C3,

A3+B2+B6+C3,

A3+B3+B6+C3,

A3+B4+B7+C3,

A3+B6+B7+C3,

A4+B1+B4+C3,

A4+B2+B3+C3,

A4+B2+B8+C3,

A4+B3+B8+C3,

A4+B5+B6+C3,

A4+B7+B8+C3,

A5+B1+B6+C3,

A5+B2+B5+C3,

A5+B3+B5+C3,

A5+B4+B6+C3,

A5+B5+B8+C3,

A2+A3+B2+C3,

A2+A3+B7+C3,

A2+A4+B4+C3,

A3+A4+B1+C3,

A3+A4+B6+C3,

A4+A5+B3+C3,

A4+A5+B8+C3,

A1+A3+A5+C4,

A1+A2+B4+C4,

A1+A3+B1+C4,

A1+A3+B2+C4,

A1+A3+B7+C4,

A1+A4+B4+C4,

A1+A5+B1+C4,

A1+A5+B6+C4,

A1+B1+B4+C4,

A1+B2+B3+C4,

A1+B2+B8+C4,

A1+B3+B8+C4,

A1+B5+B6+C4,

A1+B7+B8+C4,

A2+B1+B6+C4,

A2+B2+B5+C4,

A2+B3+B5+C4,

A2+B4+B6+C4,

A2+B5+B8+C4,

A3+B1+B3+C4,

A3+B1+B8+C4,

A3+B2+B7+C4,

A3+B3+B7+C4,

A3+B4+B8+C4,

A3+B6+B8+C4,

A4+B1+B5+C4,

A4+B2+B4+C4,

A4+B3+B4+C4,

A4+B4+B5+C4,

A4+B5+B7+C4,

A5+B1+B2+C4,

A5+B1+B7+C4,

A5+B2+B6+C4,

A5+B3+B6+C4,

A5+B4+B7+C4,

A5+B6+B7+C4,

A1+A3+B3+C4,

A1+A3+B8+C4,

A1+A4+B5+C4,

A1+A5+B2+C4,

A1+A5+B7+C4,

A1+B1+B5+C4,

A1+B2+B4+C4,

A1+B3+B4+C4,

A1+B4+B5+C4,

A1+B5+B7+C4,

A2+B1+B2+C4,

A2+B1+B7+C4,

A2+B2+B6+C4,

A2+B3+B6+C4,

A2+B4+B7+C4,

A2+B6+B7+C4,

A3+B1+B4+C4,

A3+B2+B3+C4,

A3+B2+B8+C4,

A3+B3+B8+C4,

A3+B5+B6+C4,

A3+B7+B8+C4,

A4+B1+B6+C4,

A4+B2+B5+C4,

A4+B3+B5+C4,

A4+B4+B6+C4,

A4+B5+B8+C4,

A5+B1+B3+C4,

A5+B1+B8+C4,

A5+B2+B7+C4,

A5+B3+B7+C4,

A5+B4+B8+C4,

A5+B6+B8+C4,

A1+A3+B4+C4,

A1+A4+B1+C4,

A1+A4+B6+C4,

A1+A5+B3+C4,

A1+A5+B8+C4,

Al+B 1+B6+C4,

A1+B2+B5+C4,

A1+B3+B5+C4,

A1+B4+B6+C4,

A1+B5+B8+C4,

A2+B1+B3+C4,

A2+B1+B8+C4,

A2+B2+B7+C4,

A2+B3+B7+C4,

A2+B4+B8+C4,

A2+B6+B8+C4,

A3+B1+B5+C4,

A3+B2+B4+C4,

A3+B3+B4+C4,

A3+B4+B5+C4,

A3+B5+B7+C4,

A4+B1+B2+C4,

A4+B1+B7+C4,

A4+B2+B6+C4,

A4+B3+B6+C4,

A4+B4+B7+C4,

A4+B6+B7+C4,

A5+B1+B4+C4,

A5+B2+B3+C4,

A5+B2+B8+C4,

A5+B3+B8+C4,

A5+B5+B6+C4,

A5+B7+B8+C4,

A1+A3+B5+C4,

A1+A4+B2+C4,

A1+A4+B7+C4,

A1+A5+B4+C4,

A1+B1+B2+C4,

A1+B1+B7+C4,

A1+B2+B6+C4,

A1+B3+B6+C4,

A1+B4+B7+C4,

A1+B6+B7+C4,

A2+B1+B4+C4,

A2+B2+B3+C4,

A2+B2+B8+C4,

A2+B3+B8+C4,

A2+B5+B6+C4,

A2+B7+B8+C4,

A3+B1+B6+C4,

A3+B2+B5+C4,

A3+B3+B5+C4,

A3+B4+B6+C4,

A3+B5+B8+C4,

A4+B1+B3+C4,

A4+B1+B8+C4,

A4+B2+B7+C4,

A4+B3+B7+C4,

A4+B4+B8+C4,

A4+B6+B8+C4,

A5+B1+B5+C4,

A5+B2+B4+C4,

A5+B3+B4+C4,

A5+B4+B5+C4,

A5+B5+B7+C4,

A2+A3+B1+C4,

A1+A3+B6+C4,

A1+A4+B3+C4,

A1+A4+B8+C4,

A1+A5+B5+C4,

A1+B1+B3+C4,

A1+B1+B8+C4,

A1+B2+B7+C4,

A1+B3+B7+C4,

A1+B4+B8+C4,

A1+B6+B8+C4,

A2+B1+B5+C4,

A2+B2+B4+C4,

A2+B3+B4+C4,

A2+B4+B5+C4,

A2+B5+B7+C4,

A3+B1+B2+C4,

A3+B1+B7+C4,

A3+B2+B6+C4,

A3+B3+B6+C4,

A3+B4+B7+C4,

A3+B6+B7+C4,

A4+B1+B4+C4,

A4+B2+B3+C4,

A4+B2+B8+C4,

A4+B3+B8+C4,

A4+B5+B6+C4,

A4+B7+B8+C4,

A5+B1+B6+C4,

A5+B2+B5+C4,

A5+B3+B5+C4,

A5+B4+B6+C4,

A5+B5+B8+C4,

A2+A3+B2+C4,

A2+A3+B3+C4, A2+A3+B4+C4, A2+A3+B5+C4, A2+A3+B6+C4, A2+A3+B7+C4,

A2+A3+B8+C4, A2+A4+B1+C4, A2+A4+B2+C4, A2+A4+B3+C4, A2+A4+B4+C4,

A2+A4+B5+C4, A2+A4+B6+C4, A2+A4+B7+C4, A2+A4+B8+C4, A3+A4+B1+C4,

A3+A4+B2+C4, A3+A4+B3+C4, A3+A4+B4+C4, A3+A4+B5+C4, A3+A4+B6+C4,

A3+A4+B7+C4, A3+A4+B8+C4, A4+A5+B1+C4, A4+A5+B2+C4, A4+A5+B3+C4,

A4+A5+B4+C4, A4+A5+B5+C4, A4+A5+B6+C4, A4+A5+B7+C4, A4+A5+B8+C4,

A1+A2+A3+C5, A1+A2+A4+C5, A1+A2+A5+C5, A1+A3+A4+C5, A1+A3+A5+C5,

A1+A4+A5+C5, A1+A2+B1+C5, A1+A2+B2+C5, A1+A2+B3+C5, A1+A2+B4+C5,

A1+A2+B5+C5, A1+A2+B6+C5, A1+A2+B7+C5, A1+A2+B8+C5, A1+A3+B1+C5,

A1+A3+B2+C5, A1+A3+B3+C5, A1+A3+B4+C5, A1+A3+B5+C5, A1+A3+B6+C5,

A1+A3+B7+C5, A1+A3+B8+C5, A1+A4+B1+C5, A1+A4+B2+C5, A1+A4+B3+C5,

A1+A4+B4+C5, A1+A4+B5+C5, A1+A4+B6+C5, A1+A4+B7+C5, A1+A4+B8+C5,

A1+A5+B1+C5, A1+A5+B2+C5, A1+A5+B3+C5, A1+A5+B4+C5, A1+A5+B5+C5,

A1+A5+B6+C5, A1+A5+B7+C5, A1+A5+B8+C5, A1+B1+B2+C5, A1+B1+B3+C5,

A1+B1+B4+C5, A1+B1+B5+C5, A1+B1+B6+C5, A1+B1+B7+C5, A1+B1+B8+C5,

A1+B2+B3+C5, A1+B2+B4+C5, A1+B2+B5+C5, A1+B2+B6+C5, A1+B2+B7+C5,

A1+B2+B8+C5, A1+B3+B4+C5, A1+B3+B5+C5, A1+B3+B6+C5, A1+B3+B7+C5,

A1+B3+B8+C5, A1+B4+B5+C5, A1+B4+B6+C5, A1+B4+B7+C5, A1+B4+B8+C5,

A1+B5+B6+C5, A1+B5+B7+C5, A1+B5+B8+C5, A1+B6+B7+C5, A1+B6+B8+C5,

A1+B7+B8+C5, A2+B1+B2+C5, A2+B1+B3+C5, A2+B1+B4+C5, A2+B1+B5+C5,

