JP2022070853A - HUMANIZED ANTIBODIES AGAINST AMYLOID β - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide chimeric and humanized antibodies against amyloid β, and methods and compositions for therapeutic and diagnostic use in treatment of amyloidosis, a group of disorders and abnormalities associated with amyloid protein such as Alzheimer's disease.

SOLUTION: The present invention provides a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof, which specifically binds to at least one epitope on the β-amyloid protein, where the epitope comprises at least two consecutive amino acid residues predominantly involved in binding to the antibody.

SELECTED DRAWING: Figure 12

COPYRIGHT: (C)2022,JPO&INPIT

Description

The present invention relates to methods and compositions for the diagnosis and treatment of amyloidosis, a group of disorders and abnormalities associated with amyloid proteins such as Alzheimer's disease.

Amyloidosis is not a single disease unit, but a group of diverse progressive disease processes characterized by extracellular tissue deposition of waxy starch-like proteins called amyloid that accumulate in one or more organs or body systems. .. As amyloid deposits accumulate, they begin to interfere with the normal functioning of the organ or body system. There are at least 15 types of amyloidosis. The predominant types are primary amyloidosis with no known history, secondary amyloidosis following any other condition, and hereditary amyloidosis.

Secondary amyloidosis is a chronic infection such as tuberculosis, bacterial infection, familial Mediterranean fever, bone infection (osteomyelitis), rheumatoid arthritis, inflammation of the small intestine (granulomatous ileitis), Hodgkin's disease, and Hansen's disease. It occurs among inflammatory diseases.

Amyloid deposits include amyloid P (pentagonal) component (AP), a glycoprotein associated with normal serum amyloid P (SAP), and sulfated glycosaminoglycan (GAG), a connective tissue complex carbohydrate. Is done. Amyloid protein fibril, which accounts for about 90% of amyloid material and contains one of several types of protein. These proteins can be folded into so-called "β-pleated" sheet fibrils, a unique protein configuration that presents the binding sites of Congo Red and provides the unique staining properties of amyloid proteins.

Many age-related diseases are based on or associated with amyloid-like proteins, which are characterized by the accumulation of extracellular deposits of amyloid or amyloid-like material that contribute to the pathology and progression of the disease. These disorders include neurological disorders such as Alzheimer's disease (AD), Lewy body dementias, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Netherlands); Guam Parkinson-dementia complex, but these Includes, but is not limited to, diseases. Other diseases based on or related to amyloid-like proteins include progressive nuclear palsy, multiple sclerosis; Creutzfeldt-Jakob disease, Parkinson's disease, HIV-related dementia, ALS (amyotrophic lateral sclerosis). , Adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors, and others including leukoplakia.

Although the pathologies of the development of these diseases can be diverse, their characteristic deposits often contain many common molecular components. To a large extent, this is due to the local activation of pro-inflammatory pathways resulting in the concomitant deposition of activated complement components, acute phase reactants, immune modulators and other inflammatory mediators. It can be determined (McGeer et al., 1994).

Alzheimer's disease (AD) is a neurological disorder primarily thought to be caused by amyloid plaques, an accumulation of abnormal protein deposits in the brain. The most common type of amyloid found in the brain of affected individuals is composed primarily of Aβ fibrils. Scientific evidence demonstrates that the production and accumulation of patchy β-amyloid protein leads to neuronal cell death, which contributes to the onset and progression of AD. Loss of nerve cells in strategically important brain regions, in turn, leads to neurotransmitter depletion and memory impairment. Proteins that are the major contributors to the accumulation of plaques include amyloid precursor protein (APP) and two presenilins (presenilin I and presenilin II). Sequential cleavage of amyloid precursor protein (APP), which is constitutively expressed and catabolized in most cells, by enzymes β and γ secretase results in the release of 39-43 amino acid Aβ peptides. Degradation of APPs appears to increase their tendency to aggregate in a patchy manner. The strong tendency to build aggregates is especially in the Aβ (1-42) fragment, due to the two highly hydrophobic amino acid residues at its C-terminus. The Aβ (1-42) fragment is therefore primarily involved in and responsible for the initiation of amyloid plaque formation in AD and is therefore considered highly pathogenic. Therefore, there is a demand for agents that prevent the formation of amyloid plaques and diffuse the presence of plaques in AD.

Symptoms of AD appear slowly, and the first symptoms are only mild amnesia. At this stage, individuals may forget the names of recent events, activities, families or things and may not be able to solve simple math problems. As the disease progresses, the symptoms become more easily noticeable and severe enough to force people with AD or their families to seek medical help. Mid-term symptoms of AD include forgetting to do simple tasks such as grooming, which causes problems with speaking, comprehension, and reading and writing. Patients with late-stage AD may become anxious or aggressive, may even wander out of the home, and ultimately require full-scale nursing care.

Currently, the only definitive means for diagnosing AD is to identify plaques and tangles in brain tissue at postmortem autopsy of an individual. For this reason, doctors can only make a diagnosis of "suspected AD" or "almost certain AD" while the person is alive. Using current methods, physicians can correctly diagnose AD with a probability of up to 90 percent, using multiple tools for diagnosing "almost certain" AD. The doctor asks questions about the patient's general health, medical history, and history of problems learned in daily activities. Behavioral tests on memory, problem-solving, attention, calculation, and language provide information on cognitive impairment, and medical tests such as blood, urine or spinal fluid tests, and brain scans provide more detailed information. Can be done.

Management of AD consists of treatment with medication and treatment without medication. Treatments aimed at altering the underlying processes of the disease (delaying or reversing progression) have been largely unsuccessful. Drugs that ameliorate the deficiency (deficiency) or dysfunction of chemical messengers (neurotransmitters) in nerve cells, especially cholinesterase inhibitors (ChEI) such as tacrine and rivastigmine, have been shown to improve symptoms. ChEI interferes with the enzymatic degradation of neurotransmitters, thereby increasing the amount of chemical messenger that can be used to transmit neurosignals in the brain.

For some early and mid-stage patients with this disease, tacrine (COGNEX®, Morris Plains, NJ), donepezil (ARICEPT®, Tokyo, JP), rivastigmine (EXELON®, East). Drugs such as Hanover, NJ), or galantamine (REMINYL®, New Brunswick, NJ) may help prevent some symptoms from getting worse over a limited period of time. Another drug, memantine (NAMENDA®, New York, NY), has been approved for the treatment of moderate to severe AD. Drugs for dealing with the psychological symptoms of AD are also available. Also, some drugs may help control the behavioral symptoms of AD such as insomnia, agitation, wandering, anxiety and depression. Treating these symptoms calms the patient and makes caregiving easier for the caregiver. Unfortunately, however, the disease continues to progress and has little average effect on mental function, despite major therapeutic advances that have consistently shown that this group of drugs is superior to placebo. .. ChEI also has side effects, including gastrointestinal dysfunction, hepatotoxicity and weight loss.

Another disease based on or associated with the accumulation and deposition of amyloid-like proteins is macular degeneration.

Macular degeneration is a common eye that causes alteration of the macula in the central region of the retina, a paper-thin tissue in the back of the eye where light-sensitive cells send visual signals to the brain. It is a disease. A sharp and clear "straight ahead" vision is processed by the macula. Damage to the macula results in the development of blind spots and blurred vision or disturbance of the visual field. Age-related macular degeneration (AMD) is one of the leading causes of visual impairment in the United States and is the leading cause of legal blindness in Caucasians for people over the age of 65. Approximately 1.8 million Americans over the age of 40 have advanced AMD, and another 7.3 million with intermediate AMD are also at significantly higher risk of vision loss. The government estimates that by 2020, there will be 2.9 million people with advanced AMD. Patients with AMD are often surprised and disappointed to find that little is known about the cause and treatment of this blindness disorder.

There are two types of macular degeneration: dry macular degeneration and wet macular degeneration. The dry form, in which macular cells begin to slowly disintegrate, is diagnosed in 85 percent of cases of macular degeneration. Dry AMD usually affects both eyes, but one eye may have diminished vision and the other may not be affected. Geode, a subretinal yellow deposit, is a common early sign of dry AMD. The risk of developing advanced dry or wet AMD increases as the number or size of the cavities increases. Dry AMD can progress and cause vision loss without conversion to a wet form of the disease. However, early dry AMD may also suddenly change to a wet form.

Wet type accounts for only 15 percent of cases, but 90 percent cause blindness and are considered advanced AMD (no early or intermediate wet AMD is present). Wet AMD always has a dry-type disease first. As the dry form worsens, abnormal blood vessels begin to grow on the back of the macula in some people. These blood vessels are extremely fragile and leak fluid and blood (hence the name "wet" macular degeneration), causing rapid damage to the macula.

Dry AMD often causes a slight blurring of the visual field at first. Subsequently, the center of the visual field becomes particularly hazy, and this area grows as the disease progresses. Symptoms may go unnoticed if only one eye is affected. In wet AMD, straight lines may appear wavy or a sudden loss of central vision may occur.

Diagnosis of macular degeneration typically involves observation of the posterior part of the eye using a procedure called fundus examination, which helps diagnose mydriatic eye, visual acuity, and AMD. If suspected, fluorescent fundus angiography may be performed. If dry AMD reaches advanced stages, there is currently no cure to prevent vision loss. However, special high-dose formulations of antioxidants and zinc may delay or prevent the progression of intermediate AMD to the advanced phase. Macugen® (Pegaptanib Sodium Injection), laser photocoagulation and photodynamic therapy can reduce abnormal vascular growth and bleeding in the macula, which helps some people with moist AMD. become. However, these techniques do not restore already diminished vision. If vision is already impaired, there are low vision aids that can help improve quality of life.

One of the earliest signs of age-related macular degeneration (AMD) is the accumulation of extracellular deposits known as geodes between the basal layer of retinal pigment epithelium (RPE) and the Bruch's membrane (BM). be. A recent study by Anderson et al. Confirmed that the geode contains amyloid β (Experimental Eye Research 78 (2004) 243-256).

Ongoing research continues with research to explore environmental, genetic, and dietary factors that can contribute to AMD. Retinal cell transplantation, drugs that may prevent or slow the progression of the disease, radiation therapy, genetic therapy, transplantation of computer chips into the retina that may help stimulate vision, and the growth of new blood vessels under the macula. New treatment strategies are also being explored, including drugs that are thought to prevent.

One important factor to consider when developing a new drug is ease of use for the target patient. Oral drug delivery, especially tablets, capsules and softgels, accounts for 70% of all dosage forms consumed, for the convenience of the patient. Drug developers agree that patients prefer oral delivery to injections or other, more invasive forms of drug administration. Formulations that provide sparse dosing intervals (ie, once-daily or sustained release) are also preferred. The ease of administration of antibiotics in oral dosage form results in improved compliance for the patient being treated.

What is needed is Alzheimer's disease (AD), Levy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); neurological disorders such as Guam Parkinson-dementia complex; Advanced nuclear palsy, multiple sclerosis; Kreuzfeld-Jakob disease, Parkinson's disease, HIV-related dementia, ALS (muscle atrophic lateral sclerosis), adult-onset diabetes; senile heart amyloidosis; endocrine tumors, and yellow spots Amyloid, including, but not limited to, secondary amyloidosis and age-related amyloidosis, including, but not limited to, other diseases based on or associated with amyloid-like proteins such as other diseases, including degeneration. An effective method and composition for preventing or coping with a group of diseases and disorders associated with amyloidosis associated with plaque formation. Particularly in need are agents capable of countering the physiological manifestations of the disease, such as the formation of plaques associated with the aggregation of fibers of amyloid or amyloid-like peptides.

Anti-amyloid antibodies induced by inoculation with Aβ _1-42 mixed with Freund's complete or incomplete adjuvant have been reported to reduce amyloid loading in transgenic mice with human Alzheimer's disease (Schenk et al., 1999). ). Intraperitoneal inoculation of NORBA transgenic mice reconstituted in liposomes with tetrapalmitylated Aβ _1-16 elicited effective titers of anti-amyloid antibody, which allowed amyloid fibrils and plaques in vitro and in vivo. It was reported to be lysed (Nicolau et al., 2002).

The expected mechanism of amyloid plaques and lysis of fibers was first proposed by Bard et al. (2000), who, based on their own data, opsonized the plaques with antibodies, followed by microglial macrophages. Concluded that it would be destroyed by. De Mattos et al. (2001) showed that mAbs targeting the central domain of β-amyloid bind to plasma amyloid and completely isolate them. They argued that the presence of these mAbs in the bloodshed shifts the equilibrium of Aβ between the brain and plasma to facilitate peripheral removal and catabolism instead of deposition in the brain.

Long-term human treatment with rodent antibodies is detectable about 8-12 days after dosing and may result in an antiglobulin response that peaks at about 20-30 days. In the face of such an antiglobulin response, treatment should be discontinued after about 10 days at the longest, which leads to a rapid initiation of the secondary antiglobulin response, so retreatment at a later date Usually excluded. Although the rodent antibody shares a considerable degree of sequence conservation with the sequence of the human antibody, it is sufficient for the rodent antibody to be immunogenic in humans between the rodent antibody and the human antibody. There are many sequence differences.

This problem may be overcome by making antibodies directly in humans or by creating "humanized" antibodies (also known as "reshaped antibodies"). Humanized antibodies have variable region amino acid sequences containing CDRs from rodents interspersed in human or human-like framework sequences. Since the specificity of humanized antibodies is provided by rodent-derived CDRs, their residues should be used essentially unchanged and do not significantly interfere with the antibody's affinity and specificity for its target antigen. , Only minor modifications are allowed. Framework residues may be derived from any primate, or in particular any human variable region, or a combination thereof, and the resulting designed variable region is reshaped. Can be regarded.

Appropriate selection of framework regions is important to maximize the likelihood that affinity will be retained by the reshaped antibody. It is known that framework sequences function to keep CDRs in their correct spatial orientation for interaction with antigens, and that framework residues can sometimes even participate in antigen binding. be. In order to maintain the affinity of the antibody for that antigen, it is advantageous to select the human framework sequence that most closely resembles the sequence of the rodent framework. It may then be necessary to replace one or more amino acids in the human framework sequence with the corresponding residues in the rodent framework to avoid loss of affinity. This replacement may be assisted by computer modeling.

The present invention can be presented to an antibody in the form of monomers, dimers, trimmers, etc., in the form of polymers, in the form of aggregates, fibers, filaments, or in condensed mottled form, various β-amyloid antigens. A highly specific and highly effective antibody that has the ability to specifically recognize and specifically bind to a specific epitope of origin, particularly a chimeric antibody containing a fragment thereof, more particularly a portion containing the fragment. Provided are novel methods and compositions comprising antibodies that have been fully or fully humanized. The antibodies enabled by the teachings of the present invention are neurological disorders such as Alzheimer's disease (AD), Levy body dementia, Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guam Parkinson-dementia complex. ; And progressive nuclear palsy, multiple sclerosis; Kreuzfeld-Jakob disease, hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson's disease, HIV-related dementia, ALS (muscle atrophic lateral sclerosis), adult-onset diabetes Geriatric heart amyloidosis; endocrine tumors and, to example, other diseases such as, but not limited to, amyloid-like proteins such as, including, but not limited to, amyloidosis. It is particularly useful in the treatment of amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis.

In some embodiments, the invention is on a β-amyloid protein comprising at least one or two contiguous amino acid residues in which one, at least two, and at least three binding sites are each primarily involved in antibody binding. A chimeric antibody or fragment thereof or a humanized antibody that recognizes and binds to at least one distinct binding site, particularly at least two distinct binding sites, and more particularly at least three distinct binding sites. Or about the fragment.

In particular, a chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to the invention comprises at least two contiguous amino acid residues in which at least two of the three distinct binding sites are primarily involved in antibody binding. , And at least one of the three distinct binding sites binds to at least two, in particular at least three distinct binding sites on the β-amyloid protein, which contain at least one amino acid residue.

At least two distinct binding sites containing at least two contiguous amino acid residues that are primarily involved in antibody binding are located very close to each other on the antigen and do not participate in antibody binding or are said to be at least two contiguous sites. Separated by at least one amino acid residue involved to a significantly lesser extent when compared to the amino acid residue and / or adjacent to the at least one amino acid residue, thus forming a conformational discontinuous epitope. do.

At least three distinct binding sites, each containing at least two contiguous amino acid residues and at least one amino acid residue, which are primarily involved in antibody binding, are located very close to each other on the epitope and antibody binding. Separated by and / or adjacent to the amino acid residue by at least one amino acid residue that is not involved in or is significantly less involved when compared to said amino acid residue. They are primarily involved in antibody binding and therefore form stereostructural discontinuous epitopes.

In particular, a chimeric antibody or a chimeric antibody thereof that recognizes and binds to at least one distinct binding site on β-amyloid protein, in particular at least two distinct binding sites, more particularly at least three distinct binding sites. Provided is a fragment or a humanized antibody or a fragment thereof. Here, each of the at least one or at least two distinct binding sites comprises at least two contiguous amino acid residues primarily involved in antibody binding and the at least two contiguous sites representing the first binding site. The amino acid residue is -Phe-Phe- embedded in the following core sequence (SEQ ID NO: 9):
Xaa ₃ --Phe --Phe --Xaa ₄ --Xaa ₅ --Xaa ₆
During the ceremony
Xaa ₃ is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile;
Xaa ₄ is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser, and Ile;
Xaa ₅ is an amino acid residue selected from the group consisting of Glu and Asp, and
Xaa ₆ is an amino acid residue selected from the group consisting of Glu and Asp, and the amino acid residues Xaa ₃ , Xaa ₄ , Xaa ₅ , and Xaa ₆ are not involved in antibody binding or -Phe-Phe- It is significantly less involved when compared to the binding site.

In another aspect of the invention
Xaa ₃ is Val or Leu, especially Val;
Xaa ₄ is Ala or Val, especially Ala;
Xaa ₅ is Glu or Asp, especially Glu;
Xaa ₆ is Glu or Asp, especially Asp,
A chimeric antibody or a fragment thereof or a humanized antibody or a fragment thereof is provided.

In particular, a chimeric antibody or a chimeric antibody thereof that recognizes and binds to at least one distinct binding site on β-amyloid protein, in particular at least two distinct binding sites, more particularly at least three distinct binding sites. Provided is a fragment or a humanized antibody or a fragment thereof. Here, each of the distinct binding sites comprises at least one and at least two contiguous amino acid residues that are primarily involved in antibody binding, and at least two contiguous amino acid residues representing the first binding site. , Is embedded in the core sequence below -Phe-Phe- and at least one amino acid residue is -His- embedded in the core sequence:
-Xaa ₁ _ His --Xaa ₃ --Xaa ₄ --Xaa ₅ --Xaa ₆ --Phe --Phe --Xaa ₇ --Xaa ₈ --Xaa ₉ -
During the ceremony
Xaa ₁ is an amino acid residue selected from the group consisting of His, Asn, Gln, Lys, and Arg;
Xaa ₃ is an amino acid residue selected from the group consisting of Asn and Gln;
Xaa ₄ is an amino acid residue selected from the group consisting of His, Asn, Gln, Lys, and Arg;
Xaa ₅ is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser, and Ile;
Xaa ₆ is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile;
Xaa ₇ is an amino acid residue selected from the group consisting of Ala, Val, Leu, and Ile;
Xaa ₈ is an amino acid residue selected from the group consisting of Glu and Asp;
Xaa ₉ is an amino acid residue selected from the group consisting of Glu and Asp, and amino acid residues Xaa ₁ , Xaa ₃ , Xaa ₆ , Xaa ₇ , Xaa ₈ and Xaa ₉ are not involved in antibody binding, respectively. Or, it is significantly less involved when compared to the -His- and -Phe-Phe- binding sites.

In another aspect of the invention
Xaa ₃ is Gln or Asn, especially Gln;
Xaa ₄ is Lys;
Xaa ₅ is Leu;
Xaa ₆ is Val or Leu, especially Val;
Xaa ₇ is Ala or Val, especially Ala;
Xaa ₈ is Glu or Asp, especially Glu;
Xaa ₉ is Asp or Glu, especially Asp,
A chimeric antibody or a fragment thereof or a humanized antibody or a fragment thereof is provided.

In another aspect of the invention, it recognizes and binds to at least one distinct binding site on the β-amyloid protein, in particular at least two distinct binding sites, more particularly at least three distinct binding sites. Provided is a chimeric antibody or a fragment thereof or a humanized antibody or a fragment thereof. Here, each of the at least one or at least two distinct binding sites comprises at least two contiguous amino acid residues primarily involved in antibody binding and at least two contiguous amino acids representing a second binding site. The residue is -Lys-Leu- embedded in the following core sequence (SEQ ID NO: 10):
Xaa ₁ --Xaa ₂ --Lys --Leu --Xaa ₃
During the ceremony
Xaa ₁ is an amino acid residue selected from the group consisting of His, Asn, Gln, Lys, and Arg;
Xaa ₂ is an amino acid residue selected from the group consisting of Asn and Gln;
Xaa ₃ is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile; and amino acid residues Xaa ₂ and Xaa ₃ are not involved in antibody binding or-. It is significantly less involved when compared to the Lys-Leu-binding site.

In another aspect of the invention, it recognizes and binds to at least one distinct binding site on the β-amyloid protein, in particular at least two distinct binding sites, more particularly at least three distinct binding sites. Provided is a chimeric antibody or a fragment thereof or a humanized antibody or a fragment thereof. Here, the separate binding sites each contain at least one and at least two contiguous amino acid residues that are primarily involved in antibody binding, and the at least one and at least two contiguous amino acids are involved in antibody binding. It is separated by at least one amino acid residue that is significantly less involved when compared to amino acid residues that are not involved or are primarily involved in antibody binding, each embedded within the core sequence below. -His- and -Lys-Leu-:
His --Xaa ₂ --Lys --Leu --Xaa ₃ --Xaa ₄ --Xaa ₅ --Xaa ₆ --Xaa ₇ --Xaa ₈ -
During the ceremony
Xaa ₂ is an amino acid residue selected from the group consisting of Asn and Gln;
Xaa ₃ is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile;
Xaa ₄ is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile;
Xaa ₅ is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile;
Xaa ₆ is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser, and Ile;
Xaa ₇ is an amino acid residue selected from the group consisting of Glu and Asp.
Xaa ₈ is an amino acid residue selected from the group consisting of Glu and Asp.
And the amino acid residues Xaa ₂ , Xaa ₃ , Xaa ₆ , Xaa ₇ , and Xaa ₈ are not involved in antibody binding, respectively, or are significantly less when compared to the -His- and -Lys-Leu- binding sites. Involved.

In another aspect of the invention
Xaa ₂ is Gln or Asn, especially Gln;
Xaa ₃ is Val or Leu, especially Val;
Xaa ₄ is Phe;
Xaa ₅ is Phe;
Xaa ₆ is Ala or Val, especially Ala;
Xaa ₇ is Glu or Asp, especially Glu;
Xaa ₈ is Asp or Glu, especially Asp,
A chimeric antibody or a fragment thereof or a humanized antibody or a fragment thereof is provided.

In another aspect of the invention, the at least two distinct binding sites on a β-amyloid protein, each containing at least two contiguous amino acid residues that are primarily involved in antibody binding. Provided are a chimeric antibody or a fragment thereof or a humanized antibody or a fragment thereof that recognizes a site and binds to the binding site. Here, the at least two contiguous amino acids are separated by at least one amino acid residue that is not involved in antibody binding or is significantly less involved than the contiguous amino acid residues, and the contiguous amino acid residue. The groups are -Phe-Phe- and -Lys-Leu-, which represent the first and second binding sites embedded in the following core sequences, respectively:
Xaa ₁ --Xaa ₂ --Lys --Leu --Xaa ₃ --Phe --Phe -Xaa ₄ --Xaa ₅ --Xaa ₆
During the ceremony
Xaa ₁ is an amino acid residue selected from the group consisting of His, Asn, Gln, Lys, and Arg;
Xaa ₂ is an amino acid residue selected from the group consisting of Asn and Gln;
Xaa ₃ is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile;
Xaa ₄ is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser, and Ile;
Xaa ₅ is an amino acid residue selected from the group consisting of Glu and Asp.
Xaa ₆ is an amino acid residue selected from the group consisting of Glu and Asp, and the amino acid residues Xaa ₂ , Xaa ₃ , Xaa ₄ , Xaa ₅ , and Xaa ₆ are not involved in antibody binding, respectively. It is significantly less involved when compared to the -Lys-Leu- and -Phe-Phe- binding sites.

In another aspect of the invention, it recognizes and binds to at least one distinct binding site on the β-amyloid protein, in particular at least two distinct binding sites, more particularly at least three distinct binding sites. Provided is a chimeric antibody or a fragment thereof or a humanized antibody or a fragment thereof. Here, each of the distinct binding sites comprises at least one and at least two contiguous amino acid residues that are primarily involved in antibody binding, and the at least one and at least two contiguous amino acids are involved in antibody binding. Each amino acid residue that is not or is separated by at least one amino acid residue that is significantly less involved when compared to the amino acid residue that is primarily involved in antibody binding, and is primarily involved in antibody binding. , -His- and -Phe-Phe- and -Lys-Leu- embedded in the following core sequences:
His --Xaa ₂ --Lys --Leu --Xaa ₃ --Phe --Phe --Xaa ₄ _ Xaa ₅ _ Xaa ₆
During the ceremony
Xaa ₂ is an amino acid residue selected from the group consisting of Asn and Gln;
Xaa ₃ is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile;
Xaa ₄ is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser, and Ile;
Xaa ₅ is an amino acid residue selected from the group consisting of Glu and Asp.
Xaa ₆ is an amino acid residue selected from the group consisting of Glu and Asp, and the amino acid residues Xaa ₂ , Xaa ₃ , Xaa ₄ , Xaa ₅ , and Xaa ₆ are not involved in antibody binding, respectively, or-. It is significantly less involved when compared to the His-, -Lys-Leu-, and -Phe-Phe- binding sites.

In another aspect of the invention
Xaa ₂ is Gln or Asn, especially Gln;
Xaa ₃ is Val or Leu, especially Val;
Xaa ₄ is Ala or Val, especially Ala;
Xaa ₅ is Glu or Asp, especially Glu;
Xaa ₆ is Asp or Glu, especially Asp,
A chimeric antibody or a fragment thereof or a humanized antibody or a fragment thereof is provided.

In another aspect of the invention, there is provided a chimeric antibody or fragment thereof or a humanized antibody or fragment thereof that recognizes and binds to at least two distinct binding sites on β-amyloid protein. Here, each of the at least two distinct binding sites comprises at least two contiguous amino acid residues that are primarily involved in antibody binding, and the at least two contiguous amino acids are not or contiguous in antibody binding. Representing the first and second binding sites embedded in the following core sequences, separated by at least one amino acid residue involved to a significantly lesser extent than the amino acid residue, -Phe-Phe, respectively. -And -Lys-Leu-:
Xaa ₁ --Xaa ₂ --Lys --Leu --Xaa ₃ --Phe --Phe -Xaa ₄ --Xaa ₅ --Xaa ₆
During the ceremony
Xaa ₁ is an amino acid residue selected from the group consisting of His, Asn, Gln, Lys, and Arg;
Xaa ₂ is an amino acid residue selected from the group consisting of Asn and Gln;
Xaa ₃ is an amino acid residue selected from the group consisting of Val, Ala, Leu, Met, Phe, norleucine, and Ile.
Xaa ₄ is an amino acid residue selected from the group consisting of Ala, Val, Leu, and Ile;
Xaa ₅ is an amino acid residue selected from the group consisting of Glu and Asp.
Xaa ₆ is an amino acid residue selected from the group consisting of Glu and Asp, and the amino acid residues Xaa ₂ , Xaa ₃ , Xaa ₄ , Xaa ₅ , and Xaa ₆ are not involved in antibody binding, respectively, or-. It is significantly less involved when compared to the Lys-Leu- and -Phe-Phe binding sites.

In another aspect of the invention
Xaa ₁ is His or Arg, especially His;
Xaa ₂ is Gln or Asn, especially Gln;
Xaa ₃ is Val or Leu, especially Val;
Xaa ₄ is Ala or Val, especially Ala;
Xaa ₅ is Glu or Asp, especially Glu;
Xaa ₆ is Asp or Glu, especially Asp,
A chimeric antibody or a fragment thereof or a humanized antibody or a fragment thereof is provided.

In certain embodiments of the invention, there is provided a chimeric antibody or fragment thereof or a humanized antibody or fragment thereof that recognizes and binds to at least two distinct binding sites on β-amyloid protein. Here, each of the at least two distinct binding sites is an antibody, which is-Phe --Phe --Ala --Glu-, in particular-Phe --Phe --Ala-, in particular-Phe --Phe-and-Lys --Leu-. Contains at least two contiguous amino acid residues that are primarily involved in the binding of, and each of the at least two distinct binding sites is the amino acid sequence set forth in SEQ ID NO: 7 -Val --Phe --Phe --Ala --Glu --Asp. -And the amino acid sequence His --Gln --Lys --Leu --Val-shown in SEQ ID NO: 8.

In certain embodiments of the invention, it recognizes and binds to at least one distinct binding site on a β-amyloid protein, in particular at least two distinct binding sites, more particularly at least three distinct binding sites. , Chimera antibody or fragment thereof or humanized antibody or fragment thereof. Here, the at least one or at least two distinct binding sites are-Phe-Phe-and-Lys-Leu-, and-His-respectively, at least one and at least two that are primarily involved in the binding of the antibody. Each contains two contiguous amino acid residues, each with a separate binding site embedded in the amino acid sequence -Val --Phe --Phe --Ala --Glu-and the amino acid sequence --His --Gln --Lys --Leu --Val. ..

In another aspect of the invention, the chimeric antibody or fragment thereof or the humanized antibody or fragment thereof has at least two distinct binding sites, respectively, in at least two contiguous sequences in the amino acid sequences set forth in SEQ ID NOs: 7 and 8, respectively. Consecutive amino acid residues, in particular -Phe-Phe- and -Lys-Leu-, contain at least two distinct bindings on the β-amyloid protein that are primarily involved in the binding of the β-amyloid protein. Includes an antigen recognition site and an antigen binding site that recognizes the site and binds to the binding site.

In a more specific embodiment of the invention, four separate binding sites each contain two binding sites, each containing one amino acid residue, and two consecutive amino acid residues that are primarily involved in antibody binding. Provided are an antibody or fragment thereof according to the invention that binds to four distinct binding sites on a β-amyloid protein, including two binding sites comprising. Here, the four distinct binding sites are located very close to each other on the β-amyloid protein, and the four binding sites do not participate in or bind to antibody binding, but the four distinct binding sites. It is separated by at least one amino acid residue that is significantly less involved when compared to one amino acid residue and two consecutive amino acid residues in the binding site, and thus two in four separate binding sites. Contiguous amino acid residues form a three-dimensionally discontinuous epitope.

