EA043617B1 - HUMANIZED AND DEIMMUNIZED ANTIBODIES - Google Patents

Description

Field of technology to which the invention relates

The invention relates to humanized and deimmunized antibodies that bind to an epitope at the N-terminus of the pyroglutamate variant of beta-amyloid peptide (Άβ N3pE), and to the prophylactic and therapeutic treatment of diseases and conditions that are associated with the accumulation and deposition of amyloid peptides, such as amyloidosis, being a group of disorders and anomalies associated with the pyroglutamate variant of amyloid peptide, such as Alzheimer's disease, Down syndrome, cerebral amyloid angiopathy and other related aspects. More particularly, it relates to the use of monoclonal antibodies of the present invention to bind the pyroglutamate variant of beta-amyloid peptide in the blood plasma, brain and cerebrospinal fluid to prevent the accumulation or to regress the deposition of Άβ N3pE within the brain and in various tissues in the periphery and to reduce severity of amyloidosis. The present invention further relates to diagnostic assays for diagnosing amyloidosis using the antibodies of the present invention.

State of the art

Amyloidosis is not a single disease, but rather a group of heterogeneous progressive pathological processes characterized by extracellular tissue deposits of a waxy starch-like protein called amyloid, which accumulates in one or more organs or systems of the body. As amyloid deposits accumulate, they prevent an organ or body system from performing normal functions. There are at least 15 different types of amyloidosis. The main forms are primary amyloidosis without a known preexisting condition, secondary amyloidosis after certain other conditions, and hereditary amyloidosis.

Secondary amyloidosis occurs with chronic infectious or inflammatory disease such as tuberculosis, a bacterial infection called familial Mediterranean fever, bone infections (osteomyelitis), rheumatoid arthritis, inflammation of the small intestine (granulomatous ileitis), Hodgkin's disease, and leprosy.

Amyloid deposits include amyloid P (pentagonal) component (AP), normal serum amyloid P-associated glycoprotein (SAP), and sulfated glycosaminoglycans (GAGs), which are complex carbohydrates of connective tissue. Amyloid protein fibrils, which account for approximately 90% of the amyloid material, contain one of several different types of proteins. These proteins are capable of folding into so-called beta-pleated sheet fibrils, a unique protein configuration that has binding sites for Congo red dye, resulting in the unique staining properties of the amyloid protein.

Many age-related diseases are caused by or associated with amyloid-like proteins, and they are particularly characterized by the formation of extracellular deposits of amyloid or amyloid-like material that contribute to the pathogenesis as well as the progression of the disease. These diseases include, but are not limited to, neurological disorders such as mild cognitive impairment (MCI), Alzheimer's disease (AD) such as sporadic Alzheimer's disease (SAD) or familial forms of Alzheimer's dementia (FAD) such as familial British dementia (FBD) and familial Alzheimer's disease (FBD). Danish dementia (FDD), neurodegeneration in Down syndrome, dementia with Lewy bodies, hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guam complex, which is parkinsonism-dementia. Other diseases that are caused by or associated with amyloid-like proteins include progressive supranuclear gaze palsy, multiple sclerosis; Creutzfeldt-Jakob disease, Parkinson's disease, HIV-related dementia, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; age-related cardioamyloidosis; endocrine tumors and others, including macular degeneration.

Although the pathogenesis of these diseases may differ, their characteristic deposits often contain many common molecular components. This may be largely due to local activation of proinflammatory signaling pathways, which leads to the simultaneous deposition of activated complement components, acute phase reactants, immunomodulators and other inflammatory mediators (McGeer et al., Tohoku J Exp Med. 174(3): 269 -277 (1994)).

Recent accumulating evidence demonstrates the involvement of Ae peptide variants with N-terminal modification in Alzheimer's disease. Targeted biopsies demonstrate the presence of Ae 1-40 and Ae 1-42 not only in the brains of patients with Alzheimer's disease, but also in senile plaques in unaffected individuals. However, Ae N3pE-40/Ae N3pE-42 with N-terminal truncation and pyroGlu modification is incorporated almost exclusively into plaques of Alzheimer's disease patients, making this Ae variant a suitable diagnostic marker and a potential target for drug development.

Several commercial manufacturers now offer ELISA kits that can detect Ae 1-40/1-42 and Ae N3pE-40/Ae N3pE-42 in the low picogram (pg) range.

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The brains of patients with Alzheimer's disease (AD) are morphologically characterized by the presence of neurofibrillary tangles and Ae-peptide deposits in the neocortical structures of the brain (Selkoe, D. J. & Schenk, D. Alzheimer's disease: molecular understanding predicts amyloid-based therapeutics. Annu. Rev. Pharmacol. Toxicol 43, 545-584 (2003). Ae peptides are released from amyloid precursor protein (APP) after sequential cleavage by β- and γ-secretase. Cleavage by γsecretase results in the formation of Ae-peptides 1-40 and Ae-peptides 1-42, which differ in their C-termini and exhibit different activities regarding aggregation, fibril formation and neurotoxicity (Shin, R.W. et al., Amyloid beta-protein ( Abeta) 1-40 but not Abeta 1-42 contributes to the experimental formation of Alzheimer disease amyloid fibrils in rat brain. J. Neurosci. 17, 8187-8193 (1997); Iwatsubo, T. et al., Visualization of Abeta 42 (43) and Abeta 40 in senile plaques with end-specific Abeta monoclonals: evidence that an initially deposited species is Abeta 42(43).Neuron 13, 45-53 (1994);Iwatsubo, T., Mann, D.M., Odaka, A., Suzuki, N. & Ihara, Y. Amyloid beta protein (Abeta) deposition: Abeta 42(43) precedes Abeta 40 in Down syndrome. Ann. Neurol. 37, 294-299 (1995); Hardy, J. A. & Higgins , G. A. Alzheimer's disease: the amyloid cascade hypothesis. Science 256, 184-185 (1992); Roener, S., Ueberham, U., Schliebs, R., Perez-Polo, J. R. & Bigl, V. The regulation of amyloid precursor protein metabolism by cholinergic mechanisms and neurotrophin receptor signaling. Prog. Neurobiol. 56, 541-569 (1998).

Most Ae peptides deposited in diffuse plaques have an N-terminal truncation or modification. Studies by Piccini and Saido have shown that the basic structure of senile plaques and vascular deposits consists of 50% pyroGlu-modified peptides (Piccini et al., J Biol Chem. 2005 Oct 7; 280(40):34186-92; Saido et al. al. Neuron 1995 Feb;14(2):457-66). Peptides modified with pyroGlu are more cytotoxic than other forms of Ae molecules, and they are resistant to aminopeptidases (Russo et al., J Neurochem. 2002 Sep; 82(6):14809). Thus, molecules of the pyroGlu-Ae form have a longer half-life, as a result of which the accumulation of these molecules of this form and the formation of neurotoxic oligomers as well as aggregates is advantageous (Saido, Neurobiol Aging. 1998 Jan-Feb; 19(1 Suppl):S69 -75). Due to the cyclization of glutamate to pyroGlu, charged amino acids will be lost, which sharply reduces the solubility of the peptide and causes an increased tendency to aggregation. In vitro studies have shown that initial oligomerization, such as Ae3(pE), is much faster compared to unmodified peptides (Schilling et al., Biochemistry. 2006 Oct 17;45(41): 12393-9). Ae-peptides N3pE-42 coexist with Ae-peptides 1-40/1-42 (Saido, T.S. et al., Dominant and differential deposition of distinct beta-amyloid peptide species, Abeta N3pE, in senile plaques. Neuron 14, 457-466 (1995); Saido, T. C., Yamao, H., Iwatsubo, T. & Kawashima, S. Amino- and carboxyl-terminal heterogeneity of beta-amyloid peptides deposited in the human brain. Neurosci. Lett. 215, 173-176 (1996)), and based on a number of observations, this may play a significant role in the pathogenesis of AD. For example, it has been shown that the specific neurotoxicity of Ae-peptides N3pE-42 (Russo, S. et al. Pyroglutamate-modified amyloid betapeptides--AbetaN3(pE)--strongly affect cultured neuron and astrocyte survival. J. Neurochem. 82, 1480-1489 (2002) and pE-modification of N-truncated Ae-peptides confers resistance to destruction by most aminopeptidases, as well as Ae-degrading endopeptidases (Russo, S. et al., Pyroglutamate-modified amyloid beta-peptides--AbetaN3(pE )--strongly affect cultured neuron and astrocyte survival. J. Neurochem. 82, 1480-1489 (2002); Saido, T. C. Alzheimer's disease as proteolytic disorders: anabolism and catabolism of betaamyloid. Neurobiol. Aging 19, S69-S75 (1998) Cyclization of glutamic acid to pE results in loss of N-terminal charge, which accelerates the aggregation of Ae N3pE compared with unmodified Aepeptides (He, W. & Barrow, C.J. The Abeta 3-pyroglutamyl and 11-pyroglutamyl peptides found in senile plaque have greater beta-sheet forming and aggregation propensities in vitro than full-length A beta. Biochemistry 38, 10871-10877 (1999); Schilling, S. et al. On the seeding and oligomerization of pGlu-amyloid peptides (in vitro). Biochemistry 45, 12393-12399 (2006)). Thus, reducing the formation of Ae N3pE-42 should lead to destabilization of the peptides, making them more accessible for degradation, and, in turn, preventing the formation of higher molecular weight Ae aggregates and increasing neuronal survival.

However, for a long time it was not known exactly how pE modification of Ae peptides occurs. Recently, it was discovered that glutaminyl cyclase (QC) is able to catalyze the formation of Ae N3pE-42 under slightly acidic conditions and that specific QC inhibitors prevent the formation of Ae N3pE-42 in vitro (Schilling, S., Hoffmann, T., Manhart, S., Hoffmann, M. & Demuth, H.-U. Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions. FEBS Lett. 563, 191-196 (2004); Cynis, H. et al. Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells. Biochim. Biophys. Acta 1764, 1618-1625 (2006).

All the facts indicate that pyroGlu Ae is a kind of seed for the initialization of fibril formation. In another study (Piccini et al., 2005, supra), volunteers with plaque deposits but without specific AD pathology could be distinguished from AD patients by the characteristic number of Ae molecular forms. Thus, the number of modified pyroGlu pepti

- 2,043,617 N-terminal truncations were significantly higher in the brains of AD patients.

Posttranslational formation of pyroGlu at position 3 or 11 of the Άβ peptide implies cyclization of the N-terminal glutamate residue. Glutaminyl cyclase (QC) plays an important role in the formation of pyroGlu peptides. QC is widely distributed in the plant and animal world and, in particular, is involved in the maturation of peptide hormones. Cyclization of glutamine by releasing ammonia and glutamate by releasing water to pyroGlu is accomplished by QC. Unlike glutamine cyclization, glutamate cyclization does not occur spontaneously. QC catalyzes the efficient (unwanted) side reaction from glutamate to pyroGlu. The resulting pyroGlu residue protects the protein from proteolytic degradation. There are several literature sources that show that QC plays an important role in the formation of pyroGlu-Άβ:

1) Several studies have shown that QC catalyzes the formation of pyroGlu residues from glutamate at the N-terminus of Άβ (Cynis et al., Biochim Biophys Acta. 2006 Oct; 1764(10): 1618-25, Schilling et al., FEBS Lett 2004 Apr 9;563(l-3): 191-6);

2) both Άβ-peptides and QC are expressed in large quantities in the hippocampus and cortex. These brain regions are at particular risk for damage in AD (Pohl et al., Proc Natl Acad Sci USA. 1991 Nov 15;88(22):10059-63, Selkoe Physiol Rev. 2001 Apr;81(2):741- 66);

3) APP is cleaved by β-secretase during transport to the plasma membrane, due to which the N-terminus of Ae with a free glutamate residue can be obtained (Greenfield et al., Proc Natl Acad Sci USA. 1999 Jan 19; 96(2):742 -7). Co-localization of processed APP and QC was determined in secretory vesicles. Thus, in the weakly acidic environment of vesicles, accelerated modification of the glutamate residue to pyroglutamate can occur;

4) other neurodegenerative diseases (familial Danish (FDD) or British dementia (FBD)) are also associated with N-terminally modified pyroGlu peptides, for example Bri2, but at the same time they are not related to Ae in their primary structure (Vidal R et al., 1999 Proc. Natl. Acad. Sci. U.S.A. 97, 4920-4925).

It is possible that QC-catalyzed formation of pyroGlu-Ae is involved in the development and progression of neurodegenerative diseases. The formation of N-terminally modified amyloid peptides is clearly a fundamental factor in the process of Ae aggregation and may be the onset of disease. Inhibition of pyroGlu-Ae formation by inhibition of QC may represent a therapeutic approach. QC inhibitors would be able to prevent the formation of pyroGlu-Ap, reduce the concentration of the pyroglutamate variant of Ae in the brain, and thus delay the oligomerization of Ae peptides. Schilling et al. show that QC expression is upregulated in the cortex of AD patients and correlates with the appearance of pyroGlu-modified Ae peptide. Oral administration of a QC inhibitor resulted in decreased levels of pyroglutamate-modified AerE(3-42) in two different transgenic mouse models of AD and in a new Drosophila model (Schilling et al., 2008 Biol. Chem. (389), 983-991).

Lewy body dementia (LBD) is a neurodegenerative disorder that can occur in people over 65 years of age, and it typically causes symptoms of cognitive impairment and abnormal behavior changes. Symptoms may include cognitive impairment, neurological signs, sleep disturbance and autonomic failure. In most cases, cognitive impairment is a characteristic feature of LBD. Patients have recurrent episodes of confusion that gradually worsen. Changes in cognitive abilities are often associated with shifts in attention and vigilance. Cognitive impairment and fluctuations in thinking may change over a period of minutes, hours or days. Lewy bodies are formed from phosphorylated and non-phosphorylated neurofilament proteins; they contain the synaptic protein alpha-synuclein, as well as ubiquitin, which is involved in eliminating damaged or abnormal proteins. In addition to Lewy bodies, Lewy neurites, which are inclusion bodies in the cellular processes of nerve cells, may also be present. Amyloid plaques can form in the brains of patients affected by DLB, but they tend to be smaller than in patients with Alzheimer's disease. Neurofibrillary tangles, another micropathological feature of AD, are not the main characteristic of LBD but are often present in addition to amyloid plaques.

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of upper and lower motor neurons. Some patients with ALS may have dementia or aphasia (ALS-D). The dementia most commonly is frontotemporal dementia (FTD), and many of these cases have ubiquitin-positive, tau-negative inclusions in neurons of the dentate gyrus and superficial layers of the frontal and temporal lobes.

Inclusion body myositis (IBM) is a disabling disease, usually affecting people over 50 years of age, in which muscle fibers become inflamed and atrophy, but the brain is spared and patients retain full intelligence. Levels of two enzymes involved in the production of e-amyloid protein were found to be elevated

- 3 043617 inside muscle cells of patients with this most common progressive muscle disease of older people, in which amyloid-β levels are also elevated.

Another disease that is caused by or associated with the accumulation and deposition of amyloid-like protein is macular degeneration. Macular degeneration is a common eye disease that causes damage to the macula, which is the central area of the retina (the paper-thin tissue in the fundus of the eye where light-sensitive cells send visual signals to the brain). Crisp, clear central vision is processed by the macula. Damage to the macula leads to the development of blind spots and blurred or distorted vision. Age-related macular degeneration (AMD) is the leading cause of visual impairment in the United States, and in people over 65 years of age, it is the leading cause of civil blindness in Caucasians. Approximately 1.8 million Americans aged 40 years and older have advanced AMD, and an additional 7.3 million people with intermediate AMD are at significant risk of vision loss. The government estimates that by 2020 there will be 2.9 million people with advanced AMD. Victims of AMD are often surprised and upset to learn how little is known about the causes and treatment of this blinding condition.