A2+B1+B6+C5, A2+B1+B7+C5, A2+B1+B8+C5, A2+B2+B3+C5, A2+B2+B4+C5,

A2+B2+B5+C5, A2+B2+B6+C5, A2+B2+B7+C5, A2+B2+B8+C5, A2+B3+B4+C5,

A2+B3+B5+C5, A2+B3+B6+C5, A2+B3+B7+C5, A2+B3+B8+C5, A2+B4+B5+C5,

A2+B4+B6+C5, A2+B4+B7+C5, A2+B4+B8+C5, A2+B5+B6+C5, A2+B5+B7+C5,

A2+B5+B8+C5, A2+B6+B7+C5, A2+B6+B8+C5, A2+B7+B8+C5, A3+B1+B2+C5,

A3+B1+B3+C5, A3+B1+B4+C5, A3+B1+B5+C5, A3+B1+B6+C5, A3+B1+B7+C5,

A3+B1+B8+C5, A3+B2+B3+C5, A3+B2+B4+C5, A3+B2+B5+C5, A3+B2+B6+C5,

A3+B2+B7+C5, A3+B2+B8+C5, A3+B3+B4+C5, A3+B3+B5+C5, A3+B3+B6+C5,

A3+B3+B7+C5, A3+B3+B8+C5, A3+B4+B5+C5, A3+B4+B6+C5, A3+B4+B7+C5,

A3+B4+B8+C5, A3+B5+B6+C5, A3+B5+B7+C5, A3+B5+B8+C5, A3+B6+B7+C5,

A3+B6+B8+C5, A3+B7+B8+C5, A4+B1+B2+C5, A4+B1+B3+C5, A4+B1+B4+C5,

A4+B1+B5+C5, A4+B1+B6+C5, A4+B1+B7+C5, A4+B1+B8+C5, A4+B2+B3+C5,

A4+B2+B4+C5,

A4+B3+B4+C5,

A4+B4+B5+C5,

A4+B5+B7+C5,

A5+B1+B2+C5,

A5+B1+B7+C5,

A5+B2+B6+C5,

A5+B3+B6+C5,

A5+B4+B7+C5,

A5+B6+B7+C5,

A2+A3+B3+C5,

A2+A3+B8+C5,

A2+A4+B5+C5,

A3+A4+B2+C5,

A3+A4+B7+C5,

A4+B2+B5+C5,

A4+B3+B5+C5,

A4+B4+B6+C5,

A4+B5+B8+C5,

A5+B1+B3+C5,

A5+B1+B8+C5,

A5+B2+B7+C5,

A5+B3+B7+C5,

A5+B3+B4+C5,

A5+B4+B5+C5,

A5+B4+B8+C5, A5+B5+B6+C5, A5+B5+B7+C5,

A5+B6+B8+C5, A5+B7+B8+C5, A2+A3+B1+C5,

A2+A3+B4+C5, A2+A3+B5+C5, A2+A3+B6+C5,

A2+A4+B1+C5, A2+A4+B2+C5, A2+A4+B3+C5,

A2+A4+B6+C5, A2+A4+B7+C5, A2+A4+B8+C5,

A3+A4+B3+C5, A3+A4+B4+C5, A3+A4+B5+C5,

A3+A4+B8+C5, A4+A5+B1+C5, A4+A5+B2+C5, A4+A5+B3+C5,

A4+A5+B4+C5, A4+A5+B5+C5, A4+A5+B6+C5, A4+A5+B7+C5, A4+A5+B8+C5,

A4+B2+B6+C5,

A4+B3+B6+C5,

A4+B4+B7+C5,

A4+B6+B7+C5,

A5+B1+B4+C5,

A5+B2+B3+C5,

A5+B2+B8+C5,

A5+B3+B8+C5,

A4+B2+B7+C5,

A4+B3+B7+C5,

A4+B4+B8+C5,

A4+B6+B8+C5,

A5+B1+B5+C5,

A5+B2+B4+C5,

A4+B2+B8+C5,

A4+B3+B8+C5

A4+B5+B6+C5,

A4+B7+B8+C5,

A5+B1+B6+C5

A5+B2+B5+C5,

A5+B3+B5+C5,

A5+B4+B6+C5

A5+B5+B8+C5,

A2+A3+B2+C5,

A2+A3+B7+C5

A2+A4+B4+C5,

A3+A4+B1+C5,

A3+A4+B6+C5

A1+A2+A3+C6,

A1+A4+A5+C6,

A1+A2+B5+C6,

A1+A3+B2+C6,

A1+A3+B7+C6,

A1+A4+B4+C6,

A1+A5+B1+C6,

A1+A5+B6+C6,

A1+B1+B4+C6,

A1+B2+B3+C6,

A1+B2+B8+C6,

A1+B3+B8+C6,

A1+B5+B6+C6,

A1+B7+B8+C6,

A2+B1+B6+C6,

A2+B2+B5+C6,

A1+A2+A4+C6,

A1+A2+B1+C6,

A1+A2+B6+C6,

A1+A3+B3+C6,

A1+A3+B8+C6,

A1+A4+B5+C6,

A1+A5+B2+C6,

A1+A5+B7+C6,

A1+B1+B5+C6,

A1+B2+B4+C6,

A1+B3+B4+C6,

A1+B4+B5+C6,

A1+B5+B7+C6,

A2+B1+B2+C6,

A2+B1+B7+C6,

A2+B2+B6+C6,

A1+A2+A5+C6,

A1+A2+B2+C6,

A1+A2+B7+C6,

A1+A3+B4+C6,

A1+A4+B1+C6,

A1+A4+B6+C6,

A1+A5+B3+C6,

A1+A5+B8+C6,

A1+B1+B6+C6,

A1+B2+B5+C6,

A1+B3+B5+C6,

A1+B4+B6+C6,

A1+B5+B8+C6,

A2+B1+B3+C6,

A2+B1+B8+C6,

A2+B2+B7+C6,

A1+A3+A4+C6,

A1+A2+B3+C6,

A1+A2+B8+C6,

A1+A3+B5+C6,

A1+A4+B2+C6,

A1+A4+B7+C6,

A1+A5+B4+C6,

Al+B 1+B2+C6,

A1+B1+B7+C6,

A1+B2+B6+C6,

A1+B3+B6+C6,

A1+B4+B7+C6,

A1+B6+B7+C6,

A2+B1+B4+C6,

A2+B2+B3+C6,

A2+B2+B8+C6,

A1+A3+A5+C6,

A1+A2+B4+C6,

A1+A3+B1+C6,

A1+A3+B6+C6,

A1+A4+B3+C6,

A1+A4+B8+C6,

A1+A5+B5+C6,

Al+B 1+B3+C6,

Al+B 1+B8+C6,

A1+B2+B7+C6,

A1+B3+B7+C6,

A1+B4+B8+C6,

A1+B6+B8+C6,

A2+B1+B5+C6,

A2+B2+B4+C6,

A2+B3+B4+C6,

A2+B3+B5+C6,

A2+B4+B6+C6,

A2+B5+B8+C6,

A3+B1+B3+C6,

A3+B1+B8+C6,

A3+B2+B7+C6,

A3+B3+B7+C6,

A3+B4+B8+C6,

A3+B6+B8+C6,

A4+B1+B5+C6,

A4+B2+B4+C6,

A4+B3+B4+C6,

A4+B4+B5+C6,

A4+B5+B7+C6,

A5+B1+B2+C6,

A5+B1+B7+C6,

A5+B2+B6+C6,

A5+B3+B6+C6,

A5+B4+B7+C6,

A5+B6+B7+C6,

A2+A3+B3+C6,

A2+A3+B8+C6,

A2+A4+B5+C6,

A3+A4+B2+C6,

A3+A4+B7+C6,

A2+B3+B6+C6,

A2+B4+B7+C6,

A2+B6+B7+C6,

A3+B1+B4+C6,

A3+B2+B3+C6,

A3+B2+B8+C6,

A3+B3+B8+C6,

A3+B5+B6+C6,

A3+B7+B8+C6,

A4+B1+B6+C6,

A4+B2+B5+C6,

A4+B3+B5+C6,

A4+B4+B6+C6,

A4+B5+B8+C6,

A5+B1+B3+C6,

A5+B1+B8+C6,

A5+B2+B7+C6,

A5+B3+B7+C6, A5+B3+B8+C6, A5+B4+B5+C6,

A5+B4+B8+C6, A5+B5+B6+C6, A5+B5+B7+C6,

A5+B6+B8+C6, A5+B7+B8+C6, A2+A3+B1+C6,

A2+A3+B4+C6, A2+A3+B5+C6, A2+A3+B6+C6,

A2+A4+B1+C6, A2+A4+B2+C6, A2+A4+B3+C6,

A2+A4+B6+C6, A2+A4+B7+C6, A2+A4+B8+C6,

A3+A4+B3+C6, A3+A4+B4+C6, A3+A4+B5+C6,

A3+A4+B8+C6, A4+A5+B1+C6, A4+A5+B2+C6, A4+A5+B3+C6,

A4+A5+B4+C6, A4+A5+B5+C6, A4+A5+B6+C6, A4+A5+B7+C6, A4+A5+B8+C6.