In particular, the first two contiguous amino acid residues that are primarily involved in antibody binding are -Lys-Leu- embedded within the core sequence below, and at least two contiguous second. The amino acid residue is -Phe-Phe- embedded in the core sequence, the first single amino acid residue is -His- embedded in the core sequence, and the second single. One amino acid residue is -Asp- embedded in the core sequence:
--Xaa ₁ --His --Xaa ₂ --Lys --Leu --Xaa ₃ --Phe --Phe --Xaa ₄ --Xaa ₅ --Asp --Xaa ₆
During the ceremony
Xaa ₁ is an amino acid residue selected from the group consisting of His, Asn, Gln, Lys, and Arg, especially His;
Xaa ₂ is an amino acid residue selected from the group consisting of Asn and Gln, especially Gln;
Xaa ₃ is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile, especially Val;
Xaa ₄ is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser, and Ile, especially Ala;
Xaa ₅ is an amino acid residue selected from the group consisting of Glu and Asp, especially Glu;
Xaa ₆ is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile, especially Val; and amino acid residues Xaa ₁ , Xaa ₂ , Xaa ₃ , Xaa ₄ , Xaa ₅ , Xaa ₆ are not or are involved in antibody binding, but significantly less when compared to the -His-, -Asp-, -Lys-Leu, and -Phe-Phe-binding sites. Involved in degree.

In certain embodiments, the present invention relates to an antibody or fragment thereof according to the invention that binds to four distinct binding sites on β-amyloid protein. Here, the four distinct binding sites include two binding sites, each containing one amino acid residue, and two binding sites, each containing two consecutive amino acid residues, predominantly for antibody binding. The first two contiguous amino acid residues involved are -Lys-Leu- embedded in the following core sequence, and the second at least two contiguous amino acid residues are in the core sequence. The embedded -Phe-Phe-, the first single amino acid residue is -His- embedded in the core sequence, and the second single amino acid residue is in the core sequence. Embedded in -Asp-:
--Xaa ₁ --His --Xaa ₂ --Lys --Leu --Xaa ₃ --Phe --Phe --Xaa ₄ --Xaa ₅ --Asp --Xaa ₆
During the ceremony
Xaa ₁ is an amino acid residue selected from the group consisting of His, Asn, Gln, Lys, and Arg, especially His;
Xaa ₂ is an amino acid residue selected from the group consisting of Asn and Gln, especially Gln;
Xaa ₃ is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile, especially Val;
Xaa ₄ is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser, and Ile, especially Ala;
Xaa ₅ is an amino acid residue selected from the group consisting of Glu and Asp, especially Glu;
Xaa ₆ is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile, especially Val; and amino acid residues Xaa ₁ , Xaa ₂ , Xaa ₃ , Xaa ₄ , Xaa ₅ , Xaa ₆ are not or are involved in antibody binding, but significantly more when compared to the -His-, -Asp-, -Lys-Leu-, and -Phe-Phe- binding sites. Involved to a lesser extent.

In a specific embodiment of the invention, recognition and binding sites as defined above herein are between amino acid residues 12-24, particularly between residues 14-23, more particularly amino acid residues. It forms a three-dimensional discontinuous epitope localized in the region of β-amyloid protein between 14 and 20. Here, at least two distinct recognition and binding sites, each containing at least two amino acid residues, are located at positions 16 and 17 and positions 19 and 20, respectively, and at least one containing at least one amino acid residue. The distinct recognition and binding sites are located at positions 14 where the residues are primarily involved in the binding of β-amyloid proteins, and the distinct recognition and binding sites are amino acid residues, especially residues 21 and 22. Separated by one amino acid residue located at positions 15 and 18, where the amino acid residues are on at least one adjacent side and the amino acid residues are not directly involved in the binding of the antigen, or at least to a substantially lesser extent. Has been done.

In yet another aspect of the invention, at least three distinct recognition and binding sites are amino acid residues, in particular residues 12 and 13, and residues 21 and 22 flanking on both sides and directly upon antigen binding. It is separated by one amino acid residue located at positions 15 and 18, which is not involved, or at least to a substantially lesser extent.

In a specific embodiment, contiguous amino acid residues primarily involved in the binding of β-amyloid protein, in particular -Lys-Leu- at positions 16 and 17, and -Phe-Phe- at positions 19 and 20 are described below. It is embedded in the core area.

In another specific embodiment, amino acid residues that are primarily involved in the binding of β-amyloid protein, in particular -Lys-Leu- at positions 16 and 17, and -Phe-Phe- at positions 19 and 20, and position 14 -His- is embedded in the following core area.

In another aspect of the invention, at least one CDR of non-human origin, in particular, embedded in one or more human or primate-derived framework regions, respectively, in the variable regions of the light and heavy chains. Provided are two CDRs of non-human origin, more particularly three CDRs of non-human origin, and optionally a humanized antibody or fragment thereof, comprising a constant region derived from an antibody of human or primate origin. The humanized antibody or fragment thereof is an epitope containing the following amino acid sequence (SEQ ID NO: 11) and is isolated or β-amyloid protein as part of β-amyloid plaque, especially β-amyloid monomer. Peptides, more particularly β-amyloid polymer peptides, and even more particularly β-amyloid fibrils, fibrils, or filaments can be specifically recognized and bound:
Xaa ₁ --Xaa ₂ --Lys --Leu --Xaa ₃ --Phe --Phe -Xaa ₄ _ Xaa ₅ _ Xaa ₆
During the ceremony
Xaa ₁ is an amino acid residue selected from the group consisting of His, Asn, and Gln, especially His;
Xaa ₂ is an amino acid residue selected from the group consisting of Asn and Gln, especially Gln;
Xaa ₃ is an amino acid residue selected from the group consisting of Val, Leu, and Ile, especially Val.
Xaa ₄ is an amino acid residue selected from the group consisting of Ala and Val, especially Ala;
Xaa ₅ is an amino acid residue selected from the group consisting of Glu and Asp, especially Glu;
Xaa ₆ is an amino acid residue selected from the group consisting of Glu and Asp, especially Asp.

In yet another embodiment of the invention, at least one CDR of non-human origin, embedded in one or more human or primate-derived framework regions, respectively, in the variable regions of the light and heavy chains. Provided are two CDRs of non-human origin, in particular three CDRs of non-human origin, and optionally a humanized antibody or fragment thereof, comprising a constant region derived from an antibody of human or primate origin. The humanized antibody or fragment thereof is an epitope containing the following amino acid sequence, isolated or as part of β-amyloid plaque, β-amyloid protein, especially β-amyloid monomer peptide, more particularly β. -Can specifically recognize and bind amyloid polymer peptides, and more particularly β-amyloid fibers, fibrils, or filaments:
His --Xaa ₂ --Lys --Leu --Xaa ₃ --Phe --Phe --Xaa ₄ _ Xaa ₅ _ Xaa ₆
During the ceremony
Xaa ₂ is an amino acid residue selected from the group consisting of Asn and Gln, especially Gln;
Xaa ₃ is an amino acid residue selected from the group consisting of Val, Leu, and Ile, especially Val;
Xaa ₄ is an amino acid residue selected from the group consisting of Ala and Val, especially Ala;
Xaa ₅ is an amino acid residue selected from the group consisting of Glu and Asp, especially Glu;
Xaa ₆ is an amino acid residue selected from the group consisting of Glu and Asp, especially Glu; and the amino acid residues Xaa ₂ , Xaa ₃ , Xaa ₄ , Xaa ₅ , Xaa ₆ are not involved in antibody binding or It is significantly less involved when compared to the -His- and -Lys-Leu- and -Phe-Phe- binding sites.

In a specific embodiment of the invention, CDRs of non-human origin are obtained from donor antibodies made against antigen fragments that do not contain a separate binding site, especially from mouse donor antibodies. The conversion of this epitope region is a supermolecule containing, for example, an antigenic peptide corresponding to the amino acid sequence of β-amyloid peptide modified with a hydrophilic moiety such as polyethylene glycol (PEG), in particular β-amyloid peptide Aβ _1-16 . The use of the antigenic construct may at least partially result in the hydrophilic moiety, eg, lysine, glutamic acid, and cysteine or the hydrophilic moiety, as described herein in the immunization process below. Covalently attached to each of the ends of an antigenic peptide through at least one of any other suitable amino acids or amino acid analogs, particularly one or two amino acids, which can serve as a linking device for coupling to the peptide fragment. are doing. When PEG is used as the hydrophilic moiety, any suitable for functioning as a fixing element for embedding the free PEG terminal in the bilayer of the liposomes described herein with phosphatidylethanolamine, for example an antigenic construct. Covalently bind to other compounds in.

In particular, CDRs of non-human origin were published on December 1, 2005 by Braunschweig, Mascheroder Weg 1B, 38124 Branuschweig's Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), under the provisions of the Budapest Convention, Accusion Number: DSM ACC2750. It is obtained from a mouse donor antibody that exhibits the characteristic properties of ACI-O1-Ab7C2 deposited under (also named "mC2" throughout this application).

In certain embodiments of the invention, CDRs of non-human origin were assigned to Braunschweig, Mascheroder Weg 1B, 38124 Branuschweig's Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on December 1, 2005, under the provisions of the Budapest Treaty. Session number: Obtained from the mouse donor antibody ACI-O1-Ab7C2 deposited under DSM ACC2750 (also named "mC2" throughout this application).

The use of Lipid A as part of the immunization protocol may also contribute to the conversion of epitope regions.

In a specific embodiment, the invention represents a heavy chain variable region (HCVR) CDR2 incorporated into a human or primate-derived framework region, SEQ ID NO: 2 and SEQ ID NO: 3 and. Concerning a humanized antibody or fragment thereof comprising at least one peptide having an amino acid sequence selected from the group of sequences consisting of SEQ ID NO: 4 representing CDR1 of the light chain variable region (LCVR).

In another embodiment, the invention is a heavy chain variable region (HCVR) incorporated within a heavy chain framework region of human or primate origin, SEQ ID NO: 2 and SEQ ID NO: 3 representing CDR3. With respect to a humanized antibody or fragment thereof comprising at least one peptide having an amino acid sequence selected from the group consisting of sequences consisting of.

In yet another embodiment, the invention comprises a peptide having the amino acid sequence of SEQ ID NO: 4 representing CDR1 of the light chain variable region (LCVR) incorporated into a light chain framework region of human or primate origin. Containing, including humanized antibodies or fragments thereof.

In particular, the invention comprises a light chain comprising at least one peptide having the amino acid sequence of SEQ ID NO: 4 representing CDR1 of the light chain variable region (LCVR) incorporated into a framework region of human or primate origin. Regarding variable regions (LCVR).

In another specific embodiment, the invention comprises SEQ ID NO: 2 representing CDR2 of a heavy chain variable region (HCVR) and SEQ ID NO: 2 representing CDR3 incorporated into a framework region of human or primate origin. It relates to a heavy chain variable region (HCVR) containing at least one peptide having an amino acid sequence selected from the group of sequences consisting of three.

The invention further relates to a humanized antibody or fragment thereof comprising at least two peptides incorporated into a framework region of human or primate origin. These peptides are different and of SEQ ID NO: 1 for CDR1 of the heavy chain variable region (HCVR), SEQ ID NO: 2 for CDR2, and SEQ ID NO: 3 for CDR3 and of the light chain variable region (LCVR). An amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3 is shown, and the same CDR may be present twice in the antibody. not. In particular, if at least two CDRs present are both CDRs of the light chain variable region (LCVR), then at least one of the CDRs must be CDR1 represented by SEQ ID NO: 4.

From SEQ ID NO: 1, Representing CDR1 of the Heavy Chain Variable Region (HCVR), SEQ ID NO: 2, and SEQ ID NO: 3 representing CDR3 incorporated into the heavy chain framework region of human or primate origin. Humanized antibodies or fragments thereof comprising at least two peptides having an amino acid sequence selected from the group of sequences, particularly humanized antibodies or fragments thereof in which the same CDR cannot be present twice in the antibody. Included by.

In particular, the present invention incorporates a heavy chain framework region of human or primate origin with SEQ ID NO: 1, representing CDR1 of a heavy chain variable region (HCVR), SEQ ID NO: 2, and CDR3. SEQ ID NO:: Containing at least two peptides having an amino acid sequence selected from the group of sequences consisting of 3 heavy chain variable regions (HCVR).

In a further embodiment, the invention comprises SEQ ID NO: 4, representing CDR1 of a light chain variable region (LCVR) incorporated into a light chain framework region of human or primate origin: 4, SEQ ID NO: 5, representing CDR2, And a fragment of a humanized antibody or fragment thereof comprising at least two peptides having an amino acid sequence selected from the group consisting of SEQ ID NO: 6 representing CDR3.

In particular, the present invention incorporates a light chain framework region of human or primate origin with SEQ ID NO: 4, representing CDR1 of a light chain variable region (LCVR), SEQ ID NO: 5, and CDR3. SEQ ID NO:: Having at least two peptides with an amino acid sequence selected from the group of sequences consisting of: the same CDR cannot be present twice in an antibody, and in particular at least one of the CDRs is a sequence. Number: Regarding the light chain variable region (LCVR), which must be CDR1 represented by 4.

The invention also comprises SEQ ID NOs: 1, Representing CDR1 of Heavy Chain Variable Regions (HCVR), SEQ ID NO: 2, and CDR3 incorporated into heavy chain framework regions of human or primate origin. Representing a humanized antibody or fragment thereof comprising a peptide having the amino acid sequence of SEQ ID NO: 3 in the order shown above.

In particular, the present invention incorporates a heavy chain framework region of human or primate origin with SEQ ID NO: 1, representing CDR1 of a heavy chain variable region (HCVR), SEQ ID NO: 2, and CDR3. Containing a peptide having the amino acid sequence of SEQ ID NO: 3 in particular in the order shown above, relating to a heavy chain variable region (HCVR).

SEQ ID NO: 4, representing CDR1 of the light chain variable region (LCVR) incorporated into the light chain framework region of human or primate origin: 4, SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3. Also included by the invention are humanized antibodies or fragments thereof comprising peptides having the amino acid sequence of, in particular the order shown above.

In particular, the present invention incorporates a light chain framework region of human or primate origin with SEQ ID NO: 4, representing CDR1 of a light chain variable region (LCVR), SEQ ID NO: 5, and CDR3. Representing a light chain variable region (LCVR) comprising peptides having the amino acid sequence of SEQ ID NO: 6 in the order shown above.

The invention also represents a heavy chain variable region (HCVR) CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2, and a sequence representing CDR3 incorporated into a human or primate-derived framework region. It has an amino acid sequence selected from the group consisting of a sequence consisting of number: 3, and SEQ ID NO: 4 representing CDR1 of the light chain variable region (LCVR), SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3. With respect to a humanized antibody or fragment thereof comprising at least three peptides, in particular a humanized antibody or fragment thereof in which the same CDR cannot be present twice in the antibody.

In another embodiment, the invention comprises SEQ ID NO: 1, representing CDR1 of a heavy chain variable region (HCVR), in which the antibody is integrated into a framework region of human or primate origin: 1, SEQ ID NO: 1. From a group of sequences consisting of 2, and SEQ ID NO: 3, which represents CDR3, and SEQ ID NO: 4, which represents CDR1 of the light chain variable region (LCVR), SEQ ID NO: 5, which represents CDR2, and SEQ ID NO: 6 which represents CDR3. With respect to a humanized antibody or fragment thereof comprising at least four peptides having an amino acid sequence of choice, in particular a humanized antibody or fragment thereof in which the same CDR cannot be present twice in the antibody.

In yet another embodiment, the invention comprises SEQ ID NO: 1, representing CDR1 of a heavy chain variable region (HCVR) incorporated into a framework region of human or primate origin: 1, SEQ ID NO: 2, representing CDR2, And SEQ ID NO: 3 representing CDR3, and SEQ ID NO: 4 representing CDR1 of the light chain variable region (LCVR), SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3 selected from the group of sequences. With respect to a humanized antibody or fragment thereof comprising at least 5 peptides having an amino acid sequence, particularly the humanized antibody or fragment thereof in which the same CDR cannot be present twice in the antibody.

In yet another embodiment, the invention comprises SEQ ID NO: 1, representing CDR1 of a heavy chain variable region (HCVR) incorporated into a framework region of human or primate origin: 1, SEQ ID NO: 2, representing CDR2, And a peptide having the amino acid sequences of SEQ ID NO: 3, representing CDR3, and SEQ ID NO: 4, representing CDR1 of the light chain variable region (LCVR), SEQ ID NO: 5, representing CDR2, and SEQ ID NO: 6 representing CDR3. , With respect to humanized antibodies or fragments thereof.

In a specific embodiment, the invention has at least one amino acid sequence of SEQ ID NO: 2 representing CDR2 of a heavy chain variable region (HCVR) incorporated into a heavy chain framework region of human or primate origin. Containing a humanized antibody, heavy chain variable region (HCVR), or fragment thereof, comprising one peptide.

In another specific embodiment, the invention has the amino acid sequence of SEQ ID NO: 3 representing CDR3 of a heavy chain variable region (HCVR) incorporated into a heavy chain framework region of human or primate origin. Concerning a humanized antibody, heavy chain variable region (HCVR), or fragment thereof, comprising at least one peptide.

In another specific embodiment, the invention represents SEQ ID NOs: 1 and CDR2 representing CDR1 of a heavy chain variable region (HCVR) incorporated into a heavy chain framework region of human or primate origin. Number: For a humanized antibody, heavy chain variable region (HCVR), or fragment thereof, comprising at least two peptides having an amino acid sequence of 2.

In another specific embodiment, the invention represents SEQ ID NO: 1 and a sequence representing CDR3 of a heavy chain variable region (HCVR) incorporated into a heavy chain framework region of human or primate origin. Number: For a humanized antibody, heavy chain variable region (HCVR), or fragment thereof, comprising at least two peptides having an amino acid sequence of 3.

In another specific embodiment, the invention represents SEQ ID NOs: 2 and CDR3 representing CDR2 of a heavy chain variable region (HCVR) incorporated into a heavy chain framework region of human or primate origin. Number: For a humanized antibody, heavy chain variable region (HCVR), or fragment thereof, comprising at least two peptides having an amino acid sequence of 3.

In another specific embodiment, the invention represents SEQ ID NO: 4 and CDR2 representing CDR1 of a light chain variable region (LCVR) incorporated into a heavy chain framework region of human or primate origin. Number: For a humanized antibody, light chain variable region (LCVR), or fragment thereof, comprising at least two peptides having an amino acid sequence of 5.

In another specific embodiment, the invention represents SEQ ID NO: 4 and CDR3 representing CDR1 of a light chain variable region (LCVR) incorporated into a heavy chain framework region of human or primate origin. Number: For a humanized antibody, light chain variable region (LCVR), or fragment thereof, comprising at least two peptides having an amino acid sequence of 6.

Both heavy chain variable regions (HCVRs) and light chain variable regions (LCVRs) of a mouse C2 antibody each confer at least one of its CDR regions to at least two CDR regions of a humanized antibody, a humanized antibody or The fragments are further included by the present invention. Thus, the resulting humanized antibody or fragment thereof may include:
-At least the amino acid sequence of SEQ ID NO: 1 representing CDR1 (HCVR) combined with the amino acid sequence of SEQ ID NO: 4 representing CDR1 (LCVR);
-At least the amino acid sequence of SEQ ID NO: 2 representing CDR2 (HCVR) combined with the amino acid sequence of SEQ ID NO: 4 representing CDR1 (LCVR);
-At least the amino acid sequence of SEQ ID NO: 3 representing CDR3 (HCVR) combined with the amino acid sequence of SEQ ID NO: 4 representing CDR1 (LCVR);
-At least the amino acid sequence of SEQ ID NO: 1 representing CDR1 (HCVR) combined with the amino acid sequence of SEQ ID NO: 5 representing CDR2 (LCVR);
-At least the amino acid sequence of SEQ ID NO: 2 representing CDR2 (HCVR) combined with the amino acid sequence of SEQ ID NO: 5 representing CDR2 (LCVR);
-At least the amino acid sequence of SEQ ID NO: 2 representing CDR2 (HCVR) combined with the amino acid sequence of SEQ ID NO: 6 representing CDR3 (LCVR);
-At least the amino acid sequence of SEQ ID NO: 1 representing CDR1 (HCVR) combined with the amino acid sequence of SEQ ID NO: 6 representing CDR3 (LCVR);
-At least the amino acid sequence of SEQ ID NO: 3 representing CDR3 (HCVR) combined with the amino acid sequence of SEQ ID NO: 5 representing CDR2 (LCVR);
-At least the amino acid sequence of SEQ ID NO: 3 representing CDR3 (HCVR) combined with the amino acid sequence of SEQ ID NO: 6 representing CDR3 (LCVR).

In yet another embodiment, the invention comprises a constant region of a light chain and / or a heavy chain of human or primate origin, as described herein above in a chimeric antibody or fragment thereof or a humanized antibody or fragment thereof. Regarding.

In a further embodiment, the invention comprises at least one of the amino acids representing the CDR regions of the light and / or heavy chains set forth in SEQ ID NOs: 1-6, particularly at least one but at most five. More particularly at least one but at most four, and even more particularly at least one but at most three, especially at least one but at most two, so that the antibody maintains its full functionality. With respect to chimeric antibodies or fragments thereof or humanized antibodies or fragments thereof that are altered through conservative substitutions.

In particular, the present invention is in CDR2 of the light chain variable region (LCVR) set forth in SEQ ID NO: 5, in particular Lys at Kabat position 50 by amino acid residues selected from the group consisting of Arg, Gln, and Glu. With respect to a chimeric antibody or fragment thereof or a humanized antibody or fragment thereof that has been replaced by Arg.

In particular, the present invention is light, in CDR2 set forth in SEQ ID NO: 5, where Lys at Kabat position 50 is replaced, in particular by Arg, with an amino acid residue selected from the group consisting of Arg, Gln, and Glu. Regarding the chain variable region (LCVR).

In another embodiment, the invention is in CDR2 of the light chain variable region (LCVR) set forth in SEQ ID NO: 5, particularly by amino acid residues selected from the group consisting of Asn and Thr where Ser at Kabat position 53. With respect to a chimeric antibody or fragment thereof or a humanized antibody or fragment thereof, which has been replaced by Asn.

In particular, the present invention is in CDR2 set forth in SEQ ID NO: 5, where Ser at Kabat position 53 is replaced by an amino acid residue selected from the group consisting of Asn and Thr, particularly by Asn. Regarding (LCVR).

In certain embodiments of the invention, the chimera has an amino acid sequence in which the heavy chain variable region (HCVR) is 90%, particularly 95%, and more particularly 98% identical to the sequences set forth in SEQ ID NOs: 15 and 16, respectively. Provided is an antibody or a fragment thereof or a humanized antibody or a fragment thereof.

In another embodiment of the invention, the light chain variable region (LCVR) has an amino acid sequence that is 90%, particularly 95%, more particularly 98% identical to the sequences set forth in SEQ ID NOs: 12 and 13, respectively. A chimeric antibody or a fragment thereof or a humanized antibody or a fragment thereof is provided.

In yet another embodiment of the invention, at least two, in particular three, of the CDR regions of the heavy chain variable regions (HCVRs) are 90%, particularly 95%, with the corresponding CDR regions set forth in SEQ ID NOs: 1-3. , More particularly a humanized antibody or fragment thereof having an amino acid sequence that is 98% identical.

In a further aspect of the invention, at least two, in particular three, of the CDR regions of the light chain variable regions (LCVRs) are 90%, particularly 95%, more with the corresponding CDR regions set forth in SEQ ID NOs: 4-6. Particularly provided are humanized antibodies or fragments thereof having amino acid sequences that are 98% identical.

In yet another embodiment, the invention has heavy chain variable regions (HCVRs) of 90%, 91%, 92%, 93%, 94%, 95%, respectively, with the sequences shown in SEQ ID NOs: 15 and 16, respectively. The present invention relates to a chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to the present invention as described above herein, which has an amino acid sequence that is 96%, 97%, 98%, or 99% identical.

In yet another embodiment, the invention comprises light chain variable regions (LCVRs) as 90%, 91%, 92%, 93%, 94%, 95%, respectively, with the sequences set forth in SEQ ID NOs: 12 and 13, respectively. The present invention relates to a chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to the present invention as described above herein, which has an amino acid sequence that is 96%, 97%, 98%, or 99% identical.

In yet another embodiment, the invention relates to at least one of the CDR regions of a heavy chain variable region (HCVR), in particular at least two, in particular three, with the corresponding CDR regions set forth in SEQ ID NOs: 1-3. Chimeras according to the invention as described herein above, having amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. With respect to antibodies or fragments thereof or humanized antibodies or fragments thereof.

In yet another embodiment, the invention relates to at least one of the CDR regions of the light chain variable regions (LCVRs), in particular at least two, in particular three, with the corresponding CDR regions set forth in SEQ ID NOs: 4-6. Chimeras according to the invention as described herein above, having amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. With respect to antibodies or fragments thereof or humanized antibodies or fragments thereof.

In yet another embodiment, the invention derives from at least one of the amino acids representing the acceptor framework sequences obtained from the human germline V _H and V _K sequences, respectively, from the corresponding region of the mouse antibody ACI-01-Ab7C2. The present invention relates to a humanized antibody as described above herein, which is altered through substitutions for amino acids or conservative substitutions thereof.

In particular, in the present invention, the Trp at Kabat position 47 in the acceptor framework sequence obtained from the human germline V _H sequence of the KABAT subgroup V _H III of the heavy chain variable region, respectively, is as shown in SEQ ID NO: 15. Humanizations containing heavy chain variable regions and this heavy chain variable region that have been replaced by amino acids selected from the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe, especially Leu and Ile, especially Leu. Regarding antibodies.

The present invention further presents the Ser and Arg at Kabat position 94 in the acceptor framework sequence obtained from the human germline V _H sequence of the KABAT subgroup V _H III of the heavy chain variable region, as set forth in SEQ ID NO: 15. It relates to a heavy chain variable region and a humanized antibody containing this heavy chain variable region, which are replaced by amino acids selected from the group consisting of Thr, among others, by Ser, respectively.

In yet another embodiment, the invention has Trp at Kabat position 47 in the acceptor framework sequence obtained from the human germline V _H sequence of the KABAT subgroup V _H III of the heavy chain variable region, respectively, with SEQ ID NO: 15. Amino acids selected from the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe as shown in, especially Leu and Ile, especially Arg at Kabat position 94, are replaced by SEQ ID NO: 15. With respect to a heavy chain variable region and a humanized antibody containing this heavy chain variable region, which has been replaced by an amino acid selected from the group consisting of Ser and Thr shown in S.

The present invention further comprises Lys, Arg, Gln, and Asn for Gln at Kabat position 45 in the acceptor framework sequence obtained from the human germline V _K sequence of the KABAT subgroup V _K II of the light chain variable region. It relates to a light chain variable region and a humanized antibody containing this light chain variable region, which has been replaced by amino acids selected from the group, in particular Lys and Arg, and in particular by Lys, respectively.

The present invention further describes Tyr at Kabat position 87 in the acceptor framework sequence obtained from the human germline V _K sequence of the KABAT subgroup V _K II of the light chain variable region, Phe, Leu, Val, Ile, and Ala. It relates to a light chain variable region and a humanized antibody containing this light chain variable region, respectively, which has been replaced by an amino acid selected from the group consisting of, especially by Leu and Phe, in particular by Phe.

The present invention further comprises Lys at Kabat position 50 in the CDR2 region obtained from the mouse monoclonal antibody, in particular the mouse antibody ACI-01-Ab7C2, as shown in SEQ ID NO: 12, consisting of Arg, Gln, His, and Asn. It relates to a light chain variable region and a humanized antibody containing this light chain variable region, respectively, which has been replaced by an amino acid selected from the group, in particular by Arg.

In yet another embodiment, the invention has Ala, Val, respectively, as Asn at Kabat position 53 in the CDR2 region obtained from the mouse monoclonal antibody, in particular the mouse antibody ACI-01-Ab7C2, as shown in SEQ ID NO: 12. Amino acids selected from the group consisting of, Leu, Ser, and Ile; among other things relating to the light chain variable region and the humanized antibody comprising this light chain variable region, which has been replaced by Ser.

In yet another embodiment, the invention has Trp at Kabat position 47 in the acceptor framework sequence obtained from the human germline V _H sequence of the KABAT subgroup V _H III of the heavy chain variable region, respectively. From the human germline V _H sequence of the KABAT subgroup V _H III, which is replaced by amino acids selected from the group consisting of Ile, Val, Met, Ala, and Phe, especially Leu and Ile, especially Leu, and in the heavy chain variable region. Arg at Kabat position 94 in the resulting acceptor framework sequence is replaced by an amino acid selected from the group consisting of Ser and Thr shown in SEQ ID NO: 15, especially by Ser, and the KABAT subgroup of the light chain variable region. The Tyr at Kabat position 87 in the acceptor framework sequence obtained from the human germline V _K sequence of V _K II is particularly Leu and by amino acids selected from the group consisting of Phe, Leu, Val, Ile, and Ala. With respect to humanized antibodies, which have been replaced by Phe, in particular by Phe.

In yet another embodiment, the invention is Trp at Kabat position 47 in the acceptor framework sequence obtained from the human germline V _H sequence of the KABAT subgroup V _H III of the heavy chain variable region set forth in SEQ ID NO: 15, respectively. However, it relates to a heavy chain variable region and a humanized antibody containing this heavy chain variable region, which has been replaced by Leu.

In yet another embodiment, the invention comprises the Arg at Kabat position 94 in the acceptor framework sequence obtained from the human germline V _H sequence of the KABAT subgroup V _H III of the heavy chain variable region, respectively. With respect to the heavy chain variable region and the humanized antibody containing this heavy chain variable region, which has been replaced by Ser shown in 15.

In yet another embodiment, the invention has Trp at Kabat position 47 in the acceptor framework sequence obtained from the human germline V _H sequence of the KABAT subgroup V _H III of the heavy chain variable region, respectively, Leu and Ile, Arg at Kabat position 94 in the acceptor framework sequence obtained from the human germline V _H sequence of the KABAT subgroup V _H III of the heavy chain variable region, which was replaced by Leu in particular, by Ser shown in SEQ ID NO: 15. It relates to a heavy chain variable region that has been replaced and a humanized antibody containing this heavy chain variable region.

In yet another embodiment, Phe replaces Tyr at Kabat position 87 in the acceptor framework sequence obtained from the human germline V _K sequence of the KABAT subgroup V _K II of the light chain variable region, respectively. Containing a light chain variable region and this heavy chain variable region.

In yet another embodiment, the invention has Trp at Kabat position 47 in the acceptor framework sequence obtained from the human germline V _H sequence of the KABAT subgroup V _H III of the heavy chain variable region, respectively, Leu and Ile, Arg at Kabat position 94 in the acceptor framework sequence obtained from the human germline V _H sequence of the KABAT subgroup V _H III of the heavy chain variable region, which was replaced by Leu in particular, as shown in SEQ ID NO: 15. The Tyr at Kabat position 87 in the acceptor framework sequence obtained from the human germline V _K sequence of the KABAT subgroup V _K II of the light chain variable region has been replaced by Phe. The present invention relates to a chain variable region and a humanized antibody containing this heavy chain variable region.

In certain embodiments, the present invention has Trp at Kabat position 47 in the acceptor framework sequence obtained from the human germline V _H sequence of the KABAT subgroup V _H III of the heavy chain variable region, respectively, Leu, Norleucine, Ile, Amino acids selected from the group consisting of Val, Met, Ala, and Phe, especially Leu and Ile, especially Arg at Kabat position 94, consisting of Ser and Thr as shown in SEQ ID NO: 15. Lys at Kabat position 50 in the CDR2 region, which is replaced by an amino acid selected from the group, in particular Ser, and obtained from the mouse monoclonal antibody, in particular the mouse antibody ACI-01-Ab7C2, consists of Arg, Gln, His, and Asn. With respect to the heavy chain variable region and the humanized antibody containing this heavy chain variable region, which has been replaced by amino acids selected from the group, in particular by Arg.