There are two forms of macular degeneration: dry macular degeneration and wet macular degeneration. The dry form, in which the cells of the macula begin to slowly deteriorate, is diagnosed in 85 percent of cases of macular degeneration. Both eyes are usually affected by dry AMD, although one eye may go blind while the other eye remains unaffected. Drusen, which are yellow deposits under the retina, are typical early signs of dry AMD. The risk of developing late stage dry AMD or wet AMD increases as the number or size of drusen increases. It is likely that dry AMD will progress to cause vision loss without progressing to wet AMD; however, it is also possible that early on the dry form of AMD will suddenly change to the wet form.

The wet form, although it accounts for only 15 percent of cases, is blinding in 90 percent and is considered late-stage AMD (there is no early or intermediate stage wet AMD). Wet AMD is always preceded by a dry form of the disease. As the dry form worsens, some people develop abnormally developing blood vessels growing behind the macula. These vessels are very fragile and will leak fluid and blood (hence wet macular degeneration), causing rapid damage to the macula.

Dry AMD often initially causes mild blurred vision. The center of the visual field in particular may then become blurred, and this area grows as the disease progresses. If only one eye is affected, there may be no symptoms. In wet AMD, straight lines may appear jagged and loss of central vision may occur quickly.

Diagnosis of macular degeneration usually involves a thorough vision test and eye examination, testing visual acuity and examining the fundus using a procedure called ophthalmoscopy to help diagnose AMD, and fluorescein angiography may also be performed if wet AMD is suspected. If you develop advanced stages of dry AMD, there is currently no treatment to prevent vision loss. However, a specific formulation with high doses of antioxidants and zinc may delay or prevent progression from intermediate to advanced AMD. Using Macugen® (pegaptanib sodium injection), laser photocoagulation, and photodynamic therapy can control abnormal blood vessel growth and bleeding in the macula, which is helpful for some people with wet AMD; however, vision that has already been lost cannot be restored using these techniques. If vision has already been lost, there are low vision aids that can help improve your quality of life.

One of the earliest signs of age-related macular degeneration (AMD) is the accumulation of extracellular deposits, known as drusen, between the basement membrane of the retinal pigment epithelium (RPE) and Bruch's membrane (BM). Recent studies by Anderson et al have confirmed that drusen contain beta-amyloid. (Experimental Eye Research 78 (2004) 243 - 256).

Pyroglutamate variants of Ae peptides have been shown to play a key role in Ae peptide accumulation and plaque formation in Alzheimer's disease. Due to their hydrophobic potential, these peptides have been shown to promote aggregation and plaque formation. Additionally, in a transgenic mouse model expressing Ae N3pE-42 in neurons, this peptide was shown to be neurotoxic in vivo and lead to neuronal loss (Wirths et al. (2009) Acta Neuropatho/118, 487-496).

Antibodies with specificity for the N-terminal pyroglutamate of Ae peptides are thought to be preferred due to their specificity only for pathogenic forms of Ae molecules that carry pyroglutamate at the N-terminus, but without recognition of APP or other forms of Ae molecules without the N-terminal pyroglutamate . Thus, it is believed that the risk of potential side effects, such as uncontrollable inflammation of brain tissue, will be reduced by the use of antibodies of the present invention compared with antibodies directed against other forms of

- 4,043,617 Αβ molecules that are not pyroglutamate variants.

Antibodies targeting N3pE Ae peptides are known (Acero et al. (2009) J Neuroimmunol 213,

39-46; Saido et al. (1996) Neuron 14, 457-466; US patent documents No. 7122374 and WO 2012/136552).

However, there is a need for humanized and deimmunized antibodies with specificity for N3pE Ae peptides that can be used in human therapy and which have a beneficial effect on amyloidosis, in particular on cognitive function in diseases and conditions in which Ae N3pE may be involved. such as clinical or preclinical stage of Alzheimer's disease, Down syndrome and clinical or preclinical stage of cerebral amyloid angiopathy.

The parent antibody for the antibody of the present invention is a variant of clone No. 6 disclosed in WO 2017/009459, which has a light chain variable region with the amino acid sequence:

DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDGKTYLNWFQQRPGQSPRRLTYLVSK

LDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFPFTFGGGTKVEIK (SEQ

ID NO: 1), which is disclosed under SEQ ID NO: 14 in WO 2017/009459;

and which has a heavy chain variable region with the amino acid sequence: QVQLVQSGAEVKKSGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGLINPSN

GVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREAKREWDETYWGQ

GTLVTVSS (SEQ ID NO: 49), which is disclosed under SEQ ID NO: 27 in WO 2017/009459.

However, with this variant of clone no. 6, as disclosed in WO 2017/009459, it was not possible to ensure production in accordance with the CMC. In particular, and despite several attempts, stable cell clones were not obtained for CHO-DG44 cells, with only weak transient expression (with insufficient antibody production) of this variant clone no. 6 being observed and the stable expression being a prerequisite was not achieved for production in accordance with CMC.

Brief description of the invention

Therefore, it was an object of the present invention to provide humanized and deimmunized antibodies with improved properties to overcome the disadvantages of prior art antibodies.

In general, the present invention provides new methods and compositions that provide highly specific and highly effective antibodies, including chimeric antibodies and fragments thereof, including partially or fully humanized antibodies and fragments thereof, having the ability to specifically recognize and bind to specific epitopes from a number of β-amyloid antigens, in particular Ae-peptides N3pE, which can be presented to the antibody in monomeric, dimeric, trimeric, etc. or polymeric form, in the form of an aggregate, fibers, filaments or in compacted plaque form.

In particular, the object of the present invention is achieved by using an antibody or a functional variant thereof, wherein the variable portion of the light chain of said antibody comprises, consists essentially of, or consists of the amino acid sequence:

DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDGKTYLNWFQQRPGQSPRRLTYLVSK LDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFPFTFGGGTKVEIK (SEQ ID NO: 1), and wherein the variable portion of the heavy chain of said antibody comprises, essentially consists of, or consists of the amino acid sequence:

QVQLVQSGAEVVKPGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGLINPSD

GVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREAKREWDETYWGQ GTLVTVSS (SEQ Ш NO: 2).

The heavy chain variable portion (SEQ ID NO: 2) contains three mutations at positions K12V, S14P and N55D compared to the parent antibody disclosed in WO 2017/009459.

Surprisingly, it has been found that introduction of these three point mutations results in a humanized and deimmunized antibody that is suitable for CMC production in high yield. It was possible to obtain antibody-producing cell lines from CHO-DG44 cells for antibodies of the present invention containing these three point mutations. Additionally, it was possible to provide improved transient expression in CHO-DG44, resulting in higher yields of the antibody of the present invention containing the K12V, S14P and N55D mutations compared to the parent antibody, while maintaining the favorable binding characteristics of the antibody. Finally, the antibody of the present invention containing the K12V, S14P and N55D mutations can be stably expressed at high levels of expression allowing production in accordance with CMC.

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The present invention provides humanized and deimmunized antibodies or fragments thereof that have a beneficial effect on diseases and conditions associated with amyloidosis in which Άβ N3pE may be involved.

In another embodiment, the present invention provides antibodies and fragments thereof that bind to N3pE Άβ peptides in the bloodstream and tissue, particularly in the brain. The antibodies of the present invention are capable of binding free molecules of N3pE Άβ-peptides or even bound forms of N3pE Άβ-peptides.

Thus, the present invention further provides antibodies that alter the clearance of soluble and bound forms of N3pE Άβ-peptides in the central nervous system, such as in the brain, and in the bloodstream, such as in blood plasma.

In a further embodiment, the present invention provides antibodies and fragments thereof, wherein the antibodies specifically bind to the pyroglutamate-bearing N-terminus of Άβ N3pE.

In a further embodiment, the present invention provides antibodies and fragments thereof, wherein the antibodies show increased selectivity for Άβ peptide oligomers and/or fibrils. Antibodies of the present invention exhibit multiple times, such as 10, 25, 50, 100, 150, 200, 250 or greater than 250 times lower binding constant value (K _D value) with respect to binding to oligomers and/or fibrils Άβ (1-42) than comparable monoclonal antibodies known from the prior art, in particular compared to the parent antibody disclosed in WO 2017/009459. Accordingly, antibodies of the present invention that are formulated to bind selectively to N3pE Άβ peptides are more specific for N3pE Άβ peptides and show reduced cross-reactivity to Άβ peptides other than N3pE Άβ peptides.

In yet another embodiment, the present invention further relates to host cells transformed with vectors or containing nucleic acid molecules that express antibodies of the present invention or fragments thereof.

In addition, the present invention provides pharmaceutical compositions containing the antibodies of the present invention and fragments thereof.

The present invention further relates to the use of antibodies of the present invention and fragments thereof useful for binding to and clearing or removing Άβ N3pE in humans and thereby diagnosing, preventing and treating diseases and conditions characterized by amyloidosis or Άβ N3pE toxicity.

In a specific embodiment, antibodies of the present invention that are capable of binding to and clearing or clearing Άβ N3pE peptides in biological fluids and tissues are useful for the prevention and/or treatment of conditions associated with the formation of Άβ N3pE plaques, such as diffuse, neuritic and cerebrovascular plaques in the brain.

Administration of antibodies of the present invention, including immunologically reactive fragments thereof, can result in clearance or removal of Άβ N3pE from the aforementioned plaques or other biological complexes. Thus, the antibody of the present invention will be readily transported into the bloodstream, other body fluids and sites where the aforementioned plaques and/or other biological complexes are formed, or anywhere else where Άβ N3pE exhibits damaging effects.

In addition, removal of Άβ N3pE from plaques or other biological complexes using the antibodies of the present invention may result in solubilization of insoluble forms of plaques and thus lead to the removal of intact plaques from diseased tissue, such as brain tissue. This, in turn, may lead to improved cognitive function in patients diagnosed with a neurodegenerative disease such as mild cognitive impairment (MCI), Alzheimer's disease (AD), such as sporadic Alzheimer's disease (SΆD) or familial forms of Alzheimer's dementia (FΆD). , such as familial British dementia (FBD) and familial Danish dementia (FDD) or others, neurodegeneration in Down syndrome, dementia with Lewy bodies, hereditary cerebral haemorrhage with amyloidosis (Dutch type); Guam complex, which is parkinsonism-dementia. In particular, the present invention provides an antibody of the present invention for use in the treatment of a condition selected from prodromal AD, mild ΆD, moderate ΆD and severe ΆD.

In another embodiment, the present invention provides an antibody of the present invention for use in slowing cognitive decline in a patient diagnosed with a condition selected from clinical or preclinical Alzheimer's disease, Down syndrome, and clinical or preclinical cerebral amyloid angiopathy.

Binding of antibodies of the present invention to Άβ N3pE in the bloodstream or other body fluids may further result in the removal of circulating or soluble forms of Άβ N3pE. As discussed above, Άβ N3pE is highly hydrophobic and has a high affinity for other, such as non-pyroglutamate, Αβ peptides, resulting in the formation of oligomeric and supramolecular structures such as amyloid plaques. These oligomeric structures in particular have been shown to be highly neurotoxic. The formation of oligomeric structures leads to cell damage and death of neuronal cells. Thus, removal of circulating or soluble forms of Ae N3pE, or even oligomers containing Ae N3pE, results in the prevention of cell damage and/or neurotoxicity. Thus, the present invention also provides methods for preventing a neurodegenerative disease such as mild cognitive impairment (MCI), Alzheimer's disease (AD) such as sporadic Alzheimer's disease (SAD) or familial forms of Alzheimer's dementia (FAD) such as familial British dementia (FBD) and familial Danish dementia (FDD) or others, neurodegeneration in Down syndrome, dementia with Lewy bodies, hereditary cerebral haemorrhage with amyloidosis (Dutch type); Guam complex, which is parkinsonism-dementia. In particular, the present invention provides methods for treating a condition selected from prodromal AD, mild AD, moderate AD and severe AD.

In another embodiment, the present invention provides a method for slowing cognitive decline in a patient diagnosed with a condition selected from clinical or preclinical Alzheimer's disease, Down syndrome, and clinical or preclinical cerebral amyloid angiopathy.

The present invention further provides methods for the prevention and/or treatment of other diseases that are caused by or associated with amyloid-like proteins, in particular Ae N3pE, such as progressive supranuclear gaze palsy, multiple sclerosis; Creutzfeldt-Jakob disease, Parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), dementia associated with adult-onset diabetes; age-related cardioamyloidosis and others, including macular degeneration.

The present invention further provides a highly sensitive and yet reliable detection technique that allows the quantification of Ae variants, in particular Ae N3pE, in biological samples, such as cerebrospinal fluid or serum samples, preferably in blood serum samples or tissue samples. This represents a significant challenge given the low levels of these N3pE Ae peptides in the blood. However, the availability of such an accessible detection technique is a prerequisite for studying the effectiveness of small molecule inhibitors in screening and drug development programs.

Antibodies provided in accordance with the teachings of the present invention are particularly useful for the diagnosis of amyloidosis, a group of diseases and disorders associated with the formation of amyloid plaques, including secondary amyloidosis and age-related amyloidosis, including without limitation, neurological disorders such as Alzheimer's disease (AD), dementia with Lewy bodies, Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam parkinsonism dementia complex, as well as other diseases that are based on or associated with amyloid-like proteins, such as progressive supranuclear palsy, multiple sclerosis; Creutzfeldt-Jakob disease, Dutch hereditary cerebral hemorrhage, Parkinson's disease, HIV-related dementia, ALS (amyotrophic lateral sclerosis), adult-onset diabetes-related dementia; age-related cardiac amyloidosis and others, including macular degeneration, for example.

Description of graphic materials

In fig. 1 shows the effect of point mutations in the heavy chain on transient expression (A) and target binding (B) for selected antibody variants of the present invention. All antibodies tested contain the light chain variable portion of SEQ ID NO: 1 and the K324A mutation in the heavy chain.

Ab 1: contains two mutations K12V and S14P in the variable part of the heavy chain compared to the original sequence under SEQ ID NO: 49;

Ab 2: contains the N55D mutation in the variable part of the heavy chain compared to the original sequence under SEQ ID NO: 49;

Ab 3: is the original sequence of SEQ ID NO: 49 of the variable heavy chain without any mutations.

In fig. 2 shows the effect of point mutations in the heavy chain on transient expression (A) and target binding (B) for selected antibody variants of the present invention. All antibodies tested contain the light chain variable portion of SEQ ID NO: 1 and the K324A mutation in the heavy chain.

Ab 1: contains two mutations K12V and S14P in the variable part of the heavy chain compared to the original sequence under SEQ ID NO: 49;

Ab 2: contains the N55D mutation in the variable part of the heavy chain compared to the original sequence under SEQ ID NO: 49;

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Ab 3: contains three mutations K12V, S14P and N55D in the variable part of the heavy chain compared to the original sequence under SEQ ID NO: 49.

In fig. 3 shows CHO-DG44 clones expressing the light chain variable antibody of SEQ ID NO: 1 and the heavy chain variable antibody of SEQ ID NO: 2, seven, ten and 13 days after seeding the wells of a 24-well plate from selected colonies from a 96-well plate.

In fig. Figure 4 shows the expression titers for CHO-DG44 after gene amplification by MTX (day 7).

Detailed Description of the Invention

Definitions.

The term antibody is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) derived from at least two intact antibodies, and antibody fragments to the extent that they exhibit the desired biological activity. The antibody may be IgM, IgG (eg, IgG1, IgG2, IgG3, or IgG4), IgD, IgA, or IgE, for example. However, it is preferable that the antibody is not an IgM antibody.

Antibody fragments include a portion of an intact antibody, typically the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments: diabodies; single-chain antibody molecules and polyspecific antibodies derived from antibody fragments.

The term monoclonal antibody as used in this invention refers to an antibody obtained from a population of essentially homogeneous antibodies, that is, the individual antibodies making up the population are identical except for possible naturally occurring mutations that may be present in minute quantities. Monoclonal antibodies are highly specific, directed against a single antigenic site. Additionally, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibodies can often be useful in that they are synthesized in a hybridoma culture that is not contaminated with other immunoglobulins. The term monoclonal indicates the nature of the antibody, which is obtained from an essentially homogeneous population of antibodies, and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be produced by conventional methods using recombinant DNA. Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.