A2+B3+B7+C6,

A2+B4+B8+C6,

A2+B6+B8+C6,

A3+B1+B5+C6,

A3+B2+B4+C6,

A3+B3+B4+C6,

A3+B4+B5+C6,

A3+B5+B7+C6,

A4+B1+B2+C6,

A4+B1+B7+C6,

A4+B2+B6+C6,

A4+B3+B6+C6,

A4+B4+B7+C6,

A4+B6+B7+C6,

A5+B1+B4+C6,

A5+B2+B3+C6,

A5+B2+B8+C6,

A2+B3+B8+C6,

A2+B5+B6+C6,

A2+B7+B8+C6,

A3+B1+B6+C6,

A3+B2+B5+C6,

A3+B3+B5+C6,

A3+B4+B6+C6,

A3+B5+B8+C6,

A4+B1+B3+C6,

A4+B1+B8+C6,

A4+B2+B7+C6,

A4+B3+B7+C6,

A4+B4+B8+C6,

A4+B6+B8+C6,

A5+B1+B5+C6,

A5+B2+B4+C6,

A5+B3+B4+C6,

A2+B4+B5+C6,

A2+B5+B7+C6

A3+B1+B2+C6,

A3+B1+B7+C6

A3+B2+B6+C6

A3+B3+B6+C6,

A3+B4+B7+C6

A3+B6+B7+C6

A4+B1+B4+C6,

A4+B2+B3+C6

A4+B2+B8+C6

A4+B3+B8+C6,

A4+B5+B6+C6,

A4+B7+B8+C6

A5+B1+B6+C6,

A5+B2+B5+C6,

A5+B3+B5+C6

A5+B4+B6+C6,

A5+B5+B8+C6,

A2+A3+B2+C6

A2+A3+B7+C6,

A2+A4+B4+C6,

A3+A4+B1+C6

A3+A4+B6+C6

The skilled person will know that there are a number of peptides and proteins known in the art that are able to bind to receptors expressed on the luminal side of brain vasculature. The sequences or CDRs of these peptides or proteins are non-limiting examples of binding sites that can be employed in the multispecific binding molecules and are listed in Table 8.

Table 8; peptide- and protein-based BBB shuttles

Proposed target	shuttle	Sequence/CDR	Reference
LRP1	Angiopep-2	: VT'ïi.Si A&p*· i xk KNN c K	Olla-Salvia et al. Chem. Soc. Rev. 2016,45:4690-4707
LRP2, LDLR	ApoB (3371-3409)	-isVSP At vYb i.LETTS . A Λ V'	Olla-Salvia et al. Chem. Soc. Rev. 2016,45:4690-4707
LRP1, LRP2, LDLR	ApoE (159- 167)		Olla-Salvia et al. Chem. Soc. Rev. 2016,45:4690-4707
LDLR	peptide-22		Olla-Salvia et al. Chem. Soc. Rev. 2016, 45:4690-4707
T1R1	THR		Olla-Salvia et al. Chem. Soc. Rev. 2016, 45:4690-4707
TfRl	THR retro enantio	;\'<V	Olla-Salvia et al. Chem. Soc. Rev. 2016,45:4690-4707
TFR1,	CRT		Olla-Salvia et al. Chem. Soc. Rev. 2016,45:4690-4707
Leptin receptors	Leptin30	UNWm	Olla-Salvia et al. Chem. Soc. Rev. 2016, 45:4690-4707
nAchR	RVG29	m WH3i WGT TV. SS i'·'.:?·	Olla-Salvia et al. Chem. Soc. Rev. 2016,45:4690-4707
nAchR	CDX	ΐ >>. <?ίτχί h;:.·:	Olla-Salvia et al. Chem. Soc. Rev. 2016,45:4690-4707
KCa Channel	Apaniin	OiSs O JAS:·· S3 > jQQS-f ,W.	Olla-Salvia et al. Chem. Soc. Rev. 2016,45:4690-4707
KCa channel	MiniAP-4	rf.is^li&SSS.S^AS.TSiX]	Olla-Salvia et al. Chem. Soc. Rev. 2016,45:4690-4707
GSH transporter	GSH		Olla-Salvia et al. Chem. Soc. Rev. 2016,45:4690-4707
GM1	G23	HLEiETrEWsysA:	Olla-Salvia et al. Chem. Soc. Rev. 2016, 45:4690-4707
Unknown	«7		Olla-Salvia et al. Chem. Soc. Rev. 2016,45:4690-4707
Unknown	TGN		Olla-Salvia et al. Chem. Soc.
			Rev. 2016,45:4690-4707

AMT	TAT(47-57)		Olla-Salvia et al. Chem. Soc. Rev. 2016,45:4690-4707
AMT Passive diffusion	SynB I (phenylproli ne)4-NH2		Olla-Salvia et al. Chem. Soc. Rev. 2016,45:4690-4707 Olla-Salvia et al. Chem. Soc. Rev. 2016.45:4690-4707
Passive diffusion	Diketopipera zine	tlfesylp r«ti wsk A	Olla-Salvia et al. Chem. Soc. Rev. 2016,45:4690-4707
Cdc50A	FC5
TfR		SYYMH (CDR-1 (HC)) EIAPTNGRTNY1EKPKS (CDR-2 (HC)) GTRAYHY (CDR-3 (HC)) RASDNLYSNLA (CDR-1 (LQ) DATNLAD (CDR-2 (LQ) QHPWGTPLT (CDR-3 (LQ) EVQL VQSGAE VKKPGAS VKV SCKASGYTFTSYWMHWVRQA PGQRLEWIGE1APTNGRTNY IEKFKSRATLTVDKSASTAY MELSSLRSEDTAVYYCARGT RAYHYWGQGTMVTVSS (HC) DIQMTQSPSSLSASVGDRVT ITCRASDNLYSNLAWYQQKP GKSPKLLVYDATNLADGVPS RFSGSGSGTDYTLTISSLQP EDFATYYCQHFWGTPLTFGQ GTKVEIK (LC)	US9708406B2
TfR	AB405/AB2 21	gftfsnygmh MIYYDSSKMNYADTVKG PTSHYVVDV QASQD1GNWLA GATS LAD LQAYNTPWT	WO 2015191934 A2
Basigin	Anti-BsgA	E VQL VES GGGL VLPGRSMKL S C A AS GF TFRT YYM AW VRQ APTKGLEWVASISIGGDNTYYRDSVMGRF TISRDDAKSTL HLQMD LRSEDT AT Y YC	WO 2016094566 A2

		VRLRGYED YWGQGVM VT VS S (HC) EIVLTQSPATMPASPGEKVTLTCRASSSIRY IYWYOOKSGTSPKLWTYDTSKLASG VP RFSGSGSGTSYSLTISSMETEDTATYYCQQ GRSYPLTFGSGTKLEIK (LC)
Basigin	Anti-BsgB	evqlvesggglvqpgrslklscvasgftf xnywmfwirq APGKGLEWFASlTN^TijGS/LW.YPDSVKGRF 'nSRDNAQSTL YLQTNSLRPEDTATY YCA RR IX.iS YYPY Y a.yfplwgpgtt vrvss :hc NTVMTQSPTSMFIS VGDRVTMNCKASRS V gtnvdwyqq ktgosplllfygasryigvpdrfiosgsgf DFTLÏ'ISMQ AEDL A V Y YCLQ.YN.YNW AF GGGTKLELK (LC)	WO 2016094566 A2
Basigin	Anti-BsgC	EVOLVESGGSLVOPGRSMKVSCAASGFTF IKmiAWVRQAPTKGLEWVASISTGGGN TYYRDSVKGRFriSRDNAKSTLYLQMDSL rsedtaitycaltuny^yadyvm&aw GQGASVTVSS (HC) DiQMrQSPASLSASEXiETVSniCLASLG^ SLAWYOQKPGKSPOLLIYGASSi..ODGVPS RFSGSGSGTQFSLKISGMQPED egiyycoogykypftfgsgtkleik (LC)	WO 2016094566 A2
Basigin	Anti-BsgD	E VQL VES GGGL VQPGRSMKL S C A AS .GF.IESNYYM aw VROAPTKGLEWVAS1STGGGYTYYRDSVK GRFHSRDLAKST LYLQMDSLRSEDTATYHCARSllNYRNYG DYVMDAWGOGASVTVxSS (HC) DIOMTOSPASLSASLGETVSIECLASEGISN ^WYQQKPGKxSPQLLIYDASSLQVGVPxS RFSGSGSGTQYSLK1SGLQPEDEGVYYCÖQ GYKYPFTFGSGTKLEIK (LC)	WO 2016094566 A2
Basiging	Anti-BsgE	QV QLKESGPG L V QPSQTLSLTCS VSGLSLT TSSLSWIRQPPGKGLEWMGG^^	WO 2016094566 A2