In certain embodiments, the present invention has Trp at Kabat position 47 in the acceptor framework sequence obtained from the human germline V _H sequence of the KABAT subgroup V _H III of the heavy chain variable region, respectively, Leu, norleucine, Ile, Amino acids selected from the group consisting of Val, Met, Ala, and Phe, especially those replaced by Leu and Ile, especially Leu, and Arg at Kabat position 94 selected from the group consisting of Ser and Thr shown in SEQ ID NO: 15. The Asn at Kabat position 53 in the CDR2 region obtained from the mouse monoclonal antibody, in particular the mouse antibody ACI-01-Ab7C2, is composed of Ala, Val, Leu, Ser, and Ile. Amino acids selected from the group; especially with respect to the heavy chain variable region and the humanized antibody containing this heavy chain variable region, which has been replaced by Ser.

In a specific embodiment, the invention relates to a light chain variable region of SEQ ID NO: 12.

In another specific embodiment of the invention, there is provided a humanized antibody comprising a light chain variable region of SEQ ID NO: 12.

In a specific embodiment, the invention relates to a light chain variable region comprising the signal sequence set forth in SEQ ID NO: 13.

In another specific embodiment of the invention, there is provided a humanized antibody comprising a complete light chain variable region comprising the signal sequence set forth in SEQ ID NO: 13.

In another specific embodiment of the invention, there is provided a humanized antibody comprising a light chain variable region of SEQ ID NO: 12 and a light chain constant region of SEQ ID NO: 14.

In another specific embodiment of the invention, there is provided a humanized antibody comprising a complete light chain variable region of SEQ ID NO: 13 and a light chain constant region of SEQ ID NO: 14.

In a specific embodiment, the invention relates to a heavy chain variable region of SEQ ID NO: 15.

In another specific embodiment of the invention, there is provided a humanized antibody comprising a heavy chain variable region of SEQ ID NO: 15.

In a specific embodiment, the invention relates to a heavy chain variable region comprising the signal sequence set forth in SEQ ID NO: 16.

In another specific embodiment of the invention, there is provided a humanized antibody comprising a complete heavy chain variable region comprising the signal sequence set forth in SEQ ID NO: 16.

In another specific embodiment of the invention, there is provided a humanized antibody comprising a heavy chain variable region of SEQ ID NO: 15 and a heavy chain constant region of SEQ ID NO: 17.

In another specific embodiment of the invention, there is provided a humanized antibody comprising a heavy chain variable region of SEQ ID NO: 16 and a heavy chain constant region of SEQ ID NO: 17.

In certain embodiments, the humanized antibodies described herein according to the invention are Aβ monomeric peptides having at least 30, particularly at least 35, more particularly at least 38, and even more particularly at least 40 amino acid residues. Or an Aβ polymer-soluble amyloid peptide containing multiple Aβ monomer units, and in particular an Aβ polymer-soluble amyloid peptide containing multiple Aβ _1-42 monomeric peptides and / or multiple Aβ _1-42 monomeric units. Especially with a molar concentration ratio of antibody to Aβ1-42 of up to 1: 1000, especially up to 1: 500, more particularly up to 1: 300, and even more particularly up to 1: 200, especially up to Coincubation at a molar concentration ratio of antibody to Aβ1-42 from 1:10 to 1: 100 inhibits aggregation of Aβ monomers to the polymer polymer fibrils.

In particular, co-incubation of the antibody according to the invention with the soluble amyloid peptide of the amyloid monomer and / or polymer for 24 to 60 hours, especially 30 to 50 hours, more particularly 48 hours, especially 24 hours, Perform at temperatures of 28 ° C to 40 ° C, especially 32 ° C to 38 ° C, and more particularly 37 ° C.

In a specific embodiment of the invention, coincubation of the amyloid monomer and / or polymer with the soluble amyloid peptide is performed for 24 hours at a temperature of 37 ° C.

In particular, antibodies, in particular any functionally equivalent antibody or humanized antibody according to the invention comprising any functional portion thereof, comprises an Aβ _1-42 monomeric peptide and / or multiple Aβ _1-42 monomeric units. Aβ monomer by coincubation with an Aβ polymer soluble amyloid peptide that binds to an Aβ polymer soluble amyloid peptide containing and / or contains an Aβ _1-42 monomeric peptide and / or multiple Aβ _1-42 monomeric units. Inhibits aggregation of the body and / or polymer to the polymer polymer fibrils.

In some embodiments, an antibody, particularly any functionally equivalent antibody or humanized antibody according to the invention comprising any functional portion thereof, has a molar concentration ratio of up to 1: 1000 antibody to Aβ1-42, in particular 1: 1. Aβ monomer and / or when compared to each amyloid peptide monomer (control) incubated in a buffer at a molar ratio of 10 to 1: 100, especially at a molar ratio of 1:10. Aggregation of Aβ-soluble polymers containing multiple Aβ monomer units into polymer polymer fibrils is at least 50%, especially at least 60%, especially at least 65%, more particularly at least 75%, and even more particularly at least. Inhibits 80%, especially at least 85% -90%, or more.

In a specific embodiment of the invention, an antibody, in particular any functionally equivalent antibody or humanized antibody according to the invention comprising any functional portion thereof, has a molar concentration ratio of 1: 100 antibody to Aβ1-42. Inhibits at least 30% aggregation of Aβ-soluble polymers containing Aβ monomers and / or multiple Aβ monomer units to the polymeric polymer fibrils.

In another specific embodiment of the invention, a humanized antibody according to the invention comprising an antibody, particularly any functionally equivalent antibody or functional portion thereof, has a molar concentration ratio of 1:10 antibody to Aβ1-42. At least 80% of the aggregation of Aβ-soluble polymers containing Aβ monomers and / or multiple Aβ monomer units to the polymer fibrils is inhibited.

Binding of the antibodies described herein according to the invention to amyloid-producing monomeric peptides and / or polymeric peptides, in particular the amyloid form (1-42), is a monomeric amyloid-producing peptide and / or. Alternatively, it causes inhibition of aggregation of the polymer amyloid-producing peptide on the polymer fibrils or filaments. Through inhibition of aggregation of amyloid-producing monomeric and / or polymer peptides, the antibodies according to the invention become insoluble due to changes in amyloid plaques, especially secondary conformations, and in affected animals or humans. It can prevent or delay the formation of amyloid morphology (1-42), which is known to be a major part of amyloid plaques in the brain.

The ability of an antibody to inhibit aggregation according to the invention is determined by any suitable method known in the art, particularly by density gradient ultracentrifugation, followed by SDS-PAGE sedimentation analysis on a preformed gradient, and / or thioflavin. It can be determined by T (Th-T) fluorescence assay.

In some embodiments, the invention is by aggregation of Aβ monomeric peptides having at least 30, particularly at least 35, more particularly at least 38, and even more particularly at least 40 amino acid residues, particularly Aβ _1-42 monomeric peptides. Preformed polymer polymer amyloid fibrils or filaments with a molar concentration ratio of 1: 5 to 1: 1000, especially 1: 10 to 1: 500, more particularly 1: 10 to 1: 1. By co-incubating at a ratio of 300, and even more particularly at a ratio of 1:10 to 1: 100, at least 20%, especially at least 30%, more particularly at least 35%, and even more particularly at least 40%. Antibodies described herein, including any functionally equivalent antibody or functional portion thereof that disaggregates fibrils or filaments of polymer amyloid preformed at least 50% or greater thereof, in particular. Regarding humanized antibodies.

In a specific embodiment of the invention, the anti-aggregation and deagglomeration abilities of an antibody are determined by density gradient ultracentrifugation, followed by SDS-PAGE sedimentation analysis on a preformed gradient, respectively.

In another specific embodiment of the invention, the anti-aggregation and de-aggregation abilities of an antibody are determined by thioflavin T (Th-T) fluorescence assay, respectively.

In another specific embodiment, the antibody according to the invention comprises a fibril or filament of a polymer polymer amyloid preformed with amyloid for 12 to 36 hours, particularly 18 to 30 hours, more particularly 24 hours, 28 hours. Coincubation is carried out at a temperature of ° C. to 40 ° C., particularly 32 ° C. to 38 ° C., more particularly 37 ° C.

In particular, coincubation of the preformed polymer polymer amyloid with fibril or filament is performed for 24 hours at a temperature of 37 ° C.

In a specific embodiment of the invention, an antibody, in particular any functionally equivalent antibody or humanized antibody according to the invention comprising any functional portion thereof, has a molar concentration ratio of 1: 100 antibody to Aβ1-42. The preformed polymer fibrils or filaments can be deagglomerated by at least 24%.

In another specific embodiment of the invention, a humanized antibody according to the invention comprising an antibody, particularly any functionally equivalent antibody or functional portion thereof, has a molar concentration ratio of 1:10 antibody to Aβ1-42. The preformed polymer amyloid fibrils or filaments can be deaggregated by at least 32%.

Through the disaggregation of fibrils or filaments of amyloid-producing polymers, the antibodies according to the invention can prevent or delay the formation of amyloid plaques, which results in alleviation of disease-related symptoms and delay or reversal of their progression.

Accordingly, any functionally equivalent antibody described herein or an antibody comprising a functional portion thereof, particularly a humanized antibody, suffering from a disease or condition that results in an increase in the concentration of Aβ in the brain. It is one further aspect of the invention to provide an antibody capable of reducing the total amount of Aβ in the brain of animals, especially mammals, especially humans.

In another embodiment, the invention is bi-effective in that it exhibits both aggregation inhibitory and disaggregation properties, particularly in combination with a high degree of conformational sensitivity. The present invention relates to a humanized antibody as described above herein.

In particular, the present invention relates to soluble amyloid peptides of amyloid monomers and / or polymers, particularly β- such as, for example, Aβ monomeric peptides 1-39; 1-40, 1-41 or 1-42. Amyloid monomeric peptides and / or polymer-soluble β-amyloid peptides containing multiple Aβ monomeric units, particularly Aβ _1-42 monomeric and / or multiple Aβ _1-42 monomeric units. Co-incubation of the containing Aβ polymer with a soluble amyloid peptide inhibits the aggregation of the Aβ monomer to the polymer polymer fibrils or filaments and further amyloid monomeric peptides, especially the Aβ monomeric peptide 1-. 39; fibrils of preformed high molecular weight polymer amyloids formed by aggregation of β-amyloid monomeric peptides such as 1-40, 1-41 or 1-42, especially Aβ _1-42 monomeric peptides. Alternatively, by co-incubation with a filament, the fibrils or filaments of the preformed polymer can be deaggregated, according to the invention, the chimeric antibody or fragment thereof as described above herein or the humanized antibody or fragment thereof. Regarding.

In another aspect, the invention provides an increase in random coil conformation at the expense of β-sheet conformation and improved solubilization of preformed polymer polymer amyloid fibrils or filaments, α-helices and / Or a random coil configuration, especially a β-sheet configuration to a random coil configuration at a given location in the molecule, especially in the environment of the Tyr 10 and Val 12 of the Aβ protein. The present invention relates to a chimeric antibody or fragment thereof as described above, or a humanized antibody or fragment thereof, which is capable of inducing conversion of. In particular, the reduction in β-sheet conformation is at least 30%, especially at least 35%, and more particularly at least 40, compared to each preformed amyloid polymer fibril or filament (control) incubated in buffer. % And above.

The ability of an antibody to induce transfer in secondary structure is determined by solid-state 13C NMR spectroscopy, especially by measuring the integrated intensity of the Tyr 10 and Val 12 Cβ conformations in the Aβ _1-42 peptide.

In a further aspect of the invention, at least about 1 × 10 ^-7 M to at least about 1 × 10 ^-12 M, particularly at least about 1 × 10 ^-8 M to at least about 1 × 10 ^-11 M, more particularly at least. High binding affinity with K _D in the range of about 1 × 10 ^-9 M to at least about 1 × 10 ^-10 M, and even more particularly at least about 1 × 10 ^-8 M to at least about 2 × 10 ^-8 M. At least one light chain or fragment thereof or at least one heavy chain or a heavy chain thereof that binds to an Aβ monomer by sex, but preferably shows no significant cross-reactivity with amyloid precursor protein (APP). Provided are a chimeric antibody or fragment thereof or a humanized antibody or fragment thereof as described above herein according to the present invention, which comprises a fragment.

In another aspect of the invention, at least about 1 × 10 ^-7 M to at least about 1 × 10 ^-12 M, particularly at least about 1 × 10 ^-8 M to at least about 1 × 10 ^-11 M, more particularly. High binding with K _D in the range of at least about 1 × 10 ^-9 M to at least about 1 × 10 ^-10 M, and even more particularly at least about 2 × 10 ^-9 M to at least about 5 × 10 ^-9 M. At least one light chain or fragment thereof or at least one that binds to a fiber, fibril, or filament of Aβ with affinity, but preferably shows no significant cross-reactivity with amyloid precursor protein (APP). Provided are a chimeric antibody or fragment thereof or a humanized antibody or fragment thereof as described above herein, according to the invention, comprising two heavy chains or fragments thereof.

In another embodiment, the antibody or fragment thereof as described herein above according to the invention is at least 2-fold, particularly at least 4-fold, particularly at least 10-fold, particularly at least 15-fold more than the binding affinity for the Aβ monomer. It exhibits a double, more particularly at least 20-fold, and particularly at least 25-fold higher binding affinity for Aβ fibers, fibrils, or filaments.

In yet another embodiment, it substantially binds to aggregated Aβ, including Aβ plaques in mammalian, especially human brain, but preferably has no significant cross-reactivity with amyloid precursor protein (APP). Provided are a chimeric antibody or fragment thereof or a humanized antibody or fragment thereof, not shown herein, as described above.

In another aspect of the invention, substantially binding to soluble polymer amyloid, particularly amyloid β (Aβ), including Aβ monomers in mammalian, especially human brain, is preferably amyloid precursor protein. Provided are a chimeric antibody or fragment thereof or a humanized antibody or fragment thereof, as described herein above, which does not exhibit any significant cross-reactivity with (APP).

Further provided are chimeric antibodies or fragments thereof or humanized antibodies or fragments thereof, as described herein above, which significantly reduce the Aβ plaque load in the brain of mammals, especially humans. This causes the fibers to disaggregate to soluble polymer and monomeric forms, either by binding to plaques of the antibody or by inducing conformational transformation, in tissues and / or body fluids, especially in the brain. Between the insoluble and aggregated states of amyloid, especially amyloid β (Aβ), by binding to and stabilizing the deaggregated and solubilized amyloid morphology, especially the amyloid β (Aβ) morphology. It is possible to achieve either by shifting the equilibrium of the to its soluble form. Therefore, through the activity of the antibody according to the invention, it favors removal and catabolism in tissues and / or body fluids, especially in the periphery rather than in the brain. Therefore, the advantageous effect of the antibody according to the present invention can be obtained without binding of the antibody to the plaque.

Through this stabilizing activity, the antibodies according to the invention can neutralize the toxic effects of polymer and less aggregated soluble amyloid proteins, especially amyloid β (Aβ) proteins, in tissues and / or body fluids. .. Therefore, in a specific embodiment of the invention, the antibody according to the invention may achieve its beneficial effect without necessarily binding to aggregated amyloid β in the brain.

In a further aspect of the invention, from mouse donor antibodies having at least 5-fold, particularly at least 8-fold, more particularly at least 10-fold, particularly at least 15-fold affinity for Aβ, especially 2005. Deposited on 1 December with Braunschweig, Mascheroder Weg 1B, 38124 Braunschweig's Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under accession number: DSM ACC2750 (named "mC2" throughout this application). And at least one light chain that incorporates at least one, especially two, more particularly three CDR regions, obtained from the mouse antibody ACI-01-Ab7C2 (named hC2 for humanized C2 antibody) or its Provided are humanized antibodies or fragments thereof as described above herein, according to the invention, comprising a fragment or at least one heavy chain or fragment thereof.

In certain embodiments, the antibodies of the invention are of any isotype and subtype (eg, IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1, and IgA2) (eg, two full length light chains and two). A complete antibody (with a full-length heavy chain); in particular an IgG4 isotype antibody; or, in another embodiment, it is an antigen-binding fragment of the complete antibody (eg, Fab, F (ab') ₂ , and Fv). possible.

Accordingly, the invention also relates to antigen-binding fragments of the antibodies described herein. In certain embodiments of the invention, the fragment comprises a Fab immunoglobulin expression library product and an epitope binding fragment of any of the antibodies and fragments mentioned above, a Fab fragment, a Fab'fragment, an F (ab) ₂ fragment, And selected from the group consisting of F _v fragments.

In another embodiment, the antibody or antigen binding fragment of the present invention is bound to polyethylene glycol. In yet another embodiment, the constant region of the antibody of the invention is modified to reduce biological effector function via at least one constant region as compared to an unmodified antibody. In yet another embodiment, the antibody or antigen binding fragment of the present invention comprises an Fc region having a modified effector function.

The invention further relates to a nucleotide molecule according to the invention, comprising a nucleotide sequence encoding a chimeric antibody or fragment thereof or a humanized antibody or fragment thereof, as previously disclosed herein.

In particular, the present invention presents a nucleotide sequence encoding a series of continuous amino acid molecules set forth in SEQ ID NOs: 2 and 3, which represent complementarity determining regions (CDRs) 2 and 3 of the heavy chain variable region (HCVR), respectively. Or related to nucleotide molecules, including complementary sequences.

More particularly, the invention presents a nucleotide or complementary sequence encoding a series of continuous amino acid molecules set forth in SEQ ID NO: 4, which indicates complementarity determining regions (CDRs) 1 of the light chain variable region (LCVR). With respect to nucleotide molecules, including.

In another aspect of the invention, a nucleotide molecule comprising the nucleotide sequence or complementary sequence set forth in SEQ ID NO: 18 and SEQ ID NO: 19, which encodes the amino acid sequences of CDR2 and CDR3 of the heavy chain variable region (HCVR), respectively. I will provide a.

In another aspect of the invention, there is provided a nucleotide molecule comprising the nucleotide sequence or complementary sequence set forth in SEQ ID NO: 20, which encodes the nucleotide sequence of CDR1 of the light chain variable region (LCVR).

In another aspect of the invention, there is provided a nucleotide molecule comprising the nucleotide sequence or complementary sequence of SEQ ID NO: 21 encoding the light chain variable region.

In another aspect of the invention, there is provided a nucleotide molecule comprising a nucleotide sequence of SEQ ID NO: 22 or a complementary sequence encoding a complete light chain variable region comprising a signal sequence.

In another aspect of the invention, there is provided a nucleotide molecule comprising a nucleotide sequence encoding a light chain variable region of SEQ ID NO: 22 and a light chain constant region of SEQ ID NO: 23. The invention also includes complementary strands of the nucleotide molecule.

In another aspect of the invention, there is provided a nucleotide molecule comprising a nucleotide sequence of SEQ ID NO: 24 encoding a heavy chain variable region. The invention also includes complementary strands of the nucleotide molecule.

In another aspect of the invention, there is provided a nucleotide molecule comprising a nucleotide sequence of SEQ ID NO: 25 encoding a complete heavy chain variable region comprising a signal sequence. The invention also includes complementary strands of the nucleotide molecule.

In another aspect of the invention, there is provided a nucleotide molecule comprising a nucleotide sequence encoding a heavy chain variable region of SEQ ID NO: 25 and a heavy chain constant region of SEQ ID NO: 26. The invention also includes complementary strands of the nucleotide molecule.

Also, a nucleotide sequence that hybridizes to one of the nucleotide sequences of the invention encoding the above antibody, either isolated or as part of a longer nucleotide molecule, particularly to its complementary strand. Included by the present invention.

In particular, the invention hybridizes to any of the nucleotide sequences set forth in SEQ ID NOs: 18-26 and 29-32, especially to its complementary strand, under conventional hybridization conditions, especially stringent hybridization conditions. Regarding the nucleotide sequence to hybridize.

In another aspect of the invention, there is provided an expression vector according to the invention comprising a nucleic acid molecule as previously mentioned herein.

In another aspect of the invention, there is provided a cell according to the invention comprising an expression vector comprising a nucleic acid as previously mentioned herein.

In yet another embodiment, the invention comprises an antibody according to the invention, particularly a therapeutically effective amount, a functionally equivalent antibody or any derivative or functional portion thereof, as described herein herein. The present invention relates to a composition comprising a chimeric antibody or a fragment thereof or a humanized antibody or a fragment thereof, particularly a composition which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

In another aspect of the invention, the composition comprises a therapeutically effective amount of the antibody.

Antibodies, in particular monoclonal antibodies according to the invention, particularly chimeric antibodies or fragments thereof as described herein herein, comprising any functionally equivalent antibody or derivative or functional portion thereof in a therapeutically effective amount. Alternatively, a mixture comprising a humanized antibody or fragment thereof, and optionally additional bioactive substances and / or pharmaceutically acceptable carriers and / or diluents and / or excipients is further included.

In particular, the invention relates to mixtures in which the further bioactive substance is a compound used in the drug treatment of amyloid or amyloid-like proteins associated with amyloid or amyloid-like proteins involved in Alzheimer's disease, amyloidosis.

In another aspect of the invention, the other bioactive substance or compound is also a therapeutic agent that can be used in the treatment of amyloidosis caused by amyloid β or in the treatment of other neurological disorders. May be.

Other bioactive substances or compounds may exert their biological effects by the same or similar mechanism of action as the antibodies according to the invention, or by unrelated mechanisms of action, or with multiple relationships and / Or may be exerted by an unrelated mechanism of action.

In general, other bioactive compounds include neurotransmission activators, psychotherapeutic agents, acetylcholine esterase inhibitors, calcium channel antagonists, bioamines, benzodiazepine tranquilizers, acetylcholine synthesis, storage or release activators, acetylcholine. Includes postsynaptic receptor agonists, monoamine oxidase-A or -B inhibitors, N-methyl-D-aspartate glutamate receptor antagonists, non-steroidal anti-inflammatory agents, antioxidants, and serotoninergic receptor antagonists It can be.

More particularly, the invention is a compound effective against oxidative stress, an anti-apparent compound, along with the antibody according to the invention and optionally a pharmaceutically acceptable carrier and / or diluent and / or excipient. , Inhibitors of DNA repair such as metal chelating agents, pyrenzepine and metabolites, 3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS), α-secretase activator, N containing β- and γ-secretase inhibitors, tau proteins, neurotransmitters, β-sheet breakers, attractants of cellular components that remove / deplete amyloid β, and pyroglutamine-oxidized amyloid β3-42. Inhibitors of end-cleaving amyloid β, anti-inflammatory molecules, or cholineresterase inhibitors (ChEI) such as taclin, rivastigmin, donepezil, and / or galantamine, M1 agonists, and other drugs including any amyloid or tau modifiers. , As well as a mixture comprising at least one compound selected from the group consisting of nutritional supplements.

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

In a further embodiment, the mixture according to the invention may contain niacin or memantine along with the antibody according to the invention, and optionally a pharmaceutically acceptable carrier and / or diluent and / or excipient.

In yet another embodiment of the invention, antibodies, in particular monoclonal antibodies according to the invention, particularly chimeric antibodies or fragments thereof or humanized antibodies or fragments thereof as described herein, and optionally further pharmaceutically acceptable. Hallucinations, delusions, thought disorders (where prominent thought clutter, derailment, derailed thoughts appear), and absurd or confused behavior, as well as anxiety, with carriers and / or diluents and / or excipients "Atypical antipsychotics" for the treatment of positive and negative psychotic symptoms, including illness, non-tonal feelings, apathy, and social withdrawal, eg, clozapine, ziplacidone, risperidone, aliprazole, or oranzapine. Provide a mixture containing.

In one particular aspect of the invention, the compositions and mixtures according to the invention and as described herein above contain therapeutically effective amounts of antibodies and bioactive substances, respectively.

Other compounds that can be suitably used in mixtures in combination with the antibodies according to the invention are described in WO 2004/058258 (particularly see pages 16-17), which are therapeutic targets (36-39). Pages), alcan sulfonic acid and alkanol sulfate (pages 39-51), cholineresterase inhibitors (pages 51-56), NMDA receptor antagonists (pages 56-58), estrogen (pages 58-59), non-steroidal anti-inflammatory Drugs (pages 60-61), antioxidants (pages 61-62), peroxysome growth-activating receptor (PPAR) agonists (pages 63-67), cholesterol-lowering agents (pages 68-75); amyloid inhibitors (pages 75) -Pages 77), amyloid formation inhibitors (pages 77-78), metal chelating agents (pages 78-79), antipsychotics and antidepressants (pages 80-82), nutritional supplements (pages 83-89) and brain Contains compounds (see pages 89-93) and prodrugs (pages 93 and 94) that enhance the bioavailability of bioactive substances within, and this document is incorporated herein by reference.

In another embodiment, the invention comprises antibodies, in particular monoclonal antibodies according to the invention, in particular chimeric antibodies or fragments thereof as described herein above or humanized antibodies or fragments thereof, and / or bioactive substances according to the invention. Concers with mixtures containing in therapeutically effective amounts.

The present invention further relates to Alzheimer's disease (AD), Levy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); neurological disorders such as Guam Parkinson-dementia complex; and progressive nucleus. Paralysis, multiple sclerosis; Kreuzfeld-Jakob disease, Parkinson's disease, HIV-related dementia, ALS (muscle atrophic lateral sclerosis), adult-onset diabetes; senile heart amyloidosis; including endocrine tumors, and yellow spot degeneration, A group associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including, but not limited to, other diseases based on or associated with amyloid-like proteins such as other diseases. Antibodies, in particular monoclonal antibodies according to the invention, particularly chimeric antibodies or fragments thereof as described herein herein With respect to the use of humanized antibodies or fragments thereof, and / or functional portions thereof, and / or pharmaceutical compositions or mixtures containing the antibody.

Alzheimer's disease (AD), Levy body dementia, Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); neurological disorders such as Guam Parkinson-dementia complex; as well as progressive nuclear paralysis, multiple sclerosis Diseases; Kreuzfeld Jacob's disease, Parkinson's disease, HIV-related dementia, ALS (muscle atrophic lateral sclerosis), adult-onset diabetes; senile heart amyloidosis; endocrine tumors, and other diseases including luteal degeneration. In a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including, but not limited to, other diseases based on or associated with amyloid-like protein. Antibodies for use in certain methods to prevent, treat or mitigate the effects of amyloidosis, in particular monoclonal antibodies according to the invention, in particular chimeric antibodies or fragments thereof as described herein herein. Or a method for the preparation of a pharmaceutical composition or mixture comprising a humanized antibody or fragment thereof, and / or a functional portion thereof, and / or a particularly therapeutically effective amount of the antibody and / or the functional portion thereof. Also included in the invention are methods comprising the step of formulating an antibody, in particular a monoclonal antibody according to the invention, particularly a pharmaceutically acceptable form of a chimeric antibody or fragment thereof according to the invention or a humanized antibody or fragment thereof. Is done.

Animals affected by the following disorders with antibodies and / or functional portions thereof, in particular humanized antibodies and / or functional moieties thereof, or compositions or mixtures containing such antibodies and / or functional moieties thereof. Or by administration to humans, Alzheimer's disease (AD), Levy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); neurological disorders such as Guam Parkinson-dementia complex; Advanced nuclear palsy, multiple sclerosis; Kreuzfeld-Jakob disease, Parkinson's disease, HIV-related dementia, ALS (muscle atrophic lateral sclerosis), adult-onset diabetes; senile heart amyloidosis; endocrine tumor, and luteal degeneration Amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including, but not limited to, other diseases based on or associated with amyloid-like proteins such as other diseases, including disease. A method for preventing, treating, or ameliorating the effects of a group of diseases and disorders associated with amyloidosis, further comprising the step of administering a therapeutically effective amount of the antibody. Included by.

Neurological disorders such as Alzheimer's disease (AD), especially cognitive memory capacity, by administering to animals, especially mammals or humans, antibodies, especially the pharmaceutical compositions according to the present invention. Methods for the treatment of a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including but not limited to diseases or conditions characterized by loss. It is also an object of the present invention to provide.

In a specific embodiment, the invention retains cognitive memory capacity by administering to an animal, in particular a mammal or a human, an antibody, in particular the pharmaceutical composition as described herein herein. Provided are methods for restoring the cognitive memory capacity of animals, especially mammals or humans, particularly suffering from memory impairment, to increase or increase.

Neurological disorders such as Alzheimer's disease (AD), particularly cognitive, using the therapeutic compositions and methods of producing such compositions, as well as according to the invention, using the antibodies as described herein. For the treatment of a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including but not limited to diseases or conditions characterized by loss of memory. It is also an object of the present invention to provide the method of the present invention.

In particular, the invention relates to the treatment of animals, especially mammals or humans, suffering from an amyloid-related condition characterized by loss of cognitive memory, which results in retention of cognitive memory.

The invention further comprises a method of diagnosing an amyloid-related disease or condition in a patient comprising the step of detecting immunospecific binding of an antibody or active fragment thereof to an epitope of an amyloid protein in a sample or in situ.
(A) Antibodies that bind a sample or specific body part or part suspected of containing an amyloid protein to an epitope of an amyloid protein, particularly a monoclonal antibody according to the invention, particularly according to the invention, as described herein. The step of contacting a street chimeric antibody or fragment thereof or a humanized antibody or fragment thereof and / or a functional moiety thereof;
(B) The step of binding the antibody and / or its functional portion to an amyloid protein to form an immunological complex;
A method comprising (c) detecting the formation of the immunological complex; and (d) correlating the presence or absence of the immunological complex with the presence or absence of an amyloid protein in a sample or specific body part or site. Regarding.

A method of determining the degree of amyloid formative burden in tissues and / or body fluids.
(A) Steps to obtain a sample representative of the tissue and / or body fluid under investigation;
(B) Using an antibody, in particular a monoclonal antibody according to the invention, in particular a chimeric antibody or fragment thereof as described above herein, or a humanized antibody or fragment thereof, and / or a functional portion thereof, according to the invention. The step of inspecting the sample for the presence of amyloid protein;
Also included are (c) determining the amount of antibody bound to the protein; and (d) calculating the plaque load in the tissue and / or body fluid.

In particular, the present invention determines the formation of an immunological complex in step (c) such that the presence or absence of an immunological complex correlates with the presence or absence of an amyloid protein. It relates to a method for determining the degree of amyloid load.

In another aspect of the invention, an antibody, particularly a monoclonal antibody according to the invention, particularly a chimeric antibody or fragment thereof or a humanized antibody or fragment thereof, and / or a functional portion thereof, as described herein above, according to the invention. Provided are test kits for the detection and diagnosis of amyloid-related diseases or conditions, including.

In particular, the present invention relates to amyloid proteins such that the presence or absence of one or more antibodies according to the invention and / or the functional portion thereof, as well as the presence or absence of an immunological complex, correlates with the presence or absence of the amyloid protein. For the detection and diagnosis of amyloid-related diseases or conditions, including instructions for using antibodies for the purpose of binding to form an immunological complex and detecting the formation of the immunological complex. Regarding the test kit.

In another aspect, the invention provides an antibody or variant thereof comprising a variable region as set forth in SEQ ID NO: 27. In some embodiments, the cell line expresses the antibody.

In another aspect, the invention provides an antibody gene or variant thereof comprising a variable region as set forth in SEQ ID NO: 29. In some embodiments, the cell line expresses the antibody.