Monoclonal antibodies in this invention specifically include chimeric antibodies (immunoglobulins) in which part of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain(s) ) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies to the extent that they exhibit the required biological activity.

Humanized forms of non-human (eg, murine) antibodies are immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding antibody subsequences) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies are predominantly human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. In some cases, framework region (FR) residues of a human immunoglobulin Fv are replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences.

These modifications are made to further refine and optimize the effectiveness of the antibodies. In general, a humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the CDR regions correspond to non-human immunoglobulin regions, and all or substantially all of the FR regions correspond to non-human immunoglobulin regions. sections from the human immunoglobulin sequence. An optimally humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically human immunoglobulin. For more information, see Jones et al.

- 8 043617

Nature, 321:522-525 (1986), Reichmann et al., Nature. 332:323-329 (1988) and Presta, Curr. Op. Struct. Biel.,

2:593-596 (1992).

In addition to humanization, deimmunization includes other changes such as the removal of T-cell epitopes.

The term therapeutically effective amount, as used in this invention and in the accompanying claims, means that the amount of antibody administered is a sufficient amount to achieve the intended purpose, such as, in this case, at least the removal of circulating or soluble forms of the pyroglutamate variant of beta-amyloid peptide (Άβ N3pE) and variants thereof, but preferably removing or removing the N3pE Άβ peptide from plaques or other biological complexes. Or more preferably, reducing plaque load and/or removing entire plaques from diseased tissue, such as brain tissue.

Single chain Fv or sFv antibody fragments contain the VH and VL domains of the antibody, these domains being present on the same polypeptide chain. Typically, the Fv polypeptide further contains a polypeptide linker between the VH and VL domains, which allows the sFv to form the required structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term diabodies refers to small antibody fragments with two antigen-binding sites, these fragments containing a heavy chain variable domain (VH) linked to a light chain variable domain (VD) in the same polypeptide chain (VH-VD). When using a linker that is too short, to enable pairing between two domains on the same chain, the domains are forced to pair with complementary domains of the other chain and create two antigen-binding sites. Diabodies are described more fully in Hollinger et al., Proc. Natl. Acad. Sol. USA, 90:64446448 (1993).

An isolated antibody is an antibody that has been identified and separated and/or extracted from a component of its natural environment. Contaminants from its natural environment are materials that will interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of the antibody, as determined by the Lowry method, and most preferably greater than 99% by weight, (2) to an extent sufficient to obtain at least 15 residues N-terminal or internal amino acid sequence using a spinning beaker sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. An isolated antibody includes the antibody in situ in recombinant cells, since at least one component of the antibody's natural environment will not be present. Typically, however, the isolated antibody will be obtained through at least one purification step.

As used herein, the expressions cell, cell line, and cell culture are used interchangeably, and all such designations include progeny. Thus, the words transformants and transformed cells include the primary cell under study and the culture obtained from it, without regard to the number of transfers. It is also clear that all offspring may not be exactly identical in DNA content due to intentional or unintentional mutations. Included are mutant progeny that have the same function or biological activity as the original transformed cell. Where different designations are intended, this will be clear from the context.

As used herein, the terms polypeptide, peptide and protein are used interchangeably and are defined to mean a biomolecule consisting of amino acids linked by a peptide bond.

When peptides or amino acid sequences are mentioned in this invention, each amino acid residue is represented by a one- or three-letter designation corresponding to the trivial name of the amino acid, according to the following list:

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Amino acid Single letter character Three letter symbol Alanin A Ala Arginine R Arg Asparagine Ν Asn Aspartic acid D Asp Cysteine WITH Cys Glutamine Q Gin Glutamic acid E Glu Glycine G Gly G istidin N His Isoleucine I He Leucine L Leu Lysine TO Lys Methionine m Met Phenylalanine F Phe Proline R Pro Serin S Ser Threonine T Thr Tryptophan W Trp Tyrosine Υ Tyr Valin V Vai

As used herein, the singular number is defined to mean one or more and includes the plural unless inappropriate for context.

The phrase diseases and disorders caused by or associated with amyloid or amyloid-like proteins includes, without limitation, diseases and disorders caused by the presence or activity of amyloid-like proteins in a monomeric, fibrillar or polymeric state, or any combination of these three. Such diseases and disorders include, but are not limited to, amyloidosis, endocrine tumors, and macular degeneration.

The term amyloidosis refers to a group of diseases and disorders associated with the formation of amyloid plaques, including, but not limited to, secondary amyloidosis and age-related amyloidosis, diseases such as, but not limited to, neurological disorders such as Alzheimer's disease (AD), including diseases or conditions characterized by capacity loss cognitive memory, such as, for example, mild cognitive impairment (MCI), sporadic Alzheimer's disease, dementia with Lewy bodies, Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guam complex, which is parkinsonism-dementia, familial forms of Alzheimer's disease, such as familial British dementia (FBD) and familial Danish dementia (FDD), and other diseases that are caused by or associated with amyloid-like proteins or associated with them, such as progressive supranuclear gaze palsy, multiple sclerosis; Creutzfeldt-Jakob disease, Parkinson's disease, HIV-related dementia, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), adult-onset diabetes and age-related cardiac amyloidosis, and various eye diseases including macular degeneration, drusen-related optic neuropathy and cataracts due to beta-amyloid deposition.

Amyloid β, Ae or β-amyloid is a recognized term in the art and refers to β-amyloid proteins and peptides, amyloid-β precursor protein (APP), as well as modifications thereof,

- 10 043617 fragments and any functional equivalents. In particular, as used herein, β-amyloid refers to any fragment produced by proteolytic cleavage of APP, but especially those fragments that are involved in or associated with amyloid pathology, including, but not limited to, Λβ _1-38 , Λβ _1-40 , Λβ _1-42 . The amino acid sequences of these Λβpeptides are as follows:

Αβ 1-42 (SEQ ID NO: 37):

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-PheAla-Glu-Asp-Val-Gly-Ser- Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-IleAla,

Αβ 1-40 (SEQ ID NO: 38):

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-PheAla-Glu-Asp-Val-Gly-Ser- Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val,

Αβ 1-38 (SEQ ID NO: 39):

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-PheAla-Glu-Asp-Val-Gly-Ser- Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly.

pGlu-Λβ or Λβ N3pE refers to N-terminally truncated forms of Λβ that begin with a glutamic acid residue at position 3 in the amino acid sequence of Λβ, and where this glutamic acid residue cyclizes to form a pyroglutamic acid residue. In particular, as used herein, pGlu-Λβ or Λβ N3pE refers to those fragments that are involved in or associated with amyloid pathology, including, but not limited to, pGlu-Λβ _3-38 , pGlu-Λβ _3-40 , p-Glu-Λβ _3-42 .

The sequences of the N-terminally truncated forms Λβ, Λβ _3-38 , Λβ _3-40 , Λβ _3-42 are as follows:

Αβ 3-42 (SEQ ID NO: 40):

Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-GluAsp-Val-Gly-Ser-Asn-Lys- Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala,

Αβ 3-40 (SEQ ID NO: 41):

Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-GluAsp-Val-Gly-Ser-Asn-Lys- Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val,

Αβ 3-38 (SEQ ID NO: 42):

Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-GluAsp-Val-Gly-Ser-Asn-Lys- Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly.

The present invention relates to antibodies specific for human Λβ peptides that are N-terminally truncated by cleavage or loss of amino acids No. 1 and 2 from the N-terminus, and in which the exposed N-terminal amino acid No. 3 is thereby modified to form pyroglutamate, and which therefore carry a pyroglutamate residue at position 3 from the N-terminus (hereinafter referred to as N3pE Λβ-peptides, or N3pE-AP peptides, or Λβ-peptide pyroglutamate variants).

In a first aspect, the present invention provides an antibody wherein the light chain variable portion of said antibody comprises, is substantially comprised of, or consists of the amino acid sequence:

DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDGKTYLNWFQQRPGQSPRRLTYLVSK

LDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFPFTFGGGTKVEIK (SEQ ID NO: 1), and wherein the heavy chain variable portion of said antibody comprises, is substantially comprised of, or consists of the amino acid sequence:

QVQLVQSGAEVVKPGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGLINPSD

GVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREAKREWDETYWGQ GTLVTVSS (SEQ Ш NO: 2).

In a preferred embodiment of the present invention, an antibody having a light chain variable portion of said antibody that contains, essentially consists of, or consists of the amino acid sequence of SEQ ID NO: 1 contains the following CDR regions in the light chain:

CDRl V _L : SSQSLLYSDGKTYLN (SEQ Ш NO: 3);

CDR2 V _L : LVSKLDS (SEQ ID NO: 4) and

CDR3 V _L : VQGTHFP (SEQ ID NO: 5).

In a further preferred embodiment of the present invention, an antibody having a heavy chain variable portion of said antibody that contains, essentially consists of, or consists of the amino acid sequence of SEQ ID NO: 2 contains the following CDR regions in the heavy chain:

CDRl V _H : GYSFTGHTMN (SEQ ID NO: 6);

CDR2 V _H : LINPSDGVTRYNQKFQG (SEQ ID NO: 7) and

CDR3 V _H : EAKREWDETY (SEQ ID NO: 8).

The present invention further provides light chains and heavy chains of the antibodies of the present invention.

In a preferred embodiment, the antibody of the present invention has a light chain, wherein the light chain comprises, consists essentially of, or consists of the amino acid sequence:

DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDGKTYLNWFQQRPGQSPRRLTYLVSK

LDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFPFTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 17).

In a further preferred embodiment, the antibody of the present invention has a heavy chain, wherein the heavy chain comprises, is substantially comprised of, or consists of the amino acid sequence:

QVQLVQSGAEVVKPGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGLINPSD GVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREAKREWDETYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQ S SGL YSL S S VVT VP S S SLGTQT YICNVNHKP SNTK VDKK VEPK S CDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 19).

C1q, and two serine proteases, C1r and C1s, form the C1 complex, which is the first component of the complement-dependent cytotoxicity (CDC) pathway. C1q is a hexavalent molecule with a molecular weight of approximately 460,000 and a tulip-like structure in which six collagen stalks are connected to six globular head regions (Burton and Woof, Advances in Immunol 51:1-84; 1992). Binding of IgG1 molecules to C1q initiates complement activation and then leads to complement-mediated cell lysis. The antibodies of the present invention are useful for the treatment of inflammatory diseases and conditions, i.e. the antibodies of the present invention must have anti-inflammatory properties.

The effector functions of the antibodies of the present invention may also be mediated by the interaction of the Fc region of the antibody with Fc receptors (FcR), which are specialized cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of red blood cells and various other cellular targets (eg, tumor cells) coated with the appropriate antibody through antibody-dependent cell-mediated mediated cytotoxicity (ADCC) (Van de Winkel and Anderson, J. Leuk. Bioi. 49:511-24; 1991).

In this regard, the present invention further provides antibodies that still bind to Fc receptors to perform their effector functions. But it is preferable that the antibodies of the present invention do not exhibit complement-dependent cytotoxicity. More preferably, the antibodies of the present invention do not activate the complement system, but rather inhibit complement-mediated cell lysis.

Thus, in a preferred embodiment, the antibodies of the present invention have a human IgG Fc region that includes one or more amino acid substitutions, preferably 3 or 2 amino acid substitutions, most preferably a single amino acid substitution.

Amino acid substitutions can be accomplished using conventional methods, such as site-directed mutagenesis of the Fc region of the human IgG1 antibodies of the present invention.

In a more preferred embodiment, the antibodies of the present invention have a human IgG Fc region that includes an amino acid substitution at position 324 as shown in SEQ ID NO: 18 [position 324 corresponds to position 322 according to the EU numbering scheme, Kabat et al., Sequences of Proteins of Immunological Interest, ^5th Ed., US Department of Health and Human Services, NIH Publication No. 91-3242, National Institutes of Health, Bethesda, MD (1991); Edelman et al., PNAS USA 63:78-85 (1969)]. The amino acid substitution is preferably K324A.

In the most preferred embodiment, the antibody of the present invention has a heavy chain that contains, essentially consists of, or consists of the amino acid sequence of SEQ ID NO:

QVQLVQSGAEVVKPGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGLINPSD

GVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREAKREWDETYWGQ

GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

HTFP AVLQ S SGL YSL S SWT VP S S SLGTQT YICNVNHKP SNTK VDKK VEPK SCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTIS KA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18).

Further preferred according to the present invention are antibodies containing, consisting essentially of, or consisting of the following combinations of light chain and heavy chain and light chain and heavy chain variable portions:

A) light chain variable portion: SEQ ID NO: 1 heavy chain variable portion: SEQ ID NO: 2 light chain: SEQ ID NO: 17 heavy chain: SEQ ID NO: 19;

B) light chain variable: SEQ ID NO: 1 heavy chain variable: SEQ ID NO: 2 light chain: SEQ ID NO: 17 heavy chain: SEQ ID NO: 18.

Antibody according to B) is more preferred. The heavy chain provides for the amino acid substitution K324A.

Preferred antibodies in accordance with the present invention are humanized forms of mouse monoclonal antibodies that are produced by the Aβ 6-1-6 hybridoma cell line (DSM Accession No. ACC 2924), which is described in WO 2010/009987.

The light and heavy chain sequences for the antibodies of the present invention may vary. The immunoglobulins may have two pairs of light chain/heavy chain complexes, at least one chain containing one or more murine complementarity determining regions (CDRs) operably linked to human framework segments.

In a preferred embodiment, the antibody of the present invention contains two light chains and two heavy chains, wherein the amino acid sequence of each light chain is SEQ ID NO: 17, and the amino acid sequence of each heavy chain is SEQ ID NO: 18 or SEQ ID NO: 19.

More preferably, the antibody of the present invention contains two light chains and two heavy chains, wherein the amino acid sequence of each light chain is SEQ ID NO: 17, and the amino acid sequence of each heavy chain is SEQ ID NO: 19.

Most preferably, the antibody of the present invention contains two light chains and two heavy chains, wherein the amino acid sequence of each light chain is SEQ ID NO: 17, and the amino acid sequence of each heavy chain is SEQ ID NO: 18.

In another embodiment, the present invention is directed to recombinant nucleic acid molecules encoding antibodies of the present invention containing heavy and light chain CDRs as set forth in the present invention.

The human framework region of antibodies of the present invention is determined by comparing the amino acid sequence of the framework or variable region CDR of a non-human immunoglobulin with the corresponding sequences in a sequence collection containing human immunoglobulin variable regions. A sequence with a high percentage of identical amino acids is selected.

In a preferred embodiment of the present invention, the light chain variable portion having the amino acid sequence according to SEQ ID NO: 1 is encoded by a nucleic acid molecule that contains, consists essentially of, or consists of the nucleic acid sequence:

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Gacgtggtgatgactcagtctccactctccctgcccgtcacccttggacagccggcctccatctcctgcaagtcaagtcagagcctcct gcactccgacggcaagacctacttgaactggttccagcagaggccaggccagtctccaaggcgcctgacctatctggtgtctaagctg gactctggggtcccagacagattcagcggcag tgggtcaggcactgacttcacactgaagatcagcagggtggaggctgaggatgtc ggagtctactactgcgtgcaaggtacacacttcccattcacgttcggcggagggaccaaggtggaaatcaaa (SEQ ID NO: 9).

In a further preferred embodiment of the present invention, the heavy chain variable portion having the amino acid sequence according to SEQ ID NO: 2 is encoded by a nucleic acid molecule that contains, consists essentially of, or consists of the nucleic acid sequence:

caggtgcagctcgtgcagtctggggctgaggtggtgaagccaggtgcctcagtgaaggtctcctgcaaggcatctggttactcattcac tggtcacaccatgaactgggtgcgacaggcccctggacaagggcttgagtggatgggactcatcaatccttccgatggtgttactaggt acaaccagaagttccaggcagagt caccatcaccagggacacgtccacgaccaccgttcacatggagctgaccagcctgacatctg aggacacggccacctactactgtacgagagaggcgaaacgggagtgggacgagacttactggggccagggaaccctggtcaccgt ctcctca (SEQ ID NO: 10).