		NSPlKSRLSlSRDTSKSOrFLKMNSLQTEDT AMYFC ARNGVYHNYWYFDFWGPGTMVTV SS (HC) QFTLTQPKSVSGSLRSTITIPC^SGmGH NYVSWYOQHL GRPPÏNVIYADDQRPS E VSDRFSGSIDS SSN SASLTITNLQM DDEADYFCQSYDSNVDIVFGGGTKLTVI·.. (LC)
Glutl	Anti-Glutl	QVQLQQPGSVLVRPGASVKkSCKASgYTF TGS,WUl.W AKQRPGQGLE W1GE1HPYSGNT NYNERFKGKATLTVDTPSSTAYVDLRSLT FED5AVY YCAKEGGWFLRIYGMDYWGO GTSVTVSS (HC) DIVLTQSPSSLSASLGDTnTl'CHASi^INV WI.SWYQQKPGNIPKL.IJYKASN'LI-ISGVPS RFSGSGSGTGFTLTISSLQPEDIATYYCQQG QTFPYTFGGGTRLEIK (LC)	WO 2016094566 A2

Therapeutic use

In one embodiment, of the invention the multispecific binding molecules are used for the diagnosis, prevention or treatment of neurodegenerative disorders, 5 selected from the group comprising AD, CAA, CTE, MSA, LBD, CBD, PSP, HD, ALS, FTD, FTDP-17 and PD.

In particular embodiments, the antibodies or antigen-binding fragments thereof disclosed herein may be used alone, or in combination with therapeutics known in the art for the treatment of NDDs.

Methods of Making Antibodies and Antigen-binding Fragments

Thereof

The present invention includes methods for making multispecific antibodies or antigen-binding fragments thereof. The skilled person will know that there are a variety of methods that allow the production of multispecific binding molecules such as multispecific antibodies. See Brinkmann, U., Kontermann, R.E, MAbs, 2017, 9 (2), 182-212.

Methods for producing and screening for monospecific antibodies using hybridoina technology are routine and well known in the art. In a non-limiting example, mice can be immunized with an antigen of interest or a cell expressing such an antigen. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. B-cells are cultured, as described by Steenbakkers et al., 1994, Mol. Biol. Rep. 19: 125-134.

B-cell clones from reactive supernatants are then immortalized, e.g. by mini-electrofusion following published procedures (Steenbakkers et al., J. Immunol. Meth. 1992, 152: 69-77; Steenbakkers et al., 1994, Mol. Biol. Rep. 19:12534). Hybridomas are selected and cloned by limiting dilution.

The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding the antigen. Ascites fluid, which generally contains high levels of antibodies, can be generated by inoculating mice intraperitoneally with positive hybridoma clones.

Monoclonal antibodies derived from animals other than rats and mice offer unique advantages. Many protein targets relevant to signal transduction and disease are highly conserved between mice, rats and humans, and can therefore be recognized as self-antigens by a mouse or rat host, making them less immunogenic. This problem may be avoided when using rabbit as a host animal. See, e.g., Rossi et al., Am. J. Clin. Pathol., 2005, 124, 295-302.

Adjuvants that can be used in the methods of antibody generation include, but are not limited to, protein adjuvants; bacterial adjuvants, e.g., whole bacteria (BCG, Corynebacterium parvum, Salmonella minnesota) and bacterial components including cell wall skeleton, trehalose dimycolate, monophosphoryl lipid A, methanol extractable residue (MER) of tubercle bacillus, complete or incomplete Freund's adjuvant; viral adjuvants; chemical adjuvants, e.g., aluminum hydroxide, iodoacetate and cholesteryl hemisuccinateor; naked DNA adjuvants.

Other adjuvants that can be used in the methods of the invention include, Cholera toxin, paropox proteins, MF-59 (Chiron Corporation; See also Bieg et al. (1999) “GAD65 And Insulin B Chain Peptide (9-23) Are Not Primary Autoantigens In The Type 1 Diabetes Syndrome Of The BB Rat,” Autoimmunity, 31(1):15-24, which is incorporated herein by reference), MPI,R (Corixa Corporation; See also Lodmell et al. (2000) “DNA Vaccination Of Mice Against Rabies Virus: Effects Of The Route Of Vaccination And The Adjuvant Monophosphoryl Lipid A (MPL),” Vaccine, 18: 10591066; Johnson et al. (1999) “3-O-Desacyl Monophosphoryl Lipid A Derivatives: Synthesis And Immunostimulant Activities,” Journal of Medicinal Chemistry, 42: 4640-4649; Baldridge et al. (1999) “Monophosphoryl Lipid A (MPL) Formulations For The Next Generation Of Vaccines,” Methods, 19: 103-107, all of which are incorporated herein by reference), RC-529 adjuvant (Corixa Corporation; the lead compound from Corixa's aminoalkyl glucosaininide 4-phosphate (AGP) chemical library, see also www.corixa.com), and DETOX™ adjuvant (Corixa Corporation; DETOX™ adjuvant includes MPLk adjuvant (monophosphoryl lipid A) and mycobacterial cell wall skeleton; See also Eton et al. (1998) “Active Immunotherapy With Ultraviolet B-Irradiated Autologous Whole Melanoma Cells Plus DETOX In Patients With Metastatic Melanoma,” Clin. Cancer Res. 4(3):619-627; and Gupta et al. (1995) “Adjuvants For Human Vaccines—Current Status, Problems And Future Prospects,” Vaccine, 13(14): 1263-1276, both of which are incorporated herein by reference).

Antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized using conventional methodologies with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Eonberg et al. (1995) “Human Antibodies From Transgenic Mice,” Int. Rev. Immunol. 13:65-93, which is incorporated herein by reference in its entirety). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Publication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix/Aingen(Freemont, Calif.) and Medarex/BMS (Princeton, N.J.), Kymab (Cambridge, UK) and Merus (Utrecht, Netherlands) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

The antibodies disclosed herein may also be produced recombinantly (e.g., in an E. colifYI expression system, a mammalian cell expression system or a lower eukaryote expression system). In this embodiment, nucleic acids encoding the antibody immunoglobulin molecules of the invention (e.g., Vn or Vl) may be inserted into a pET-based plasmid and expressed in the E. coliiVl system. For example, the present invention includes methods for expressing an antibody or antigen-binding fragment thereof or immunoglobulin chain thereof in a host cell (e.g., bacterial host cell such as E.coli such as BE21 or BE21DE3) comprising expressing T7 RNA polymerase in the cell which also includes a polynucleotide encoding an immunoglobulin chain that is operably linked to a T7 promoter. For example, in an embodiment of the invention, a bacterial host cell, such as a E. coli, includes a polynucleotide encoding the T7 RNA polymerase gene operably linked to a lac promoter and expression of the polymerase and the chain is induced by incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside).

Monoclonal antibody preparations can be produced using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS, pp. 563-681 (Elsevier, N.Y., 1981) (both of which are incorporated by reference in their entireties). The term “monoclonal antibody as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. One example of a method for recombinant production of antibodies is disclosed in U.S. Patent No. 4,816,567.

Thus, the present invention includes recombinant methods for making an antibody or antigen-binding fragment thereof of the present invention, or an immunoglobulin chain thereof, comprising introducing a polynucleotide encoding one or more immunoglobulin chains of the antibody or fragment (e.g., heavy and/or light immunoglobulin chain); culturing the host cell (e.g., CHO or Pichia or Pichia past,oris) under condition favorable to such expression and, optionally, isolating the antibody or fragment or chain from the host cell and/or medium in which the host cell is grown.

Antibodies of the present invention can also be synthesized by any of the methods set forth in U.S. Patent No. 6,331,415.

Eukaryotic and prokaryotic host cells, including mammalian cells as hosts for expression of the antibodies or fragments or immunoglobulin chains disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichiapastoris, Pichiafinlandica, Pichia tvehalophila, Pichia koclamae, Pichia membra,naefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa, Pichia sp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp., Yarrowia lipolytica, and Neurospora crassa. When recombinant expression vectors encoding the heavy chain or antigen-binding portion or fragment thereof, the light chain and/or antigen-binding fragment thereof are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody or fragment or chain in the host cells or secretion of the into the culture medium in which the host cells are grown.