In another aspect, the invention provides a method for deaggregating preformed β-amyloid fibrils, comprising the step of interacting the hC2 antibody with preformed β-amyloid fibrils.

In another aspect, the invention provides a humanized antibody or fragment thereof according to any of the claims described, which protects neurons from Aβ-induced degradation.

In another aspect, the invention provides a method for preventing Aβ-induced neuronal degradation, comprising treating a neuron with an effective amount of a humanized antibody or fragment thereof as disclosed herein.

In another aspect, the invention provides the use of humanized antibodies or fragments thereof as described herein for the preparation of pharmaceuticals to prevent neuronal degeneration upon exposure to Aβ oligomers.

The above and other objects, features and advantages of the present invention will be clarified by examining the detailed description of the aspects disclosed below and the appended claims.
[Invention 1001]
A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof that specifically binds to at least one epitope on β-amyloid protein.
The epitope comprises at least two contiguous amino acid residues that are primarily involved in binding to the antibody, wherein the at least two contiguous amino acid residues are the following core sequences (SEQ ID NO: 10) :.
Xaa ₁ --Xaa ₂ --Lys --Leu --Xaa ₃
Embedded in-Lys-Leu-,
During the ceremony
Xaa ₁ is an amino acid selected from the group consisting of His, Asn, Gln, Lys, and Arg.
Xaa ₂ is an amino acid selected from the group consisting of Asn and Gln;
Xaa ₃ is an amino acid selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile.
Chimeric antibody or fragment thereof or humanized antibody or fragment thereof.
[Invention 1002]
A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof that specifically binds to at least one epitope on β-amyloid protein.
The epitope comprises at least two contiguous amino acid residues that are primarily involved in binding to the antibody, wherein the at least two contiguous amino acid residues are the following core sequences (SEQ ID NO: 9) :.
Xaa ₃ --Phe --Phe --Xaa ₄ --Xaa ₅ --Xaa ₆
Embedded in-Phe-Phe-,
During the ceremony
Xaa ₃ is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile;
Xaa ₄ is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser, and Ile;
Xaa ₅ is an amino acid residue selected from the group consisting of Glu and Asp, and
Xaa ₆ is an amino acid residue selected from the group consisting of Glu and Asp,
Chimeric antibody or fragment thereof or humanized antibody or fragment thereof.
[Invention 1003]
A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof that specifically binds to at least two epitopes on β-amyloid protein.
Each of the at least two epitopes contains at least two contiguous amino acid residues that are primarily involved in antibody binding, and the at least two contiguous amino acids are either not involved in antibody binding or from the contiguous amino acid residues. Also separated by at least one amino acid residue involved to a significantly smaller extent, the contiguous amino acid residues are each in the following core sequence:
Xaa ₁ --Xaa ₂ --Lys --Leu --Xaa ₃ --Phe --Phe --Xaa ₄ _ Xaa ₅ _ Xaa ₆
Embedded in-Phe-Phe- and -Lys-Leu-,
During the ceremony
Xaa ₁ is an amino acid selected from the group consisting of His, Asn, Gln, Lys, and Arg;
Xaa ₂ is an amino acid selected from the group consisting of Asn and Gln;
Xaa ₃ is an amino acid selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile;
Xaa ₄ is an amino acid selected from the group consisting of Ala, Val, Leu, Ser, and Ile;
Xaa ₅ is an amino acid selected from the group consisting of Glu and Asp, and
Xaa ₆ is an amino acid selected from the group consisting of Glu and Asp,
The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to the present invention 1001 or 1002.
[Invention 1004]
Xaa ₁ is an amino acid selected from the group consisting of His and Arg;
Xaa ₂ is an amino acid selected from the group consisting of Asn and Gln;
Xaa ₃ is an amino acid selected from the group consisting of Val and Leu;
Xaa ₄ is an amino acid selected from the group consisting of Ala and Val;
Xaa ₅ is an amino acid selected from the group consisting of Glu and Asp;
Xaa ₆ is an amino acid selected from the group consisting of Glu and Asp,
The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to any one of 1001 to 1003 of the present invention.
[Invention 1005]
Xaa ₁ is His;
Xaa ₂ is Gln;
Xaa ₃ is Val;
Xaa ₄ is Ala;
Xaa ₅ is Glu;
Xaa ₆ is Asp,
The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to the present invention 1004.
[Invention 1006]
A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof that specifically binds to at least two epitopes on β-amyloid protein.
The at least two epitopes each contain at least two contiguous amino acid residues that are primarily involved in the binding of antibodies that are-Phe -Phe- and --Lys --Leu-, respectively, and at least two distinct binding sites. Represents the amino acid sequence -Val --Phe --Phe --Ala --Glu --Asp-shown in SEQ ID NO: 7 and the amino acid sequence His --Gln --Lys --Leu --Val-shown in SEQ ID NO: 8, respectively.
The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to any one of 1001 to 1005 of the present invention.
[Invention 1007]
A human containing at least one CDR of non-human origin and one or more human-derived or primate-derived framework regions, and optionally a constant region derived from a human or primate-sourced antibody, within the variable region. Antibodies or fragments thereof
β-amyloid protein, β-amyloid monomer peptide, polymer-soluble amyloid peptide containing multiple β-amyloid monomer units, β-amyloid fibers, fibrils, or filaments, and isolated or β-amyloid. To the β-amyloid polymer peptide as part of the plaque, the following amino acid sequence (SEQ ID NO: 11):
Xaa ₁ --Xaa ₂ --Lys --Leu --Xaa ₃ --Phe --Phe- Xaa ₄ _ Xaa ₅ _ Xaa ₆
Can specifically bind to epitopes containing
During the ceremony
Xaa ₁ is an amino acid residue selected from the group consisting of His, Asn, and Gln, especially His;
Xaa ₂ is an amino acid residue selected from the group consisting of Asn and Gln, especially Gln;
Xaa ₃ is an amino acid residue selected from the group consisting of Val, Leu, and Ile, especially Val;
Xaa4 is an amino acid residue selected from the group consisting of Ala and Val, especially Ala;
Xaa5 is an amino acid residue selected from the group consisting of Glu and Asp, especially Glu, and
Xaa6 is an amino acid residue selected from the group consisting of Glu and Asp, especially Asp.
Humanized antibody or fragment thereof.
[Invention 1008]
The humanized antibody or fragment thereof according to any one of the present inventions 1001 to 1007, wherein the CDR of non-human origin is obtained from a donor antibody prepared against an antigen fragment containing no epitope.
[Invention 1009]
Framework regions of human or primate origin, as well as SEQ ID NOs: 2 representing CDR2 of heavy chain variable regions (HCVRs) and SEQ ID NOs: 3 representing CDR3s, and SEQ ID NOs of CDR1s of light chain variable regions (LCVRs). At least one CDR with an amino acid sequence selected from the group of sequences consisting of: 4
A humanized antibody or fragment thereof, comprising a variable region having.
[Invention 1010]
It has an amino acid sequence selected from a group consisting of a heavy chain framework region derived from humans or primates, and a sequence number consisting of SEQ ID NO: 2 representing CDR2 and SEQ ID NO: 3 representing CDR3 of the heavy chain variable region (HCVR). , At least one CDR
The humanized antibody or fragment thereof according to the present invention 1009, which comprises a heavy chain variable region comprising.
[Invention 1011]
One CDR with the amino acid sequence of SEQ ID NO: 4, representing CDR1 of the light chain framework region of human or primate origin, and the light chain variable region (LCVR).
The humanized antibody or fragment thereof according to the present invention 1009, which comprises a light chain variable region comprising.
[Invention 1012]
Framework regions of human or primate origin, as well as SEQ ID NO: 1 for CDR1 of heavy chain variable region (HCVR), SEQ ID NO: 2 for CDR2, and SEQ ID NO: 3 for CDR3, and light chain variable region. At least two CDRs with an amino acid sequence selected from the group of sequences consisting of SEQ ID NO: 4, representing CDR1 of (LCVR), SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3.
The humanized antibody or fragment thereof according to the present invention 1009, which comprises a variable region comprising.
[Invention 1013]
Humanized antibody,
A sequence consisting of a heavy chain framework region of human or primate origin, as well as SEQ ID NO: 1 representing CDR1 of the heavy chain variable region (HCVR), SEQ ID NO: 2 representing CDR2, and SEQ ID NO: 3 representing CDR3. At least two CDRs with amino acid sequences selected from the group
The humanized antibody or fragment thereof according to the present invention 1012, wherein the humanized antibody comprising a heavy chain variable region having the same CDR cannot be present twice in the antibody.
[Invention 1014]
Humanized antibody,
A sequence consisting of a light chain framework region of human or primate origin, as well as SEQ ID NO: 4, representing CDR1 of the light chain variable region (LCVR), SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3. At least two CDRs with an amino acid sequence selected from the group
The humanized antibody or fragment thereof according to the present invention 1012, wherein the humanized antibody comprising a light chain variable region having the same CDR cannot be present twice in the antibody.
[Invention 1015]
The amino acid sequences of the heavy chain framework region from humans or primates, as well as SEQ ID NO: 1 for CDR1 of the heavy chain variable region (HCVR), SEQ ID NO: 2 for CDR2, and SEQ ID NO: 3 for CDR3. CDRs with the order shown in particular
The humanized antibody or fragment thereof according to the present invention 1009, which comprises a heavy chain variable region comprising.
[Invention 1016]
The amino acid sequences of the light chain framework region of human or primate origin, as well as SEQ ID NO: 4, representing CDR1 of the light chain variable region (LCVR), SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3. CDRs with the order shown in particular
The humanized antibody or fragment thereof according to the present invention 1009, which comprises a light chain variable region comprising.
[Invention 1017]
Humanized antibody,
Framework regions of human or primate origin, as well as SEQ ID NO: 1 for CDR1 of heavy chain variable region (HCVR), SEQ ID NO: 2 for CDR2, and SEQ ID NO: 3 for CDR3, and light chain variable region. At least three CDRs with an amino acid sequence selected from the group of sequences consisting of SEQ ID NO: 4, representing CDR1 of (LCVR), SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3.
The humanized antibody or fragment thereof according to the present invention 1009, wherein the humanized antibody comprising a variable region having the same CDR cannot be present twice in the antibody.
[Invention 1018]
Humanized antibody,
Framework regions of human or primate origin, as well as SEQ ID NO: 1 for CDR1 of heavy chain variable region (HCVR), SEQ ID NO: 2 for CDR2, and SEQ ID NO: 3 for CDR3, and light chain variable region. At least four CDRs with an amino acid sequence selected from the group consisting of SEQ ID NO: 4, representing CDR1 of (LCVR), SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3.
The humanized antibody or fragment thereof according to the present invention 1009, wherein the humanized antibody comprising a variable region having the same CDR cannot be present twice in the antibody.
[Invention 1019]
Humanized antibody,
Framework regions of human or primate origin, as well as SEQ ID NO: 1 for CDR1 of heavy chain variable region (HCVR), SEQ ID NO: 2 for CDR2, and SEQ ID NO: 3 for CDR3, and light chain variable region. At least 5 CDRs with an amino acid sequence selected from the group of sequences consisting of SEQ ID NO: 4, representing CDR1 of (LCVR), SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3.
The humanized antibody or fragment thereof according to the present invention 1009, wherein the humanized antibody comprising a variable region having the same CDR cannot be present twice in the antibody.
[Invention 1020]
Framework regions of human or primate origin, as well as SEQ ID NO: 1 for CDR1 of heavy chain variable region (HCVR), SEQ ID NO: 2 for CDR2, and SEQ ID NO: 3 for CDR3, and light chain variable region. CDR with amino acid sequence of SEQ ID NO: 4, representing CDR1 of (LCVR), SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3
The humanized antibody or fragment thereof according to the present invention 1009, which comprises a variable region comprising.
[Invention 1021]
The humanized antibody or fragment thereof according to the present invention 1010, wherein at least one CDR is a CDR having the amino acid sequence of SEQ ID NO: 2, which represents CDR2 of the heavy chain variable region (HCVR).
[Invention 1022]
The humanized antibody or fragment thereof according to the present invention 1010, wherein the at least one CDR is a CDR having the amino acid sequence of SEQ ID NO: 3 representing CDR3 of the heavy chain variable region (HCVR).
[Invention 1023]
The humanized antibody or fragment thereof according to the present invention 1013, wherein at least two CDRs are CDRs having the amino acid sequences of SEQ ID NO: 1 representing CDR1 and SEQ ID NO: 2 representing CDR2 of the heavy chain variable region (HCVR).
[Invention 1024]
The humanized antibody or fragment thereof according to the present invention 1013, wherein at least two CDRs are CDRs having the amino acid sequences of SEQ ID NO: 1 representing CDR1 and SEQ ID NO: 3 representing CDR3 of the heavy chain variable region (HCVR).
[Invention 1025]
The humanized antibody or fragment thereof according to the present invention 1013, wherein at least two CDRs are CDRs having the amino acid sequences of SEQ ID NO: 2 representing CDR2 and SEQ ID NO: 3 representing CDR3 of the heavy chain variable region (HCVR).
[Invention 1026]
The humanized antibody or fragment thereof according to the present invention 1014, wherein at least two CDRs are CDRs having the amino acid sequences of SEQ ID NO: 4 representing CDR1 of the light chain variable region (LCVR) and SEQ ID NO: 5 representing CDR2.
[Invention 1027]
The humanized antibody or fragment thereof according to the present invention 1014, wherein at least two CDRs are CDRs having the amino acid sequences of SEQ ID NO: 4 representing CDR1 and SEQ ID NO: 6 representing CDR3 of the light chain variable region (LCVR).
[Invention 1028]
The humanized antibody or fragment thereof according to the present invention 1012, wherein at least two CDRs are selected from the group of CDRs according to any one of the inventions 1023-1027.
[Invention 1029]
A humanized antibody or fragment thereof according to any of 1009-1028 of the present invention, comprising a constant region of a light chain and / or a heavy chain of human or primate origin.
[Invention 1030]
A chimeric antibody or a chimeric antibody thereof in which at least one of the amino acids in the CDR regions of the light and heavy chains shown in SEQ ID NOs: 1 to 6 is changed by conservative substitution so that the antibody maintains its full functionality. Fragments or humanized antibodies or fragments thereof.
[Invention 1031]
Within the amino acid sequence of CDR2 of the light chain variable region (LCVR) shown in SEQ ID NO: 5, Lys at Kabat position 50 is replaced by Arg, Gln, or Glu, especially by Arg, and / or Asn at Kabat position 53 is Ala. , Val, Leu, Ser, and Ile; among other things, the chimeric antibody or fragment thereof or humanized antibody or fragment thereof according to the present invention 1030, which is replaced by Ser.
[Invention 1032]
The humanized antibody or fragment thereof according to any one of the present inventions 1009 to 1031, wherein the framework sequence is the human germline V _K sequence of the KABAT subgroup V _K II.
[Invention 1033]
The humanized antibody or fragment thereof according to any one of 1009 to 1031 of the present invention, wherein the framework sequence is the human germline V _H sequence of the KABAT subgroup V _H III.
[Invention 1034]
The present invention comprises altering at least one of the amino acids in a human or primate-derived framework region by substitution with or conservatively substituted with an amino acid from the corresponding region of the mouse humanized antibody ACI-01-Ab7C2. The humanized antibody or fragment thereof according to any one of 1001 to 1033.
[Invention 1035]
A book that replaces Trp at Kabat position 47 in the framework region of heavy chain variable regions of human or primate origin with amino acids selected from the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe. The humanized antibody or fragment thereof according to Invention 1034.
[Invention 1036]
Arg at Kabat position 94 in the framework region of the heavy chain variable region of human or primate origin shown in SEQ ID NO: 15 is replaced by an amino acid selected from the group consisting of Ser and Thr, particularly Ser, the present invention 1034. The humanized antibody or fragment thereof described.
[Invention 1037]
Trp at Kabat position 47 in the framework region of the heavy chain variable region of human or primate origin shown in SEQ ID NO: 15 was selected from the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe. Arg at Kabat position 94 in the framework region of the heavy chain variable region of human or primate origin, which is replaced by amino acids, especially Leu and Ile, especially Leu, and is set forth in SEQ ID NO: 15, consisting of Ser and Thr. The humanized antibody or fragment thereof according to the present invention 1034, which is replaced by a more selected amino acid, especially Ser.
[Invention 1038]
Tyr at Kabat position 87 in the framework region of the light chain variable region of human or primate origin shown in SEQ ID NO: 12 by an amino acid selected from the group consisting of Phe, Leu, Val, Ile, and Ala. The humanized antibody or fragment thereof according to the present invention 1034, which is particularly replaced by Leu and Phe, in particular by Phe.
[Invention 1039]
Trp at Kabat position 47 in the framework region of the heavy chain variable region of human or primate origin shown in SEQ ID NO: 15 was selected from the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe. Arg of Kabat position 94 in the framework region of the heavy chain variable region of human or primate origin shown in SEQ ID NO: 15, which is replaced by amino acids, especially Leu and Ile, especially Leu, consisting of Ser and Thr. Tyr at Kabat position 87 in the framework region of the light chain variable region of human or primate origin shown in SEQ ID NO: 12, replaced by more selected amino acids, especially Ser, in Phe, Leu, Val, Ile, The humanized antibody or fragment thereof according to the present invention 1034, which is replaced by an amino acid selected from the group consisting of and Ala, particularly by Leu and Phe, especially by Phe.
[Invention 1040]
Trp at Kabat position 47 in the framework region of the heavy chain variable region of human or primate origin shown in SEQ ID NO: 15 was selected from the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe. The humanized antibody or fragment thereof according to the present invention 1035, which is replaced by amino acids, especially Leu and Ile, especially Leu.
[Invention 1041]
The present invention replaces Arg at Kabat position 94 in the framework region of the heavy chain variable region of human or primate origin shown in SEQ ID NO: 15 with an amino acid selected from the group consisting of Ser and Thr, especially by Ser. 1036 The humanized antibody or fragment thereof.
[Invention 1042]
The invention 1037 replaces Trp at Kabat position 47 in the framework region of the heavy chain variable region of human or primate origin shown in SEQ ID NO: 15 with an amino acid selected from the group consisting of Leu and Ile, especially Leu. The humanized antibody or fragment thereof described.
[Invention 1043]
The humanized antibody or fragment thereof according to the present invention 1038, wherein the Tyr at Kabat position 87 in the framework region of the light chain variable region of human or primate origin shown in SEQ ID NO: 12 is replaced by Phe.
[Invention 1044]
SEQ ID NO: 15: Trp at Kabat position 47 in the framework region of the human or primate-derived heavy chain variable region shown in 15 is replaced with an amino acid selected from the group consisting of Leu and Ile, especially Leu, and SEQ ID NO: Arg at Kabat position 94 in the framework region of the heavy chain variable region of human or primate origin shown in: 15 is replaced by Ser, and the light chain variable region of human or primate origin shown in SEQ ID NO: 12 The humanized antibody or fragment thereof according to the present invention 1039, wherein the Tyr at Kabat position 87 in the framework region is replaced by Phe.
[Invention 1045]
1. Chimera antibody or fragment thereof or humanized antibody or fragment thereof.
[Invention 1046]
Described in any of 1001-1044 of the invention, wherein the light chain variable region (LCVR) has an amino acid sequence that is 90%, particularly 95%, more particularly 98% identical to the sequences set forth in SEQ ID NOs: 12 and 13, respectively. Chimera antibody or fragment thereof or humanized antibody or fragment thereof.
[Invention 1047]
Amino acids in which at least two, especially three, of the CDR regions of a heavy chain variable region (HCVR) are 90%, particularly 95%, and more particularly 98% identical to the corresponding CDR regions shown in SEQ ID NOs: 1-3. The humanized antibody or fragment thereof according to any one of the present inventions 1001 to 1044 having a sequence.
[Invention 1048]
Amino acids in which at least two, especially three, of the light chain variable region (LCVR) CDR regions are 90%, particularly 95%, and more particularly 98% identical to the corresponding CDR regions set forth in SEQ ID NOs: 4-6. The humanized antibody or fragment thereof according to any one of the present inventions 1001 to 1044 having a sequence.
[Invention 1049]
A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof, comprising the light chain variable region of SEQ ID NO: 13.
[Invention 1050]
The chimeric antibody or fragment thereof or humanized antibody or fragment thereof according to the present invention 1049, further comprising the light chain constant region of SEQ ID NO: 14.
[Invention 1051]
A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof, comprising a light chain variable region of SEQ ID NO: 13 and a light chain constant region of SEQ ID NO: 14.
[Invention 1052]
A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof, which comprises a heavy chain variable region of SEQ ID NO: 16.
[Invention 1053]
The chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to 1052 of the present invention, further comprising a heavy chain constant region of SEQ ID NO: 17.
[Invention 1054]
A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof, comprising a heavy chain variable region of SEQ ID NO: 16 and a heavy chain constant region of SEQ ID NO: 17.
[Invention 1055]
The chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to any one of 1050, 1051, 1053, and 1054 of the present invention, wherein the N-terminal Lys of the heavy chain constant region has been removed.
[Invention 1056]
The chimeric antibody or fragment thereof or humanized antibody or fragment thereof according to any one of the present inventions, wherein the antibody is an _IgG4 isotype comprising IgG1, IgG2, IgG3, or mutated IgG4, in particular an IgG4 isotype.
[Invention 1057]
Any of the inventions described above, wherein the antibody is bi-effective in that it exhibits both aggregation inhibitory and deaggregation properties, especially in combination with a high degree of conformational sensitivity. The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to the above.
[Invention 1058]
Β-Amyloid monomer peptides such as, for example, Aβ monomeric peptides 1-39; 1-40, 1-41 or 1-42, with soluble amyloid peptides of amyloid monomers and / or polymers. And / or a polymer containing the Aβ monomer unit of multiples Aβ polymer containing a soluble β-amyloid peptide, particularly Aβ _1-42 monomer and / or the Aβ _1-42 monomer unit of Aβ 1-42. By co-incubation with a soluble amyloid peptide, the antibody inhibits the aggregation of the Aβ monomer to the fibrils or filaments of the polymer polymer and the amyloid monomer peptide, in particular the Aβ monomer peptide 1-. 39; fibrils of preformed high molecular weight polymer amyloids formed by aggregation of β-amyloid monomeric peptides such as 1-40, 1-41, or 1-42, especially Aβ _1-42 monomeric peptides. Alternatively, by co-incubation with the filament, the antibody can further disaggregate the fibrils or filaments of the preformed polymer.
The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to any one of the present inventions.
[Invention 1059]
The chimeric antibody or fragment thereof or humanized antibody or fragment thereof according to any one of the present inventions, which substantially binds to Aβ aggregated in a mammalian brain, particularly a human brain.
[Invention 1060]
The chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to any one of the present inventions, which reduces the Aβ plaque load in a mammalian brain, particularly a human brain.
[Invention 1061]
A nucleic acid molecule comprising the nucleotide sequence encoding the chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to any one of 1001 to 1060 of the present invention.
[Invention 1062]
Nucleic acid molecule comprising a nucleotide sequence encoding an antibody variable region comprising SEQ ID NOs: 2 and 3 representing complementarity determining regions (CDRs) 2 and 3 of heavy chain variable regions (HCVR).
[Invention 1063]
A nucleic acid molecule comprising a nucleotide sequence encoding an antibody variable region comprising SEQ ID NO: 4, representing complementarity determining regions (CDRs) 1 of the light chain variable region (LCVR).
[Invention 1064]
The nucleic acid molecule of the present invention 1062, comprising the nucleotide sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 19.
[Invention 1065]
The nucleic acid molecule of the present invention 1063, comprising the nucleotide sequence set forth in SEQ ID NO: 20.
[Invention 1066]
Nucleic acid molecule containing the nucleotide sequence encoding the light chain variable region of SEQ ID NO: 22.
[Invention 1067]
A nucleic acid molecule comprising a nucleotide sequence encoding a light chain variable region of SEQ ID NO: 22 and a light chain constant region of SEQ ID NO: 23.
[Invention 1068]
Nucleic acid molecule containing the nucleotide sequence encoding the heavy chain variable region of SEQ ID NO: 25.
[Invention 1069]
A nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable region of SEQ ID NO: 25 and a heavy chain constant region of SEQ ID NO: 26.
[Invention 1070]
An expression vector containing the nucleotide sequence according to any one of the present inventions 1060 to 1069.
[Invention 1071]
A cell comprising the expression vector according to any one of 1060 to 1068 of the present invention or the expression vector according to 1070 of the present invention.
[Invention 1072]
A composition comprising an antibody according to any of 1001-1060 of the present invention comprising a therapeutically effective amount of any functionally equivalent antibody or functional portion thereof.
[Invention 1073]
The composition of the present invention 1072, which is a pharmaceutical composition optionally further comprising a pharmaceutically acceptable carrier.
[Invention 1074]
The composition according to any of 1072 to 1073 of the present invention, comprising a therapeutically effective amount of the antibody.
[Invention 1075]
The chimeric antibody or fragment thereof or humanized antibody or fragment thereof according to any one of 1001 to 1060 of the present invention, which comprises a therapeutically effective amount of any functionally equivalent antibody or functional portion thereof, and optionally further organisms. A mixture comprising an active substance and / or a pharmaceutically acceptable carrier and / or a diluent and / or an excipient.
[Invention 1076]
The mixture according to the invention 1075, wherein the further bioactive substance is a compound used in the treatment of amyloid or amyloidosis, a group of diseases and disorders associated with amyloid or amyloid-like proteins, such as the Aβ protein involved in Alzheimer's disease.
[Invention 1077]
DNA repair of compounds of the invention and optionally pharmaceutically acceptable carriers and / or diluents and / or excipients, such as compounds against oxidative stress, anti-aplastic compounds, metal chelating agents, pyrenzepine and metabolites. Inhibitors, 3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS), α-secretase activators, β- and γ-secretase inhibitors, tau proteins, nerves Transmitter, β-sheet breaker, attractant of cellular components that remove / deplete amyloid β, inhibitor of N-terminal cleavage type amyloid β including pyroglutamine-oxidized amyloid β3-42, anti-inflammatory molecule, Or at least selected from the group consisting of cholineresterase inhibitors (ChEI) such as taclin, rivastigmin, donepezil, and / or galantamine, M1 agonists, and other drugs including any amyloid or tau modifiers, as well as nutritional supplements. The mixture according to the present invention 1076, which comprises one compound.
[Invention 1078]
The mixture according to the present invention 1077, wherein the compound is a cholinesterase inhibitor (ChEI).
[Invention 1079]
The mixture according to any one of the present inventions 1075 to 1077, wherein the compound is a compound selected from the group consisting of tacrine, rivastigmine, donepezil, galantamine, niacin, and memantine.
[Invention 1080]
The mixture according to any of the present inventions 1075-1079, comprising a therapeutically effective amount of an antibody and / or a bioactive substance.
[Invention 1081]
Alzheimer's disease (AD), Levy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guam Parkinson-neurological disorders such as dementia complex; as well as progressive nuclear paralysis, multiple sclerosis Diseases; Kreuzfeld Jacob's disease, Parkinson's disease, HIV-related dementia, ALS (muscle atrophic lateral sclerosis), adult-onset diabetes; senile heart amyloidosis; endocrine tumors, and other diseases including luteal degeneration. A group associated with amyloid plaque formation, including, but not limited to, secondary amyloidosis and age-related amyloidosis, including, but not limited to, other diseases based on or associated with amyloid-like protein. A chimeric antibody or fragment thereof, or a humanized antibody or fragment thereof, and / or any of 1001 to 1060 of the present invention for preparing a drug for treating or alleviating the effects of amyloidosis, which is a disease and disorder of the disease. Use of the functional portion thereof and / or the pharmaceutical composition according to any one of 1072 to 1074 of the present invention, or the mixture according to any of 1075 to 1080 of the present invention.
[Invention 1082]
The use according to the invention 1081, wherein the amyloid-related disease or condition is Alzheimer's disease.
[Invention 1083]
The use of the present invention 1081, wherein the amyloid-related condition is characterized by a loss of cognitive memory capacity in animals, particularly mammals or humans.
[Invention 1084]
The use of the present invention 1081, wherein treatment of an animal, particularly a mammal or a human, suffering from an amyloid-related condition characterized by loss of cognitive memory capacity results in an increase in cognitive memory capacity.
[Invention 1085]
The use of the present invention 1081, wherein treatment of an animal, particularly a mammal or a human, suffering from an amyloid-related condition characterized by loss of cognitive memory capacity results in a complete recovery of cognitive memory capacity.
[Invention 1086]
Alzheimer's disease (AD), Levy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guam Parkinson-neurological disorders such as dementia complex; as well as progressive nuclear paralysis, multiple Sclerosis; Kreuzfeld Jacob's disease, Parkinson's disease, HIV-related dementia, ALS (muscle atrophic lateral sclerosis), adult-onset diabetes; senile heart amyloidosis; endocrine tumors, and other diseases including luteal degeneration, etc. A group of diseases associated with amyloid plaque formation, including, but not limited to, secondary amyloidosis and age-related amyloidosis, including, but not limited to, other diseases based on or associated with amyloid-like protein. Either the pharmaceutical composition according to any one of 1072 to 1074 of the present invention or 1075 to 1080 of the present invention for use in a method for preventing, treating, or alleviating the effects of amyloidosis, which is a disorder. A method for preparing the mixture according to the above, comprising the step of formulating the antibody of the present invention in a pharmaceutically acceptable form.
[Invention 1087]
The method of the present invention 1086, wherein the antibody is included in the composition in a therapeutically effective amount.
[Invention 1088]
The method of the present invention 1086, wherein the amyloid-related disease or condition is Alzheimer's disease.
[Invention 1089]
The method of the present invention 1086, wherein the amyloid-related condition is characterized by a loss of cognitive memory capacity in animals, particularly mammals or humans.
[Invention 1090]
The method of the invention 1086, wherein treatment of an animal, particularly a mammal or human, suffering from an amyloid-related condition characterized by loss of cognitive memory capacity results in an increase in cognitive memory capacity.
[Invention 1091]
The method of the present invention 1086, wherein the amyloid-related condition impairs cognitive memory capacity in animals, particularly mammals or humans.
[Invention 1092]
Treatment of animals, especially mammals or humans, suffering from amyloid-related conditions characterized by loss of cognitive memory capacity reverses cognitive memory capacity and, in particular, complete recovery of cognitive memory capacity. Bringing, the method of the present invention 1086.
[Invention 1093]
Alzheimer's disease (AD), Levy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guam Parkinson-neurological disorders such as dementia complex; as well as progressive nuclear paralysis, multiple Sclerosis; Kreuzfeld Jacob's disease, Parkinson's disease, HIV-related dementia, ALS (muscle atrophic lateral sclerosis), adult-onset diabetes; senile heart amyloidosis; endocrine tumors, and other diseases including luteal degeneration, etc. A group of diseases associated with amyloid plaque formation, including, but not limited to, secondary amyloidosis and age-related amyloidosis, including, but not limited to, other diseases based on or associated with amyloid-like protein. The effects of the disorder, amyloidosis, according to any of 1001 to 1060 of the present invention, or a fragment thereof or a humanized antibody or a fragment thereof, and / or a functional portion thereof, and / or 1072 to 1074 of the present invention. Preventing, treating, or treating by administering to any animal or human affected by such a disorder the pharmaceutical composition according to any, or the mixture according to any of the present invention 1075-1080. A method of alleviating, comprising the step of administering the antibody in a therapeutically effective amount.
[Invention 1094]
The method of the present invention 1093, wherein the amyloid-related disease or condition is Alzheimer's disease.
[Invention 1095]
The method of the present invention 1093, wherein the amyloid-related condition is characterized by a loss of cognitive memory capacity in animals, particularly mammals or humans.
[Invention 1096]
The method of the present invention 1093, wherein treatment of an animal, particularly a mammal or human, suffering from an amyloid-related condition characterized by loss of cognitive memory capacity results in increased retention of cognitive memory capacity.
[Invention 1097]
The method of the present invention 1093, wherein the amyloid-related condition impairs cognitive memory capacity in animals, particularly mammals or humans.
[Invention 1098]
The method of the present invention 1093, wherein treatment of an animal, particularly a mammal or human, suffering from an amyloid-related condition characterized by loss of cognitive memory capacity results in a complete recovery of cognitive memory capacity.
[Invention 1099]
A method of diagnosing an amyloid-related disease or condition in a patient.
(a) A step of contacting a sample suspected to contain amyloid protein or a specific body part or body part with the antibody according to any one of 1001 to 1060 of the present invention;
(b) Step of binding the antibody to an amyloid protein;
(c) Steps to detect antibodies bound to the protein; and
(d) A method comprising correlating the presence or absence of antibody binding with the presence or absence of amyloid protein in a sample or specific body part or site.
[Invention 1100]
A method of determining the degree of amyloid formative burden in tissues and / or body fluids.
(a) Steps to obtain a sample representative of the tissue and / or body fluid under investigation;
(b) A step of inspecting the sample for the presence of an amyloid protein using the antibody according to any one of the present inventions 1001 to 1060;
(c) Steps to determine the amount of antibody bound to the protein;
(d) A method comprising the step of calculating the plaque load in the tissue and / or body fluid.
[Invention 1101]
A test kit for detecting and diagnosing amyloid-related diseases and conditions, which comprises the antibody according to any one of the present inventions 1001 to 1060.
[Invention 1102]
A container containing one or more antibodies according to the present invention, and an immunological complex formed by binding to the amyloid protein so that the presence or absence of the immunological complex correlates with the presence or absence of the amyloid protein. The test kit of the present invention 1101, which comprises an instruction manual for using the antibody for the purpose of detecting the formation of a scientific complex.
[Invention 1103]
A framework region of human or primate origin, and at least one CDR with the amino acid sequence of SEQ ID NO: 4, representing CDR1 of the light chain variable region (LCVR).
Light chain variable region (LCVR), including.
[Invention 1104]
At least one amino acid sequence selected from the group consisting of a framework region derived from humans or primates, and a sequence number consisting of SEQ ID NO: 2 representing CDR2 and SEQ ID NO: 3 representing CDR3 in the heavy chain variable region (HCVR). Two CDRs
Heavy chain variable region (HCVR), including.
[Invention 1105]
A sequence consisting of a heavy chain framework region derived from humans or primates, and a heavy chain variable region (HCVR) consisting of SEQ ID NO: 1 representing CDR1, SEQ ID NO: 2 representing CDR2, and SEQ ID NO: 3 representing CDR3. At least two CDRs with amino acid sequences selected from the group
Heavy chain variable region (HCVR), including.
[Invention 1106]
A sequence consisting of a light chain framework region of human or primate origin, and a light chain variable region (LCVR) of SEQ ID NO: 4, representing CDR1, SEQ ID NO: 5, representing CDR2, and SEQ ID NO: 6 representing CDR3. At least two CDRs with amino acid sequences selected from the group
Light chain variable region (LCVR), including.
[Invention 1107]
Heavy chain framework regions of human or primate origin, and in particular in the order shown above, represent CDR1 of the heavy chain variable region (HCVR), SEQ ID NO: 1, Representing CDR2, and SEQ ID NO: 2, and CDR3. CDR with the amino acid sequence of SEQ ID NO: 3
Heavy chain variable region (HCVR), including.
[Invention 1108]
Incorporated human or primate-derived light chain framework regions and light chain variable regions (LCVRs), SEQ ID NO: 4 for CDR1, SEQ ID NO: 5 for CDR2, and SEQ ID NO: 6 for CDR3. CDRs having the amino acid sequences of
Light chain variable region (LCVR), including.
[Invention 1109]
SEQ ID NO: 12 light chain variable region (LCVR).
[Invention 1110]
Provided is a humanized antibody or fragment thereof comprising the light chain variable region (LCVR) of SEQ ID NO: 12.
[Invention 1111]
Light chain variable region (LCVR) containing the signal sequence shown in SEQ ID NO: 13.
[Invention 1112]
A humanized antibody or fragment thereof comprising a complete light chain variable region (LCVR) comprising the signal sequence set forth in SEQ ID NO: 13.
[Invention 1113]
A humanized antibody or fragment thereof comprising the light chain variable region (LCVR) of SEQ ID NO: 12 and the light chain constant region of SEQ ID NO: 14.
[Invention 1114]
A humanized antibody or fragment thereof comprising the complete light chain variable region (LCVR) of SEQ ID NO: 13 and the light chain constant region of SEQ ID NO: 14.
[Invention 1115]
Heavy chain variable region (HCVR) of SEQ ID NO: 15.
[Invention 1116]
A humanized antibody or fragment thereof comprising the heavy chain variable region (HCVR) of SEQ ID NO: 15.
[Invention 1117]
Heavy chain variable region (HCVR) containing the signal sequence shown in SEQ ID NO: 16.
[Invention 1118]
A humanized antibody or fragment thereof comprising a complete heavy chain variable region (HCVR) containing the signal sequence set forth in SEQ ID NO: 16.
[Invention 1119]
A humanized antibody or fragment thereof comprising a heavy chain variable region (HCVR) of SEQ ID NO: 15 and a heavy chain constant region of SEQ ID NO: 17.
[Invention 1120]
A humanized antibody or fragment thereof comprising a heavy chain variable region (HCVR) of SEQ ID NO: 16 and a heavy chain constant region of SEQ ID NO: 17.
[Invention 1121]
The chimeric antibody or fragment thereof according to any of the present inventions, which substantially binds to the Aβ monomer but preferably does not show any significant cross-reactivity with amyloid precursor protein (APP). Humanized antibody or fragment thereof.
[Invention 1122]
At least about 1 × 10 ^-7 M to at least about 1 × 10 ^-12 M, especially at least about 1 × 10 ^-8 M to at least about 1 × 10 ^-11 M, and more particularly at least about 1 × 10 ^-8 M. To Aβ monomers with a binding affinity represented by KD in the range of at least about 1 × 10 ^-10 _M , and more particularly at least about 1 × 10 ^-8 M to at least about 5 × 10 ^-8 M. The chimeric antibody or fragment thereof or humanized antibody or fragment thereof according to 1122 of the present invention that binds but preferably exhibits no significant cross-reactivity with amyloid precursor protein (APP).
[Invention 1123]
The chimeric antibody according to any of the present invention, which substantially binds to a fiber, fibril, or filament of Aβ, but preferably exhibits no significant cross-reactivity with amyloid precursor protein (APP). Or a fragment thereof or a humanized antibody or a fragment thereof.
[Invention 1124]
At least about 1 × 10 ^-7 M to at least about 1 × 10 ^-12 M, especially at least about 1 × 10 ^-8 M to at least about 1 × 10 ^-11 M, and more particularly at least about 1 × 10 ^-9 M. Aβ fibers, fibrils with binding affinity represented by KD in the range of at least about 1 × 10 ^-10 _M , and more particularly at least about 2 × 10 ^-9 M to at least about 8 × 10 ^-9 M. , Or a fragment thereof or a humanized antibody or fragment thereof according to 1123 of the present invention, which binds to a filament, but preferably shows no significant cross-reactivity with amyloid precursor protein (APP).
[Invention 1125]
The chimeric antibody or fragment thereof according to any one of the present inventions, which substantially binds to the soluble polymer amyloid β but preferably does not exhibit any significant cross-reactivity with amyloid precursor protein (APP). Or a humanized antibody or fragment thereof.
[Invention 1126]
Substantially binds to Aβ monomer and / or Aβ fibers, fibrils, or filaments, and / or soluble polymer amyloid β, but is preferably markedly cross-reactive with amyloid precursor protein (APP). The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to any one of the present inventions, which does not show any of the above.
[Invention 1127]
The chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to 1126 of the present invention, in which the binding affinity increases in ascending order.
[Invention 1128]
The present invention exhibits a binding affinity for Aβ fibers, fibrils, or filaments that is at least 10-fold, particularly at least 15-fold, more particularly at least 20-fold, and particularly at least 25-fold higher than the binding affinity for Aβ monomers. 1127 The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof.
[Invention 1129]
Substantially binding to aggregated Aβ, including Aβ plaques in mammalian, especially human brain, but preferably showing no significant cross-reactivity with amyloid precursor protein (APP), said invention. The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to any one of the above.
[Invention 1130]
Substantially binding to soluble fibers containing Aβ monomers in mammalian, especially human brain, but preferably showing no significant cross-reactivity with amyloid precursor protein (APP), said. A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to any one of the present invention.
[Invention 1131]
By inducing a conformational transformation, the fibers are deaggregated to soluble polymer and monomeric forms and bound to deaggregated and solubilized Aβ forms in tissues and / or body fluids, especially in the brain. Moreover, according to any one of the present inventions, the equilibrium between the soluble state of Aβ and the aggregated state of Aβ can be transferred to the soluble state by stabilizing the Aβ form. Chimeric antibody or fragment thereof or humanized antibody or fragment thereof.
[Invention 1132]
β- to α-helix and / or random coil conformations, especially random coil conformations, and even more particularly at given locations in the molecule, especially in the environment of Tyr 10 and Val 12 for Aβ proteins. Antibodies or fragments thereof capable of inducing transfer of sheet conformation, with increased random coil conformation at the expense of β-sheet conformation, and fibrils or filaments of preformed high molecular weight polymer amyloid. The chimeric antibody or fragment thereof or humanized antibody or fragment thereof according to 1131 of the present invention, which results in improved solubilization.
[Invention 1133]
The reduction in β-sheet conformation is at least 30%, especially at least 35%, and more particularly at least compared to the fibrils or filaments (controls) of each preformed amyloid polymer incubated in buffer. The chimeric antibody or fragment thereof or humanized antibody or fragment thereof according to 1132 of the present invention, which reaches 40% or more.
[Invention 1134]
Substantially binds to soluble Aβ protein species that are polymeric and less aggregated, including monomeric forms, thus modifying the peripheral removal and catabolism of these Aβ protein species and their toxic effects. The chimeric antibody or fragment thereof according to 1131 of the present invention or a humanized antibody or fragment thereof, which is to be lowered.
[Invention 1135]
At least one light chain or fragment thereof or at least one heavy chain incorporating at least one, particularly two, and more particularly three CDR regions obtained from a mouse donor antibody, particularly from the mouse antibody ACI-01-Ab7C2. Antibodies containing the fragments or fragments thereof that have at least 5-fold, particularly at least 8-fold, more particularly at least 10-fold, especially at least 15-fold higher affinity for Aβ antigens than mouse donor antibodies. The humanized antibody or fragment thereof according to any one of the present inventions having.
[Invention 1136]
The use according to any of the inventions 1083-1085, wherein the humanized antibody or fragment thereof does not necessarily bind to aggregated amyloid β in the brain.
[Invention 1137]
The method of any of the present inventions 1089-1091, wherein the humanized antibody or fragment thereof does not necessarily bind to aggregated amyloid β in the brain.
[Invention 1138]
The use of the present invention 1081, wherein treatment of an animal, particularly a mammal or a human, suffering from an amyloid-related condition characterized by loss of cognitive memory capacity results in retention of cognitive memory capacity.
[Invention 1139]
The method of the invention 1086, wherein treatment of an animal, particularly a mammal or human, suffering from an amyloid-related condition characterized by loss of cognitive memory capacity results in retention of cognitive memory capacity.
[Invention 1140]
A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to any one of the present inventions, wherein the equilibrium between the soluble Aβ and the aggregated Aβ can be transferred to its soluble form. ..
[Invention 1141]
By deaggregating the fibers to soluble polymer and monomeric forms, the equilibrium between soluble Aβ and aggregated Aβ can be transferred to that soluble form. A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to any one of the inventions.
[Invention 1142]
Although it does not substantially bind to Aβ in human tissues and / or body fluids, especially in the brain, it induces conformational transformations to disaggregate fibers into soluble polymer and monomeric forms and tissue. And / or the equilibrium between the soluble and aggregated Aβ by binding to and / or stabilizing the deaggregated and solubilized Aβ morphology in body fluids, especially in the brain. The chimeric antibody or fragment thereof or humanized antibody or fragment thereof according to any one of the present inventions, which is capable of transferring the antibody to its soluble form.
[Invention 1143]
The humanized antibody according to any one of the present inventions, which exhibits a binding affinity that is at least 5-fold, 10-fold, particularly at least 15-fold, more particularly at least 20-fold, and particularly at least 100-fold higher than the binding affinity of a mouse antibody. Or a fragment of it.
[Invention 1144]
The humanized antibody or fragment thereof according to any of the present invention, wherein the affinity, specificity, and stability of the antibody or fragment thereof are modified by changes in the glycosylation profile.
[Invention 1145]
An antibody or variant thereof comprising the variable region listed in SEQ ID NO: 27.
[Invention 1146]
A cell line expressing the antibody according to the present invention 1145.
[Invention 1147]
An antibody gene or variant thereof, comprising the variable region set forth in SEQ ID NO: 29.
[Invention 1148]
A cell line expressing the antibody according to the present invention 1147.
[Invention 1149]
A method for deagglomerating preformed β-amyloid fibrils, comprising the step of interacting the hC2 antibody with preformed β-amyloid fibrils.
[Invention 1150]
A humanized antibody or fragment thereof according to any of the present inventions, which protects neurons from Aβ-induced degradation.
[Invention 1151]
A method of preventing Aβ-induced neuronal degradation, comprising treating a neuron with an effective amount of the humanized antibody or fragment thereof according to any of the present invention.
[Invention 1152]
Use of a humanized antibody or fragment thereof according to any of the inventions described above for the preparation of a pharmaceutical for preventing neuronal degeneration due to exposure to Aβ oligomers.