In a further preferred embodiment of the present invention, the CDR regions of the light chain of an antibody of the present invention are encoded by nucleic acid molecules having the nucleic acid sequences:

CDRl Vl: tcaagtcagagcctcctgcactccgacggcaagacctacttgaac (SEQ ID NO: 11);

CDR2 V _L : ctggtgtctaagctggactct (SEQ ID NO: 12) and

CDR3 V _L : gtgcaaggtacacacttccca (SEQ ID NO: 13).

In a further preferred embodiment of the present invention, the heavy chain CDR regions of the antibody of the present invention are encoded by nucleic acid molecules having the nucleic acid sequences:

CDRl V _H : ggttactcattcactggtcacaccatgaac (SEQ ID NO: 14);

ICDR2 V _H : ctcatcaatccttccgatggtgttactaggtacaaccagaagttcc

Agggc (SEQ IDNO: 15) and

CDR3 _VH : gaggcgaaacgggagtgggacgagacttac (SEQ ID NO: 16).

In a further preferred embodiment of the present invention, the light chain is encoded by a nucleic acid molecule that contains, essentially consists of, or consists of the amino acid sequence:

Gacgtggtgatgactcagtctccactctccctgcccgtcacccttggacagccggcctccatctcctgcaagtcaagtcagagcctcct gcactccgacggcaagacctacttgaactggttccagcagaggccaggccagtctccaaggcgcctgacctatctggtgtctaagctg gactctggggtcccagacagattcagcggcag tgggtcaggcactgacttcacactgaagatcagcagggtggaggctgaggatgtc ggagtctactactgcgtgcaaggtacacacttcccattcacgttcggcggagggaccaaggtggaaatcaaaaggaccgtggccgca ccctctgtgttcatcttcccccccagcgacgagcagctgaagagcggcactgcatct gtcgtgtgtctgctgaacaacttctacccaagg gaggcgaaagtgcagtggaaggtagacaacgccttgcaatccggcaactcccaggagagcgtgaccgagcaggacagcaaagact caacctacagcctgagcagtactttgaccctgtctaaggccgattacgagaagcacaaggtgtacgcctgcgaggtaacccacca gg gactgagctctcccgtgaccaagagcttcaacaggggcgagtgc (SEQ ID NO: 20).

In a further preferred embodiment of the present invention, the heavy chain is encoded by a nucleic acid molecule that contains, essentially consists of, or consists of the amino acid sequence:

- 14 043617 caggtgcagctcgtgcagtctggggctgaggtggtgaagccaggtgcctcagtgaaggtctcctgcaaggcatctggttactcattcac tggtcacaccatgaactgggtgcgacaggcccctggacaagggcttgagtggatgggactcatcaatccttccgatggtgttactaggt acaacc agaagttccagggcagagtcaccatcaccagggacacgtccacgaccaccgttcacatggagctgaccagcctgacatctg aggacacggccacctactactgtacgagaggcgaaacgggagtgggacgagacttactggggccagggaaccctggtcaccgt ctcctcagccagcactaagggcccgagcgtgttccccctcgcccctagcag taagagcaccagcggtggcacggcggcacttggct gcttggttaaggacttcccagagcccgtgaccgtgtcctggaactctggggcacttaccagtggcgtgcacaccttccccgctgtac tgcagagcagcggcttgtacagcttgtcttccgtcgtaacggtgcccagcagcagcttgggaacccagaccta catctgcaacgtaaac cacaagccatccaacaccaaggtagacaaaaaggtcgaacccaagtcctgcgacaagacccacacctgtccaccctgtcctgcaccc gagctcctgggaggtcccagcgttttcctgttccctccaaagccaaaggataccctgatgatcagcaggacccccgaggtgacctgcg tggtggtggacg tgagccacgaagaccctgaggtcaagttcaactggtacgttgatggggtggaggtacacaatgccaagaccaaac ctcgagaggagcaatacaacagcacctaccgagttgtgagcgtgcttaccgtgctgcaccaggactggctgaacggcaaggagtaca agtgcgctgtgagcaacaaggctctgccggctcccatcgagaagaccat cagcaaggccaagggccagcccagggagccacaggt ttacacgttgcccccctcaagggacgagttgaccaagaaccaggttccctcacgtgccttgtgaagggcttctaccccagcgacatcg ccgtggaatggggagagcaacgggcagcccgagaacaactacaagacgaccccccctgttctggacagcgacggctctttct tcctgt attcaaagctcaccgtggacaaaagcaggtggcagcagggtaatgtgttctcctgcagcgtgatgcacgaggccctgcataaccacta cacccaaaagagcttgagcctctcccccggtaag (SEQ ID NO: 21).

In a further preferred embodiment of the present invention, the heavy chain is encoded by a nucleic acid molecule that contains, essentially consists of, or consists of the amino acid sequence:

caggtgcagctcgtgcagtctggggctgaggtggtgaagccaggtgcctcagtgaaggtctcctgcaaggcatctggttactcattcac tggtcacaccatgaactgggtgcgacaggcccctggacaagggcttgagtggatgggactcatcaatccttccgatggtgttactaggt acaaccagaagttccaggcagagt caccatcaccagggacacgtccacgaccaccgttcacatggagctgaccagcctgacatctg aggacacggccacctactactgtacgagagaggcgaaacgggagtgggacgagacttactggggccagggaaccctggtcaccgt ctcctcagccagcactaagggcccgagcgtgttccccctcgcccctagcagtaagagcaccagcggtggca cggcggcacttggct gcttggttaaggactacttcccagagcccgtgaccgtgtcctggaactctggggcacttaccagtggcgtgcacaccttccccgctgtac tgcagagcagcggcttgtacagcttgtcttccgtcgtaacggtgcccagcagcagcttgggaacccagacctacatctgcaacgtaaac caca agccatccaacaccaaggtagacaaaaaggtcgaacccaagtcctgcgacaagacccacacctgtccaccctgtcctgcaccc gagctcctgggaggtcccagcgttttcctgttccctccaaagccaaaggataccctgatgatcagcaggacccccgaggtgacctgcg tggtggtggacgtgagccacgaagaccctgagg tcaagttcaactggtacgttgatggggtggaggtacacaatgccaagaccaaac ctcgagaggagcaatacaacagcacctaccgagttgtgagcgtgcttaccgtgctgcaccaggactggctgaacggcaaggagtaca agtgcaaggtgagcaacaaggctctgccggctccatcgagaagaccatcagcaaggccaagggccagccca gggagccacagg tttacacgttgcccccctcaagggacgagttgaccaagaaccaggtttccctcacgtgccttgtgaagggcttctaccccagcgacatcg ccgtggaatggggagagcaacgggcagcccgagaacaactacaagacgaccccccctgttctggacagcgacggctctttcttcctgt attcaaagctc accgtggacaaaagcaggtggcagcagggtaatgtgttctcctgcagcgtgatgcacgaggccctgcataaccacta cacccaaaagagcttgagcctctcccccggtaag (SEQ ID NO: 22).

Additionally preferred according to the present invention are antibodies that are encoded by a combination of nucleic acid molecules containing, essentially consisting of, or consisting of:

C) light chain variable: SEQ ID NO: 9 heavy chain variable: SEQ ID NO: 10 light chain: SEQ ID NO: 20 heavy chain: SEQ ID NO: 22;

D) light chain variable: SEQ ID NO: 9 heavy chain variable: SEQ ID NO: 10 light chain: SEQ ID NO: 20 heavy chain: SEQ ID NO: 21.

Combination D) is most preferred because it encodes a humanized and deimmunized antibody that includes the K324A amino acid substitution in the heavy chain.

The above nucleic acid molecules can be integrated into expression vectors well known in the art. Transfection of these expression vectors into a suitable host, host selection, and expression, collection and purification of light chains, heavy chains, light/heavy chain dimers or intact antibodies, binding fragments or other forms of immunoglobulin are procedures well known in the art.

One skilled in the art may select a vector based on desired properties, for example to obtain the vector in a specific cell, such as a mammalian cell or a bacterial cell.

Any of a variety of inducible promoters or enhancers may be incorporated into the eyelids

- 15 043617 torus for expressing an antibody of the present invention or a nucleic acid that can be controlled. Such inducible systems include, for example, the tetracycline-inducible system (Gossen & Bizard, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992); Gossen et al., Science, 268:17664769 (1995); Clontech , Palo Alto, Calif.); heavy metal-inducible metallothionein gene promoter; an insect steroid hormone sensitive to ecdysone or related steroids such as muristerone (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996); Yao et al., Nature, 366:476- 479 (1993); Invitrogen, Carlsbad, Calif.); murine mammary tumor virus (MMTV) induced by steroids such as glucocorticoid and estrogen (Lee et al., Nature, 294:228-232 (1981); and heat shock protein gene promoters induced by temperature changes; neuron-specific gene promoter rat enolase (Forss-Petter, et al., Neuron 5; 197-197 (1990)); human β-actin gene promoter (Ray, et al., Genes and Development (1991) 5:2265-2273); gene promoter human platelet-derived growth factor B (PDGF-B) chain (Sasahara, et al., Cell (1991) 64:217-227); rat sodium channel gene promoter (Maue, et al., Neuron (1990) 4:223-231 ); the promoter of the human copper-zinc superoxide dismutase gene (Ceballos-Picot, et al., Brain Res. (1991) 552:198-214) and promoters for members of the mammalian POU domain regulatory gene family (Xi et al., (1989) Nature 340:35-42).

Regulatory elements, including promoters or enhancers, can be constitutive or regulated depending on the nature of the regulation. Regulatory sequences or regulatory elements are operably linked to one of the nucleic acid molecule sequences of the present invention such that the physical and functional relationship between the nucleic acid molecule sequence and the regulatory sequence allows the nucleic acid molecule sequence to be transcribed. Vectors useful for expression in eukaryotic cells may include, for example, regulatory elements including the CAG promoter, SV40 early promoter, cytomegalovirus (CMV) promoter, steroid-inducible mouse mammary tumor virus (MMTV) promoter, Pgtf, murine virus promoter Moloney leukemia (MMLV), thy-1 promoter and the like.

If necessary, the vector may contain a selectable marker. As used herein, the term selectable marker refers to a genetic element that provides a selectable phenotype to a cell into which the selectable marker is introduced. The selectable marker is typically a gene whose gene product provides resistance to an agent that inhibits cell growth or kills the cell. Various selectable markers can be used in the DNA constructs of the present invention, including, for example, the Neo, Hyg, hisD, Gpt and Ble genes, as described, for example, in Ausubel et al. (Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999)) and US Patent No. 5,981,830. Drugs useful for screening for the presence of a selectable marker include, for example, G418 for Neo, hygromycin for Hyg , histidinol for hisD, xanthine for Gpt and bleomycin for Ble (see Ausubel et al., supra, (1999); US patent No. 5981830). The DNA constructs of the present invention may include a positive selectable marker, a negative selectable marker, or both (see, for example, US Pat. No. 5,981,830).

Various mammalian cell culture systems can also be used to express the recombinant protein. Examples of mammalian expression systems include the COS-7 monkey kidney fibroblast lines described by Gluzman, Cell, 23: 175 (1981). Other cell lines capable of expressing a compatible vector include, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors typically include an origin of replication, a suitable promoter and enhancer, as well as any required ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcription termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from SV40 splice and polyadenylation sites can be used to provide the required non-transcribed genetic elements.

Polypeptides can be recovered and isolated from recombinant cell cultures by methods that include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Isolation can be simplified if the polypeptide is expressed on the surface of cells, but this is not a requirement. It may also be desirable to isolate cleavage products that are cleaved following expression of a longer form of the polypeptide. Protein refolding steps known in the art can be used, if desired, to obtain the complete conformation of the mature protein. High performance liquid chromatography (HPLC) can be used for the final purification steps.

Human constant region DNA sequences can be isolated according to well known procedures from a variety of human cells.

The present invention relates in particular to antibodies which are characterized in that they

- 16 043617 bind to N3pE Αβ-peptides with high affinity. The present invention also relates to antibodies which are characterized in that they bind to N3pE Άβ-peptides or immunologically active fragments thereof with high affinity. Said high affinity in the context of the present invention means an affinity with a _KD value of 10 ^-5 M, 10 ^-6 M or 10 ^-7 M or more, preferably a KD value of 10 ^-8 M or more, and even more preferably a KD value of 10 ^-9 M -10 ^-12 M. As a consequence, the antibodies of the present invention bind to monomeric Άβ N3pE with higher affinity than already known antibodies.

Preferably, the binding epitope for the antibodies of the present invention when binding to Άβ N3pE is an epitope that carries a pyroglutamate at the N-terminus. More preferably, the binding epitope for the antibody of the present invention is selected from the group consisting of pEFRHDSGYEVHHQKLV (SEQ ID NO: 23), pEFRHDSGYEVHHQKL (SEQ ID NO: 24), pEFRHDSGYEVHHQK (SEQ ID NO: 25), pEFRHDSGYEVHHQ (SEQ ID NO: 26) , pEFRHDSGYEVHH (SEQ ID NO: 27), pEFRHDSGYEVH (SEQ ID NO: 28), pEFRHDSGYEV (SEQ ID NO: 29), pEFRHDSGYE (SEQ ID NO: 30), pEFRHDSGY (SEQ ID NO: 31), pEFRHDSG (SEQ ID NO: 32), pEFRHDS (SEQ ID NO: 33), pEFRHD (SEQ ID NO: 34), pEFRH (SEQ ID NO: 35) and pEFR (SEQ ID NO: 36).

Most preferably, the antibodies of the present invention do not bind to binding epitopes that do not carry a pyroglutamate at the N-terminus.

Even more preferably, when binding to the above and subsequently mentioned binding epitopes, the antibodies of the present invention always bind to sequences or portions of sequences that contain pyroglutamate at the N-terminus. The antibodies of the present invention do not bind to sequences or portions of sequences that do not contain a pyroglutamate at the N-terminus.

In addition, the antibody of the present invention can also bind to the Άβ N3pE variant. In the context of the present invention, the Άβ N3pE variant is, in particular, ρΕ-Αβ ₃ . ₃₈ , ρΕ-Αβ _3.4ο , ρΕ-Αβ _3.42 ·

Additional N3pE Άβ peptide variants are all Άβ N3pE variants that have been shown to accumulate in the brain following or preceding Alzheimer's disease. They are pE-Άβ _3-x peptides, where x is defined as an integer between 19 and 42, for example in pE-Άβ _3-42 above, 42 would be the integer for x.

In the context of the present invention, a functional variant of the antibody of the present invention is an antibody that retains binding abilities, in particular high affinity binding abilities to the pE-Άβ _3- peptide. The provision of such functional variants is known in the art and covers the above-mentioned possibilities that have been indicated in the definition of antibodies and fragments thereof.

In a further embodiment, the antibody is an antibody fragment as defined above.

- 17 043617

In a further preferred embodiment, the antibody of the present invention is a humanized and deimmunized antibody that contains complementarity determining regions (CDRs) from the above antibodies. Preferably the antibody may be labeled; valid labels are those labeled above, and in particular those known to those skilled in the art of diagnostic antibody use.

In another embodiment, the antibodies may be immobilized on a solid phase.

In another embodiment, antibodies in accordance with the present invention and previously described in this invention or fragments thereof exhibit a binding affinity to the Άβ N3pE oligomer, fiber, fibril or filament that is at least 2-fold, in particular at least 4-fold, in particular at least 10 times, in particular at least 15 times, more particularly at least 20 times, but especially at least 25 times the binding affinity for the Άβ N3pE monomer.