A variety of host-expression vector systems may be utilized to express the antibodies of the invention. Such host-expression systems represent vehicles by which the coding sequences of the antibodies may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibodies of the invention in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing immunoglobulin coding sequences; yeast (e.g., Saccharomyces, Pi,chid) transformed with recombinant yeast expression vectors containing immunoglobulin coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the immunoglobulin coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing immunoglobulin coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphotic cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (rat retinal cells developed by Crucell) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al. (1983) “Easy Identification Of cDNA Clones,” EMBO J. 2:1791-1794), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye et al. (1985) “Up-Promoter Mutations In The Lpp Gene Of Escherichia coli,” Nucleic Acids Res. 13:3101-3110; Van Heeke et al. (1989) “Expression Of Human Asparagine Synthetase In Escherichia coli,” J. Biol. Chern. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione Stransferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose

100 beads followed by elution in the presence of free gluta-thione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera friigiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts, (see e.g., see Logan et al. (1984) “Adenovirus Tripartite Leader Sequence Enhances Translation Of mRNAs Late After Infection,” Proc. Natl. Acad. Sci. (U.S.A.) 81:3655-3659). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bitter et al. (1987) “Expression And Secretion Vectors For Yeast,” Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing

101 (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the posttranslational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express an antibody of the invention may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibodies of the invention. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibodies of the invention.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al. (1977) “Transfer Of Purified Herpes Virus Thymidine Kinase Gene To Cultured Mouse Cells,” Cell 11:223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al. (1962) “Genetics Of Human Cess Line. IV. DNA-Mediated Heritable Transformation Of A Biochemical Trait,” Proc. Natl. Acad. Sci. (U.S.A.) 48:20262034), and adenine phosphoribosyltransferase (Lowy et al. (1980) “Isolation Of

102

Transforming DNA: Cloning The Hamster Aprt Gene,” Cell 22:817-823) genes can be employed in tk- hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al. (1980) “Transformation Of Mammalian Cells With An Amplfiable Dominant-Acting Gene,” Proc. Natl. Acad. Sci. (U.S.A.) 77:3567-3570; O'Hare et al. (1981) “Transformation Of Mouse Fibroblasts To Methotrexate Resistance By A Recombinant Plasmid Expressing A Prokaryotic Dihydrofolate Reductase,” Proc. Natl. Acad. Sci. (U.S.A.) 78:15271531); gpt, which confers resistance to mycophenolic acid (Mulligan et al. (1981) “Selection For Animal Cells That Express The Escherichia coli Gene Coding For Xanthine-Guanine Phosphoribosyltransferase, Proc. Natl. Acad. Sci. (U.S.A.) 78:2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Tachibana et al. (1991) “Altered Reactivity Of Immunoglobutin Produced By Human-Human Hybridoma Cells Transfected By pSV2-Neo Gene,” Cytotechnology 6(3):219-226; Tolstoshev (1993) “Gene Therapy, Concepts, Current Trials And Future Directions,” Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993) “The Basic Science Of Gene Therapy,” Science 260:926-932; and Morgan et al. (1993) “Human gene therapy,” Ann. Rev. Biochein. 62:191-217). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY; Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CURRENT PROTOCOLS IN HUMAN GENETICS, John Wiley & Sons, NY.; Colbere-Garapin et al. (1981) “A New Dominant, Hybrid Selective Marker For Higher Eukaryotic Cells,” J. Mol. Biol. 150:1-14; and hygro, which confers resistance to hygromycin (Santerre et al. (1984) “Expression Of Prokaryotic Genes For Hygromycin B And G418 Resistance As Dominant,-Selection Markers In Mouse L Cells,” Gene 30:147-156).

The expression levels of an antibody of the invention can be increased by vector amplification (for a review, see Bebbington and Hentschel, “The Use Of Vectors Based On Gene Amplification For The Expression Of Cloned Genes In Mainmaian Cells,” in DNA CLONING, Vol. 3. (Academic Press, New York, 1987)).

103

When a marker in the vector system expressing an antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, production of the antibody will also increase (Crouse et al. (1983) “Expression And Amplification Of Engineered Mouse Dihydrofolate Reductase Minigenes,” Mol. Cell. Biol. 3:257-266).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot (1986) “Expression And Amplification Of Engineered Mouse Dihydrofolate Reductase Minigenes,” Nature 322:562-565; Kohler (1980) “Immunoglobulin Chain Loss In Hybridoma Lines,” Proc. Natl. Acad. Sci. (U.S.A.) 77:2197-2199). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Antibodies and antigen-binding fragments thereof and immunoglobulin chains can be recovered from the culture medium using standard protein purification methods. Further, expression of antibodies and antigen-binding fragments thereof and immunoglobulin chains of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4. Thus, in an embodiment of the invention, the mammalian host cells (e.g., CHO) lack a glutamine synthetase gene and are grown in the absence of glutamine in the medium wherein, however, the polynucleotide encoding the immunoglobulin chain comprises a glutamine synthetase gene which complements the lack of the gene in the host cell.

104

The present invention includes methods for purifying an antibody or antigen-binding fragment thereof of the present invention comprising introducing a sample comprising the antibody or fragment to a purification medium (e.g., cation exchange medium, anion exchange medium, hydrophobic exchange medium, affinity purification medium (e.g., protein-A, protein-G, protein-A/G, protein-L)) and either collecting purified antibody or fragment from the flow-through fraction of said sample that does not bind to the medium; or, discarding the flow-through fraction and eluting bound antibody or fragment from the medium and collecting the eluate. In an embodiment of the invention, the medium is in a column to which the sample is applied. In an embodiment of the invention, the purification method is conducted following recombinant expression of the antibody or fragment in a host cell, e.g., wherein the host cell is first lysed and, optionally, the lysate is purified of insofubfe materials prior to purification on a medium.

In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal. Therefore, the particular glycosylation pattern of an antibody will depend on the particular cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein, comprise the instant invention, independent of the glycosylation pattern the antibodies may have. Similarly, in particular embodiments, antibodies with a glycosylation pattern comprising only nonfucosylated iV-glycans may be advantageous, because these antibodies have been shown to typically exhibit more potent efficacy than their fucosylated counterparts both in vitro and in vivo (See for example, Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Patent Nos. 6,946,292 and 7,214,775). These antibodies with non-fucosylated A^T-glycans are not likely to be immunogenic because their carbohydrate structures are a normal component of the population that exists in human serum IgG.

The present invention further includes antigen-binding fragments of the antibodies disclosed herein. The antibody fragments include F(ab)2 fragments, which may be produced by enzymatic cleavage of an IgG by, for example, pepsin.

105

Fab fragments may be produced by, for example, reduction of F(ab)2 with dithiothreitol or mercaptoethylamine.

Immunoglobulins may be assigned to different classes depending on the amino acid sequences of the constant domain of their heavy chains. In some embodiments, different constant domains may be appended to humanized Vl and Vh regions derived from the CDRs provided herein. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgGl, IgG2, IgG3 and IgG4; IgAl and IgA2. The invention comprises antibodies and antigen-binding fragments of any of these classes or subclasses of antibodies.

In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region, e.g. a human constant region, such as Dl, D2, D3, or □ 4 human heavy chain constant region or a variant thereof. In another embodiment, the antibody or antigen-binding fragment comprises a light chain constant region, e.g. a human light chain constant region, such as lambda or kappa human light chain region or variant thereof. By way of example, and not limitation the human heavy chain constant region can be D4 and the human light chain constant region can be kappa. In an alternative embodiment, the Fc region of the antibody is D4 with a Ser228Pro mutation (Angal S. et al., 1993, Mol Immunol. 30: 105-108 position 241 is based on the Kabat numbering system).

In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgGl subtype. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgG2 subtype. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgG4 subtype.

Experimental and Diagnostic Uses

Multispecific antibodies (e.g., humanized antibodies) and antigenbinding fragments thereof may also be useful in diagnostic assays for targets involved in NDD as discussed herein, e.g., detecting its expression in specific cells,

106 tissues, serum or CSF. Such diagnostic methods may be useful in various disease diagnoses.

The present invention includes ELISA assays (enzyme-linked immunosorbent assay) incorporating the use of a multispecific antibody or antigenbinding fragment thereof disclosed herein.

For example, such a method comprises the following steps:

(a) coat a substrate (e.g., surface of a microtiter plate well, e.g., a plastic plate) with a multispecific antibody or antigen-binding fragment thereof;

(b) apply a sample to be tested for the presence of a pathological protein involved in any NDD described herein to the substrate;

(c) wash the plate, so that unbound material in the sample is removed;

(d) apply detectably labeled antibodies (e.g., enzyme-linked antibodies) which are also specific to the antigen;

(e) wash the substrate, so that the unbound, labeled antibodies are removed;

(f) if the labeled antibodies are enzyme linked, apply a chemical which is converted by the enzyme into a fluorescent signal; and (g) detect the presence of the labeled antibody.

Detection of the label associated with the substrate indicates the presence of the pathological protein.

In a further embodiment, the labeled antibody or antigen-binding fragment thereof is labeled with peroxidase which react with ABTS (e.g., 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)) or 3,3’,5,5’-Tetramethylbenzidine to produce a color change which is detectable. Alternatively, the labeled antibody or fragment is labeled with a detectable radioisotope (e.g., ³H) which can be detected by scintillation counter in the presence of a scintillant.

107

A multispecific antibody or antigen-binding fragment thereof of the invention may be used in a Western blot or immune-protein blot procedure. Such a procedure forms part of the present invention and includes e.g.:

(1) optionally transferring proteins from a sample to be tested for the presence of pathological protein (e.g., from a PAGE or SDS-PAGE electrophoretic separation of the proteins in the sample) onto a membrane or other solid substrate using a method known in the art (e.g., semi-dry blotting or tank blotting); contacting the membrane or other solid substrate to be tested for the presence of bound target or a fragment thereof with a multispecific antibody or antigen-binding fragment thereof of the invention.