(Example 2) Expression cassette of mouse light chain variable region of chimeric antibody. (Example 2) Expression cassette of mouse heavy chain variable region of chimeric antibody. (Example 5.2) Comparison of the mouse heavy chain variable region with the closest mouse germline sequence. (Example 8) Activity of purified humanized C2 antibody. (Example 9) Of the antibody produced by transient expression of the C2 modified CDRL2 construct linked to the C2 chimeric heavy chain compared to the chimeric antibody C2ChVHAF / ChVK and the purified antibody produced by transient transfection. Binding activity. (Example 11) Results of immunohistochemical binding assay using chimeric antibody AF and humanized antibody H4K1. (Example 12) Functionality of mC2 for amyloid fibers. (Example 12) Binding affinity of humanized C2 in ELISA. (Example 14) Three-dimensional structure-specific binding of mC2 to different classes of amyloid proteins. The pellet preparation in the description for this figure refers to Aβ _1-42 fibers and the supernatant preparation refers to amyloid monomers. Humanized C2 VK sequences compared to mouse and human acceptor sequences DPK 15 and J _K 1. Humanized C2 _VH sequences compared to mouse and human acceptor sequences DP54 and J H6. Complete DNA and protein sequences of the light chain variable region of the C2 humanized antibody, C2HuVK1. Complete DNA and protein sequences of the light chain constant region (human C kappa) of humanized C2 antibody. FIG. 13-2 is a diagram showing the continuation of FIG. 13-1. FIG. 13-3 is a diagram showing the continuation of FIG. 13-2. FIG. 13-4 is a diagram showing the continuation of FIG. 13-3. FIG. 13-5 is a diagram showing the continuation of FIG. 13-4. Complete DNA and protein sequences of the heavy chain constant region of humanized C2 antibody (human IgG4 ser228-pro). FIG. 14-2 is a diagram showing the continuation of FIG. 14-1. Figures 15A-15C are the results of the (Example 15) epitope mapping experiment. (Example 13) Results of aggregation assay experiment. (Example 13) Results of deagglomeration assay experiment. (Example 16) Results of a neuroprotective experiment using the humanized antibody C2.

Definitions The terms "polypeptide,""peptide," and "protein," as used herein, are defined to mean biomolecules that are compatible and consist of amino acids linked by peptide bonds. To.

As used herein, the terms "one (a)", "one (an)", and "the" mean "one or more" and if the context is inappropriate. Except for, it is defined as including multiple.

The term "disease and disorder caused by or associated with amyloid or amyloid-like protein" refers to the state of a monomer, fibril, or polymer, or any combination of three amyloid-like. Diseases and disorders caused by the presence or activity of proteins include, but are not limited to. Such diseases and disorders include, but are not limited to, amyloidosis, endocrine tumors, and macular degeneration.

The term "amyloidosis" includes Alzheimer's disease (AD), Levy body dementia, including diseases or conditions characterized by cognitive loss of memory, such as mild cognitive impairment (MCI). Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); neurological disorders such as Guam Parkinson-dementia complex; and progressive nuclear palsy, multiple sclerosis; Kreuzfeld-Jakob disease, Parkinson's disease, HIV-related Includes dementia, ALS (muscle atrophic lateral sclerosis), inclusion myopathy (IBM), adult-onset diabetes, and senile heart amyloidosis; and jaundice degeneration, druse-related optic neuropathy, and cataracts due to β-amyloid deposition Includes, but is not limited to, secondary amyloidosis such as disease and age-related amyloidosis, including, but not limited to, other diseases based on or associated with amyloid-like protein such as various eye diseases. Refers to a group of diseases and disorders associated with amyloid plaque formation, not limited to these.

As used herein, the term "detecting" or "detected" means using known methods for the detection of biomolecules, such as immunochemical or histological methods, under investigation. Refers to qualitatively or quantitatively determining the presence or concentration of biomolecules.

A "polymer-soluble amyloid" is an amyloid peptide or amyloid-like peptide that is soluble in the mammalian or human body, and more particularly in the brain, or a modified or cleaved amyloid peptide, or an oligomeric structure or polymer. Refers to multiple aggregated monomers of other derivatives of amyloid peptides that form the structure and are soluble in, especially in the mammalian or human body, more particularly in the brain, amyloid β (Aβ) or modified or cleaved. Refers to a plurality of aggregated monomers of the amyloid β (Aβ) peptide or a derivative thereof.

"Amyloid β, Aβ or β-amyloid" is a term recognized in the art, amyloid β protein and peptide, amyloid β precursor protein (APP), and modifications, fragments and any of them. Refers to a functional equivalent. In particular, the amyloid β used herein is any fragment produced by proteolytic cleavage of APP, in particular Aβ _1-38 , Aβ _1-39 , Aβ _1-40 , Aβ _1-41 Aβ _1-42 . , And, but not limited to, Aβ _1-43 , meaning fragments involved in or associated with amyloid pathology.

The structures and sequences of the amyloid β peptides described above are well known to those of skill in the art, and methods of producing the peptides or extracting them from the brain and other tissues are described, for example, in Glenner and Wong, Biochem Biophys Res Comm 129, 885. -890 (1984). In addition, amyloid β peptide is also commercially available in various forms.

By "isolated" is meant a biomolecule that does not contain at least some of its naturally occurring components.

As used herein, the term "antibody" or "antibody" is a term recognized in the art and is a molecule or active fragment of a molecule that binds to a known antigen, in particular an immunoglobulin molecule and an immunoglobulin molecule. Is interpreted to refer to the immunologically active portion of the molecule, i.e., a molecule containing a binding site that specifically binds to an antigen. Immunoglobulins are one or more polypeptides that are substantially encoded by a myriad of immunoglobulin variable region genes, as well as immunoglobulin kappa and lambda, alpha, gamma, delta, epsilon, and mu constant region genes. It is a protein containing. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, respectively, which in turn define immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE. Heavy chain subclasses are also known. For example, an IgG heavy chain in humans can be one of the IgG1, IgG2, IgG3, and IgG4 subclasses. The immunoglobulins according to the invention can be any class (IgG, IgM, IgD, IgE, IgA, and IgY) or subclasses (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules.

As used herein, "specifically binding" with respect to an antibody means that the antibody binds to its target antigen with greater affinity than it binds to a structurally different antigen.

Typical immunoglobulin structural units are known to contain tetramers. Each tetramer is composed of two identical polypeptide chain pairs, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each strand defines a variable region of about 100-110 or more amino acids that are primarily involved in antigen recognition. The terms variable light chain ( _VL ) and variable heavy chain (V _H ) refer to these light chains and heavy chains, respectively.

Antibodies are present as full-length unchanged antibodies or as many well-characterized fragments produced by digestion with various peptidases or chemicals. Thus, for example, pepsin is a dimer of Fab, a light chain that digests an antibody below the disulfide bond in the hinge region and is itself linked to V _H -CH ₁ by a disulfide bond, F (ab'). Produces ₂ . F (ab') ₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F (ab') ₂ dimer to a Fab'monomer. Fab'monomers are Fab fragments that essentially have part of the hinge region (see Fundamental Immunology, WE Paul, Raven Press, NY (1993) for a more detailed description of other antibody fragments). .. Although various antibody fragments are defined in terms of digestion of unchanged antibodies, one of ordinary skill in the art will either chemically or by utilizing recombinant DNA methodologies. It is thought that one of them correctly understands that de novo synthesis is possible. Accordingly, as used herein, the term antibody refers to an antibody fragment that is either produced by modification of the entire antibody or synthesized by de novo, or an antibody obtained by using recombinant DNA methodologies. And fragments are also included.

"Antibodies" shall include, within the scope of the invention, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single-stranded antibodies, bispecific antibodies, salified antibodies, human and humanized antibodies, and active fragments thereof. .. Examples of active fragments of molecules that bind to known antigens include isolated light and heavy chains, including the products of the Fab immunoglobulin expression library, as well as epitope-binding fragments of any of the antibodies and fragments described above. Contains Fab, Fab / c, Fv, Fab', and F (ab') ₂ fragments.

These active fragments can be derived from the antibodies of the invention by a number of techniques. For example, the monoclonal antibody can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. Subsequently, an appropriate fraction containing the Fab fragment can be collected and concentrated by membrane filtration and the like. Further descriptions of common techniques for the isolation of active fragments of antibodies are described, for example, in Khaw, B. A. et al. J. Nucl. Med. 23: 1011-1019 (1982); Rousseaux et al. Methods Enzymology, See 121: 663-69, Academic Press, 1986.

Antibodies produced by recombination include conventional full-length antibodies, known active antibody fragments derived from digestion by proteolysis, unique active antibody fragments such as Fv or single chain Fv (scFV), domain-deficient antibodies, and the like. May be good. Fv antibodies are about 50 Kd in size and contain variable regions of light and heavy chains. Single-chain Fv (“scFv”) polypeptides can be covalently expressed from nucleic acids containing VH and VL coding sequences, either directly linked or linked by a linker encoding the peptide. VH :: VL heterodimer linked to. See Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. Converts naturally aggregated but chemically separated light and heavy polypeptide chains from the antibody V region into scFv molecules that are thought to fold into a three-dimensional structure that is substantially similar to the antigen binding site. Many structures to do. See, for example, US Pat. Nos. 5,091,513, 5,132,405, and 4,956,778.

The binding site refers to the portion of the antibody molecule involved in antigen binding. The antigen binding site is formed by amino acid residues in the N-terminal variable (“V”) region of the heavy (“H”) and light (“L”) chains. Antibody variable regions are referred to as "hypervariable regions" or "complementarity determining regions" (CDRs) that intervene between more conserved adjacent stretches known as "framework regions" (FRs) 3 Includes two very different stretches. In the antibody molecule, the three hypervariable regions of the light chain (LCDR1, LCDR2, and LCDR3) and the three hypervariable regions of the heavy chain (HCDR1, HCDR2, and HCDR3) are antigen-binding surfaces in three-dimensional space with respect to each other. Or it is arranged to form a pocket. Therefore, the antibody binding site corresponds to the amino acids that make up the CDR of the antibody and any framework residue that makes up the binding site pocket.

The identity of amino acid residues in a particular antibody that constitutes a binding site can be determined using methods well known in the art. For example, the antibody CDR may be identified as a hypervariable region originally defined by Kabat et al. ("Sequences of Proteins of Immunological Interest", E. Kabat et al., US Department of Health and Human Services; Johnson, G and Wu, TT (2001) Kabat Database and its applications: future directions. Nucleic Acids Research, 29: 205-206; see http://immuno.bme.nwa.edu). The location of the CDR may also be identified as the structural loop structure originally described by Chothia and others (Chothia and Lesk, J. Mol. Biol. 196, 901 (1987), Chothia et al., Nature. See 342, 877 (1989), and Tramontano et al., J. Mol. Biol. 215, 175 (1990)). Alternatively, the "AbM definition" or Macallum et al. ("Antibody-antigen interactions: contact analysis and binding site topography", eclectic of Kabat and Chothia and obtained using Oxford Molecular's AbM antibody modeling software (now Accelrys). , J Mol Biol. 1996 Oct 11; 262 (5): 732-45) contains the "contact definition" of CDR. The following chapters identify CDRs based on various known definitions.

The general guidelines for identifying CDRs in antibodies from sequences alone are as follows.

LCDR1: LCDR1:
Start-around 24 residues.
The previous residue is always Cys.
The last residue is always Trp. Typically, TRP is followed by TYR-GLN, but LEU-GLN, PHE-GLN, or TYR-LEU can also follow.
The length is 10 to 17 residues.

LCDR2:
Start--after 16 residues at the end of L1.
The previous sequence is usually ILE-TYR, but it can also be VAL-TYR, ILE-LYS, or ILE-PHE.
The length is usually 7 residues.

LCDR3:
Start-usually after 33 residues at the end of L2.
The previous residue is Cys.
The last residue is PHE-GLY-X-GLY.
The length is 7-11 residues.

HCDR1: HCDR1:
Start-before and after residue 26 (after 4 residues of CYS) [Chothia / AbM definition]. The Kabat definition starts after 5 residues.
The previous array is CYS-XXX.
The last residue is TRP, typically followed by VAL, but also ILE, or ALA.
The length is 10-12 residues according to the AbM definition, but the Chothia definition excludes the last 4 residues.

HCDR2:
Start--after 15 residues at the end of CDR-H1 in the Kabat / AbM definition.
The previous sequence is typically LEU-GLU-TRP-ILE-GLY (SEQ ID NO: 1), but there can be many variants.
The last residue is LYS / ARG-LEU / ILE / VAL / PHE / THR / ALA-THR / SER / ILE / ALA.
The length is 16-19 residues according to the Kabat definition (AbM definition ends earlier than 7 residues).

HCDR3:
Start-after 33 residues at the end of CDR-H2 (after 2 residues of CYS).
The previous sequence is CYS-XX (typically CYS-ALA-ARG).
The rear array is TRP-GLY-X-GLY.
The length is 3 to 25 residues.

Specific that constitutes part of the binding site by having a side chain that is outside the CDR but is still part of the binding site lining (ie, it is available for connection through the binding site). The identity of amino acid residues in an antibody can be determined using methods well known in the art such as molecular modeling and X-ray crystallography. See, for example, Riechmann et al., (1988) Nature, 332 :; 323-327.

A chimeric antibody is an antibody in which one or more regions of the antibody are derived from an animal of one species and one or more regions of the antibody are derived from an animal of a different species. Preferred chimeric antibodies are antibodies that contain regions derived from primate immunoglobulins. Chimeric antibodies for clinical use in humans are typically understood to have constant regions derived from humans as well as variable regions derived from non-human animals such as rodents. In contrast, humanized antibodies use CDRs derived from non-human antibodies, along with most or all of the variable framework regions derived from human immunoglobulins and all constant regions derived from human immunoglobulins. Human chimeric antibodies are typically understood to have variable regions derived from rodents. A typical human chimeric antibody has a human heavy chain constant region and a human light chain constant region, as well as variable regions of both heavy and light chains derived from rodent antibodies. The chimeric antibody may contain slight changes to the native amino acid sequence of the human constant region and the native rodent variable region sequence. Chimeric and humanized antibodies, CDR conjugation approaches (see, eg, US Pat. No. 5,843,708; 6,180,370; 5,693,762; 5,585,089; 5,530,101), chain shuffling strategies (eg, US Pat. 5,565,332; Rader et al., Proc. Natl. Acad. Sci. USA (1998) 95: 8910-8915), molecular modeling strategies (see US Pat. No. 5,639,641), etc. It may be prepared in.

A "humanized antibody" as used herein in the case of a two-strand antibody is an antibody in which at least one strand has been humanized. Humanized antibody chains have variable regions in which one or more of the framework regions are human. A single-chain humanized antibody is an antibody in which a chain has a variable region in which one or more of the framework regions are human. The non-human portion of the variable region of a humanized antibody chain or fragment thereof is derived from a non-human source, particularly a non-human antibody of rodent origin. Non-human contributions to humanized antibodies typically come in the form of at least one CDR region interspersed within a framework region derived from one (or more) human immunoglobulins. In addition, framework support residues may be modified to preserve binding affinity.

The humanized antibody may further comprise a constant region (eg, at least one constant region or portion thereof in the case of a light chain, and preferably three constant regions in the case of a heavy chain). The constant region of a humanized antibody, if present, is usually human.

Methods for obtaining "humanized antibodies" are well known to those of skill in the art (eg, Queen et al., Proc. Natl Acad Sci USA, 86: 120029-10032 (1989), Hodgson et al., Bio / Technoloy. , 9: 421 (1991)).

"Humanized antibodies" can also be obtained by novel genetically engineered approaches that allow the production of mature human-like polyclonal antibodies in large animals such as rabbits and mice. See, for example, US Pat. No. 6,632,976.

As used herein, the term constant region (CR) refers to a constant region gene in an immunoglobulin. The constant region gene encodes the portion of the antibody molecule that imparts effector function. For chimeric human and humanized antibodies, typically non-human (eg, mouse) constant regions are replaced by human constant regions. The constant region of the chimeric or humanized antibody is typically derived from human immunoglobulin. The heavy chain constant region can be selected from five isotypes: alpha, delta, epsilon, gamma, or mu. In addition, heavy chains of various subclasses (such as the IgG subclass of heavy chains) are involved in different effector functions and can therefore produce antibodies with the desired effector function by selecting the desired heavy chain constant region. can. The constant regions that can be used within the scope of the invention are the Fc regions of gamma 1 (IgG1), in particular gamma 1 (IgG1) isotypes, gamma 3 (IgG3), and in particular gamma 4 (IgG4). The light chain constant region can be kappa or lambda type, especially kappa type. In some embodiments, the light chain constant region is a human kappa constant chain (Heiter et al. (1980) Cell 22: 197-207) and the heavy constant chain is a human IgG4 constant chain.

The term "monoclonal antibody" is also well known in the art and refers to an antibody that is the product of a single cloned antibody-producing cell. Monoclonal antibodies are typically made by fusing normal short-lived antibody-producing B cells with rapidly proliferating cells such as cancer cells (sometimes called "immortal" cells). The resulting hybrid cells or hybridomas grow rapidly to produce antibody-producing clones.

For the purposes of the present invention, "monoclonal antibody" should be construed to include antibodies produced by mother clones that have not yet reached full monoclonality.