In yet another embodiment, there are provided antibodies or fragments thereof previously described in this invention that essentially bind to aggregated Άβ, including Άβ plaques that contain Άβ N3pE, in a mammal, particularly in the human brain, but preferably not exhibit any significant cross-reactivity with amyloid precursor protein (APP).

In another aspect, the present invention provides antibodies or fragments thereof as previously described herein, wherein the antibodies essentially bind to oligomeric or polymeric amyloid that contains Άβ N3pE, in particular amyloid β (Άβ) in a mammal, especially in the human brain , but preferably do not exhibit significant cross-reactivity with amyloid precursor protein (APP).

The present invention also relates to compositions containing said antibodies, and to the use of said compositions in the treatment of amyloidosis, especially in the treatment of a neurodegenerative disease in a mammal, particularly in humans. Said neurodegenerative disease is particularly selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (ΆD), such as sporadic Alzheimer's disease (SAD), or familial forms of Alzheimer's dementia (FΆD), such as familial British dementia (FBD). ) and familial Danish dementia (FDD), neurodegeneration in Down syndrome. Preferably, said neurodegenerative disease is Alzheimer's disease.

Thus, in a preferred embodiment, the present invention is directed to a method of treating and/or preventing conditions characterized by the formation of plaques containing Άβ N3pE in mammals, preferably in humans, the method comprising administering, preferably peripherally, to a human in need of such treatment, a therapeutically or prophylactically effective amount of a monoclonal antibody of the present invention or an immunologically reactive fragment thereof, wherein the antibody specifically binds to an epitope of the N3pE Άβ peptide that carries pyroglutamate at the N-terminus.

In another embodiment, the present invention is directed to a method of inhibiting the formation of amyloid plaques and clearing or removing amyloid plaques in mammals, preferably humans, the method comprising administering to a human subject in need of such suppression an effective amount of an antibody that binds to Άβ N3pE in bloodstream, body fluids or tissues, especially in the brain, and more preferably leads to the clearance of Άβ N3pE from blood plasma and brain.

Accordingly, the present invention also provides methods for reversing cognitive decline, improving cognitive performance, treating cognitive decline, and preventing cognitive decline in a subject diagnosed with mild cognitive impairment (MCI), Alzheimer's disease (ΆD), such as sporadic Alzheimer's disease (SΆD). or familial forms of Alzheimer's dementia (FAD), such as familial British dementia (FBD) and familial Danish dementia (FDD), Down syndrome neurodegeneration, and clinical or preclinical cerebral amyloid angiopathy, preferably Alzheimer's disease, comprising administering to the subject an effective amount of an antibody of the present invention.

The present invention also provides the use of an antibody of the present invention for the manufacture of a medicament for the treatment, prevention or regression of mild cognitive impairment (MCI), Alzheimer's disease (ΆD), such as sporadic Alzheimer's disease (SAD) or familial forms of Alzheimer's dementia (FAD), such as e.g. familial British dementia (FBD) and familial Danish dementia (FDD), neurodegeneration in Down syndrome and clinical or preclinical cerebral amyloid angiopathy, preferably Alzheimer's disease, or for regression of cognitive decline, improvement of cognitive function, treatment of cognitive decline and prevention decline in cognitive abilities in a subject diagnosed with mild cognitive impairment (MCI), Alzheimer's disease (ΆΌ), such as sporadic Alzheimer's disease (SΆD), or familial forms of Alzheimer's dementia (FAD), such as familial British dementia (FBD), and familial Danish dementia (FDD), neurodegeneration in Down syndrome and clinical or preclinical cerebral amyloid angiopathy, preferably Alzheimer's disease.

The present invention further provides the antibodies disclosed herein for use in the prevention, treatment or regression of mild cognitive impairment (MCI), Alzheimer's disease (AD), such as sporadic Alzheimer's disease (SAD) or familial forms of Alzheimer's dementia (FAD), such as for example familial British dementia (FBD) and familial Danish dementia (FDD), neurodegeneration in Down syndrome and clinical or preclinical cerebral amyloid angiopathy, preferably Alzheimer's disease; for the treatment, prevention or regression of cognitive decline, improvement of cognitive function, treatment of cognitive decline and prevention of cognitive decline in a subject diagnosed with mild cognitive impairment (MCI), Alzheimer's disease (AD), such as sporadic Alzheimer's disease (SAD) or familial forms of Alzheimer's dementia (FAD), such as familial British dementia (FBD) and familial Danish dementia (FDD), neurodegeneration in Down syndrome and clinical or preclinical cerebral amyloid angiopathy, preferably Alzheimer's disease; or suppression of amyloid plaque formation or effects due to Ae N3pE in mammals, preferably in humans.

In a specific embodiment, the present invention provides a method of preserving or increasing cognitive memory capacity, but in particular restoring cognitive memory capacity in a mammal, particularly a human, suffering from memory impairment, by administering to the animal, particularly the mammal or a human, an antibody of the present invention or a pharmaceutical a composition containing an antibody in accordance with the present invention and described in this invention above.

The present invention also provides methods for assessing the response of a human subject to treatment with an antibody that binds Ae N3pE or a variant thereof, comprising:

a) administering an antibody of the present invention or a fragment thereof to a subject; and

b) measuring the concentration of Ae N3pE in a biological sample obtained from the subject.

The present invention also provides a method of treating a human with an antibody that binds Ae N3pE or a variant thereof, comprising:

a) administering a first amount of an antibody or fragment thereof to a subject;

b) measuring the concentration of Ae N3pE in a biological sample obtained from the subject over a period of 3 hours to two weeks after administration of the first dose;

c) optionally calculating a second amount of antibody or fragment thereof based on the result obtained in step b), wherein the second amount is the same or different from the first amount, and

d) introducing a second amount of antibody or fragment thereof.

The present invention also includes a method for assessing in a mammal, preferably a human subject, the effectiveness of an antibody that binds to Ae N3pE, or a fragment thereof, in inhibiting or preventing the formation of Ae N3pE-mediated amyloid plaque, in reducing the burden of Ae N3pE-containing plaques, in relation to reducing the effects of toxic Ae N3pE and variants thereof, or for treating a condition or disease associated with plaques containing Ae N3pE, comprising:

a) obtaining the first biological sample from the subject;

b) measuring the initial concentration of Ae N3pE in the first sample;

c) administering an antibody of the present invention or a fragment thereof to a subject;

d) obtaining a second biological sample from the subject within a period of 3 hours to two weeks after administration of the antibody or fragment thereof; and

e) measuring the concentration of Ae N3pE in the second biological sample; wherein the potency is related to the amount of Ae N3pE bound to the antibody in the blood and the concentration of Ae N3pE, in particular the reduction in its concentration, in the second biological sample compared to the first biological sample.

The biological sample can be any sample, such as from a human. In one specific example, the sample is a tissue sample, a body fluid sample, or a cell sample. In one embodiment, the biological sample is selected from the group consisting of blood, serum, urine, cerebrospinal fluid (CSF), blood plasma, lymph, saliva, sweat, pleural fluid, synovial fluid, tear fluid, bile, and pancreatic secretions. In a further embodiment, the biological sample is blood plasma. In a preferred embodiment, the biological sample is CSF.

A biological sample can be obtained from a subject using a method well known to one skilled in the art. Specifically, a blood sample can be obtained from the subject and a blood sample

- 19 043617 can be separated into serum and plasma using standard methods. The subject from which the biological sample is obtained is preferably a subject suspected of having the disease or condition of amyloidosis, preferably Alzheimer's disease, at risk of developing Alzheimer's disease and/or at risk of developing or having some other form of dementia. Specifically, the sample is obtained from a subject suspected of having mild cognitive impairment (MCI) and/or who is in the early stages of Alzheimer's disease.

The effectiveness of the antibodies of the present invention in the diagnosis, prevention and/or treatment of amyloidosis, such as mild cognitive impairment, Alzheimer's disease, familial British dementia or familial Danish dementia and, for example, neurodegeneration in Down syndrome, can be tested in existing animal models of Alzheimer's disease.

Suitable animal models of Alzheimer's disease are reviewed in McGowan et al. TRENDS in Genetics, Vol. 22, No. May 2006, pp 281-289, and selected from PDAPP, Tg2576, APP23, TgCRND8, ^P SE ^N 1M146V ^{or P} SE ^N 1M146L, PSAPP, APPDutch, ^BRI -^40 and BRI-A^ ^2, JNPL ^{3, Tau} P301S, ^Tau V337M, ^Tau R406W, rTg4510, Htau, TAPP, 3xTgAD, described below.

PDAPP: the first transgenic model of mutant APP with persistent plaque formation. Mice express human APP cDNA with the Indiana mutation (APP _V717F ). Plaque formation begins at 6–9 months in hemizygous PDAPP mice. There is synaptic loss but no obvious cell loss or NFT pathology. This model is widely used in vaccine therapy strategies.

Tg2576: mice express mutant APPSWE under the control of the hamster prion protein gene promoter. Plaque formation is observed from 9 months of age. These mice have cognitive deficits but lack cell loss or NFT pathology. This model is one of the most widely used transgenic models in the field of Alzheimer's disease.

APP23: mice express mutant APPswe under the control of the Thy1 promoter. Severe cerebrovascular amyloid, amyloid deposits are observed from 6 months of age, and some loss of hippocampal neurons is associated with the formation of amyloid plaques.

TgCRND8: mice express several APP mutations (Swedish plus Indiana). Cognitive deficits coincide with the rapid development of extracellular plaques at ~3 months of age. Cognitive deficits can be reversed with vaccine therapy against Ae.

PSEN _1m146v or PSEN _1m146l (lines 6.2 and 8.9, respectively): These models were the first in vivo demonstration that mutant PSEN1 selectively increases Ab42. No obvious plaque formation is observed.

PSAPP (Tg2576xPSEN _1M146L , PSEN1-A246E+APP _swe ): Digenic transgenic mice with the addition of a mutant PSEN1 transgene that significantly accelerates amyloid pathology compared to single transgenic APP mutant mice, demonstrating that PSEN1-induced elevation of Av42 enhances plaque formation.

APP _Dutch : Mice express APP with the Dutch mutation, which causes hereditary cerebral hemorrhage with Dutch type amyloidosis in humans. Mice with APP _Dutch develop severe congophilic amyloid angiopathy. The addition of a mutant PSEN1 transgene redistributes amyloid pathology to the parenchyma, suggesting distinct roles for Av40 and Av42 in vascular and parenchymal amyloid pathology.

BRI-Ae40 and BRI-Ae42: mice express distinct Ae isoforms without overexpressing APP. Only mice expressing Ae42 develop senile plaques and CAA, whereas BRI-AP40 mice do not develop plaques, indicating that Ae42 is essential for plaque formation.

JNPL3: mice express MART 4R0N with the P301L mutation. This is the first transgenic model with extensive tangle formation and cell loss, demonstrating that MART itself can cause cell damage and loss. JNPL3 mice develop motor impairments with age due to severe pathology and loss of motor neurons in the spinal cord.

Tau _P301S : transgenic mice expressing the shortest isoform of MART 4R with the P301S mutation. Homozygous mice develop severe paraparesis at 5–6 months of age with widespread neurofibrillary pathology in the brain and spinal cord and neuronal loss in the spinal cord.

Tau _V337M : low-level synthesis of MART 4R with the V337M mutation (1/10 of endogenous MART) under the control of the platelet-derived growth factor (PDGF) gene promoter. The development of neurofibrillary pathology in these mice suggests that the nature of MART, rather than the absolute intracellular concentration of MART, causes the pathology.

Tau _R406W : mice expressing human 4R MAP with the R406W mutation under the control of the CAMKII promoter. Mice develop MART inclusions in the forebrain from 18 months of age and have impaired associative memory.

rTg4510: TET-off induced MART transgenic mice. Abnormal MART pathology occurs from the age of one month. Mice are characterized by progressive NFT pathology and severe cell loss. Cognitive deficits are evident from 2.5 months of age. Disabling the transgene improves cognitive performance, but NT pathology worsens.

H _tau : transgenic mice expressing only human genomic MART (mouse MART knockout). H _tau mice accumulate hyperphosphorylated MART from 6 months of age and develop thioS-positive NFTs by 15 months of age.

TAPP (Tg2576xJNPL3): Enhanced forebrain MART pathology in TAPP mice compared to JNPL3, suggesting that mutant APP and/or Ae may influence subsequent MART pathology.

3xTgAD: triple transgenic model expressing mutant APP _SW E, MAPT _p301l in a PSEN knockin _1M146V (PSNE1-KI) background. Mice develop plaques from 6 months and MART pathology from 12 months of age, supporting the hypothesis that APP or Ae may directly influence neurofibrillary pathology.

Additionally, WO 2009/034158 discloses non-human transgenic animal models in which the transgene encodes at least one amyloid beta (Ae) peptide selected from the group consisting of AeN3E-42, AeN3Q-42, AeN3E- 40 and AeN3Q-40. These Ae peptides are QC and QPCTL substrates, resulting in the cyclization of N-terminal glutamine (Q) or glutamate (N) to pyroglutamate (pGlu). Thus, these transgenic animal models provide a model system for studying the effect of pGlu-Ae peptides on the development of neurodegeneration.

Antibodies to Ae pN3pE may also be useful in diagnostic assays for Ae pN3pE, for example in detecting its occurrence in specific cells, tissues or serum. Thus, the antibodies of the present invention are particularly useful in a diagnostic method for detecting amyloidosis, in particular a neurodegenerative disease selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), such as sporadic Alzheimer's disease (SAD) or familial Alzheimer's dementia (FAD), such as familial British dementia (FBD) and familial Danish dementia (FDD), neurodegeneration in Down syndrome, preferably Alzheimer's disease.

For diagnostic applications, the antibody will typically be labeled with a detectable moiety. There are numerous tags available, which can be broadly grouped into the following categories:

(a) radioactive isotopes such as 35S, 14C, 125I, 3H and 131I. Antibodies can be labeled with radioactive isotopes using techniques described, for example, in Current Protocols in Immunology, Volumes 1 and 2, Gutigen et al., Ed., Wiley-Interscience. New York, New York. Pubs. (1991), and radioactivity can be measured using scintillation counts;

(b) Fluorescent labels are available such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin and Texas red. Fluorescent tags can be conjugated to the antibody using techniques disclosed, for example, above in Current Protocols in Immunology. Fluorescence can be quantified using a fluorimeter;

(c) Various enzyme-substrate tags are available. The enzyme typically catalyzes a chemical change in a chromogenic substrate, which can be measured using a variety of techniques. For example, an enzyme may catalyze a color change in a substrate that can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Methods for quantifying changes in fluorescence are described above. A chemiluminescent substrate is electronically excited through a chemical reaction and can then emit light that can be measured (for example, using a chemiluminometer) or act as an energy donor for a fluorescent acceptor. Examples of enzymatic tags include luciferases (eg, firefly luciferase and bacterial luciferase, US Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, O-galactosidase, glucoamylase , lysozyme, saccharide oxidases (such as glucose oxidase, galactose oxidase and glucose-6phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase and the like. Methods for conjugating enzymes with antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym (ed Langone & H. Van Vunakis), Academic Press, New York, 73 : 147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) horseradish peroxidase (HRPO) with hydrogen peroxide as a substrate, where the hydrogen peroxide oxidizes a dye precursor (eg, orthophenylenediamine (OPD) or 3,3',5,5'tetramethylbenzidine hydrochloride (TMB));

(ii) alkaline phosphatase (AP) with p-nitrophenyl phosphate as the chromogenic substrate; and (iii) e-D-galactosidase (e-D-Gal) with a chromogenic substrate (eg p-nitrophenyl-e-D-galactoside) or a fluorogenic substrate 4-methylumbelliferyl-e-D-galactoside.

Many other enzyme-substrate combinations are available to those skilled in the art.

- 21 043617

Sometimes the label is indirectly conjugated to the antibody. Various techniques for achieving this will be known to one skilled in the art. For example, an antibody can be conjugated to biotin and any of the three broad categories of labels mentioned above can be conjugated to avidin, or vice versa Biotin selectively binds to avidin and thus the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve label-antibody indirect conjugation, the antibody is conjugated to a small hapten (eg digoxin) and one of the various types of labels mentioned above is conjugated to an anti-hapten antibody (eg anti-digoxin antibody). In this way, label indirect conjugation can be achieved with antibody.