Such a membrane may take the form of a nitrocellulose or vinyl-based (e.g., polyvinylidene fluoride (PVDF)) membrane to which the proteins to be tested for the presence of pathological protein in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel have been transferred (e.g., following electrophoretic separation in the gel). Before contacting the membrane with the multispecific antibody or fragment, the membrane is optionally blocked, e.g., with non-fat dry milk or the like so as to bind non-specific protein binding sites on the membrane.

(2) washing the membrane one or more times to remove unbound multispecific antibody or fragment and other unbound substances; and (3) detecting the bound multispecific antibody or fragment.

Detection of the bound antibody or fragment indicates that the pathological protein is present on the membrane or substrate and in the sample. Detection of the bound antibody or fragment may be by binding the antibody or fragment with a secondary antibody (an anti-immunoglobulin antibody) which is detectably labeled and, then, detecting the presence of the secondary antibody.

The multispecific antibodies and antigen-binding fragments thereof disclosed herein may also be used for immunohistochemistry. Such a method forms part of the present invention and comprises, e.g.,

108 (1) contacting a cell to be tested for the presence of the pathological protein with a multispecific antibody or antigen-binding fragment thereof of the invention; and (2) detecting the antibody or fragment on or in the cell.

If the antibody or fragment itself is detectably labeled, it can be detected directly. Alternatively, the antibody or fragment may be bound by a detectably labeled secondary antibody which is detected.

Pharmaceutical Compositions and Administration

To prepare pharmaceutical or sterile compositions of the multispecific antibodies and antigen-binding fragments of the invention, the antibody or antigen-binding fragment thereof is admixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984). Hence, also included in the present invention are pharmaceutical preparations comprising one of the previously described multispecific binding molecules and a pharmaceutically acceptable carrier or excipient.

Formulations of therapeutic and diagnostic agents may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

Toxicity and therapeutic efficacy of the antibodies of the invention, administered alone or in combination with another therapeutic agent, can be determined by standard pharmaceutical procedures in cell cultures or

109 experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LDso/ EDso). The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the EDso with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.

In a further embodiment, a further therapeutic agent that is administered to a subject in association with a multispecific antibody or antigenbinding fragment thereof of the invention in accordance with the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (November 1, 2002)).

The mode of administration can vary. Routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, intra-arterial, epidural or intracranial.

In particular embodiments, the multispecific antibodies or antigenbinding fragments thereof of the invention can be administered by an invasive route such as by injection. In further embodiments of the invention, an antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered intravenously, subcutaneously, intramuscularly, intraarterially, intracranially, epidurally or by inhalation, aerosol delivery. Administration by noninvasive routes (e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention.

It is preferred to administer the binding molecules of the invention through the intravenous, epidural or intracranial route.

The present invention provides a vessel (e.g., a plastic or glass vial, e.g., with a cap or a chromatography column, hollow bore needle or a syringe cylinder) comprising any of the antibodies or antigen-binding fragments of the invention or a

110 pharmaceutical composition thereof. The present invention also provides an injection device comprising any of the antibodies or antigen-binding fragments of the invention or a pharmaceutical composition thereof. An injection device is a device that introduces a substance into the body of a patient via a parenteral route, e.g., intramuscular, subcutaneous, intracranial, epidural or intravenous. For example, an injection device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g., antibody or fragment or a pharmaceutical composition thereof), a needfe for piercing skin and/or blood vessels for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore. In an embodiment of the invention, an injection device that comprises an antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition thereof is an intravenous (IV) injection device. Such a device includes the antibody or fragment or a pharmaceutical composition thereof in a cannula or trocar/needle which may be attached to a tube which may be attached to a bag or reservoir for holding fluid (e.g., saline; or lactated ringer solution comprising NaCl, sodium lactate, KC1, CaCL· and optionally including glucose) introduced into the body of the patient through the cannula or trocar/needle. The antibody or fragment or a pharmaceutical composition thereof may, in an embodiment of the invention, be introduced into the device once the trocar and cannula are inserted into the vein of a subject and the trocar is removed from the inserted cannula. The IV device may, for example, be inserted into a peripheral vein (e.g., in the hand or arm); the superior vena cava or inferior vena cava, or within the right atrium of the heart (e.g., a central IV); or into a subclavian, internal jugular, or a femoral vein and, for example, advanced toward the heart until it reaches the superior vena cava or right atrium (e.g., a central venous line). In an embodiment of the invention, an injection device is an autoinjector; a jet injector or an external infusion pump. A jet injector uses a highpressure narrow jet of liquid which penetrate the epidermis to introduce the antibody or fragment or a pharmaceutical composition thereof to a patient’s body. External infusion pumps are medical devices that deliver the antibody or fragment or a pharmaceutical composition thereof into a patient’s body in controlled amounts. External infusion pumps may be powered electrically or mechanically.

111

Different pumps operate in different ways, for example, a syringe pump holds fluid in the reservoir of a syringe, and a moveable piston controls fluid delivery, an elastomeric pump holds fluid in a stretchable balloon reservoir, and pressure from the elastic walls of the balloon drives fluid delivery. In a peristaltic pump, a set of rollers pinches down on a length of flexible tubing, pushing fluid forward. In a multi-channel pump, fluids can be delivered from multiple reservoirs at multiple rates. The present invention also provides sterilization fluids, a sharp bladed instrument, and a drilling device. Sterilization fluids may be used to sterilize the area of the head where the incision is made. A sharp bladed instrument may be a scalpel or another instrument that can be used to make such an incision in the head. The drilling device, may be a drill that can be used to make a small hole into the skull. Through this hole an injection can be made using an injection device as described above.

The pharmaceutical compositions disclosed herein may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Such needleless devices comprising the pharmaceutical composition are also part of the present invention. The pharmaceutical compositions disclosed herein may also be administered by infusion. Examples of well-known implants and modules for administering the pharmaceutical compositions include those disclosed in: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art and those comprising the pharmaceutical compositions of the present invention are within the scope of the present invention.

Furthermore, one may administer the antibody or fragment in a targeted drug delivery system, for example, in a liposome coated with a tissue

112 specific antibody or with an antibody that targets a BBB receptor. The liposomes will be targeted to and taken up selectively by the afflicted tissue. Such methods and liposomes are part of the present invention.

The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody or antigen-binding fragment, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody or fragment to effect improvement in the target disease state, while simultaneously minimizing undesired side effects. Accordingly, the amount of biologic delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. Guidance in selecting appropriate doses of therapeutic antibodies or fragments is available (see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med, 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced. In general, it is desirable that a biologic that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing any immune response to the reagent. In the case of human subjects, for example, humanized and fully human antibodies are maybe desirable.

113

Multispecific antibodies or antigen-binding fragments thereof disclosed herein may be provided by continuous infusion, or by doses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly, semiannually, annually etc. Doses may be provided, e.g., intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation. A total weekly dose is generally at least 0.05 pg/kg body weight, more generally at least 0.2 pg/kg, 0.5 pg/kg, 1 pg/kg, 10 pg/kg, 100 pg/kg, 0.25 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/ml, 10 mg/kg, 25 mg/kg, 50 mg/kg or more (see, e.g., Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al. (20003) Cancer Immunol. Imniunother. 52:151144). Doses may also be provided to achieve a pre-determined target concentration of the antibody in the subject’s serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 ug/ml or more. In other embodiments, a multispecific antibody of the present invention is administered, e.g., subcutaneously or intravenously, on a weekly, biweekly, every 4 weeks, monthly, bimonthly, or quarterly basis at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg/subject.

As used herein, the term effective amount refers to an amount of a multispecific antibody or multispecific antigen-binding fragment thereof of the invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to cause a measurable improvement in one or more symptoms of disease. An effective dose further refers to that amount of the antibody or fragment sufficient to result in at least partial amelioration of symptoms, e.g., memory loss, motor dysfunction or cognitive impairment. When applied to an individual active ingredient administered alone, an effective dose refers to that ingredient alone. When applied to a combination, an effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. An effective amount of a therapeutic will result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about, 30%; more preferably at least 40%, and most preferably by at least 50%. An effective amount can also result in an improvement in a

114 subjective measure in cases where subjective measures are used to assess disease severity.

The present invention also includes a therapeutic method in which a sample of cerebrospinal fluid (CSF) comprising the pathological protein (or a fragment thereof) is obtained from a subject diagnosed with a NDD, is purified and afterwards the purified CSF is reentered into the subject. In this method, the multispecific antibodies and antigen-binding fragments thereof are immobilized on a solid phase such a Sephadex, glass or agarose resin or filter paper, using methods well known in the art. A sample of CSF containing the pathological protein (or a fragment thereof) obtained from a subject diagnosed with an NDD is contacted with the solid phase, hereby the pathological protein is removed and the fluid is reentered into the subject. Such immobilized antibodies and fragments form part of the present invention.