"Functionally equivalent antibodies" include binding specificity for β-amyloid protein, especially for Aβ _1-42 protein, and more particularly for the 16-21 epitope region of Aβ _1-42 protein, in vitro immunoreactivity. Inhibition of aggregation of Aβ _1-42 monomers to high molecular weight polymer fibrils and / or deaggregation of preformed Aβ _1-42 polymer fibrils and / or β-sheet disruption properties, as well as prophylactic or therapeutic. Hereditary cerebral hemorrhage with amyloidosis (Dutch type); Alzheimer's disease (AD), Levy body dementia, Down syndrome, neurological disorders such as Guam Parkinson-dementia complex; and progression when administered Supranuclear palsy, multiple sclerosis; Kreuzfeld-Jakob disease, Parkinson's disease, HIV-related dementia, ALS (muscle atrophic lateral sclerosis), adult-onset diabetes; senile heart amyloidosis; endocrine tumors, and luteal degeneration Amyloid plaque formation, including but not limited to secondary amyloidosis and age-related amyloidosis, including, but not limited to, other diseases based on or associated with amyloid-like proteins such as other diseases. Refers to an antibody that substantially shares at least one major functional property with the antibodies mentioned above and described herein, including alleviating the effects of a group of diseases and disorders associated with amyloidosis. Is understood within the scope of the present invention. Antibodies are of any class such as IgG, IgM or IgA, or any subclass such as IgG1, IgG2a, and other subclasses described herein or known in the art, especially the IgG4 class. It may be a thing. In addition, it comprises other types of cells or cell lines that produce antibodies by any method, such as phage display, or produce antibodies with the desired properties, such as bacteria, insects, mammals, or humanized antibodies. , Can also be produced in any organism or cell line. Antibodies can also be formed by combining Fab moieties and Fc regions from different species.

The term "hybridize" when used refers to conventional hybridization conditions, preferably 5 x SSPE, 1% SDS, 1 x Denhardt solution as a solution and / or a hybridization temperature of 35 ° C to 70 ° C. Refers to hybridization conditions, preferably 65 ° C. After hybridization, washing is preferably performed first at 2 × SSC, 1% SDS, then at 0.2 × SSC at a temperature of 35 ° C to 70 ° C, preferably 65 ° C (with respect to the definition of SSPE, SSC, and Denhardt solutions). Sambrook et al, previously cited location, see). For example, Sambrook et al, stringent hybridization conditions as described above are particularly preferred. Particularly preferred stringent hybridization conditions are present, for example, when hybridization and washing occur at 65 ° C. as shown above. For example, non-stringent hybridization conditions in which hybridization and washing are performed at 45 ° C are less preferred, and non-stringent hybridization conditions in which hybridization and washing are performed at 35 ° C are even less preferred.

The "homology" between two sequences is determined by sequence identity. If the lengths of the two sequences to be compared to each other are different, sequence identity is preferably related to the percentage of nucleotide residues in the shorter sequence that are identical to the nucleotide residues in the longer sequence. Sequence identity is customarily determined by the use of computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix®, Genetics Computer Group, University Research Park, 575 Science Drive Madison, WI 53711). be able to. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489 to find the segment with the highest sequence identity between the two sequences. When using Bestfit or another sequence alignment program to determine if a particular sequence has, for example, 95% identity to a reference sequence of the invention, the parameters are preferably concordance. The percentage of identity is calculated over the entire length of the reference sequence and adjusted to allow a homology gap of up to 5% of the total number of nucleotides in the reference sequence. When using Bestfit, so-called optional parameters are preferably left at their preset (“default”) values. The deviations observed in the comparison between a given sequence and the above sequences of the invention may be due, for example, to additions, deletions, substitutions, insertions or recombinations. Such sequence comparisons are preferably performed by the program "fasta20u66" (version 2.0u66, September 1998, William R. Pearson and University of Virginia; W.R. Pearson (1990), Methods in Enzymology 183, 63-98, attached example. And also http://workbench.sdsc.edu/). A "default" parameter setting may be used for this purpose.

Antibodies according to the invention may be immunoglobulins or antibodies, or as alternatives, "bispecific antibodies, which are understood to have each of their identical binding sites (in the case of polyvalent). Or "bifunctional antibody".

A "bispecific antibody" or "bifunctional antibody" is an artificial hybrid antibody with two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or ligation of Fab'fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

The term "fragment" refers to a portion or portion of an antibody or antibody chain that contains fewer amino acid residues than an unchanged or complete antibody or antibody chain. Fragments can be obtained by chemical or enzymatic treatment of unchanged or complete antibodies or antibody chains. Fragments can also be obtained by recombinant means. Exemplary fragments include Fab, Fab', F (ab') 2, Fabc, and / or Fv fragments. The term "antigen-binding fragment" refers to an immunoglobulin or immunoglobulin that binds to an antigen or competes with an unchanged antibody (ie, with the unchanged antibody from which they are derived) for antigen binding (ie, specific binding). Refers to a polypeptide fragment of an antibody.

Bound fragments are produced by recombinant DNA technology or by enzymatic or chemical cleavage of unchanged immunoglobulin. Binding fragments include Fab, Fab', F (ab') ₂ , Fabc, Fv, single-chain, and single-chain antibodies.

A "fragment" is another polypeptide, at least 5 consecutive amino acid residues in the amino acid sequence, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues. Amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues Amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues It also refers to a peptide or polypeptide comprising an amino acid sequence of at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues. In a specific embodiment, the fragment of the polypeptide retains at least one function of the polypeptide.

The term "antigen" refers to an entity or fragment thereof that can bind to an antibody. An immunogen refers to an antigen that can elicit an immune response in an organism, especially an animal, more particularly a mammal, including a human. The term antigen refers to an antigenic determinant or epitope that refers to a portion of an antigen (which plays an important role in supporting contacts that are in contact with or in the antigen and are involved in antigenicity or antigenic determinants). Includes an area known as.

As used herein, the term "soluble" means partially or completely soluble in an aqueous solution.

Also as used herein, the term "immunogen" means a substance that induces the production of antibodies, T cells and other reactive immune cells directed against an immunogen antigen.

An immune response occurs to alleviate the disorder in which an individual attempts to treat by producing sufficient antibodies, T cells and other reactive immune cells against the administered immunogenic composition of the invention. It is a case of relaxation.

As used herein, the term immunogenicity refers to the magnitude of the ability of an antigen to elicit an immune response (humoral or cellular) when administered to a recipient. The present invention relates to approaches that reduce the immunogenicity of the human chimeric or humanized antibodies.

A reduced immunogenic humanized antibody refers to an original antibody, eg, a humanized antibody that exhibits reduced immunogenicity as compared to a mouse antibody.

A humanized antibody that substantially retains the binding properties of the original antibody is a human that retains the ability to specifically bind to the antigen recognized by the original antibody used to produce such a humanized antibody. Refers to a humanized antibody. Preferably, the humanized antibody is believed to exhibit the same or substantially the same antigen-binding affinity and avidity as the original antibody. Ideally, the affinity of the antibody is not considered to be less than 10%, more preferably less than about 30% of the affinity of the original antibody, and most preferably the affinity is the original antibody. Not less than 50% of. Methods for assaying antigen binding affinity are well known in the art and include maximal halving assay, competitive assay, and scatchard analysis. Suitable antigen binding assays are described herein.

A "return mutation" is a mutation introduced into a nucleotide sequence encoding a humanized antibody, which corresponds to an amino acid in the original antibody (eg, donor antibody, eg, mouse antibody). The resulting amino acid. In order to simultaneously minimize the potential immunogenicity of the resulting antibody while substantially preserving the binding properties of the original antibody, certain framework residues derived from the original antibody are used in the present invention. It may be retained throughout the humanization of the antibody. In certain embodiments of the invention, the original antibody is of mouse origin. For example, a return mutation changes a human framework residue to the original mouse residue. Examples of reversionable framework residues are canonical residues, interfacial filling residues, rare original residues near the binding site, "Vernia (which forms the platform on which the CDRs are based)". Includes, but is not limited to, residues in the "Vernier Zone" (Foote & Winter, 1992, J. Mol. Biol. 224, 487-499), and residues close to CDR3 H3.

As used herein, "conservative changes" are substantially neutral in conformation or antigenicity when compared to native proteins, respectively, with minimal tertiary structure of the mutant polypeptide. Refers to modifications that result in changes or minimal changes in the antigenic determinants of mutant polypeptides. When referring to the antibodies and antibody fragments of the invention, conservative changes mean amino acid substitutions that do not prevent the antibody from binding to the receptor. One of ordinary skill in the art will be able to predict which amino acid substitutions can be made while maintaining a high probability of being neutral in conformation and antigenicity. Such guidance is provided, for example, in Berzofsky, (1985) Science 229: 932-940 and Bowie et al. (1990) Science 247: 1306-1310. Factors that influence the ability to maintain conformation and antigenic neutrality to be considered are: (a) hydrophobic amino acids because hydrophobic residues are more likely to be located inside the protein. Substitutions are not very likely to affect antigenicity; (b) Substituted amino acids structurally mimic native amino acids, so physicochemically similar amino acid substitutions affect the conformation. And (c) modifications of the evolutionarily conserved sequence, as it is suggested that the amino acid sequence may have functional significance. Is likely to have an adverse effect on the three-dimensional structure, but is not limited to these. Those skilled in the art include microcomplement fixation methods (Wasserman et al. (1961) J. Immunol. 87: 290-295; Levine et al. (1967) Meth. Enzymol. 11: 928-936). However, but not limited to these, binding studies using well-known assays and using conformation-dependent monoclonal antibodies (Lewis et al. (1983) Biochem. 22: 948-954) show the protein conformation. It is believed that alterations can be evaluated.

In addition, the term "therapeutically effective amount" refers to the amount of antibody that, when administered to a human or animal, is sufficient to result in a therapeutic effect in the human or animal. Effective amounts will be readily determined by one of ordinary skill in the art according to routine procedures.

As used herein, the terms "treat," "prevent," "preventing," and "prevention" are the result of administration of prophylactic or therapeutic agents. It means the prevention of recurrence or onset of one or more symptoms of the disorder resulting in the subject.

Construction of Humanized Antibodies The present invention will be more easily understood by reference to the following detailed description of the particular embodiments contained herein. Although the present invention has been described with reference to the specific details of that particular aspect, such details should not be considered to limit the scope of the invention.

The present invention comprises highly specific and highly effective antibodies having the ability to specifically recognize and specifically bind to specific epitopes derived from various β-amyloid antigens and novel methods. The composition is provided. The antibodies enabled by the teachings of the present invention are neurological disorders such as Alzheimer's disease (AD), Levy body dementia, Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guam Parkinson-dementia complex. ; And progressive nuclear palsy, multiple sclerosis; Kreuzfeld-Jakob disease, Parkinson's disease, HIV-related dementia, ALS (muscle atrophic lateral sclerosis), adult-onset diabetes; senile heart amyloidosis; endocrine tumors, and Related to secondary amyloidosis and aging, including, but not limited to, other diseases that are based on or associated with amyloid-like proteins, such as, but not limited to, other diseases, including, to name just one. It is particularly useful in the treatment of amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including amyloidosis.

The fully humanized or reshaped variable regions according to the invention were implanted in CDRs from non-humans, especially rodents, especially human-derived framework sequences ("mC2" throughout this application. Braunschweig, Mascheroder Weg 1B, 38124 Branuschweig Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on December 1, 2005, under the provisions of the Budapest Treaty and the given accession number: DSM ACC2750. It may be created within the scope of the present invention by first designing a variable region amino acid sequence containing a CDR derived from the (deposited) mouse antibody ACI-01-Ab7C2. CDRs from non-humans, especially rodents, which may be obtained from the antibodies according to the invention, provide the desired specificity. Therefore, these residues should be included in the design of essentially invariant reshaped variable regions. Therefore, any modifications should be limited to a minimum and careful attention should be paid to changes in antibody specificity and affinity. On the other hand, framework residues can theoretically be obtained from any human variable region.

A human framework that is equally suitable for creating reshaped variable regions and preserving antibody affinities in order to produce reshaped antibodies that exhibit acceptable or even improved affinities. You should choose an array.

Optimal strategies have been developed to achieve this goal. Since it is known that framework sequences function to keep CDRs in their correct spatial orientation for interaction with antigens, and that framework residues can sometimes even be involved in antigen binding. This strategy derives the human framework sequences used for antibody remodeling from human variable regions that are most homologous or similar to non-human, especially rodent-derived variable regions. Aim to minimize changes that can have a negative impact on. This is also believed to maximize the likelihood that affinity will be retained in the reshaped antibody.

At its simplest level, the "optimal" strategy is to compare the donor rodent V region with all known human V region amino acid sequences and then provide an acceptor framework region for performing humanization. It involves the process of selecting the most homologous. In practice, there are some other factors that should be considered and may influence the final choice of acceptor framework areas. In this regard, molecular modeling predictions may be used prior to any experimental study attempting to maximize the affinity of the resulting reshaped antibody. In essence, the purpose of modeling is to set any key residue (if any) of the most homologous human framework, as well as rodents, to obtain the highest affinity for the reshaped antibody. It is to predict whether it should be left.

In certain aspects of the invention, CDRs are from mouse monoclonal antibodies, in particular in EP 05 02 709 2.5, a co-pending application filed on 12 December 2005, the disclosure of which is incorporated herein by reference. Obtained from the described mouse monoclonal antibody ACI-O1-Ab7C2 (named "mC2" throughout this application).

The hybridoma cell FP-12H3-C2, which produces the mouse monoclonal antibody ACI-O1-Ab7C2 (named "mC2" and for humanized C2 antibody hC2 throughout this application), was released in 2005. On December 1, in the co-pending application EP05027092.5, Braunschweig, Mascheroder Weg 1B, 38124 Braunschweig's Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), the provisions of the Budapest Treaty and the given accession number. : Deposited under DSM ACC 2750.

Β-Amyloid peptide, especially β-amyloid peptide Aβ _1-15 , modified from a mouse antibody with a hydrophobic moiety such as palmitic acid, or a hydrophilic moiety such as polyethylene glycol (PEG), or a combination of both. It may be made for supermolecular antigenic constructs containing antigenic peptides corresponding to the amino acid sequences of Aβ _1-16 and Aβ _{1-16 (Δ14)} , with hydrophobic and hydrophilic moieties such as, for example, lysine, respectively. At least one, especially one, or any other suitable amino acid or amino acid analog, such as glutamate, and cysteine, or any other suitable amino acid or amino acid analog that can act as a connector for conjugating hydrophobic and hydrophilic moieties to the peptide fragment. It is covalently attached to each of the ends of the antigenic peptide through two amino acids. When PEG is used as the hydrophilic moiety, the free PEG ends can be phosphatidylethanolamine, or any other compound suitable for functioning, for example, as a fixing element for implanting the antigenic construct into the liposome bilayer. , Covalent bond.

In particular, mouse antibodies may be made against supermolecular antigen constructs comprising an antigen peptide corresponding to the amino acid sequence of a β-amyloid peptide modified with a hydrophilic moiety, such as polyethylene glycol (PEG), which is hydrophilic. The moiety is at least one such as, for example, lysine, glutamic acid, and cysteine, or any other suitable amino acid or amino acid analog that can serve as a connecting device for conjugating hydrophobic and hydrophilic moieties to the peptide fragment. It is covalently attached to each of the ends of the antigenic peptide, especially through one or two amino acids. When PEG is used as the hydrophilic moiety, the free PEG end is phosphatidylethanolamine, or any other compound suitable for functioning as a fixing element, eg, for implanting an antigenic construct into the bilayer of liposomes. To be covalently combined.

In aspects of the invention, in a variable region, of non-human origin embedded in one or more human or primate-derived framework regions and combined with constant regions derived from human or primate source antibodies. A chimeric antibody or fragment thereof or a humanized antibody or a humanized antibody thereof, which comprises at least one of CDRs and is capable of specifically recognizing a β-amyloid monomer peptide and specifically binding to a β-amyloid monomer peptide. Provide fragments.

The CDR must be retained in the reshaped antibody that contains the residues most likely to bind to the antigen. CDRs are defined by sequences by Kabat et al, Sequence of Proteins of Immunological Interest, 5th Edition, The United States Department of Health and Human Services, The United States Government Printing Office, 1991. CDRs fall into the canonical category (Chothia et al, 1989 Nature, 342, 877-883), where key residues determine the structural conformation of the CDR loop to a large extent. These residues are almost always retained in the reshaped antibody.

In the process of preparing humanized antibodies according to the present invention, the amino acid sequences of the C2 heavy chain and light chain variable regions (V _H and V _K ) were categorized as V _H and V _K of the rodent antibody in the NCBI and Kabat databases. Compare with an array.

The closest matching mouse germline gene to C2V _K is bbl, Locus MMU231201 (Schable et al, 1999). Comparison revealed that the two amino acids differed from this germline sequence and both were located within CDRL1. It is possible to find mature mouse antibodies with similar but non-identical sequences. Some have the same CDRL2 and the same CDRL3, but CDRL1 in C2 seems to be unique. Comparison with human germline V _K sequences has shown that genes from subgroup V _K II are the best match for C2 V _K (Cox et al, 1994). Therefore, C2 V _K can be assigned to the Kabat subgroup MuV _K II array.

DPK 15 may be selected with the human J region Hu J _K 1 to provide an acceptor framework sequence for humanized V _K.

Residues at the interface between the variable light chain and the variable heavy chain are defined (Chothia et al, 1985 J. Mol. Biol., 186, 651-663). These are usually retained in the reshaped antibody. Phe at position 87 of mouse C 2 V _K is rare at the interface, where Tyr is more common in the V _K II subgroup. This indicates that this framework residue can be important for antibody activity. Tyr 87 is present in human germline and humanized C2VK.

Therefore, the humanized _VK sequence may be designed such that _C2HuVK1 consists of a mouse _C2VK CDR with a framework derived from DPK 15 and human JK1. In a specific embodiment of the invention, mouse residues may be substituted at positions 45 and / or 87 in the human framework region. Amino acid substitutions may be made at Kabat positions 50 and / or 53 from the mouse monoclonal antibody, especially in the CDR2 region obtained from the mouse antibody ACI-O1-Ab7C2. Residue 45 may be involved in supporting the conformation of the CDR. Residue 87 is located at the interface between the V _H and V _K domains. Therefore, these residues may have crucial implications for maintaining antibody binding.

The closest matching mouse germline genes to C2 VHA are _VH7183 , Locus AF120466 (Langdon et al, 2000). Comparison with the human germline V _H sequence shows that the genes from the subgroup V _H III are the best match for C2 V _H. C2 V _H AF can be assigned to the Kabat subgroup Mu V _H III D. The sequence DP54 can be selected with the human J region HuJ _H 6 to provide an acceptor framework sequence for humanized _VH .

Comparisons show that there are nine amino acid differences between the C2 VH sequence and the human acceptor germline sequences DP54 and _JH 6, most of which are located within CDRH2. Mature mouse antibodies with the same or similar (one residue different) CDRH1 or similar CDRH2 (one residue different) are found, but all three CDRs are identical to C2 _VHA F. not. The C2 antibody CDRH3 is unusually short and consists of only 3 residues. However, other antibodies with this length of CDRH3 are found in the database. That residue 47 of C2 V _H is Leu, not the more general Trp, and residue 94 is Ser, not the usual Arg, and these framework residues can be useful for antibody activity. Is shown.

Various humanized _VH sequences may be designed. C2HuVH1 consists of _C2VHA AF CDRs with frameworks derived from DP54 and _HuJH 6. In a specific embodiment of the invention, mouse residues may be substituted at position 47 or 94 or both in the human framework region. Residue 47 in framework 2 contacts both the CDR and _VK domains. Residue 94 may be involved in supporting the conformation of the CDR. Therefore, these residues may have crucial implications for maintaining antibody binding.

Different HCVR and LCVR regions may be designed, including non-human CDRs obtained from donor antibodies, such as mouse antibodies, embedded in native or modified human or primate-derived framework regions. Modifications are particularly similar to non-human residues more commonly found at this position in each subgroup, in particular due to mouse residues or more commonly found at this position in each subgroup. It may be involved in the exchange of one or more amino acid residues within the framework region by the residues having the properties of.

In modifying framework regions, framework sequences function to keep CDRs in their correct spatial orientation for interaction with antigens, and the framework residues can sometimes even be involved in antigen binding. .. In certain embodiments of the invention, human framework sequences selected so that they most closely resemble the sequences of the rodent framework in order to maximize the likelihood that the affinity will be retained in the reshaped antibody. Take steps to further adapt.

Therefore, mouse residues in the human framework region may be replaced. In particular, mouse residues are placed in the human framework region of the heavy chain variable (HCVR) region, at positions 47 or 94 or both, and in the human framework region of the light chain variable (LCVR) region, at positions 45 and / or 87. May be replaced with. Amino acid substitutions may be made at Kabat positions 50 and / or 53 in the CDR2 region obtained from mouse monoclonal antibodies, particularly mouse antibody ACI-01-Ab7C2.

Residues found at the locations shown above in the human framework region may be replaced with mouse residues found at this location in their respective subgroups. In particular, the Trp at Kabat position 47 in the human or primate-derived framework region of the heavy chain variable region set forth in SEQ ID NO: 15 is substantially steric by Leu or by substitution with similar properties. It may be replaced by amino acid residues that are neutral in structure or antigenicity and that result in minimal changes in the tertiary structure of the mutant polypeptide or in modifications that result in minimal changes in the antigenic determinants. In particular, Trp at Kabat position 47 in the human or primate-derived framework region of the heavy chain variable region shown in SEQ ID NO: 15 was selected from the group consisting of norleucine, Ile, Val, Met, Ala, and Phe. It may be further replaced by amino acids, especially by Ile. Alternative conservative substitutions that are neutral in conformation and antigenicity may be contemplated.

Arg at Kabat position 94 in the human or primate-derived framework region of the heavy chain variable region set forth in SEQ ID NO: 15 has substantially three-dimensional structure or by substitution with Ser or similar properties. It may be replaced by amino acid residues that are neutral in antigenicity and result in modifications that result in minimal changes in the tertiary structure of the mutant polypeptide or in the antigenic determinants. In particular, Arg at Kabat position 94 in the human or primate framework region of the heavy chain variable region set forth in SEQ ID NO: 15 may be replaced by Thr instead.

In another aspect of the invention, both residues may be replaced in the humanized antibody.

Gln at Kabat position 45 in the human or primate-derived framework region of the light chain variable region set forth in SEQ ID NO: 12 has substantially three-dimensional structure or by replacement with Lys or similar properties. It may be replaced by amino acid residues that are neutral in antigenicity and result in modifications that result in minimal changes in the tertiary structure of the mutant polypeptide or in the antigenic determinants. In particular, Gln at Kabat position 45 in the human or primate-derived framework region of the light chain variable region set forth in SEQ ID NO: 12 is particularly arged by an amino acid selected from the group consisting of Arg, Gln, and Asn. May be replaced by.

Leu at Kabat position 50 in the human or primate-derived framework region of the light chain variable region set forth in SEQ ID NO: 12 is substantially three-dimensional or tertiary by Lys or by substitution with similar properties. It may be replaced by amino acid residues that are neutral in antigenicity and result in modifications that result in minimal changes in the tertiary structure of the mutant polypeptide or in the antigenic determinants. In particular, Leu at Kabat position 50 in the human or primate-derived framework region of the light chain variable region set forth in SEQ ID NO: 12 is specifically arged by an amino acid selected from the group consisting of Arg, Gln, and Asn. May be replaced by.

Asn at Kabat position 53 in the human or primate-derived framework region of the light chain variable region set forth in SEQ ID NO: 12 is substantially steric by His and Gln, or by substitution with similar properties. It may be replaced by amino acid residues that are neutral in structure or antigenicity and that result in minimal changes in the tertiary structure of the mutant polypeptide or in modifications that result in minimal changes in the antigenic determinants. In particular, the Asn at Kabat position 53 in the human or primate-derived framework region of the light chain variable region set forth in SEQ ID NO: 12 is replaced by an amino acid selected from the group consisting of Gln, His, Lys, and Arg. You may.

Thr of Kabat position 87 in the human or primate-derived framework region of the light chain variable region set forth in SEQ ID NO: 12 is substantially three-dimensional or by Phe or by substitution with similar properties. It may be replaced by amino acid residues that are neutral in antigenicity and result in modifications that result in minimal changes in the tertiary structure of the mutant polypeptide or in the antigenic determinants. In particular, the Tyr at Kabat position 87 in the human or primate-derived framework region of the light chain variable region set forth in SEQ ID NO: 12 by amino acids selected from the group consisting of Leu, Val, Ile, and Ala. In particular, it may be replaced by Leu.

Variable regions obtained to contain at least one CDR of non-human origin embedded within a framework region of one or more human or primate origins, then to antibodies of human or primate origin. It may be combined with the constant region from which it is derived, and in particular with the constant region of human IgG4 or κ. The IgG4 constant region may be modified, for example, by converting serine at position 228 in the hinge region to proline (HuIgG4 Ser-Pro). This mutation stabilizes interchain disulfide bonds and prevents the formation of half molecules that can occur in native human IgG4 preparations. The IgG4 constant region may be further modified by deletion of the terminal Lys at position 439 shown in SEQ ID NO: 16.

The modified variable region may be constructed by methods known in the art, such as overlapping PCR recombination. Expression cassettes for chimeric antibodies, C2 _ChV HA and C2 ChV _K may be used as templates for mutagenesis into the required sequences of the framework region. Synthesize a set of mutagenesis primer pairs that include the region to be modified. The humanized V _H and V _K expression cassettes produced may be cloned into a suitable cloning vector known in the art, such as pUC19. After confirming that the entire DNA sequence is correct for each V _H and V _K , the modified heavy and light chain V region genes can be excised from the cloning vector as an expression cassette. These can then be transferred to suitable expression vectors such as pSVgpt and pSVhyg containing constant regions of human IgG4 Ser-Pro or κ, respectively.

Expression vector The expression vector pSVgpt is based on pSV ₂ gpt (Mulligan and Berg, 1980) and is an ampicillin resistance gene for selection in bacterial cells, gpt gene for selection in mammalian cells, mouse heavy chain immunoglobulin. Includes enhancer region, genomic sequence encoding constant region gene, and SV40 poly A sequence. The heavy chain variable region for expression is inserted as a HindIII-BamHI fragment.

The expression vector pSVhyg encodes an ampicillin resistance gene for selection in bacterial cells, a hyg gene for selection in mammalian cells, a mouse heavy chain immunoglobulin enhancer region, a kappa constant region gene, and a genomic sequence containing the kappa enhancer. And contains the SV40 poly A sequence. The light chain variable region for expression is inserted as a HindIII-BamHI fragment.

The DNA sequence should then be confirmed to be correct for humanized V _H and V _K in the expression vector.

For antibody production, humanized heavy and light chain expression vectors may be introduced into suitable production cell lines known in the art, such as NS0 cells. The introduction of the expression vector may be accomplished by electroporation-mediated cotransfection or any other suitable transformation technique available in the art. The antibody-producing cell line can then be selected and propagated, and the humanized antibody can be purified. The purified antibody can then be analyzed by standard techniques such as SDS-PAGE.

The CDRL2 sequence (“KVSNRFS”) of an antibody mouse C2 antibody with improved affinity, specificity and stability may be slightly modified without adversely affecting antibody activity. Conservative substitutions may be made by exchanging K and R at position 50 and N and S at position 53. Therefore, the two alternative CDRL2 sequences are "RVSNRFS" and "KVSSRFS", respectively. These are incorporated into the mouse _VK sequence as C2 VK-R and C2 VK-S, respectively, without any other changes.

The affinity, specificity, and stability of an antibody or fragment thereof according to the invention as described herein is modified by changes in its glycosylation profile or pattern, resulting in improved therapeutic value. be able to.

To achieve this change in glycosylation pattern, the host cell expresses a preferred range of glycosyltransferase activity that modifies glycoproteins that increase complex N-linked oligosaccharides with bisecting GlcNAc. As such, the host cell can be artificially modified. Further, modification of glycoproteins such as antibodies, including fusion proteins containing enhanced Fc-mediated cellular cytotoxicity, eg, complete antibody molecules, antibody fragments, or regions equivalent to the Fc region of immunoglobulins. Glycoform may be obtained.

Methods of obtaining antibodies with a modified glycosylation pattern are known to those of skill in the art and, for example, EP1071700, US2005272128, Ferrara et al (2006) J Biol Chem 281 (8), 5032-5036; Ferrara et al ( 2006) Biotechnology and Bioengineering 93 (5), 851-861.

Pharmaceutical Preparation and Administration Antibodies according to the invention, in particular monoclonal antibodies according to the invention, can be prepared as physiologically acceptable formulations and are pharmaceutically acceptable carriers, diluents, and / or excipients. The agent may be included using a known method. For example, an antibody according to the invention and described herein above, including any functionally equivalent antibody or functional portion thereof, in particular a monoclonal antibody comprising any functionally equivalent antibody or functional portion thereof. Is mixed with a pharmaceutically acceptable carrier, diluent and / or excipient to form a therapeutic composition. Suitable pharmaceutical carriers, diluents and / or excipients are well known in the art, including, for example, phosphate buffered saline, water, emulsions such as oil-in-water emulsions, various types of wetting agents. , Sterilized solution etc. are included.

The formulation of the pharmaceutical composition according to the present invention can be achieved according to a standard method known in the art.

The compositions of the invention can be administered to a subject in the form of a solid, liquid, or aerosol in a suitable pharmaceutically effective amount. Examples of solid compositions include pills, creams, and implantable dosing units. Pills can be administered orally. The therapeutic cream can be administered topically. Implantable dosing units can also be administered topically, eg, to the tumor site, or, eg, subcutaneously, for the purpose of systemic release of the therapeutic composition. Examples of liquid compositions include formulations suitable for intramuscular, subcutaneous, intravenous and intraarterial injection, as well as formulations for topical and intraocular administration. Examples of aerosol formulations include inhalation formulations intended for administration to the lungs.

The composition can be administered by standard routes of administration. Generally, the composition may be administered by a local, oral, rectal, intranasal, intradermal, intraperitoneal, or parenteral (eg, intravenous, subcutaneous or intramuscular) route. can. In addition, the composition can be incorporated into a sustained release matrix such as a biodegradable polymer that is a polymer implanted near a site where delivery is desired, eg, a tumor site. The method comprises a single dose, repeated doses at predetermined intervals, and continuous doses over a predetermined period of time.

As used herein, a sustained release matrix is a matrix made of a material, usually a polymer, that decomposes by hydrolysis with an enzyme or acid / base or by dissolution. When such a matrix is inserted into the body, it is affected by the action of enzymes and body fluids. Sustained release matrices are preferably liposomes, polylactide (polylactic acid), polyglycolide (polymer of glycolic acid), polylactideco-glycolide (polymer of lactic acid and glycolic acid), polyanhydrous, poly (ortho) esters. , Polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, phenylalanine, tyrosine, isoleucine and other amino acids, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicones. Selected from biocompatible materials. A preferred biodegradable matrix is one of polylactide, polyglycolide, or polylactideco-glycolide (a copolymer of lactic acid and glycolic acid).

The dose of the composition depends on various factors such as the condition to be treated, the specific composition used, as well as the patient's weight, size, gender and general health, body surface area, specific compound or composition to be administered. It is well known to those of skill in the art that it will depend on other clinical factors such as the substance, other agents administered simultaneously and the route of administration.