The antibodies of the present invention can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies A Manual of Techniques, pp.147-158 (CRC Press. Inc., 1987)

Competitive binding assays rely on the ability of a labeled standard to compete with the test sample for binding to a limited amount of antibody. The amount of Ae N3pE in the test sample is inversely proportional to the amount of standard that binds to the antibodies. To make it easier to determine the amount of bound standard, antibodies are typically insoluble before or after competition, so the standard and analyte that are bound to the antibodies can be seamlessly separated from the standard and analyte that remain unbound.

Sandwich assays involve the use of two antibodies, each capable of binding to a distinct immunogenic portion or epitope of the protein being detected. In a sandwich assay, the analyte of the test sample is bound by a first antibody that is immobilized on a solid support, and then a second antibody binds to the analyte, thereby forming an insoluble three-part complex. The second antibody itself may be labeled with a detectable fragment (direct sandwich assay) or can be measured using an anti-immunoglobulin antibody that is labeled with a detectable fragment (indirect sandwich assay). For example, one preferred type of sandwich assay is an ELISA assay in which the moiety detected is an enzyme.

For immunohistochemistry, the tissue sample may be fresh or frozen, or may be embedded in paraffin and fixed, for example, with a preservative such as formalin.

The present invention also relates to a composition that contains antibodies as defined above, wherein said composition is a composition for use in diagnostics, in particular in the diagnosis of a neurodegenerative disease selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), such as sporadic Alzheimer's disease (SAD) or familial forms of Alzheimer's dementia (FAD), such as familial British dementia (FBD) and familial Danish dementia (FDD), neurodegeneration in Down syndrome; preferably Alzheimer's disease; in particular, by detecting Ae N3pE or variants thereof in a biological sample.

Diagnostic kits.

For convenience, the antibody of the present invention may be provided in a kit, that is, a packaged combination of reagents in predetermined quantities with instructions for performing a diagnostic assay. When an antibody is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (eg, a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives may be included, such as stabilizers, buffers (eg, blocking buffer or lysis buffer), and the like. The relative amounts of the various reagents can be varied widely to provide solution concentrations of reagents that significantly optimize the sensitivity of the assay. In particular, the reagents may be presented in the form of dry powders, usually lyophilized, including excipients which, when dissolved, provide a solution of the reagent at the appropriate concentration.

The diagnostic kit of the present invention may contain an additional biologically active substance described below. Particularly preferred for use in the diagnostic kit, as further indicated, the additional biologically active substance is a glutaminyl cyclase inhibitor.

The diagnostic kit of the present invention is particularly useful in the detection and diagnosis of amyloid-associated diseases and conditions, in particular neurodegenerative diseases selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), such as sporadic Alzheimer's disease (SAD) or familial forms of Alzheimer's dementia (FAD), such as familial British dementia (FBD) and familial Danish dementia (FDD), neurodegeneration in Down syndrome; preferably Alzheimer's disease.

The present invention also relates to an antibody of the present invention or a composition containing an antibody, all of which are defined above, for use in a diagnostic method in

- 22 043617 vitro. In particular, this diagnostic method is aimed at diagnosing a neurodegenerative disease selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), such as sporadic Alzheimer's disease (SAD), or familial forms of Alzheimer's dementia (FAD), such as eg familial British dementia (FBD) and familial Danish dementia (FDD), neurodegeneration in Down syndrome; preferably Alzheimer's disease; in particular by detecting Ae N3pE or variants thereof in a biological sample.

In a particularly preferred embodiment, the present invention relates to the following method:

an in vitro or in situ diagnostic method for diagnosing an amyloid-associated disease or condition, preferably Alzheimer's disease, comprising the following steps:

contacting an antibody of the present invention with a sample, preferably selected from a sample consisting of blood serum, cerebrospinal fluid or CSF, most preferably a blood serum sample; or to a specified body part or area of the body of a subject suspected of being affected by the specified condition or disease, and detecting binding of the antibody to N3pE Ae from the sample.

More specifically, the present invention relates to a method for diagnosing an amyloid-associated disease or condition, preferably Alzheimer's disease, comprising detecting immunospecific binding of an antibody of the present invention or an immunologically active fragment thereof to Ae N3pE in a sample or in situ, comprising the steps of:

(a) bringing a sample, or a specific body part or area of the body suspected of containing amyloid protein, into contact with an antibody of the present invention or a fragment thereof;

(b) allowing the antibody and/or a functional portion thereof to contact Ae N3pE to form an immunological complex;

(c) detecting the formation of an immunological complex; and (d) correlating the presence or absence of an immunological complex with the presence or absence of Ae N3pE in the sample or a specific part or region of the body.

Also included is a method for determining the degree of amyloidogenic plaque load of tissues and/or body fluids, comprising:

(a) obtaining a representative sample of the tissue and/or body fluid being examined;

(b) testing said sample for the presence of amyloid protein using an antibody of the present invention or a chimeric antibody or fragment thereof;

(c) determining the amount of antibody bound to the protein; and (d) calculation of plaque load of tissue and/or body fluids.

In particular, the present invention relates to a method for determining the degree of amyloidogenic plaque load of tissue and/or body fluids, where the formation of the immunological complex in step c) is determined such that the presence or absence of the immunological complex correlates with the presence or absence of amyloid protein, in particular Ae N3pE .

In yet another embodiment, the present invention relates to a composition comprising an antibody in accordance with the present invention or a chimeric antibody or fragment thereof, and as previously described in this invention, including any functionally equivalent antibody or any derivative or functional parts thereof, in particular the composition which is a pharmaceutical composition, optionally further containing a pharmaceutically acceptable carrier.

In another embodiment of the present invention, the composition contains an antibody of the present invention in a therapeutically effective amount.

Additionally provided in the present invention is a mixture containing an antibody of the present invention or a chimeric antibody or fragment thereof, and as previously described in this invention, including any functionally equivalent antibody or any derivative or functional parts thereof, in a therapeutically effective amount and optionally additional biologically active substance , and/or a pharmaceutically acceptable carrier, and/or a diluent, and/or an excipient.

In particular, the present invention relates to a mixture in which the additional biologically active substance is a compound used in the treatment of amyloidosis, a group of diseases and disorders associated with amyloid or amyloid-like protein, such as Ae N3pE, involved in neurodegenerative diseases selected from the group, consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), such as sporadic Alzheimer's disease (SAD) or familial Alzheimer's dementia (FAD), such as familial British dementia (FBD) and familial Danish dementia (FDD), neurodegeneration in Down syndrome; preferably Alzheimer's disease.

In another embodiment of the present invention, another biologically active substance or compound may also be a therapeutic agent that can be used to treat amyloidosis caused by Άβ N3pE or can be used in the treatment of other neurological disorders.

Another biologically active substance or compound may exert its biological action by the same or similar mechanism as the antibody of the present invention, or by an unrelated mechanism of action, or by several related and/or unrelated mechanisms of action.

Typically, the other biologically active compound may include neuronal transmission enhancers, psychotherapeutic drugs, acetylcholinesterase inhibitors, calcium channel blockers, biogenic amines, benzodiazepine tranquilizers, acetylcholine synthesis, storage or release enhancers, postsynaptic acetylcholine receptor agonists, monoamine oxidase A or B inhibitors , N-methyl-D-aspartate glutamate receptor antagonists, non-steroidal anti-inflammatory drugs, antioxidants and serotonergic receptor antagonists.

More specifically, the present invention relates to a mixture containing at least one compound selected from the group consisting of compounds effective against oxidative stress, anti-apoptotic compounds, metal chelators, DNA repair inhibitors such as pirenzepine and metabolites, 3-amino-1 -propanesulfonic acid (3 APS), 1,3-propane disulfonate (1,3PDS), α-secretase activators, β- and γ-secretase inhibitors, tau proteins, neurotransmitter, agents that destroy β-pleated layers, attractants of cellular components, amyloid-beta clearance/reduction agents, N-terminal truncated beta-amyloid inhibitors including the pyroglutamate variant of amyloid beta 3-42 such as glutaminyl cyclase inhibitors, anti-inflammatory molecules or cholinesterase inhibitors (ChEI) such as tacrine, rivastigmine, donepezil and/or galantamine, M1 agonists and other drugs, including any amyloid- or tau-modifying drugs and dietary supplements, and dietary supplements together with the antibody of the present invention and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

The present invention further relates to a mixture in which the compound is a cholinesterase inhibitor (ChEI), in particular to a mixture in which the compound is a compound selected from the group consisting of tacrine, rivastigmine, donepezil, galantamine, niacin and memantine.

In a further embodiment, mixtures in accordance with the present invention may contain niacin or memantine together with an antibody in accordance with the present invention and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

In a further embodiment, mixtures in accordance with the present invention may contain a glutaminyl cyclase inhibitor together with an antibody in accordance with the present invention and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

Preferred glutaminyl cyclase inhibitors are described in WO 2005/075436, WO 2008/055945, WO 2008/055947, WO 2008/055950, WO 2008/065141, WO 2008/110523, WO 2008/128981, WO 2008/128982, WO 2008/128983, WO 2008/128984, WO 2008/128985, WO 2008/128986, WO 2008/128987, WO 2010/026212, WO 2011/131748, WO 2011/029920, WO 2011/107530, WO 2011/110613, WO 2012/123563 and WO 2014/140279, the disclosure of which is incorporated into this invention by reference.

In yet another embodiment, the present invention provides mixtures that contain atypical antipsychotics, such as, for example, clozapine, ziprasidone, risperidone, aripiprazole, or olanzapine, for the treatment of positive and negative psychotic symptoms, including hallucinations, delusions, thought disorders (manifested by severe incoherence , slippage, tangentiality) and bizarre or disorganized behavior, as well as anhedonia, affective flattening, apathy and social withdrawal, in association with an antibody, in particular a monoclonal antibody according to the present invention, but especially a chimeric antibody or fragment thereof, or an antibody or fragment thereof in accordance with the present invention, and as described in this invention, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

In a specific embodiment of the present invention, the compositions and mixtures in accordance with the present invention, and as described in this invention above, contain an antibody of the present invention and a biologically active substance, respectively, in a therapeutically effective amount.

Other compounds which may be suitably used in mixtures in combination with the antibody of the present invention are described in WO2008/065141 (see in particular pages 37/38), including PEP inhibitors (pp. 43/44), LiCl, dipeptidyl aminopeptidase inhibitors, preferably DP IV or DP IV like enzyme inhibitors (see pages 48/49); acetylcholinesterase (ACE) inhibitors (see page 47), PIMT enhancers, beta-secretase inhibitors (see page 41), gamma secretase inhibitors (see pages 41/42), neutral endopeptidase inhibitors, phosphodiesterase-4 inhibitors (PDE-4) (see pages 42/43), TNF-alpha inhibitors, muscarinic M1 receptor antagonists (see pages 46), NMDA receptor antagonists (see pages 47/48), sigma inhibitors 1-receptors, histamine 3-receptor antagonists (see page 43), immunomodulatory agents, immunosuppressive agents or an agent selected from the group consisting of Antegren (natalizumab), Neurelan (fampridine-SR), Campath (alemtuzumab), IR 208 , NBI 5788/MSP 771 (tiplimotide), paclitaxel, Anergix.MS (AG 284), SH636, Differin (CD 271, adapalene), BAY 361677 (interleukin-4), matrix metalloproteinase inhibitors (for example, BB 76163), interferon- tau (trophoblastin) and SAIK-MS; antibodies to beta-amyloid (see page 44), cysteine protease inhibitors (see page 44); MCP-1 antagonists (see pages 44/45), amyloid protein deposition inhibitors (see 42) and beta-amyloid synthesis inhibitors (see pages 42), which are incorporated herein by reference.

In another embodiment, the present invention relates to a mixture containing an antibody in accordance with the present invention or a chimeric antibody or fragment thereof, and as previously described in this invention, and/or a biologically active substance in a therapeutically effective amount.

Pharmaceutical compositions can be formulated with pharmaceutically acceptable carriers or diluents, as well as any other known excipients and excipients, in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, ^21st Edition, Gennaro, Ed. , Mack Publishing Co., Easton, PA, 2005.

Pharmaceutically acceptable carriers or diluents, as well as any other known excipients and excipients, should be suitable for the selected antibody of the present invention and the selected route of administration. Suitability for carriers and other components of pharmaceutical compositions is determined based on the absence of a significant adverse effect on the desired biological properties of the selected antibody or pharmaceutical composition of the present invention (for example, less than significant effect (relative inhibition of 10% or less, relative inhibition of 5% or less, etc.) .d.) for antigen binding).

The pharmaceutical composition of the present invention may also include diluents, excipients, salts, buffers, detergents (for example, a nonionic detergent such as Tween-20 or Tween-80), stabilizers (for example, sugars or free proteinogenic amino acids), preservatives, tissue fixatives, solubilizers and/or other materials suitable for inclusion in the pharmaceutical composition.

The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied to provide an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and route of administration. The dose level chosen will depend on a variety of pharmacokinetic factors, including the potency of the specific compositions of the present invention or their amide used, route of administration, time of administration, clearance rate of the particular antibody used, duration of treatment, other drugs, compounds and/or materials used in combination. with specific compositions, age, sex, weight, condition, general health and history of the patient being treated and similar factors well known in the medical field.

The pharmaceutical composition can be administered via any suitable route and method. Suitable routes for administering the antibody of the present invention in vivo and in vitro are well known in the art and can be selected by those skilled in the art.

In one embodiment, the pharmaceutical composition of the present invention is administered parenterally.

As used herein, the phrases parenteral and parenterally administered mean modes of administration other than enteral and topical administration, typically by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrathoracic injection and infusion.

In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicizing agents, antioxidants and absorption retarding agents, and the like that are physiologically compatible with the antibody of the present invention.

Examples of suitable aqueous and non-aqueous carriers that can be used in pharmaceuticals

- 25 043617 chemical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerin, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils such as olive oil , corn oil, peanut oil, cottonseed oil and sesame oil, colloidal solutions of carboxymethylcellulose, gum tragacanth and injectable organic esters such as ethyl oleate, and/or various buffers. Other carriers are well known in the pharmaceutical field.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of injectable sterile solutions or dispersions. The use of such media and means for pharmaceutically active substances is known in the art. Apart from those of any conventional media or agents that are incompatible with the antibody, their use is contemplated in the pharmaceutical compositions of the present invention.

Suitable fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants, for example (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, orthophosphoric acid, and the like.

The pharmaceutical compositions of the present invention may also contain isotonicity agents such as sugars, polyalcohols such as mannitol, sorbitol, glycerin, or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may also contain one or more auxiliary agents suitable for the chosen route of administration, such as preservatives, wetting agents, emulsifiers, dispersants, preservatives or buffers, which may increase the shelf life or effectiveness of the pharmaceutical composition. The antibodies of the present invention can be formulated with carriers that will protect the antibody from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid alone or with wax, or other materials well known in the art. Methods for preparing such compositions are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In one embodiment, the antibodies of the present invention can be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of injectable sterile solutions or dispersions. The use of such media and means for pharmaceutically active substances is known in the art. Apart from those of any conventional media or agents that are incompatible with the antibody, their use is contemplated in the pharmaceutical compositions of the present invention.

Pharmaceutical compositions for injection generally must be sterile and stable under conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier may be an aqueous or non-aqueous solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerin, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. In many cases, it is preferable to include isotonic agents such as sugars, polyalcohols such as glycerin, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that retards absorption, such as monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the antibody in the required amount into a suitable diluent with one or a combination of ingredients, such as those listed above, if necessary, followed by sterilizing microfiltration. Typically, dispersions are prepared by incorporating the antibody into a sterile carrier medium that contains the basic dispersion medium and other necessary ingredients, such as those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of production methods are vacuum drying and freeze drying (lyophilization), which provide a powder of the active ingredient plus any necessary additional ingredient from a previously sterilizing filtered solution.