In a further embodiment, nucleic acid constructs are implemented into the cell that encode for the multispecific antibodies of the invention.

For transcription from a nucleic acid construct, a regulatory sequence, such as a promoter, enhancer, terminator, splice donor and acceptor, or polyadenylation site may be used to transcribe the DNA. The RNA strand(s) may or may not be polyadenylated and the RNA strand(s) may or may not be capable of being translated into a polypeptide by the cell's translational apparatus.

The nucleic acid construct can be present as plasmid in the nucleus, where it can be translated repeatedly into the multispecific antibodies of the invention. Alternatively, the nucleic acid construct for encoding the multispecific antibodies of the present invention can be stably integrated into the chromosomal DNA of a target cell or progenitor thereof. Stable integration into the host cell DNA is a natural characteristic of various types of viruses, so that genes introduced by these vectors can be maintained for the life of that cell. Furthermore, the vector will be present in all daughter cells that result from cell division. Stable integration may be obtained by using such viruses as retroviruses, lentiviruses, adenoviruses, adeno-associated virus or herpes simples virus. Suitable retroviruses include murine leukaemia virus (MLV) and moloney murine leukemia virus (Mo

115

MuLV). The advantages of using a virus such as MLV are that there is no preexisting immunogenicity to such a virus, that there will be no immune response to viral gene products and that the virions are relatively easy to produce. However, the virus requires dividing cells for infection and may integrate randomly into the host cell genome leading to potential oncogenesis by insertional mutation. Also, it is known that the LTRs may interfere with gene expression. Suitable lentiviruses for use as the viral vector in a method of the invention include bovine lentiviruses, such as bovine immunodeficiency virus and Jembrana disease virus, equine lentiviruses such as equine infectious anemia virus, feline lentiviruses such as feline immunodeficiency virus (FIV), panther lentivirus and puma lentivirus, ovine/caprine lentiviruses such as Brazilian caprine lentivirus, caprine arthritisencephalitis virus, caprine lentivirus, Maedi-Visna virus, ovine lentivirus and Visna lentivirus and the primate lentivirus group including such viruses as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). An advantage of the use of lentivirus is that no dividing cells are required and that they may thus be used for in vivo applications to transduce non-dividing cells (e.g. memory T-lymphocytes, hemopoietic stem cells, neurons). However, when using viruses that are normally capable of causing infection in humans, these viruses need to be attenuated or made sufficiently harmless by deletion of essential genes.

Use of adeno-associated virus (AAV) vectors in a method of the invention is advantageous because these viruses both expeditiously infect non-dividing cells and may undergo site-specific integration into the host cell genome. More importantly, AAV-delivered DNA may be available as plasmids in the nucleus. Both scenarios result in long-term transduction and may therefore require only a single dose administration. AAV is a non-pathogenic dependent parvovirus with a broad host range, capable of high levels of transduction and expression in the host cell. Moreover, AAVs are particularly useful for delivering genes into the brain. For example, AAV-mediated delivery of genes encoding for anti-tau antibodies and fragments such as scFv into the brain was shown to be an effective tool for reducing tau pathology in mice. Much higher levels of antibody or antibodyfragments were observed in the brain, compared to systemic passive immunization, resulting in a marked decrease in tau accumulation. (Liu, W. et al., J. Neurosci.,

116

2016, 36 (49), 12425-12435, Ising, C. et al, J. Exp. Med„ 2017, 214 (5), 12271238).The skilled person will know how to administer the virus vector to a subject in need thereof.

Most preferred is the use of viral vectors derived from lentiviruses, adeno-associated virus (AAV), or combinations such as adenovirus-AAV hybrid vectors. The methods for constructing viral vectors and packaging those into a viral particle are known to a person skilled in the art. See Ising, C. et al, J. Exp. Med.,

2017, 214 (12), 1.; Stadler, C.R., Nature Medicine, 2017, 23. 815: Wi )2()16126993 li

Any regulatory sequences which are known or are found to cause expression of the therapeutic gene in the target cell can be used in the present invention. Such regulatory sequences may for instance be obtained from humans, animals, plants or fungi, or their associated viruses, or may be chemically synthesized, but are preferably target cell specific and include suitable eukaryotic or viral promoters operably linked to the therapeutic gene and active in directing its transcription in the target cells as well as terminators. The promoters include, but are not limited to, promoters from organ- or tissue-specific genes, or promoters of constitutively expressed genes. Examples of suitable promoters for directing the transcription of the therapeutic gene in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814) or the adenovirus 2 major late promoter the 5'-long terminal repeats from retroviruses and lentiviruses, the cytomegalus virus (CMV) immediate early promoter, and the like. These promoters and various others are easily obtainable for a person skilled in the art. Other regulatory sequences include terminator sequences and polyadenylation signals, including every sequence functioning as such in the target cells.

Kits

Further provided are kits comprising one or more components that include, but are not limited to, a multispecific antibody or antigen-binding fragment, as discussed herein in association with one or more additional components including, but not limited to a pharmaceutically acceptable carrier

117 and/or a therapeutic agent, as discussed herein. The antibody or fragment and/or the therapeutic agent can be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier, in a pharmaceutical composition.

In one embodiment, the kit includes a multispecific antibody or antigenbinding fragment thereof of the invention or a pharmaceutical composition thereof in one container (e.g., in a sterile glass or plastic vial) and a pharmaceutical composition thereof and/or a therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

In another embodiment, the kit comprises a combination of the invention, including a multispecific antibody or antigen-binding fragment thereof of the invention along with a pharmaceutically acceptable carrier, optionally in combination with one or more therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.

If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above.

The kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and patent information.

118

Detection Kits and Therapeutic Kits

As a matter of convenience, a multispecific antibody or antigen-binding fragment thereof of the invention can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic or detection assay. Where the antibody or fragment is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or lluorophore). In addition, other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.

Also provided are diagnostic or detection reagents and kits comprising one or more such reagents for use in a variety of detection assays, including for example, immunoassays such as ELISA (sandwich-type or competitive format). The kit's components may be pre-attached to a solid support, or may be applied to the surface of a solid support when the kit is used. In some embodiments of the invention, the signal generating means may come pre-associated with an antibody or fragment of the invention or may require combination with one or more components, e.g., buffers, antibody-enzyme conjugates, enzyme substrates, or the like, prior to use. Kits may also include additional reagents, e.g., blocking reagents for reducing nonspecific binding to the solid phase surface, washing reagents, enzyme substrates, and the like. The solid phase surface may be in the form of a tube, a bead, a microtiter plate, a microsphere, or other materials suitable for immobilizing proteins, peptides, or polypeptides. In particular aspects, an enzyme that catalyzes the formation of a chemiluminescent or chromogenic product or the reduction of a chemiluminescent or chromogenic substrate is a component of the signal generating means. Such enzymes are well known in the art. Kits may comprise any of the capture agents and detection reagents described herein.

119

Optionally the kit may also comprise instructions for carrying out the methods of the invention.

Also provided is a kit comprising a multispecific antibody or antigenbinding fragment thereof described herein packaged in a container, such as a vial or bottle, and further comprising a label attached to or packaged with the container, the label describing the contents of the container and providing indications and/or instructions regarding use of the contents of the container to treat one or more disease states as described herein.

In one aspect, the kit is for treating NDDs and comprises a multispecific antibody or antigen-binding fragment thereof and a further therapeutic agent or a vaccine. The kit may optionally further include a syringe for parenteral, e.g., intravenous, administration. In another aspect, the kit comprises a multispecific antibody or antigen-binding fragment thereof and a label attached to or packaged with the container describing use of the antibody or fragment with the vaccine or further therapeutic agent. In yet another aspect, the kit comprises the vaccine or further therapeutic agent and a label attached to or packaged with the container describing use of the vaccine or further therapeutic agent with the multispecific antibody or fragment. In certain embodiments, the multispecific antibody and vaccine or further therapeutic agent are in separate vials or are combined together in the same pharmaceutical composition.

The therapeutic and detection kits disclosed herein may also comprise at least one of the antibody, peptide, antigen-binding fragment, or polynucleotide disclosed herein and instructions for using the composition as a detection reagent or therapeutic agent. Containers for use in such kits may typically comprise at least one vial, test tube, flask, bottle, syringe or other suitable container, into which one or more of the detection and/or therapeutic composition(s) may be placed, and preferably suitably aliquoted. Where a second therapeutic agent is also provided, the kit may also contain a second distinct container into which this second detection and/or therapeutic composition may be placed. Alternatively, a plurality of compounds may be prepared in a single pharmaceutical composition, and may be packaged in a single container means, such as a vial, flask, syringe,

120 bottle, or other suitable single container. The kits disclosed herein will also typically include a means for containing the vial(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) are retained. Where a radiolabel, chromogenic, fluorigenic, or other type of detectable label or detecting means is included within the kit, the labeling agent may be provided either in the same container as the detection or therapeutic composition itself, or may alternatively be placed in a second distinct container means into which this second composition may be placed and suitably aliquoted. Alternatively, the detection reagent and the label may be prepared in a single container means, and in most cases, the kit will also typically include a means for containing the vial(s) in close confinement for commercial sale and/or convenient packaging and delivery.