The composition is a bioactive substance or compound, particularly a compound against oxidative stress, an anti-apyogenic compound, a metal chelating agent, an inhibitor of DNA repair such as pyrenzepine and metabolites, 3-amino-1-propanesulfonic acid (3APS). , 1,3-Propane disulfonate (1,3PDS), α-secretase activator, β- and γ-secretase inhibitors, tau proteins, neurotransmitters, β-sheet disrupting agents, amyloid β removal / depletion Atypical antipsychotics such as attractants of cellular components to cause, inhibitors of N-terminal cleaved amyloid β, including pyroglutamine-oxidized amyloid β3-42, anti-inflammatory molecules such as clozapine, ziplacidone, risperidone, alipiprazole, or olanzapine. Drugs, or other drugs, including cholineresterase inhibitors (ChEI) such as taclin, rivastigmine, donepezil, and / or galantamine, M1 agonists, and any amyloid or tau modifiers, as well as, for example, vitamin B12, cysteine, acetylcholine. Other composition containing at least one compound selected from the group consisting of nutritional supplements such as precursors, lecithin, choline, Ginkgo biloba, acetyl-L-carnitine, idebenone, propentophilin, or xanthin derivatives. It may be administered in combination with the antibody according to the invention, and optionally with pharmaceutically acceptable carriers and / or diluents and / or excipients, and procedures for treating the disease.

The proteinaceous pharmaceutically active substance may be present in an amount of 1 ng to 10 mg per dose. In general, the dosing regimen should be such that the antibodies according to the invention are in the range of 0.1 μg to 10 mg, especially 1.0 μg to 1.0 mg, more particularly 1.0 μg to 100 μg, and all within these ranges. Individual numbers are also part of the invention. If the dosing is by continuous infusion, more appropriate doses may be in the range of 1 kg body weight and 0.01 μg-10 mg per hour, as are all individual numbers within these ranges as well. It is a part.

Administration is generally considered parenteral, eg intravenous. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Non-aqueous solvents include, but are not limited to, vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and injectable organic acid esters such as ethyl oleate. The aqueous solvent may be selected from the group consisting of water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffer media. Parenteral media include sodium chloride solution, Ringer's dextrose solution, dextrose, and sodium chloride, lactated Ringer's solution, or fixed oil. Intravenous media include liquid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer dextrose) and the like. There may also be preservatives such as, for example, antibacterial agents, antioxidants, chelating agents and inert gases.

The pharmaceutical composition may further include, for example, serum albumin or immunoglobulins, particularly proteinaceous carriers such as those of human origin. Further bioactive substances may be present in the pharmaceutical compositions of the present invention, depending on their intended use.

When the binding target is located in the brain, certain embodiments of the invention provide an antibody or active fragment thereof that crosses the blood-brain barrier. Certain neurodegenerative diseases are associated with increased permeability of the blood-brain barrier, which allows the antibody or active fragment thereof to be easily introduced into the brain. For transporting molecules across the blood-brain barrier, including but not limited to physical, lipid-based, and receptor and channel-based methods, where the blood-brain barrier remains intact. There are several approaches known in the art.

Physical methods of transporting an antibody or active fragment thereof across the blood-brain barrier include completely avoiding the blood-brain barrier or creating an opening in the blood-brain barrier. Not limited. Workarounds include direct injection into the brain (see, eg, Papanastassiou et al., Gene Therapy 9: 398-406 (2002)) and implantation of a delivery device in the brain (eg, Gill et al., Nature Med. 9). : 589-595 (2003); and Gliadel Wafers ™, Guildford Pharmaceutical), but not limited to these. Methods for creating openings in the barrier include ultrasound (see, eg, US Patent Application Publication No. 2002/0038086), osmotic pressure (eg, administration of hypertonic mannitol (Neuwelt, E.A., Implication of the Blood-Brain Barrier)). and its Manipulation, Vols 1 & 2, Plenum Press, N.Y (1989))), for example, permeation with brazikinin or permeate A-7 (eg, US Pat. Nos. 5,112,596, 5,268,164, 5,506,206). (See also No. 5,686,416), as well as transfection of neurons across the blood-brain barrier with a vector containing a gene encoding an antibody or antigen-binding fragment (see, eg, US Patent Application Publication No. 2003/0083299). However, it is not limited to these.

As a lipid-based method of transporting an antibody or an active fragment thereof across the blood-brain barrier, the antibody or its activity in a liposome coupled to an antibody-binding fragment that binds to a receptor on the vascular endothelial of the blood-brain barrier. Encapsulating fragments (see, eg, US Patent Application Publication No. 20020025313), and low-density lipoprotein particles (see, eg, US Patent Application Publication No. 20040204354) or apolipoprotein E (see, eg, US Patent Application Publication No. 200040131692). (See No.) includes, but is not limited to, coating an antibody or an active fragment thereof.

Increasing the permeability of the blood-brain barrier with a glucocorticoid blocker as a receptor-based method for transporting the antibody or active fragment thereof across the blood-brain barrier (eg, US Patent Application Publication No. 2002). / 0065259, 2003/0162695, and 2005/0124533); Activate potassium channels (see, eg, US Patent Application Publication No. 2005/0089473), inhibit ABC drug transporters. (See, eg, US Patent Application Publication No. 2003/0073713); coating the antibody with transferrin and regulating the activity of one or more transferrin receptors (see, eg, US Patent Application Publication No. 2003/0129186). ), As well as cationizing the antibody (eg, US Pat. No. 5,004,697), but not limited to these.

Detection / Diagnosis In a further aspect, the invention provides methods and kits for the detection and diagnosis of amyloid-related diseases or conditions. These methods include known immunological methods commonly used to detect or quantify substances in biological samples or in situ.

Diagnosis of an amyloid-related disease or condition in a patient may be accomplished by detecting immunospecific binding of a monoclonal antibody or active fragment thereof to an epitope of an amyloid protein in a sample or in situ. This involves contacting a sample suspected of containing the amyloid protein or a specific body part or body part with an antibody that binds to an epitope of the amyloid protein, binding the antibody to the amyloid protein to form an immunological complex. Includes steps to cause, detect the formation of immunological complexes, and correlate the presence or absence of immunological complexes with the presence or absence of amyloid protein in a sample or specific body part or site.

Biological samples that can be used in the diagnosis of amyloid-related diseases or conditions include, for example, body fluids such as serum, plasma, saliva, gastric secretions, mucus, cerebrospinal fluid, lymph, or nerves, brain, heart, or. A tissue or cell sample obtained from an organism, such as a tissue of a blood vessel. Any immunoassay method known to those of skill in the art (see Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988 555-612)), to determine the presence or absence of amyloid protein in a sample, For example, assays that utilize indirect detection using secondary reagents for detection, ELISA and immunoprecipitation and aggregation assays can be used. Detailed descriptions of these assays are given, for example, to WO96 / 13590, Zrein et al. (1998) and WO96 / 29605 for Maertens and Stuyver.

For in-situation diagnosis, methods known in the art such as specific binding between an antibody according to the invention and an epitope region on an amyloid protein, such as intravenous, intranasal, intraperitoneal, cerebral. Intra-arterial injection can administer the antibody or any active functional portion thereof to the organism to be diagnosed. The antibody / antigen complex can be detected by a label attached to the antibody or functional fragment thereof.

Immunoassays used for diagnostic applications typically rely on labeled antigens, antibodies or secondary reagents for detection. These proteins or reagents may be labeled with compounds commonly known to those of skill in the art, including fluorescent, luminescent and chromogenic substances including enzymes, radioisotopes, and colored particles such as colloidal gold and latex beads. can. Of these, radioisotope labeling can be used in almost all types of assays and has a great deal of variation. Labeling with enzyme binding is especially useful when radioactivity must be avoided or when rapid results are required. Fluorescent dyes require expensive equipment to use, but provide extremely sensitive detection methods. Antibodies useful in these assays include monoclonal antibodies, polyclonal antibodies, and affinity-purified polyclonal antibodies.

Alternatively, the antibody may be indirectly labeled by reaction with a labeling substance having an affinity for immunoglobulin, such as protein A or G or a second antibody. The antibody may be bound to a second substance and detected using a labeled third substance that has an affinity for the second substance bound to the antibody. For example, the antibody may be bound to biotin and labeled avidin or streptavidin may be used to detect the antibody-biotin conjugate. Similarly, the antibody may be bound to a hapten and a labeled anti-hapten antibody may be used to detect the antibody-hapten conjugate.

Those skilled in the art will be aware of the above and other suitable labels that may be used in accordance with the present invention. Binding of these labels to an antibody or fragment thereof can be performed using standard techniques known to those of skill in the art. Typical methods are described in Kennedy, J. H., et al., 1976 (Clin. Chim. Acta 70: 1-31) and Schurs, A. H. W. M., et al. 1977 (Clin. Chim Acta 81: 1-40). ing. Coupling methods referred to in the latter include the glutaraldehyde method, the periodic acid method, the dimaleimide method, and others, all of which are incorporated herein by reference.

Current immunoassay methods utilize the dual antibody method to detect the presence of the analyte in the method. The antibody is indirectly labeled by reaction with a second antibody labeled with a detectable label. The second antibody preferably binds to the animal antibody from which the monoclonal antibody was derived. In other words, if the monoclonal antibody is a mouse antibody, the labeled second antibody is an anti-mouse antibody. When using a monoclonal antibody in the assay described below, this label is preferably antibody-coated beads, especially magnetic beads. When using polyclonal antibodies for the immunoassays described herein, the label is preferably a detectable molecule, such as a radioactive, fluorescent or electrochemically luminescent substance.

Alternative dual antibody systems, often referred to as rapid formatting systems because they are adapted for rapid determination of the presence of an analyte, can also be used within the scope of the invention. The system requires a high affinity between the antibody and the analyte. According to one aspect of the invention, the presence of amyloid protein is determined using a pair of antibodies, each specific for the amyloid protein. One of the antibody pairs is referred to herein as a "detection antibody" and the other of the antibody pairs is referred to herein as a "capture antibody". The monoclonal antibody of the present invention can be used as either a capture antibody or a detection antibody. The monoclonal antibody of the present invention can also be used as both a capture antibody and a detection antibody in a single assay. Therefore, one embodiment of the present invention uses a double antibody sandwich method to detect amyloid protein in a sample of biological fluid. In this method, the analyte (amyloid protein) is sandwiched between the detection antibody and the capture antibody, and the capture antibody is irreversibly immobilized on a solid support. The detection antibody comprises a detectable label to identify the presence of the antibody-analyte sandwich and thus the presence of the analyte.

Exemplary solid phase materials include, but are not limited to, microtiter plates, polystyrene test tubes, magnetic beads, plastic beads, or glass beads and slides well known in the field of radioimmunoassays and enzyme immunoassays. .. Methods for coupling the antibody to the solid phase are also well known to those of skill in the art. More recently, various porous materials such as nylon, nitrocellulose, cellulose acetate, glass fiber and other porous polymers have been used as solid supports.

The invention also relates to a diagnostic kit for detecting amyloid proteins in biological samples, including the compositions defined above. Furthermore, the present invention also relates to the latter diagnostic kit, which comprises, in addition to the compositions defined above, the detection reagents defined above. The term "diagnostic kit" generally refers to any diagnostic kit known in the art. More specifically, the latter term refers to the diagnostic kit described in Zrein et al. (1998).

It is yet another object of the invention to provide novel immune probes and test kits for the detection and diagnosis of amyloid-related diseases and conditions, including antibodies according to the invention. For immune probes, the antibody binds directly or indirectly to a suitable reporter molecule, such as an enzyme or radionuclide. The test kit combines one or more antibodies according to the invention with an amyloid protein to form an immunological complex, such that the presence or absence of the immunological complex correlates with the presence or absence of the amyloid protein. Includes a container containing instructions for use for the purpose of detecting the formation of immunological complexes.

Example Material The development and preparation of the mouse monoclonal antibody ACI-O1-Ab7C2 (named "mC2" throughout this application and for humanized C2 antibody hC2) is hereby referred to in its disclosure. It is described in EP 05 02 709 2.5, a co-pending application filed December 12, 2005, which is incorporated herein.

The hybridoma cell FP-12H3-C2, which produces the mouse monoclonal antibody ACI-O1-Ab7C2 (named "mC2" and for humanized C2 antibody hC2 throughout this application), was released in 2005. On December 1, in the co-pending application EP05027092.5, Braunschweig, Mascheroder Weg 1B, 38124 Braunschweig's Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), the provisions of the Budapest Treaty and the given accession number. : Deposited under DSM ACC 2750.

Hybridoma cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (penicillin / streptomycin). The isotype of the antibody produced was examined and, as expected, was found to be mouse IgG2b / kappa.

Assay ELISA for binding to amyloid β provided a reliable measure of the efficacy of C2 antibody. Positive control antibody, mouse FP-12H3-C2 antibody (Genovac lot number: AK379 / 01), and standard Chemicon antibody 1560 (lot number: 0508008791).

Selection of human constant region Since immune system recruitment is not desirable for clinical antibody candidates, the human constant region selected for heavy chains was modified to convert serine at position 228 in the hinge region to proline, human IgG4 ( It was HuIgG4 Ser-Pro). This mutation stabilizes interchain disulfide bonds and prevents the formation of half-molecules that can occur in native human IgG4 preparations. Antibodies expressed from the producing cell line are also believed to have had the terminal lysine removed. The sequences of the human constant region HuIgG4 Ser-Pro and human kappa are shown in SEQ ID NOs: 17 and 14, respectively.

Example 1 Cloning and sequencing of antibody variable regions Total RNA was prepared from 3 × 10 ⁶ hybridoma cells (1 T175 flask) using the Qiagen RNeasy minikit (catalog number: 74104). RNA was eluted in 50 μL water and examined on a 1.2% agarose gel. Cell-derived conditioned medium was retained and used to test samples by antibody activity assay.

V _H and V _K cDNAs were prepared with reverse transcriptase with mouse IgG and κ constant chain primers. First-strand cDNA was amplified by PCR with many sets of signal sequence primers. The amplified DNA was gel-purified and cloned into the vector pGem® T Easy (Promega). The resulting V _H and V _K clones were screened by PCR for expected sized inserts and the DNA sequences of the selected clones were determined by automated DNA sequencing. The location of complementarity determining regions (CDRs) in the sequence was determined with reference to other antibody sequences (Kabat EA et al., 1991). Kabat's numbering rules for antibody variable regions are used throughout this application; therefore, residue numbers may differ from the exact linear numbers.

The DNA sequence and deduced amino acid sequence for mC2V _K are shown in SEQ ID NOs: 29 and 27, respectively. Four clones gave rise to this identical productive sequence. _Unproductive and aberrant VK sequences resulting from hybridoma fusion partners were also found in many clones.

Two different productive sequences were isolated for mC2 V _H. The mC2 V _H AF sequence (see SEQ ID NO: 30) was found in a total of 29 clones, with 14 1 base pair changes in each clone. The mC2 V _H B sequence was found in a total of 8 clones. Five of these showed the major sequence and the other three clones were mutations to this. It is possible that these similar V _H B sequences arose as an artificial product of PCR amplification. An unproductive and anomalous V _H is also obtained from the C2 hybridoma, resulting from a defective VDJ connection.

To determine which is the correct active mC2 V _H , two chimeric antibodies were prepared with two different V _H sequences, AF and B, combined with mC2 V _K and tested for correct antibody activity.

Example 2 Construction of Chimeric Antibody Gene The most common form of human chimeric antibody consists of a human constant region linked to a mouse (or other non-human) variable region. Chimera antibodies have the same effector function and the same secondary detection reagents as humanized or artificially produced antibodies, firstly to confirm that the correct variable region has been identified. It provides a very useful tool for use as a control antibody in antigen binding assays, and is used to study the pharmacokinetics and other properties of human constant regions for a particular target of an antibody. You may.

Two chimeric heavy chain expression vectors consisting of mC2 VHA _{F or mC2 VH B} variable regions linked to the Hu IgG4 (Ser-Pro) constant region in the expression vector pSVgpt were constructed. It is based on pSV ₂ gpt (Mulligan and Berg, 1980) and ampicillin resistance gene for selection in bacterial cells, gpt gene for selection in mammalian cells, mouse heavy chain immunoglobulin enhancer region, constant Includes a genomic sequence encoding a regional gene, and an SV40 poly A sequence. The heavy chain variable region for expression is inserted as a HindIII-BamHI fragment.

A chimeric light chain vector consisting of C2 VK linked to the human C kappa constant region was constructed in the expression vector pSVhyg (Hieter PA et al, 1980). pSVhyg contains the ampicillin resistance gene for selection in bacterial cells, the hyg gene for selection in mammalian cells, the mouse heavy chain immunoglobulin enhancer region, the genomic sequence encoding the kappa constant region gene and containing the kappa enhancer, and Includes SV40 poly A sequence. The light chain variable region for expression is inserted as a HindIII-BamHI fragment.

An expression cassette for mouse C2 VH and VK sequences using the vectors VH-PCR1 and VK-PCR1 as templates (Riechmann et al., 1988) containing a leader signal peptide, a leader intron, and a mouse immunoglobulin promoter 5'. It was constructed by addition of an adjacency sequence, as well as a 3'adjacent sequence containing a splice site and an intron sequence. It was confirmed that the DNA sequences of VH and VK in the chimeric expression vector were correct. The DNA and amino acid sequences of the VH and VK genes in the expression cassette are shown in Figures 1 and 2.

Example 3 Expression of chimeric antibody
3.1 Expression in stable cell lines The host cell line for antibody expression was NS0, a non-immunoglobulin-producing mouse myeloma obtained from the European Collection of Animal Cell Cultures, Porton UK (ECACC No. 85110503). Heavy and light chain expression vectors were cotransfected into NS0 cells by electroporation. Colonies expressing the gpt gene were selected in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.8 μg / ml mycophenolic acid, and 250 μg / ml xanthine. Transfected cell clones were screened for human antibody production by ELISA for human IgG. Cell lines that secrete antibodies were grown, the ones that produced the most were selected, and they were submerged in liquid nitrogen and frozen. The highest producing cell lines for each antibody were grown in the same medium as above, but with only 5% FBS. Chimeric antibodies were purified using Prosep®-A (Bioprocessing Ltd). The concentration was determined by ELISA for human IgGκ antibody. Antibodies were also analyzed by SDS-PAGE.

3.2 Transient expression of chimeric antibody To facilitate testing of different chimeric antibodies, transient expression was used to rapidly produce a small amount of cell supernatant containing recombinant antibody for testing. The mC2 V _H and V _K expression cassettes were transferred to a pcDNA3.1 (Invitrogen) based vector for transient expression. The heavy chain vector contained a human IgG constant region. The light chain vector contained a human kappa constant region. Transfect both mC2 V HA and mC2 V _{H B} to human fetal kidney (HEK 298) cells with mC2 V _K according to the protocol supplied by the manufacturer using the Lipofectamine 2000 reagent (Invitrogen catalog number: 11668). It was perfect. The conditioned medium was harvested from the cells 3 days after transfection. The amount of antibody produced was determined by ELISA for human IgGκ antibody.

Example 4 Chimeric C2 antibody activity
4.1 Activity of chimeric C2 antibody produced by transient transfection
Samples of acclimatized medium from transient transfection for two different chimeric antibodies were tested by ELISA for binding to amyloid β. The results clearly show that C2 VH AF is the correct sequence. The C2 V _H AF / C2 V _K chimeric antibody binds well in this assay, but C2 V _H B / C2 V _K shows no binding. Chemicon 1560 mouse control antibody showed good binding, but binding with the supplied purified mouse C2 antibody was slight. It should be noted that the results are not directly comparable, as different secondary antibodies were used for the mouse antibody with the mouse constant region compared to the chimeric antibody with the human constant region. It was later found that the C2 hybridoma-derived conditioned medium yielded good results in this assay.

4.2 Activity of purified chimeric C2 antibody
Two different C2 chimeric antibodies were purified from a stable NS0 cell line as described and tested with amyloid β ELISA. The results obtained are consistent with those obtained with the transiently expressed antibody. The C2 ChVH AF / ChVK antibody binds well in ELISA and the C2 ChVH B / ChVK antibody does not bind at all.

Example 5 Design of humanized C2 antibody gene
The mC2 V _H and V _K amino acid sequences were compared to the rodent antibody V _H and V _K sequences in the NCBI and Kabat databases.

5.1 Light chain variable region
The closest matching mouse germline gene to mC2V _K is bbl, Locus MMU231201 (Schable et al, 1999). Only two amino acids differ from this germline sequence, both located within CDRL1. Mature mouse antibodies with similar but non-identical sequences are found. Some have the same CDRL2 and the same CDRL3, but the mC2 CDRL1 appears to be unique. You can assign mC2 V _K to the Kabat subgroup Mu V _K II. Position 87 of mC2V _K is F rather than Y, which is more common in the subgroup, indicating that this framework residue may be important for antibody activity. Comparison with human germline V _K sequences has shown that genes from subgroup V _K II are the best match for mC2 V _K (Cox et al, 1994). The sequence DPK 15 was selected with the human J region Hu J _K 1 to provide an acceptor framework sequence for humanized V _K.

Four humanized V _K sequences were designed. _C2HuVK1 consists of _mC2VK CDR with a framework derived from DPK 15 and human JK1. In versions 2, 3, and 4, mouse residues have been replaced at position 45, 87, or both in the framework. Residue 45 may be involved in supporting the conformation of the CDR. Residue 87 is located at the interface between the V _H and V _K domains. Therefore, these residues may have crucial implications for maintaining antibody binding. Table 6 shows the changes and positions made in the light chain framework region. Comparison of humanized sequences with mC 2 V _K sequences, as well as DP K 15 and human J _K 1.

5.2 Heavy chain variable region
The closest matching mouse germline genes to mC2 VHA are _VH7183 , Locus AF120466 (Langdon et al, 2000). A comparison is shown in Figure 3. Nine amino acids differ from this germline sequence and are mostly located in CDR2. Mature mouse antibodies with identical or similar (one residue different) CDR1 or similar CDR2 (one residue different) are found, but antibodies with all three CDRs identical to mC2 VHA _F not. The mC2 antibody CDR3 is unusually short and consists of only 3 residues. However, other antibodies with this length of CDR3 are found in the database. mC2 V _H AF can be assigned to the Kabat subgroup Mu V _H III D. Residue 47 of mC2 V _H is L rather than the more general W, and residue 94 is S rather than normal R, which means that these framework residues may be useful for antibody activity. It shows that. Comparison with the human germline V _H sequence shows that the genes from the subgroup V _H III are the best match for mC2 V _H. Sequence DP54 was selected with human J region HuJ _H 6 to provide an acceptor framework sequence for humanized _VH .

Four humanized _VH sequences were designed. C2HuVH1 consists of mC2 V _H AF CDR with frameworks derived from DP 54 and HuJ _H 6. In versions 2, 3, and 4, mouse residues have been replaced at position 47, 94, or both in the framework. Residue 47 in framework 2 contacts both the CDR and _VK domains. Residue 94 may be involved in supporting the conformation of the CDR. Therefore, these residues may have crucial implications for maintaining antibody binding.

Table 7 shows the changes and positions made in the heavy chain framework region.

Example 6 Construction of humanized antibody gene A modified variable region was constructed by a method called overlapping PCR recombination. Expression cassettes for the chimeric antibody C2 ChV _H AF and C2 ChV _K were used as templates for mutagenesis into the required sequences of the framework region. A set of mutagenesis primer pairs containing the region to be modified was synthesized. The produced humanized V _H and V _K expression cassettes were cloned into pUC19 and the entire DNA sequence was confirmed to be correct for each V _H and V _K. Modified heavy and light chain V region genes were excised from pUC19 as HindIII-BamHI expression cassettes. These were transferred to expression vectors pSVgpt and pSVhyg containing constant regions of human IgG4 Ser-Pro or κ, respectively, as for the chimeric antibody vector. We confirmed that the DNA sequence was correct for humanized V _H and V _K in the expression vector.

Example 7 Expression of humanized antibody
7.1 Expression in stable cell lines Humanized heavy and light chain expression vectors were electrotransfected into NS0 cells as well as for chimeric antibody expression. Antibody-producing cell lines were selected and propagated to purify humanized antibodies in exactly the same manner as for chimeric antibodies. The purified antibody was analyzed by SDS-PAGE.

7.2 Transient expression of humanized antibodies To facilitate testing of different humanized V _H and V _K constructs, the C2 humanized V _H and V _K expression cassettes are also for transient expression described in Section 7.2. Transferred to the vector of. Four humanized C2V _K constructs were cotransfected into HEK293 cells with chimeric C2V _H constructs. Similarly, four humanized C2V _H constructs, along with chimeric C2V _K constructs, were cotransfected into HEK293 cells. The conditioned medium was harvested from the cells 3 days after transfection. The amount of antibody produced was determined by ELISA for human IgGκ antibody.

Example 8 Activity of humanized C2 antibody
8.1 Activity of humanized C2 antibody produced by transient transfection A sample of acclimatized medium derived from transient transfection was tested with amyloid β ELISA. The results obtained clearly show that versions 2 and 4 of the humanized VH construct C2 HuVH AF are functional when combined with the chimeric C2 kappa chain and comparable to the chimeric C2 antibody in this assay. It is shown in. In contrast, antibodies containing versions 1 and 3 of C2 HuVH AF combined with the chimeric C2 kappa chain show no binding in this assay. This indicates that substitution of mouse residues at position 94 is essential for antibody activity. All antibodies containing the chimeric C2 heavy chain combined with the four humanized C2 kappa chains showed comparable good binding to the chimeric antibody by ELISA.

8.2 Purified humanized C2 antibody activity
Eight different humanized C2 antibodies, including all combinations of two humanized heavy chains and four humanized light chains, were purified from stable NS0 cell lines as described and tested with amyloid β ELISA (Figure). Four).

The results obtained clearly show that the C2 HuVH4 antibody works better in this assay than the C2 HuVH2 antibody. Of the C2 HuVH2 antibodies, C2 HuVH2 / HuVK3 exhibits the highest binding activity, which is approximately 2-fold reduction compared to the chimeric control antibody C2 ChVHAF / ChVK. C2 HuVH2 / HuVK2 activity is 4-5 fold reduced compared to controls. The activity of antibodies with four different humanized light chains, including C2HuVH4, is very similar. The highest activity was observed for C2HuVH4 / HuVK1 and all four antibodies were close to the control chimeric antibody in this assay.

Example 9 Modification of CDRL2
9.1 Design of light chains with modified CDR2 As mentioned above, many antibodies share the same CDRL2 sequence (“KVSNRFS”) as C2 antibodies. We decided to test whether CDRL2 could be modified slightly without adversely affecting antibody activity. Two conservative substitutions were selected: the K and R substitutions at position 50 and the N and S substitutions at position 53. Therefore, the two alternative CDRL2 sequences are "RVSNRFS" and "KVSSRFS". These were incorporated into the mouse _VK sequence as mC2 VK-R and mC2 VK-S, respectively, without any other changes.

9.2 Transient expression of modified CDRL2 antibody Two C2 light chain constructs with modified CDRL2 described in Section 11.2.1 were cloned into a light chain vector for transient expression. Each was cotransfected into HEK293 cells with a chimeric _C2VH vector. The conditioned medium was harvested from the cells 3 days after transfection. The amount of antibody produced was determined by ELISA for human IgGκ antibody.

9.3 Activity of C2 antibody with modified CDRL2
Samples of acclimatized medium from transient transfection of mC2 VK with modified _CDRL2 in combination with mC2 V _H were tested with amyloid β ELISA (Fig. 5). Both VK-R and VK-S antibodies are comparable to chimeric C2 antibodies, indicating that selected individual modifications to CDRL2 do not have a significant effect on antibody activity in this assay. ..

Example 10 Amyloid β1 to evaluate the binding specificity and affinity of affinity-determining mouse (ACI-O1-Ab-7-C2) antibody, chimeric (AF) antibody, and humanized antibody (H4K1; H4K4). BIACORE® analysis was performed using -42 monomers and fibers as antigens immobilized on CM5 chips. BIACORE® technology utilizes changes in the index of refraction at the surface layer due to the binding of the antibody to the antigen immobilized on the layer. Bonding is detected by surface plasmon resonance (SPR) of laser light refracted from the surface. Analysis of signal dynamics for binding and dissociation rates makes it possible to distinguish between non-specific and specific interactions. The concentration of the antibody used was in the range of 0.05 μM to 1.0 μM.

(Table 1) Binding specificity and affinity of mouse (ACI-O1-Ab-7-C2) antibody, chimeric (AF) antibody, and humanized antibody (H4K1; H4K4) against amyloid β1-42 monomers and fibers. sex

Example 11 Immunohistochemical binding assay
11.1 Human brain sections Brains from healthy patients, pre-AD patients with non-dementia, and AD patients were obtained from Bonn's Universitatsklinik after ethical approval. The brain was fixed in formaldehyde, the hippocampal region was dehydrated, embedded in paraffin, and 5 μm sections were cut with a microtome. Paraffin sections were stored at RT until used. For fresh material, 5 μm frozen sections were cut with a cryostat and stored at -80 ° C until the sections were used.

11.2 Immunohistochemistry Paraffin sections were deparaffinized and the slides were rehydrated by soaking in xylene and then in 100% ethanol, 90% ethanol, and 70% ethanol. Background was reduced by 30 minutes incubation in water containing 10% _H202 , ₁₀ % methanol. Antigen recovery was obtained by incubating the slides in 100% formic acid for 3 minutes. Non-specific labeling by 2-hour incubation of slides in TBS containing 10% BSA, 0.25% Triton X-100 after 3 washes in Tris buffered saline (TBS, pH 7.5). Blocked. After washing (three times in TBS), unlabeled anti-human IgG (Biomeda) was added and the slides were incubated overnight at RT in a wet chamber to block endogenous antibodies. After three more washes, primary human anti-amyloid antibody was added to the slides and incubated at RT for an additional 24 hours. After washing, alkaline phosphatase-labeled secondary anti-human IgG (Sigma) was added to the slides and incubated at RT for 2 hours. After washing, the slides were stained with Liquid permanent Red (Dakocytomation), washed with water and air dried before specimens with a non-fading mounting agent (corbitbalsam).

Frozen sections were fixed in methanol at -80 ° C for 30 minutes, H ₂ O ₂ was added to cold methanol to a final concentration of 10%, and the background was reduced by incubating at RT for 30 minutes. Non-specific by incubating the slides for 2 hours in TBS containing 10% BSA, 0.25% Triton X 100 as described above after 3 washes in Tris buffered saline (TBS, pH 7.5). The label was blocked and the same staining procedure as above was performed.

Sections were examined under a Leica DMLB microscope and photographs were taken using a Leica DC500 camera and Leica FireCam 1.2.0 software.

Both human antibodies A and C labeled brain plaques from patients with AD disease (Fig. 6). Both diffuse spots and spots with hollow cores were labeled. In addition, diffused plaques in pre-AD patients with non-dementia could also be detected with A and C antibodies. Amyloid in cerebral amyloid vasculopathy (CAA) was labeled with both antibodies, and some staining of neurons that may correspond to intracellular amyloid was also detected. No labeling was seen in control brains from healthy patients. Spots could be detected on paraffin sections pretreated with formic acid, but no spots were labeled on paraffin sections without formic acid pretreatment and frozen sections fixed in methanol. Human antibody B did not detect spots on the paraffin section, and mouse antibody did not stain either the paraffin section or the frozen section of the human brain.