Sterile injectable solutions can be prepared by incorporating the antibody in the required amount into a suitable diluent with one or a combination of the ingredients listed above, if necessary, followed by sterilizing microfiltration. Typically, dispersions are prepared by incorporating the antibody into a sterile carrier medium that contains the basic dispersion medium and other necessary ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of production methods are vacuum drying and freeze drying (lyophilization), which provide a powder of the active ingredient plus any necessary additional ingredient from a previously sterilizing filtered solution.

Dosage regimens in the above treatments and applications are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally decreased or increased as required by the therapeutic situation. Parenteral compositions can be formulated in unit dosage form for ease of administration and consistency of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit containing a predetermined amount of antibody calculated to produce the desired therapeutic effect, in combination with the required pharmaceutical carrier. The technical requirements for the unit dosage forms of the present invention are dictated by and directly depend on (a) the unique characteristics of the antibody and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the field of formulating such an antibody for the treatment of sensitivity in individuals.

Effective dosages and dosage regimens for the antibodies of the present invention depend on the disease or condition being treated and can be determined by those skilled in the art. An exemplary non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1-10 mg/kg body weight, such as about 0.1-5 mg/kg body weight, such as about 0.1-2 mg/kg body weight, such as about 0.1-1 mg/kg body weight, such as about 0.15, about 0.2, about 0.5, about 1, about 1.5 or about 2 mg/kg body weight .

A physician or veterinarian skilled in the art can readily determine and prescribe an effective amount of the required pharmaceutical composition. For example, a physician or veterinarian may begin dosing an anti-AepE3 antibody used in a pharmaceutical composition at levels lower than required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. In general, a suitable daily dose of the composition of the present invention will be that amount of antibody that is the lowest dose effective to produce a therapeutic effect. Such effective dose will generally depend on the factors described above. Administration, for example, may be intravenous, intramuscular, intraperitoneal or subcutaneous, and, for example, administered in the vicinity of the target site. If necessary, an effective daily dose of the pharmaceutical composition can be administered in two, three, four, five, six or more dosage units administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. Although the antibody of the present invention can be administered separately, it is preferable to administer the antibody in the form of a pharmaceutical composition as described above.

Examples

1. Humanization approach for creating NpE-Ab-specific antibodies.

The humanized and deimmunized antibodies of the present invention are humanized and deimmunized forms of mouse monoclonal antibodies that are produced by the Ae 6-1-6 hybridoma cell line (DSM Accession No. ACC 2924), which are described in WO 2010/009987. Humanization was carried out as described in WO 2017/009459. Working Example 1 disclosed on pages 58-60 in WO 2017/009459 is hereby incorporated by reference into the present invention.

2. RNA isolation and cDNA synthesis.

As a source for constant sequences, human B cell RNA was isolated by lysing 500 μl of whole blood with 5 ml of 1x FACS lysis solution (Becton Dickinson) for 10 minutes at room temperature. The lysate was centrifuged at 300 g for 5 min; the pellet was washed twice with PBS and then dissolved in 350 μl of Nucleo Spin ® RNA II buffer RA1 (Macherey-Nagel) and 3.5 μl of 0.5 M TCEP (SIGMA) was added. RNA was isolated according to the manufacturers' instructions. First, 10 μl of RNA was incubated with 1 μl of 0.5 μg/μl OligodT primer (Invitrogen) and 1 μl of 10 mM dNTPs for 5 min. at 65°C. Then 4 µl x first strand synthesis buffer (Invitrogen), 2 µl

- 27 043617

100 mM DTT and 0.5 μl Superscript III reverse transcriptase (Invitrogen) were added to 20 μl and the mixture was incubated for 5 min at 25°C, 50 min at 50°C, and 15 min at 70°C. Using PCR with the primer pairs shown in Table. 2, the synthesized light and heavy chain constant region cDNA can be amplified.

table 2

Primer for cloning the constant region

SEQ ID NO Name Subsequence 43 hkappa5' ACTGTGGCTGCACCATCTGTCTTC 44 'karraZ' CTAACACTCTCCCCTGTTGAAGCTC 45 hIgGlHc5’l AGGGAACCCCTGGTCACCGTCTCC 46 hIgG!Hc3’ TCATTTACCCGGGAGACAGGGAGAGG

To amplify the PNR light chain product of clone No. 6, the following forward and reverse primers were used:

RT_chim_humK16f: CAAGTCAGAGCCTCTTATATAGTG (SEQ ID NO: 47);

RT chim humK16r: GTACCTTGCACGCAGTAATAAAC (SEQ ID NO: 48).

The mouse HPRT primer was used to amplify the reference gene.

To carry out amplification, 7.5 μl of Sybergreen (Firma), 1 μl of forward primer (25 pmol/μl), 1 μl of reverse primer (25 pmol/μl), 5.5 μl of bidistilled _H2O and 1 μl of cDNA were used in a thermal cycler.

3. Expression of the recombinant antibody in CHO cells by separately cloning LC and HC into two different expression plasmids.

The antibody light and heavy chain sequences were separately cloned into two different mammalian expression vectors, pCDNA3.1 and HC-pOptiVEC, respectively. To identify the optimal combination of vectors for recombinant antibody expression in CHO cell culture, various combinations of plasmids were used to conduct transient expression in adherent CHO cells. At the second stage, it was investigated how different ratios of DNA in the LC and NS plasmid affect the expression level. When transfected with 3 μg of LC-pCDNA3.1 and 1 μg of HC-pOptiVEC, increased expression levels were found.

For further expression of the antibody in adherent CHO cells, a combination of plasmids was used, including LC-pCDNA3.1 and 1 μg HC-pOptiVEC, and the ratio of plasmid DNA was LC 3:1 HC.

Freestyle™ CHO cells in suspension culture were used in the following transfections to culture more transiently expressing cells that generate recombinant antibodies. It was first tested whether excess LC plasmid could improve antibody expression, as in the case of adherent cells. As in the case of adherent CHO cells, the ratio of LC and HC plasmid DNA was 1:1 and 3:1. Western blot analysis showed that excess LC plasmid increased antibody expression, as was the case in adherent CHO cells. When cell viability was measured, it was apparent that cell viability decreased to approximately 50% of transfected cells after 6 days. After the sixth day, no further increase in antibody levels was observed in the supernatant. Accordingly, culture supernatants were collected on day six for subsequent transfections.

To investigate how efficiently the produced antibodies were transferred into the cell supernatant, a sample of the cell lysate was subjected to SDS-PAGE and analyzed by Western blotting. GAPDH, a cytoplasmic constitutive protein, was used to reference loading comparable amounts of cell lysate protein onto an SDS gel. In the cell lysate of CHO cells expressing the antibody, there was a clear band corresponding to 120 kDa, migrating at the same size as that detected in the cell supernatant.

4. Purification of recombinant antibody using Protein G chromatography.

Antibody clone #6 was purified to examine the antigen-binding properties of the protein compared to the parent mouse antibody. Accordingly, 300 ml of chimeric and humanized antibody expressed supernatant was obtained and purified by Protein G chromatography. Since the amount of antibody expressed was very low, the yield was less than 0.1 μg/ml for a total of 25 μg of purified protein. The eluted antibody was concentrated to approximately 200 μg/ml, and 2 μg of protein was applied to SDS-PAGE, followed by Coomassie blue staining.

5. Creation of a stable cell line producing a humanized antibody.

The parent antibody for the humanized antibody of the present invention is a variant of clone No. 6 disclosed in WO 2017/009459, which has a light chain variable region with the amino acid sequence:

DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDGKTYLNWFQQRPGQSPRRLTYLVSK LDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFPFTFGGGTKVEIK (SEQ ID NO: 1), which is disclosed under SEQ ID NO: 14 in WO 2017/009459;

and which has a heavy chain variable region with the amino acid sequence: QVQLVQSGAEVKKSGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGLINPSN

GVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREAKREWDETYWGQ

GTLVTVSS (SEQ ID NO: 49), which is disclosed under SEQ ID NO: 27 in WO 2017/009459.

Two clones, C06.08 and C06.09, of the original variant of clone No. 6 were selected to create a stable cell line. To generate C06.08- and C06.09-producing Chinese hamster ovary (CHO) DG44 cells, expression plasmids containing heavy and light chain gene sequences for both antibodies were first generated. Plasmids were linearized, purified by isopropanol precipitation, redissolved in 10 mM Tris pH 8.0 and used in transfections as follows: 1 x 10 ⁶ CHO-DG44 cells were suspended in 100 μl Nucleofector V solution (Lonza) and mixed with 10 µg of linearized vector DNA. The suspension was pulsed using an Amaxa Nucleofector II electroporator (Lonza). Then, 500 μl CD CHO medium (Invitrogen) was added and the transfected cell pools were cultured for 24 h.

To create stable mini-pools (MPs), pools of transfected cells were pooled one day after transfection, transferred either to CD CHO medium and seeded at 2000 cells/well and 4000 cells/well in 96-well plates, respectively, or transferred to CD CHO medium. with 2.5 nM methotrexate (MTX) and seeded at 4000 cells/well in 96-well plates. After a culture period of 21 (S06/08) or 27 (S06/09) days, 20 MPs were transferred to 24-well plates. On day 22 or day 28 post-transfection, MPs were transferred to 12-well plates, and MPs were spread into 6-well plates on day 34 or day 48. On day 34 (D06.08) or 48 (D06.09), pools were transferred to selective medium with 30 nM MTX to induce the amplification process, and antibody expression titers were determined as measured by Octet.

Results.

table 2

Analysis of stable mini pools C06.08

Table 3

Analysis of stable mini pools C06.09

Day 1 Day 27 Days 48-52 Selected MRs MTX amplification 2000 cells/well No. 1-10 There is no cell growth; extremely low or absent antibody production 4000 cells/well No. 11-15 4000 cells/well and 2.5 nM MTX No. 15-20

As shown in table. 2 and 3, all MRs exhibited poor cell growth, extremely low or undetectable antibody production (<10 pg/cell/day), and many MRs did not tolerate MTX-29 043617 amplification. Repeating the transfection experiments with a new set of reagents resulted in similar results, whereas transfection with control vectors resulted in normal levels of protein expression. These results indicate that the genome-integrated proteins C06.08 and C06.09 may have intrinsic toxic effects on CHO-DG44 cells, for example, such proteins may cause cell death due to overexpression of misfolded C06.08 and C06.09 proteins in cell nuclei . Additional modifications to the primary sequence C06.08 and C06.09 were required, which correct protein misfolding and eliminate the induction of CHODG44 cell death.

Modifications of PBD-C06 to improve protein folding and expression.

In silico analysis of the amino acid sequences of C06 identified several stretches of amino acids (dots) in the heavy chain framework regions that were predicted to negatively affect protein folding. To test whether substitution at these points could be made with alternative amino acids that improve protein expression, four individual heavy chain mutations (K12V, S14P, N55D and F64V) were first synthesized and tested individually in transient transfections for protein expression along with the light chain under SEQ ID NO: 17. Using a target binding assay, the F64V modification was found to prevent antibody binding. In a second set of experiments, combinations of each mutation were paired and tripped into individual heavy chain genes and tested in combination with the light chain of SEQ ID NO: 17 for expression and target binding studies (FIGS. 1 and 2).

Results.

The combination of mutations K12V, S14P and N55D resulted in the best titers of transient CO6 expression and optimal target binding properties (Figs. 1 and 2), and the corresponding clone containing all three of these mutations was selected for stable transfection studies of CHO-DG44.

Robust expression of humanized antibody in CHO-DG44 cells.

Stock CHO-DG44 cells were originally obtained from Gibco, Life Technologies (FreedomTM DG44 Kit), now managed by Thermo Fisher Scientific. The CHO cell line is DHFR deficient and has been documented to comply with the current edition of the GMP regulations.

Transfection of CHO-DG44 cells with vectors expressing a humanized antibody containing the light chain of SEQ ID NO: 1 and the heavy chain of SEQ ID NO: 2 (which contains the K12V, S14P and N55D mutations) resulted in a several-fold increase in the number of CHO-DG44 clones. DG44 compared to previous transfection attempts (see above). Additionally, several clones with high titers of target antibody expression were identified following MTX exposure, showing that the K12V, S14P, and N55D mutations improved antibody folding and expression following stable integration into the CHO-DG44 genome. Long-term stable expression was confirmed under fed-batch conditions over long culture periods, confirming that selected changes (K12V, S14P, and N55D) in the heavy chain of the original clone #6 contributed to proper folding and expression in CHO-DG44 cells.

Additional stable cell lines were obtained after further vector optimization and electroporation. The original transfectants before gene amplification showed high levels of antibody expression shortly after isolation and plating into a 0.5 ml culture in a 24-well plate (Fig. 3). Production titers at day 7 increased further after the first exposure to MTX (Fig. 4), indicating that these clones, particularly those from 17, would be much more efficient producers.

Short description

The four heavy chain mutations were tested individually to determine whether they would lead to increased antibody expression in transiently transfected HEK293 cells. Three of these mutations significantly increased antibody expression, while the fourth decreased expression. Combinations of individual mutations were also tested in pairs, as well as a triple mutation, all of which were well expressed and purified by Protein A chromatography. After Biacore analysis, it was determined that a combination of four mutations reduced binding, while a combination of the specific mutations K12V, S14P and N55D was well expressed and had optimal binding properties. No changes in the light chain sequences were required to ensure high expression.

Studies were performed on stably transfected DG44 CHO cells, and higher levels of antibody expression were obtained after initial selection and subsequent gene amplification induced by stepwise increases in the amount of methotrexate (MTX). Additionally, it was determined that higher levels of antibody expression did not interfere with good cell growth. Overall, the transfection procedure resulted in the identification of several new candidates for CMC manufacturing.

5. Surface plasma resonance measurement to analyze the binding of humanized and deimmunized antibody to monomeric Άβ-peptides.

- 30 043617

Surface plasmon resonance measurements were used to examine the binding efficiency of an antibody that contains the light chain variable portion of SEQ ID NO: 1 and the heavy chain variable portion of SEQ ID NO: 2. The following procedure was used to prevent mass transfer and avidity effects from occurring during the measurement.

First, a polyclonal α-human antibody was coupled to an SPR chip, which was then loaded with antibody to such an extent that the response unit was greater than 1000.

Kinetics measurements were carried out at various concentrations (from 5 to 1000 nM) of Ae-peptide. Plots of measured concentration series are shown as an overlay plot using sensorgrams corrected to the running buffer sensorgram, aligned at the time of injection, and baseline corrected to zero before injection. The results are evaluated according to a simple 1:1 interaction model (approximation using the Langmuir equation) which transforms the rate constants kof and _kon .

The apparent kinetic constants according to the approximation using the 1:1 Langmuir equation are given in Table. 4. Due to the fact that the antibody is non-covalently bound to the chip surface, small amounts of antibody molecules were washed out during measurement. Therefore, Rmax values were approximated locally for each individual sensorgram.

Table 4

Statistical data of approximation using the Langmuir equation of the kinetic parameters of clone No. 6 of the humanized and deimmunized antibody

Αβ-peptide Kd k _a , ((GM' ¹ ) kd, (with ¹ ) K max R.I. Chi ² Αβ (reZ-18) 8.22 nM 4.79 x 10 ⁵ 3.94 x 10' ³ Global (43.9 RU) local 0.462 Αβ (3-18) 1.21 µm 3.09 x 10 ³ 3.74 x 10' ³ Global (40.8 RU) local 0.243 Αβ (1-18) 23.1 µm 173 3.99 x 10' ³ Global (57.4 RU) local 0.400 Αβ (4-18) 28.3 µm - - 48.9 RU - 0.0815

Binding to Aβ(pE3-18).