A device or apparatus for carrying out the detection or monitoring methods described herein is also provided. Such an apparatus may include a chamber or tube into which sample can be input, a fluid handling system optionally including valves or pumps to direct flow of the sample through the device, optionally filters to separate plasma or serum from blood, mixing chambers for the addition of capture agents or detection reagents, and optionally a detection device for detecting the amount of detectable label bound to the capture agent immunocomplex. The flow of sample may be passive (e.g., by capillary, hydrostatic, or other forces that do not require further manipulation of the device once sample is applied) or active (e.g., by application of force generated via mechanical pumps, electroosmotic pumps, centrifugal force, or increased air pressure), or by a combination of active and passive forces.

In further embodiments, also provided is a processor, a computer readable memory, and a routine stored on the computer readable memory and adapted to be executed on the processor to perform any of the methods described herein. Examples of suitable computing systems, environments, and/or configurations include personal computers, server computers, hand-held or laptop devices, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe

121 computers, distributed computing environments that include any of the above systems or devices, or any other systems known in the art.

122

Claims (25)

CONCLUSIONS A multispecific binding molecule, preferably an antibody, comprising at least a first binding site binding to a first target selected from the group comprising human tau protein and post-translational modified human tau protein, such as phosphorylated, acetylated, glycosylated, glycated, prolyl isomerized, nitrated, polyaminated ,ubiquitinated, simulated, oxidized and aggregated human tau protein and spliced and truncated versions thereof, α-synuclein, TDP43, mHTT, and fragments thereof, and at least a second binding site binding to a second target selected from the group comprising human tau protein. and post-translational modified human tau protein, such as phosphorylated, acetylated, glycosylated, glycated, propyl-isomerized, nitrated, polyaminated ,ubiquitinated, humoylated, oxidized and aggregated human tau protein and spliced and truncated versions thereof, Cl-tqq, β43 β43 β, alpha, DP43 α43 , C5a, IL 1B, IL6, TNF-α, APOE, IL12, IL23 and fragments thereof, wherein the first and second targets are different. A multi-specific binding molecule according to claim 1, wherein said binding molecule at least a first binding site binding to human tau protein or post-translational modified human tau protein, such as phosphorylated, acetylated, glycosylated, glycated, propyl isomerized, nitrated, polyaminated, ubiquitized, ubiquitinated, ubiquitinated comprises oxidized and aggregated human tau protein and split and truncated versions thereof and at least a second binding site binding to α-synuclein or a fragment thereof. 123 A multi-specific binding molecule according to claim 1, wherein said binding molecule at least a first binding site binding to human tau protein or post-translational modified human tau protein, such as phosphorylated, acetylated, glycosylated, glycated, propyl isomerized, nitrated, polyaminated, ubiquitinated, ubiquitinated, ubiquitinated comprises oxidized and aggregated human tau protein and split and truncated versions thereof and at least a second binding site binding to Clq or a fragment thereof. A multi-specific binding molecule according to claim 1, wherein said binding molecule comprises at least a first binding site binding to α-synuclein or a fragment thereof and at least a second binding site binding to C1q or a fragment thereof. A multi-specific binding molecule according to claim 2, wherein said binding molecule binding at least one binding site to human tau protein selected from the group comprising binding fragments of tau 13 and binding fragments of 5A6 and at least one binding site binding to α-synuclein selected from the group comprising binding fragments of Syn211 and binding fragments of H3C. A multi-specific binding molecule according to claim 3 wherein said binding molecule at least one binding site binding to tau protein selected from the group comprising binding fragments of tau 13 and binding fragments of 5A6 and at least one binding site binding to Clq selected from the group comprising binding fragments of JL- 1 and binding fragments produced by the hybridomas 23B6C8, 5B5C22, 12A5B7 and 4A4B11. 124 A multi-specific binding molecule according to claim 4, wherein said binding molecule binding at least one binding site to Clq selected from the group comprising binding fragments of JL-1 and binding fragments produced by the hybridomas 23B6C8, 5B5C22, 12A5B7 and 4A4B11 and at least one binding site binding to α -synuclein selected from the group comprising binding fragments of Syn211 and binding fragments of H3C. A multi-specific binding molecule according to claims 1-7, wherein said binding molecule comprises a portion that allows for transport of the binding molecule through the blood-brain barrier, preferably wherein said portion is a binding site. A multi-specific binding molecule according to claims 1-8, wherein said binding molecule is humanized. A multi-specific binding molecule according to claims 1-9, wherein said binding molecule is bispecific. A multi-specific binding molecule according to claims 1-9, wherein said binding molecule is trispecific. A multi-specific binding molecule according to claims 1-11, with a structure selected from the group consisting of multi-specific binding structures listed in Figure 2 from Brinkmann, et al., Mabs, 2017, 9: 182-212 and Figure 1 from Spiess, et al. . Molecular Immunology, 2015, 67: 95-106, and multispecific antibody conjugates, for example dual-variable domain (DVD) antibody, trispecific IgG2 and tetraspecific IgG2, triplet targeting triplebody, triabody, tribody, trispecific triple heads, trisp specific triple dAb, tetr aspecific dAb, multispecific dAb, circular dimeric single-chain diabody (CD-scDb), linear dimeric single-chain diabody (LD-scDb), disulfide-stabilized Fv fragment, bis-scFv, tandem tri-scFv, bispecific Fab2, Fab3, 125 chemically conjugated trimeric Fab, diaminism, tetrabody, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusions such as Bs1Ab, Bs2Ab, Bs3Ab, Trispecific C-terminal fusions, Trispecific N-terminal-fusal, T-terminalA, fusal Ts2Ab, IgAl, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgM, CODV-Ig, sdAb, trispecific Zybody, tetraspecific Zybody, pent-specific Zybody, septas-specific Zybody, septas-specific Zybody, octole-specific molecularbody into duobodies. A multispecific binding molecule according to claims 1-11, wherein the multispecific binding molecule has a structure selected from the group comprising trispecific triabodies, trispecific tribodies, IgG (H) -scFv, scFv- (H) IgG, IgG (L) -scFv, scFv- (L) IgG, 2scFv-IgG, IgG-2scFv, DVI-IgG, scFab-Fc (kih) -scFv2, scFab-Fc (kih) scFv, IgG-taFv, scFv4-IgG and TVD-Ig A multispecific binding molecule according to claims 1-13, wherein the multispecific binding molecule is an antibody and wherein the Fc region of the antibody comprises a mutation at one of the following positions: 233, 234, 235, 236, 237, 268, 269, 270, 254, 254, 294, 297, 298, 300, 318, 320, 322, 327, 329, 331. A multi-specific binding molecule according to claims 1-14 for use in the treatment or prevention of neurodegenerative disorders selected from the group comprising Alzheimer's disease, Lewy body dementia, Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis ( ALS), frontotemporal dementia, frontotemporal dementia with Parkisonism-17, multisystematrophy, corticobasal degeneration, Progressive supranuclear palsy, Peak disease, primary age-related tauopathy, argyrophilic grain disease and cerebral amyloid angiopathy. 126 A multi-specific binding molecule according to claims 1-14 for use in the treatment of Alzheimer's disease. The binding molecules for the use according to claim 16, wherein said multispecific binding molecule is a bispecific binding molecule. The binding molecules for the use of claim 16, wherein said multispecific binding molecule is a trispecific binding molecule. The binding molecules for the use of claim 16, wherein said multispecific binding molecule is a tetra-specific binding molecule. The use of a therapeutic delivery vehicle, encoding any of the multi-specific binding molecules according to claims 1-14 in the treatment of neurodegenerative disorders. A method for treating neurodegenerative disorders, comprising: administering a multispecific binding molecule according to claims 1-14 to a patient in need thereof. The use of claim 20, wherein the therapeutic delivery vehicle is an adeno-associated virus vector. A method for treating the neurodegenerative disorders, comprising: administering a nucleic acid construct encoding a multi-specific binding molecule according to claims 1-14 in a therapeutic delivery vehicle to a patient in need thereof. A diagnostic method for the detection of neurodegenerative disorders, comprising: adding a multi-specific binding molecule according to claims 1-14 to a sample obtained from a patient; determining binding of 127, said binding molecule to any of the targets selected from human tau protein and post-translational modified human tau protein, such as phosphorylated, acetylated, glycosylated, glycated, propyl-isomerized, nitrated, Polyaminated ,ubiquitinated, simulated, oxidized and aggregated human tau protein and spliced and truncated versions thereof, α-synuclein, TDP43, mHTT, Clq, C5a, IL1B, IL6, TNF-α, APOE, TREM2, IL12, IL23 and fragments thereof in said sample; and diagnosis of neurodegenerative disorder such as 10 said binding has been detected. A kit for the diagnosis of neurodegenerative disorders, comprising a multi-specific binding molecule according to any of claims 1-14 and means for the detection of said binding molecule. Title: Multispecific binding molecules for the prevention, treatment and diagnosis of neurodegenerative disorders