Qualitative and visual evaluation of the recorded sensorgrams shows a good shape for the association phase as well as for the dissociation phase. In addition, an increase in the concentration-dependent response signal and an increase in the initial slope for the association phase were observed. The maximum SPR signals were ~44 RU, which was within the optimal measurement range of binding kinetic parameters with no or negligible mass transfer effects. No baseline bias was observed, so the data were evaluated using a 1:1 Langmuir binding model, obtaining the binding properties of a given interaction and the quality of the fit (Table 4).

Binding to Αβ(3-18).

Qualitative and visual evaluation of the recorded sensorgrams shows a good shape for the association phase as well as for the dissociation phase. In addition, an increase in the concentration-dependent response signal and an increase in the initial slope for the association phase were observed. The maximum SPR signals were ~44 RU, which was within the optimal measurement range of binding kinetic parameters with no or negligible mass transfer effects. No baseline bias was observed, so the data were evaluated using a 1:1 Langmuir binding model, obtaining the binding properties of a given interaction and the quality of the fit (Table 4).

Binding to Αβ(1-18).

Qualitative and visual evaluation of the recorded sensorgrams shows a typical weak binding pattern corresponding to the peptide concentrations used. A slight shift in baseline was observed; therefore, the data were fitted using a 1:1 Langmuir coupling equation with a baseline bias model. Evaluation of these kinetic parameters (Table 4) resulted in a dissociation constant of 23.1 μM, which was higher than the highest measured concentration of Αβ(1-18). In addition, the calculated Rmax value was significantly higher than the response values for the highest concentration. Both facts indicate that the obtained binding properties are definitely in the range of K _D >10 μm.

Binding to Αβ(4-18).

Qualitative and visual evaluation of the recorded sensorgrams shows a typical shape for very weak binding, consistent with the peptide concentrations used. Due to the very rapid association and dissociation, the data could not be assessed kinetically, hence a static model was used. Evaluation of the response signals in the static model (Table 4) resulted in a dissociation constant of 28.3 μM, which was higher than the highest measured concentration of Αβ(4-18). In addition, the calculated value of Rmax significantly exceeded the values

- 31 043617 answer for the highest concentration. Both facts indicate that the obtained binding properties should definitely be in the range of KD>10 μm.

6. Binding with Av fibrils and Av oligomers.

Ab(1-42) peptide fibrils were prepared according to standard protocols at pH 8.7 and 37°C. Once completely fibrillated, the structures were aspirated into 60 μL, diluted in 140 μL running buffer, and loaded onto the sensor chip using capture antibodies at a flow rate of 1 μL/min. The system was then flushed until a stable baseline was obtained. Approximately 100 RU of Av fibrils (1-42) were captured on the sensor chip.

Oligomers of Av(1-42) peptides were prepared according to the ACUMEN protocol in Ham F12 medium overnight at 4°C and separated from aggregated Ae by centrifugation. Oligomers were then aspirated into 20 μL, diluted in 60 μL running buffer, and loaded onto the sensor chip using capture antibodies at a flow rate of 1 μL/min. The system was then flushed overnight at 100 μL/min to obtain a steady-state baseline. Approximately 300 RU of Ab(1-42) oligomers were captured on the sensor chip.

For Ab(1-42) peptide fibrils, after obtaining a stable baseline, interaction analysis was performed using 11 sequential injections (10, 30, 90, 270, 810 pM, 2.43, 7.29, 21.87, 65.61, 196 .83 and 590.49 nM) clone No. 6 of the parent antibody disclosed in WO 2017/009459, and clone No. 6 of the humanized and deimmunized antibody of the present invention with 30 μl/min, contact time of 480 s and dissociation time of 1200 s. The resulting sensorgrams were evaluated using five successive concentrations (starting with the first injection showing binding) and a single cycle kinetic model with a global approximation of _Rmax , and baseline offset, and local approximations of the volumetric effect of each injection.

For Ab(1-42) peptide oligomers, after obtaining a stable baseline, interaction analysis was performed using 11 sequential injections (10, 30, 90, 270, 810 pM, 2.43, 7.29, 21.87, 65.61, 196 .83 and 590.49 nM) clone No. 6 of the parent antibody disclosed in WO 2017/009459, and clone No. 6 of the humanized and deimmunized antibody of the present invention with 30 μl/min, contact time of 480 s and dissociation time of 3600 s. The resulting sensorgrams were evaluated using five successive concentrations (starting with the first injection showing binding) and a single cycle kinetic model with a global approximation of _Rmax , and baseline offset, and local approximations of the volumetric effect of each injection.

Results.

Table 5

Binding to fibrils of Av(1-42) peptides

Antibody K _D ka Μ'Χ' ¹ kd 10' ⁵ s ¹ Rmax Chi ² A 65.9 pM 9.9-10 ⁵ 6.53- 26 RU 0.336 IN 1.67 nM 5.97-10 ⁴ 9.99- 19.2 RU 0.602

Table 6

Binding to oligomers of Av(1-42) peptides

Antibody Kd ka mV kd 10' ⁴ s' ¹ Rmax Chi ² A 7.61 nM 1.86-10 ⁴ 1.42- 442 RU 6.31 IN 269 nM 868 2.33- 216 RU 0.284

In table 5 and 6:

A is clone No. 6 of the parent antibody disclosed in WO 2017/009459, with a light chain variable region of SEQ ID NO: 1 and a heavy chain variable region of SEQ ID NO: 49; And

B is Clone No. 6 of the humanized and deimmunized antibody of the present invention having a light chain variable region of SEQ ID NO: 1 and a heavy chain variable region of SEQ ID NO: 2.

As can be seen from these results, the present invention provides antibodies and fragments thereof, wherein the antibodies show increased selectivity for oligomers and/or fibrils of Lvpeptides. Antibodies of the present invention exhibit many times, such as 10, 25, 50, 100, 150, 200, 250 or more than 250 times lower binding constant (KD value) for binding to oligomers and/or fibrils of LV(1 -42) than comparable monoclonal antibodies known from the prior art, in particular compared to the parent antibody disclosed in WO 2017/009459. Accordingly, antibodies of the present invention that are formulated to bind selectively to N3pE LV peptides are more specific for N3pE LV peptides and show reduced cross-reactivity to LV peptides other than N3pE LV peptides.

7. Binding to Fc-gamma receptors.

The binding of two antibodies was compared that either contained the heavy chain of SEQ ID NO: 19,

- 32 043617 or its mutant variant K324A (SEQ ID NO: 18), with different Fc-gamma receptors (CD16A,

CD32A, CD32B and CD64).

The K324A mutant was obtained using site-directed mutagenesis. Binding was measured in a FACS-based bioassay in Chinese hamster ovary (CHO) cells stably expressing human CD16A, CD32A, CD32B, or CD64. Both antibodies were incubated with each cell line at 7 different concentrations for one hour, followed by washing. Receptor-bound H6 or H67 was detected using a fluorochrome-conjugated anti-Fab' antibody. Binding capacity was measured using FACS, and Kd and _Bmax were calculated using nonlinear regression.

Results: Both antibodies showed comparable binding to all receptors. 8. Binding to C1q.

The binding of two antibodies that either contained the heavy chain of SEQ ID NO: 19 or its mutant variant K324A (SEQ ID NO: 18) to C1q was compared to better characterize the effector functions of the antibodies.

A number of analytical formats for antibody binding to dq were tested, including

a) direct binding of two antibodies to the plate and then binding to C1q in solution; And

b) Streptavidin-coated plates were first incubated with biotinylated pE-Aupeptide, coupled with antibodies and then with C1q.

Thus, format a) gave the best results. The procedure is summarized below.

The ELISA plate was coated with an antibody containing the heavy chain of SEQ ID NO: 19, its mutant variant K324A (SEQ ID NO: 18) and the control K324A, which does not bind C1q, at 10, 8, 6, 4, 3, 2, 1 and 0 μg/ml in triplicate and incubated at 4°C overnight. The next day, the plate was washed three times with 1x PBS and then blocked using 1% BSA in 1x PBS at 50 µl/well. C1q (Sigma, cat. no. C1740) was added to each well at 2 μg/ml in blocking buffer and incubated for 1 h at room temperature. The plate was then washed three times with 200 μl of 1x PBS. Anti-C1q-HRP (Thermo, cat. no. PA1-84324) was added to the binding detection plate at a 1:250 dilution in blocking buffer (50 μl/well) for 1 h. The plate was washed again three times with 200 μl 1x PBS . 50 μl of TMB (Invitrogen, cat. no. 002023) was added to each well to visualize the interaction (Invitrogen, cat. no. 002023) for 2 min. 50 μl of stop solution ((1 M sulfuric acid) was added to each well before reading the absorbance at 450 nm.

Results. The antibody, which contained the wild-type heavy chain of SEQ ID NO: 19, bound to C1q. Its mutant variant K324A (containing the heavy chain of SEQ ID NO: 18) did not bind to C1q.

9. Immunohistochemistry.

Using IHC, Ae N3pE antigen can be detected in brain tissue sections. Therefore, the antibodies of the present invention were used to detect Ae N3pE.

For IHC sections, human brain tissue from the hippocampus and frontal lobe of AD patients can be used, and in addition, brain tissue sections from the hippocampus of existing animal models for Alzheimer's disease, as described in the present invention. These mouse models demonstrate elevated levels of Ae in the brain, followed by the development of neuritic plaques. Tissue sections were embedded in paraffin and serially sectioned. Sections were stained with hematoxylin to stain cell nuclei and then immunostained with anti-Ae N3pE antibodies of the present invention. The preparation and staining of tissue sections was carried out in accordance with standard techniques.

10. In vivo treatment of mice with Alzheimer's disease.

A total of 62 male mice were used in this study. Prior to immunization, four mice from an established mouse model of Alzheimer's disease (mean 5.6 mo ± 0.45) were sacrificed as controls at baseline to assess cerebral Ae plaque burden at the start of treatment. The remaining mice were divided into four groups and received the following treatment: 250 μl sterile PBS (n=12; mean 5.89 months ±0.13), 200 μg antibody of the present invention. A group of age- and sex-matched Wt littermates were injected with 250 μl PBS (n = 12; mean 5.80 mo ± 0.12) and served as behavioral controls. Mice were treated with a total volume of 250 μl (antibody or PBS) via intraperitoneal injection for 28 weeks.

Euthanasia and tissue preparation.

Mice were euthanized, perfused, and plasma collected at 6 months of age (baseline) or 13 months. The brain was removed and divided sagittally. The hippocampus, cortex, and cerebellum were dissected from one hemisphere and frozen for biochemical analysis. The other hemisphere was fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 24 h at 4°C, cryoprotected in a step gradient of sucrose solutions at 4°C, and embedded in OCT (Tissue Tek).

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Claims (15)

CLAIM 1. An antibody, where the variable portion of the light chain of said antibody consists of the amino acid sequence: DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDGKTYLNWFQQRPGQSPRRLTYLVSK LDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFPFTFGGGTKVEIK (SEQ ID NO: 1), and wherein the variable portion of the heavy chain of said antibody contains, is substantially composed of, or consists of the amino acid sequence: QVQLVQSGAEVV KPGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGLINPSD GVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREAKREWDETYWGQ GTLVTVSS (SEQ Ш NO: 2), wherein said antibody binds to the pyroglutamate-carrying N -terminal epitope in the pyroglutamate variant of beta-amyloid peptide (Ae N3pE). 2. Antibody according to claim 1, containing CDR regions: CDRl V _L : SSQSLLYSDGKTYLN (SEQ ID NO: 3); CDR2 V _L : LVSKLDS (SEQ ID NO: 4) and CDR3 V _L : VQGTHFP (SEQ ID NO: 5) in the light chain. 3. Antibody according to claim 1, containing CDR regions: CDRl V _H : GYSFTGHTMN (SEQ ID NO: 6); CDR2 V _H : LINPSDGVTRYNQKFQG (SEQ ID NO: 7) and CDR3 V _H : EAKREWDETY (SEQ ID NO: 8) in the heavy chain. 4. An antibody according to any of the preceding claims, wherein the light chain contains, consists of, or consists of the amino acid sequence: DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDGKTYLNWFQQRPGQSPRRLTYLVSK LDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFPFTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 17). 5. An antibody according to any of the preceding paragraphs, wherein the heavy chain contains, is essentially made up of, or consists of the amino acid sequence: QVQLVQSGAEVVKPGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGLINPSD GVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREAKREWDETYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18). 6. An antibody according to any of the preceding claims, wherein the heavy chain contains, consists of, or consists of the amino acid sequence: QVQLVQSGAEVVKPGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGLINPSD GVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREAKREWDETYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 19). 7. An antibody according to any of the preceding claims, wherein said antibody specifically binds to an epitope selected from the group consisting of - 34 043617 pEFRHDSGYEVHHQKLV (SEQ ID NO: 23), pEFRHDSGYEVHHQKL (SEQ ID NO: 24), pEFRHDSGYEVHHQK (SEQ ID NO: 25), pEFRHDSGYEVHHQ (SEQ ID NO: 26), pEFRHDSGYEVHH (SEQ ID NO: 27), pEFRHDSGYEV H ( SEQ ID NO: 28), pEFRHDSGYEV (SEQ ID NO: 29), pEFRHDSGYE (SEQ ID NO: 30), pEFRHDSGY (SEQ ID NO: 31), pEFRHDSG (SEQ ID NO: 32), pEFRHDS (SEQ ID NO: 33 ), pEFRHD (SEQ ID NO: 34), pEFRH (SEQ ID NO: 35) and pEFR (SEQ ID NO: 36). 8. An antibody according to any one of claims 1 to 6, wherein _said antibody of the invention binds to the N3pE variant Aβ, wherein the N3pE variant Ae is defined as pE-Aβ _{3_X} , wherein x is defined as an integer from 19 to 42. 9. Antibody according to claim 8, where the Ap N3pE variant is selected from ρΕ-Αβ ₃ .38, ρΕ-Αβ ₃ . ₄ ο, ρΕ-Αβ ₃ . ₄₂ . 10. An antibody according to any of the previous paragraphs, wherein said antibody does not bind to epitopes that do not carry a pyroglutamate at the N-terminus. 11. A pharmaceutical composition containing an antibody according to any of the previous paragraphs and an additional biologically active substance, and/or a pharmaceutically acceptable carrier, and/or a diluent, and/or an excipient. 12. The pharmaceutical composition according to claim 11, where the specified additional biologically active substance is selected from amplifiers of neural transmission, psychotherapeutic drugs, acetylcholinesterase inhibitors, calcium channel blockers, biogenic amines, benzodiazepine tranquilizers, enhancers of the synthesis, accumulation or release of acetylcholine, agonists of postsynaptic acetylcholine receptors , monoamine oxidase A or B inhibitors, N-methyl-D-aspartate glutamate receptor antagonists, non-steroidal anti-inflammatory drugs, antioxidants and serotonergic receptor antagonists. 13. The pharmaceutical composition according to claim 12, where the specified additional biologically active substance is selected from the group consisting of compounds effective against oxidative stress, anti-apoptotic compounds, metal chelators, DNA repair inhibitors, 3-amino-1propanesulfonic acid (3 APS), 1,3-propane disulfonate (1,3PDS), α-secretase activators, β- and γ-secretase inhibitors, tau proteins, neurotransmitter, agents that destroy β-pleated layers, attractants of cellular components that ensure the removal/reduction of beta-amyloid , N-terminal truncation amyloid beta inhibitors, anti-inflammatory molecules or cholinesterase inhibitors (ChEIs), M1-α gonists and other drugs. 14. Use of an antibody according to any one of claims 1 to 10 for the diagnosis of amyloidosis. 15. Use according to claim 14, wherein amyloidosis includes a neurodegenerative disease selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), such as sporadic Alzheimer's disease (SAD) or familial forms of Alzheimer's dementia (FAD) ), such as familial British dementia (FBD) and familial Danish dementia (FDD), neurodegeneration in Down syndrome and clinical or preclinical cerebral amyloid angiopathy; and/or a condition selected from prodromal AD, mild AD, moderate AD, and -