Abbreviation:
A = Chimeric antibody that binds AF (IgG4) (mC2Ch VHAF)
B = Chimeric antibody that does not bind B (IgG4) (mC2VHB)
C = Bound humanized antibody H4K1 (IgG4) (HuVH4 / HuVK1)
Mouse = ACI-01-Ab-C2 mouse antibody (IgG2b)

Example 12 Functionality of mC2 for amyloid fibrils
12.1 Modification of the three-dimensional structure of Aa1-42 fibers and initiation of deaggregation after binding of mC2 antibody To evaluate the mechanism by which the antibody deaggregates preformed β-amyloid (Aβ _1-42 ) fibers. Direct thioflavin-T (Th-T) fluorescence assay to measure aggregation and solid-state nuclear magnetic resonance (NMR) of U- ¹³ C tyrosine 10 and valine 12 labeled Aβ1-42 peptides to analyze secondary conformation. A comparison was made (Fig. 7A). The mC2 antibody solubilized 35.4% of the preformed Aβ1-42 fibers and at the same time induced the conversion of the secondary conformation from the β-sheet to a random coil. The reduction in the population of β-sheet 3D structures relative to the random coil 3D structure is only about 35%, which is in good agreement with that measured using the fluorescent Th-T assay (Fig. 7B). These data indicate that binding of the mC2 antibody triggers a transfer of secondary structure, which causes destabilization of the parallel intermolecular arrangement of β-sheets, resulting in the breakdown of elongated fibers into smaller fragments. It shows the possibility of affecting.

12.2 Three-dimensional structure-dependent binding affinity of mC2 antibody Because it is well known in the scientific literature that a percentage of antibody-antigen binding energy can be used for energy-dependent modification of the three-dimensional structure of an antigen (Blond and Goldberg). , 1987), a comparative experiment on the binding affinity of the C2 antibody for the entire Aβ _1-42 protein and for smaller 9 amino acid length peptides containing antibody epitopes (Fig. 8). For this comparison, the affinity of the humanized antibody C2 was determined by biotinylated peptide (manufactured by Mimotopes, purchased from ANAWA Trading SA) and biotinylated complete Aβ1-42 peptide (manufactured by Mimotopes) covering the complete amino acid sequence of the epitope of C2. It was analyzed by ELISA using Bachem). The analysis was performed according to the instructions of the manufacturer (Mimotopes). As shown in Figure 8 and Table 2, this antibody has a 36.0% higher affinity for peptides containing its particular epitope (amino acids 13-21 of the Aβ _1-42 sequence) than for all Aβ1-42 proteins. Combined with. This suggests that the difference in binding affinity energy was used for the energy-consuming transition of the secondary conformation of the amyloid protein, such as presenting the antigen at a more acceptable position for antibody interaction. .. This explains why the affinity of the antibody is lower for the natural state (whole amyloid protein) than for the isolated subunit.

(Table 2)

Example 13 Effect of Anti-Amyloid hC2 on Amyloid β1-42 Peptide Aggregation To assess the ability of humanized anti-human amyloid β monoclonal antibody hC2 to mediate anti-aggregation and deaggregation effects on amyloid β (Aβ), thioflavin. A T-spectral fluorescence assay was performed.

13.1 Aggregation inhibition assay
Aβ1-42 lyophilized powder was reconstituted with hexafluoroisopropanol (HFIP) to 1 mM. The peptide solution was sonicated at room temperature for 15 minutes, stirred overnight to make an aliquot and placed in a non-siliconized microcentrifuge tube. The HFIP was then evaporated under a stream of argon. The resulting peptide membrane was vacuum dried for 10 minutes and stored at -80 ° C until use.

To assay for antibody-mediated inhibition of Aβ1-42 aggregation, the hC2 antibody was prediluted in PBS and assay solutions containing the following components were made in unsiliconized incubation tubes: 3.3 or 0.33. μM pre-diluted antibody, 10 μM thioflavin T, 33 μM Aβ1-42, and 8.2% DMSO. Therefore, the final molar ratio of antibody to Aβ1-42 was 1:10 and 1: 100. A suitable control solution was also prepared. The solution was then incubated for 24 hours at 37 ° C. and spectrofluorescence (relative fluorescence unit; RFU) was read repeatedly on a Perkin-Elmer FluoroCount spectrofluorometer 6 times in a black 384-well plate (Perkin-Elmer). Spectral fluorescence was then measured and the percent disaggregation was calculated as described below.

13.2 Deaggregation Assay To assay for antibody-mediated deagglomeration of preaggregated Aβ1-42, the low molecular weight Aβ1-42 prepared above was used as a 110 μM solution in 27% DMSO and 1 × PBS. Made. The solution was then aggregated at 37 ° C. for 24 hours, after which the following was added: 3.3 or 0.33 μM pre-diluted antibody, and 10 μM thioflavin T. This resulted in a molar ratio of antibody to Aβ1-42 of 1:10 and 1: 100. The solution was then incubated for an additional 24 hours at 37 ° C. Spectral fluorescence was then measured and the percent disaggregation was calculated as described below.

13.3 Computational aggregation inhibition or deagglomeration is expressed as mean inhibition percent or deagglomeration ± mean standard deviation (SEM), respectively, according to the following equation.

13.4 Results
13.4.1 Aβ1-42 Inhibition of aggregation
Inhibition of Aβ1-42 aggregation using hC2 antibody is shown in Table 3 and FIG. At a 1: 100 antibody-to-Aβ 1-42 molar ratio, inhibition averaged 30% (two independent experiments), whereas at a 1:10 molar ratio, inhibition averaged 80%. (Two independent experiments; see Table 3).

(Table 3) hC2-mediated inhibition of Aβ1-42 aggregation at 1: 100 and 1:10 antibody to Aβ 1-42 molar ratios

13.4.2 Deagglomeration of preaggregated Aβ1-42
The deagglomeration of preaggregated Aβ1-42 using the hC2 antibody is shown in Table 4 and FIG. At a 1: 100 antibody-to-Aβ 1-42 molar ratio, disaggregation averaged 24%, whereas at a 1:10 molar ratio, inhibition averaged 32% (three times). Independent experiment; see Table 4).

(Table 4) hC2-mediated deaggregation of preaggregated Aβ1-42 aggregation at 1: 100 and 1:10 antibody to Aβ 1-42 molar ratios

Using the thioflavin T assay, the dual functional properties of the anti-Aβ humanized antibody hC2, ie, inhibition of aggregation of Aβ1-42 to the pathogenic protofibril conformation, and further preformed Aβ1-42 protofibrils. Can be shown to deaggregate. hC2 inhibited Aβ1-42 aggregation by 80% at a 1:10 antibody-to-Aβ1-42 molar ratio. The ability of hC2 to deaggregate the preaggregated protofibrils of Aβ1-42 at a molar ratio of 1:10 was shown to be 32%.

Example 14: Three-dimensional structure-specific binding of mC2 to different classes of amyloid proteins To assess the specificity of mC2 for different stages of polymerized amyloid proteins: monomeric amyloid, polymer-soluble amyloid, and fibril amyloid. To this end, ELISA coated with these different stages of polymer β-amyloid was performed (Fig. 9). The monomers are prepared by the modified method published by (Klein, 2002), the soluble polymer amyloid β is prepared by (Barghorn et al., 2005), while the fibers are prepared at a final concentration of 1 μg / μl. Preformed by incubation in Tris / HCl pH 7.4 of amyloid (Bachem, Switzerland) at 37 ° C. for 5 days, followed by centrifugation (10,000 rpm, 5 minutes). Then, the amyloid polymer was coated on an ELISA plate at a final concentration of 55 μg / ml, and binding affinity ELISA was performed by using an anti-mouse IgG monoclonal antibody (Jackson) labeled with alkaline phosphatase. As shown in Table 5, the mC2 antibody bound to the soluble polymer amyloid β with higher affinity for fiber and with the lowest affinity for monomer. These data indicate that antibody binding is affected by amyloid epitopes and by the conformation of different amyloid aggregates.

(Table 5) Three-dimensional structure-specific binding of mC2 to amyloid monomers, oligomers, and fibers

Example 15: Epitope mapping of the AC-immunized monoclonal antibody hC2 Epitope mapping of the humanized monoclonal antibody hC2 was performed by ELISA using three different peptide libraries. One library contained a total of 33 biotinylated peptides covering the complete amino acid (aa) sequence of Aβ1-42 (manufactured by Mimotopes and purchased from ANAWA Trading SA). The second library uses peptide 12 from the first peptide library (aa12-20 of Aβ) and contains biotinylated peptides in which each aa in the sequence is replaced by alanine (see Table 8 below). ), The third library contains biotinylated peptides 13, 14 or 15 (Aβ aa 13-21, 14-22, or 15-23), in which case the last amino acid is alanine, or already. It has been replaced with glycine for aa 21, which is alanine (see Table 9 below). The biotinylated complete Aβ1-42 peptide was used as a positive control (Bachem). Epitope mapping was performed according to the manufacturer's instructions. Briefly, streptavidin-coated plates (NUNC) were blocked overnight at 4 ° C with 0.1% BSA in PBS. After washing with PBS-0.05% Tween 20, the plates were coated over 1 hour at room temperature with various peptides from the library diluted to a final concentration of 10 μM in 0.1% BSA in PBS, 0.1% sodium azide. .. After washing, the plates were incubated with 2% BSA in PBS, hC2 antibody diluted to 200 ng / ml in 0.1% sodium azide or non-Aβ binding chimeric IgG4 antibody for 1 hour at room temperature. The plates were washed again and incubated with goat anti-human IgG bound with alkaline phosphatase for 1 hour at room temperature. After the final wash, the plates were incubated with a phosphatase substrate (pNPP) and read at 405 nm using an ELISA plate reader.

The humanized monoclonal antibody hC2 was shown to specifically bind to peptides 12, 13, 14, 15 and 16 of the first peptide library. These peptides contain aa 12-20, 13-21, 14-22, 15-23, and 16-24 of Aβ1-42, respectively, which indicates that the epitope is located in region 12-24 of Aβ. Suggest. A second library with alanine substitutions was used to determine the definitive aa for binding to Aβ12-20 (VHHQKLVFF). Substitution of amino acids 16, 17, 19 or 20 with alanine completely abolishes hC2 antibody binding, indicating that these aa are highly crucial for antibody binding to Aβ. When aa 15 and 18 were replaced, some hC2 antibody binding was lost.

Substitution of aa 14 with alanine also almost completely lost the binding, indicating that aa 14 is also very important for binding.

Finally, a third library was used to determine if aa 21, 22 or 23 was deterministic for binding to the epitope. Substitution of aa 23 with alanine reduced antibody binding to aa 15-23, indicating that aa 23 is also important for binding. When aa 21 was replaced with glycine, some of the binding was lost, and when aa 22 was replaced with alanine, it was slightly lost.

Example 16: Neuroprotection with hC2 antibody The ability of the antibody hC2 to protect neurons from Aβ oligomer-induced denaturation was evaluated by an in vitro assay. Mouse cortical neurons at embryonic days 16.5-17.5 were isolated, dissociated and cultured in vitro in N3-F12 medium. Cells were grown for a total of 9 days and nourished on day 3 and on the day of addition of Aβ oligomers or Aβ oligomers and the anti-Aβ antibody hC2. On day 5 (“4 days Aβ”) or day 6 (“3 days Aβ”), cells in a well were treated with 2 μM Aβ oligomers alone or with 2 μM Aβ oligomers and 50 μg / mL anti-Aβ. Treated with any of the antibodies hC2.

Aβ oligomers are prepared by dissolving Aβ1-42 (r peptide) in HFIP, from which the Aβ peptide is dispensed into a 1 Oμl aliquot at 1 mg / ml and then evaporated in a draft for 30 minutes using a peptide membrane. Stored at -80 ° C until At the time of use, the peptide membrane was dissolved in 10 μl DMSO followed by 78.6 μl HAMS F12 and the Aβ peptide solution was incubated at 4 ° C. for 24-48 hours (25 μM final concentration Aβ).

For control cells, DMSO-F12 alone was added in the same volume as Aβ-DMSO on day 5, and the cells were cultured for an additional 4 days without any additional treatment. On day 9, neurons from all culture conditions were fixed and stained with Tuj1 (anti-β-tubulin antibody) followed by FITC-labeled secondary antibody to microtubules and thus neurons. The general protrusions of the above were visualized. The results are shown in FIG.

Untreated mouse fetal cortical neurons showed normal morphology 9 days after culture (Fig. 13, leftmost panel). Treatment of cells with Aβ oligomers for 3 days induced axonal degeneration and resulted in a decrease in the total number of axons (Fig. 13, lower center panel), an even more pronounced effect on day 4 of treatment. (Fig. 13, upper center panel). In contrast, cells treated with the combination of Aβ oligomer and anti-Aβ antibody hC2 looked similar to control cells (Figure 13, upper right and lower right panels). These results indicate that the anti-Aβ antibody hC2 was able to protect fetal mouse cortical neurons from Aβ oligomer-induced denaturation.

(Table 6) Changes and positions made in the humanized C2 light chain framework region

合計8つの異なる抗体を、軽鎖ヒト化C2HuV_K1、C2HuV_K2、C2HuV_K3、C2HuV_K4、ならびに重鎖C2HuVHAF4およびC2HuVHAF2を用いて構築した。 (Table 7) Changes and positions made in the humanized C2 heavy chain framework region

A total of eight different antibodies were constructed using light chain humanized C2HuV _K 1, C2HuV _K 2, C2HuV _K 3, C2HuV _K 4, and heavy chains C2HuVHAF4 and C2HuVHAF2.

(Table 8) Summary of peptides used in the second library aa important for binding is shown in italics and underline, and aa decisive for binding is shown in italics and bold.

(Table 9) Summary of peptides used in the third library The aa important for binding is shown in italics and underline, and the aa decisive for binding is shown in italics and bold.

Bibliography list

Brief description of sequence SEQ ID NO: 1 C2 HuVH AF 4 Amino acid sequence of humanized heavy chain variable region (CDR1).
SEQ ID NO: 2 C2 HuVH AF 4 Amino acid sequence of humanized heavy chain variable region (CDR2).
SEQ ID NO: 3 C2 HuVH AF 4 Amino acid sequence of humanized heavy chain variable region (CDR3).
SEQ ID NO: 4 C2 HuVK 1 Amino acid sequence of the humanized light chain variable region (CDR1).
SEQ ID NO: 5 C2 HuVK 1 Amino acid sequence of the humanized light chain variable region (CDR2).
SEQ ID NO: 6 C2 HuVK 1 Amino acid sequence of the humanized light chain variable region (CDR3).
SEQ ID NO: 7 Amino acid sequence of Aβ epitope region 2.
SEQ ID NO: 8 Amino acid sequence of Aβ epitope region 1.
SEQ ID NO: 9 Amino acid sequence of modified Aβ epitope region 2.
SEQ ID NO: 10 Amino acid sequence of modified Aβ epitope region 1.
SEQ ID NO: 11 Amino acid sequence of the fully modified epitope region.
SEQ ID NO: 12 C2 HuVK 1 Amino acid sequence of the humanized light chain variable region.
SEQ ID NO: 13 Amino acid sequence of C2 humanized light chain.
SEQ ID NO: 14 Amino acid sequence of the humanized C2 light chain constant region.
SEQ ID NO: 15 C2 HuVH AF 4 Amino acid sequence of humanized heavy chain variable region.
SEQ ID NO: 16 C2 Amino acid sequence of humanized heavy chain.
SEQ ID NO: 17 Amino acid sequence of the modified IG gamma-4 chain C region.
SEQ ID NO: 18 C2 HuVH AF 4 Nucleotide sequence of CDR2 of the humanized heavy chain variable region.
SEQ ID NO: 19 C2 HuVH AF 4 Nucleotide sequence of CDR3 of the humanized heavy chain variable region.
SEQ ID NO: 20 C2 HuVK 1 Nucleotide sequence of CDR1 of the humanized light chain variable region.
SEQ ID NO: 21 C2 HuVK 1 Nucleotide sequence of the humanized light chain variable region.
SEQ ID NO: 22 Nucleotide sequence of C2 humanized light chain.
SEQ ID NO: 23 C2 Nucleotide sequence of the humanized light chain constant region.
SEQ ID NO: 24 C2 HuVH AF 4 Nucleotide sequence of the humanized heavy chain variable region.
SEQ ID NO: 25 C2 Humanized heavy chain nucleotide sequence.
SEQ ID NO: 26 C2 Nucleotide sequence of the humanized heavy chain constant region.
SEQ ID NO: 27 Amino acid sequence of mouse C2 light chain variable region.
SEQ ID NO: 28 Amino acid sequence of mouse C2 heavy chain variable region.
SEQ ID NO: 29 Nucleotide sequence of mouse C2 light chain variable region.
SEQ ID NO: 30 Nucleotide sequence of mouse C2 light chain.
SEQ ID NO: 31 Nucleotide sequence of mouse C2 heavy chain variable region.
SEQ ID NO: 32 Nucleotide sequence of mouse C2 heavy chain.

Claims (152)

A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof that specifically binds to at least one epitope on β-amyloid protein.
The epitope comprises at least two contiguous amino acid residues that are primarily involved in binding to the antibody, wherein the at least two contiguous amino acid residues are the following core sequences (SEQ ID NO: 10) :.
Xaa ₁ --Xaa ₂ --Lys --Leu --Xaa ₃
Embedded in-Lys-Leu-,
During the ceremony
Xaa ₁ is an amino acid selected from the group consisting of His, Asn, Gln, Lys, and Arg.
Xaa ₂ is an amino acid selected from the group consisting of Asn and Gln;
Xaa ₃ is an amino acid selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile.
Chimeric antibody or fragment thereof or humanized antibody or fragment thereof. A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof that specifically binds to at least one epitope on β-amyloid protein.
The epitope comprises at least two contiguous amino acid residues that are primarily involved in binding to the antibody, wherein the at least two contiguous amino acid residues are the following core sequences (SEQ ID NO: 9) :.
Xaa ₃ --Phe --Phe --Xaa ₄ --Xaa ₅ --Xaa ₆
Embedded in-Phe-Phe-,
During the ceremony
Xaa ₃ is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile;
Xaa ₄ is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser, and Ile;
Xaa ₅ is an amino acid residue selected from the group consisting of Glu and Asp, and
Xaa ₆ is an amino acid residue selected from the group consisting of Glu and Asp,
Chimeric antibody or fragment thereof or humanized antibody or fragment thereof. A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof that specifically binds to at least two epitopes on β-amyloid protein.
Each of the at least two epitopes contains at least two contiguous amino acid residues that are primarily involved in antibody binding, and the at least two contiguous amino acids are either not involved in antibody binding or from the contiguous amino acid residues. Also separated by at least one amino acid residue involved to a significantly smaller extent, the contiguous amino acid residues are each the following core sequence:
Xaa ₁ --Xaa ₂ --Lys --Leu --Xaa ₃ --Phe --Phe --Xaa ₄ _ Xaa ₅ _ Xaa ₆
Embedded in-Phe-Phe- and -Lys-Leu-,
During the ceremony
Xaa ₁ is an amino acid selected from the group consisting of His, Asn, Gln, Lys, and Arg;
Xaa ₂ is an amino acid selected from the group consisting of Asn and Gln;
Xaa ₃ is an amino acid selected from the group consisting of Ala, Val, Leu, norleucine, Met, Phe, and Ile;
Xaa ₄ is an amino acid selected from the group consisting of Ala, Val, Leu, Ser, and Ile;
Xaa ₅ is an amino acid selected from the group consisting of Glu and Asp, and
Xaa ₆ is an amino acid selected from the group consisting of Glu and Asp,
The chimeric antibody or fragment thereof according to claim 1 or 2, or the humanized antibody or fragment thereof. Xaa ₁ is an amino acid selected from the group consisting of His and Arg;
Xaa ₂ is an amino acid selected from the group consisting of Asn and Gln;
Xaa ₃ is an amino acid selected from the group consisting of Val and Leu;
Xaa ₄ is an amino acid selected from the group consisting of Ala and Val;
Xaa ₅ is an amino acid selected from the group consisting of Glu and Asp;
Xaa ₆ is an amino acid selected from the group consisting of Glu and Asp,
The chimeric antibody or fragment thereof according to any one of claims 1 to 3 or a humanized antibody or fragment thereof. Xaa ₁ is His;
Xaa ₂ is Gln;
Xaa ₃ is Val;
Xaa ₄ is Ala;
Xaa ₅ is Glu;
Xaa ₆ is Asp,
The chimeric antibody or fragment thereof according to claim 4 or the humanized antibody or fragment thereof. A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof that specifically binds to at least two epitopes on β-amyloid protein.
The at least two epitopes each contain at least two contiguous amino acid residues that are primarily involved in the binding of antibodies that are-Phe -Phe- and --Lys --Leu-, respectively, and at least two distinct binding sites. Represents the amino acid sequence -Val --Phe --Phe --Ala --Glu --Asp-and the amino acid sequence His --Gln --Lys --Leu --Val-shown in SEQ ID NO: 8, respectively.
The chimeric antibody or fragment thereof according to any one of claims 1 to 5, or a humanized antibody or fragment thereof. A human containing at least one CDR of non-human origin and one or more human-derived or primate-derived framework regions, and optionally a constant region derived from a human or primate-sourced antibody, within the variable region. Antibodies or fragments thereof
β-amyloid protein, β-amyloid monomer peptide, polymer-soluble amyloid peptide containing multiple β-amyloid monomer units, β-amyloid fibers, fibrils, or filaments, and isolated or β-amyloid. To the β-amyloid polymer peptide as part of the plaque, the following amino acid sequence (SEQ ID NO: 11):
Xaa ₁ --Xaa ₂ --Lys --Leu --Xaa ₃ --Phe --Phe- Xaa ₄ _ Xaa ₅ _ Xaa ₆
Can specifically bind to epitopes containing
During the ceremony
Xaa ₁ is an amino acid residue selected from the group consisting of His, Asn, and Gln, especially His;
Xaa ₂ is an amino acid residue selected from the group consisting of Asn and Gln, especially Gln;
Xaa ₃ is an amino acid residue selected from the group consisting of Val, Leu, and Ile, especially Val;
Xaa4 is an amino acid residue selected from the group consisting of Ala and Val, especially Ala;
Xaa5 is an amino acid residue selected from the group consisting of Glu and Asp, especially Glu, and
Xaa6 is an amino acid residue selected from the group consisting of Glu and Asp, especially Asp.
Humanized antibody or fragment thereof. The humanized antibody or fragment thereof according to any one of claims 1 to 7, wherein a CDR of non-human origin is obtained from a donor antibody prepared for an antigen fragment containing no epitope. Framework regions of human or primate origin, as well as SEQ ID NOs: 2 representing CDR2 of heavy chain variable regions (HCVRs) and SEQ ID NOs: 3 representing CDR3s, and SEQ ID NOs of CDR1s of light chain variable regions (LCVRs). : At least one CDR with an amino acid sequence selected from the group of sequences consisting of 4:
A humanized antibody or fragment thereof, comprising a variable region having. It has an amino acid sequence selected from a group consisting of a heavy chain framework region derived from humans or primates, and a sequence number consisting of SEQ ID NO: 2 representing CDR2 and SEQ ID NO: 3 representing CDR3 of the heavy chain variable region (HCVR). , At least one CDR
9. The humanized antibody or fragment thereof according to claim 9, which comprises a heavy chain variable region comprising. One CDR with the amino acid sequence of SEQ ID NO: 4, representing CDR1 of the light chain framework region of human or primate origin, and the light chain variable region (LCVR).
9. The humanized antibody or fragment thereof according to claim 9, which comprises a light chain variable region comprising. Framework regions of human or primate origin, as well as SEQ ID NOs: 1 for CDR1 of heavy chain variable regions (HCVR), SEQ ID NOs: 2 for CDR2, and SEQ ID NOs: 3 for CDR3, and light chain variable regions. (LCVR) At least two CDRs having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3.
9. The humanized antibody or fragment thereof according to claim 9, which comprises a variable region comprising. Humanized antibody,
A sequence consisting of a heavy chain framework region derived from humans or primates, and a heavy chain variable region (HCVR) consisting of SEQ ID NO: 1, representing CDR1, SEQ ID NO: 2, representing CDR2, and SEQ ID NO: 3 representing CDR3. At least two CDRs with amino acid sequences selected from the group
12. The humanized antibody or fragment thereof according to claim 12, wherein the humanized antibody comprising a heavy chain variable region having the same CDR cannot be present twice in the antibody. Humanized antibody,
A sequence consisting of a light chain framework region of human or primate origin, and a light chain variable region (LCVR) of SEQ ID NO: 4, representing CDR1, SEQ ID NO: 5, representing CDR2, and SEQ ID NO: 6 representing CDR3. At least two CDRs with an amino acid sequence selected from the group
12. The humanized antibody or fragment thereof according to claim 12, wherein the humanized antibody comprising a light chain variable region having the same CDR cannot be present twice in the antibody. The amino acid sequences of the heavy chain framework region from humans or primates, as well as SEQ ID NO: 1 representing CDR1 of the heavy chain variable region (HCVR), SEQ ID NO: 2 representing CDR2, and SEQ ID NO: 3 representing CDR3. CDRs with the order shown in particular
9. The humanized antibody or fragment thereof according to claim 9, which comprises a heavy chain variable region comprising. The amino acid sequences of the light chain framework region of human or primate origin, as well as SEQ ID NO: 4, representing CDR1 of the light chain variable region (LCVR), SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3. CDRs with the order shown in particular
9. The humanized antibody or fragment thereof according to claim 9, which comprises a light chain variable region comprising. Humanized antibody,
Framework regions of human or primate origin, as well as SEQ ID NOs: 1 for CDR1 of heavy chain variable regions (HCVR), SEQ ID NOs: 2 for CDR2, and SEQ ID NOs: 3 for CDR3, and light chain variable regions. (LCVR) At least 3 CDRs having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3.
The humanized antibody or fragment thereof according to claim 9, wherein the humanized antibody comprising a variable region having the same CDR cannot be present twice in the antibody. Humanized antibody,
Framework regions of human or primate origin, as well as SEQ ID NOs: 1 for CDR1 of heavy chain variable regions (HCVR), SEQ ID NOs: 2 for CDR2, and SEQ ID NOs: 3 for CDR3, and light chain variable regions. (LCVR) At least 4 CDRs having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3.
The humanized antibody or fragment thereof according to claim 9, wherein the humanized antibody comprising a variable region having the same CDR cannot be present twice in the antibody. Humanized antibody,
Framework regions of human or primate origin, as well as SEQ ID NOs: 1 for CDR1 of heavy chain variable regions (HCVR), SEQ ID NOs: 2 for CDR2, and SEQ ID NOs: 3 for CDR3, and light chain variable regions. (LCVR) At least 5 CDRs having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3.
The humanized antibody or fragment thereof according to claim 9, wherein the humanized antibody comprising a variable region having the same CDR cannot be present twice in the antibody. Framework regions of human or primate origin, as well as SEQ ID NOs: 1 for CDR1 of heavy chain variable regions (HCVR), SEQ ID NOs: 2 for CDR2, and SEQ ID NOs: 3 for CDR3, and light chain variable regions. CDR with amino acid sequences of SEQ ID NO: 4, representing CDR1 of (LCVR), SEQ ID NO: 5 representing CDR2, and SEQ ID NO: 6 representing CDR3
9. The humanized antibody or fragment thereof according to claim 9, which comprises a variable region comprising. The humanized antibody or fragment thereof according to claim 10, wherein at least one CDR is a CDR having the amino acid sequence of SEQ ID NO: 2, which represents CDR2 of the heavy chain variable region (HCVR). The humanized antibody or fragment thereof according to claim 10, wherein the at least one CDR is a CDR having the amino acid sequence of SEQ ID NO: 3, which represents CDR3 of the heavy chain variable region (HCVR). 13. The humanized antibody or fragment thereof according to claim 13, wherein at least two CDRs are CDRs having the amino acid sequences of SEQ ID NO: 1 representing CDR1 and SEQ ID NO: 2 representing CDR2 of the heavy chain variable region (HCVR). 13. The humanized antibody or fragment thereof according to claim 13, wherein at least two CDRs are CDRs having the amino acid sequences of SEQ ID NO: 1 representing CDR1 and SEQ ID NO: 3 representing CDR3 of the heavy chain variable region (HCVR). 13. The humanized antibody or fragment thereof according to claim 13, wherein at least two CDRs are CDRs having the amino acid sequences of SEQ ID NO: 2 representing CDR2 and SEQ ID NO: 3 representing CDR3 of the heavy chain variable region (HCVR). The humanized antibody or fragment thereof according to claim 14, wherein at least two CDRs are CDRs having the amino acid sequences of SEQ ID NO: 4 representing CDR1 of the light chain variable region (LCVR) and SEQ ID NO: 5 representing CDR2. The humanized antibody or fragment thereof according to claim 14, wherein at least two CDRs are CDRs having the amino acid sequences of SEQ ID NO: 4 representing CDR1 of the light chain variable region (LCVR) and SEQ ID NO: 6 representing CDR3. The humanized antibody or fragment thereof according to claim 12, wherein at least two CDRs are selected from the group of CDRs according to any one of claims 23 to 27. The humanized antibody or fragment thereof according to any one of claims 9 to 28, which comprises a constant region of a light chain and / or a heavy chain of human or primate origin. A chimeric antibody or a chimeric antibody thereof that alters at least one of the amino acids in the CDR regions of the light and heavy chains set forth in SEQ ID NOs: 1 to 6 by conservative substitution so that the antibody maintains its full functionality. Fragments or humanized antibodies or fragments thereof. Within the amino acid sequence of CDR2 of the light chain variable region (LCVR) shown in SEQ ID NO: 5, Lys at Kabat position 50 is replaced by Arg, Gln, or Glu, especially by Arg, and / or Asn at Kabat position 53 is Ala. , Val, Leu, Ser, and Ile; among other things, the chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to claim 30, which is replaced by Ser. The humanized antibody or fragment thereof according to any one of claims 9 to 31, wherein the framework sequence is a human germline V _K sequence of the KABAT subgroup V _K II. The humanized antibody or fragment thereof according to any one of claims 9 to 31, wherein the framework sequence is a human germline V _H sequence of the KABAT subgroup V _H III. Claim that at least one of the amino acids in the framework region of human or primate origin is altered by substitution with or conservative substitution with the amino acid from the corresponding region of the mouse humanized antibody ACI-01-Ab7C2. The humanized antibody or fragment thereof according to any one of 1-33. A claim to replace Trp at Kabat position 47 in the framework region of a heavy chain variable region of human or primate origin with an amino acid selected from the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe. Item 34, the humanized antibody or fragment thereof. Claim 34, claim 34, where Arg at Kabat position 94 in the framework region of the heavy chain variable region of human or primate origin set forth in SEQ ID NO: 15 is replaced by an amino acid selected from the group consisting of Ser and Thr, particularly Ser. The humanized antibody or fragment thereof described. Trp at Kabat position 47 in the framework region of the heavy chain variable region of human or primate origin shown in SEQ ID NO: 15 was selected from the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe. Arg at Kabat position 94 in the framework region of the heavy chain variable region of human or primate origin shown in SEQ ID NO: 15, which is replaced by amino acids, especially Leu and Ile, especially Leu, consisting of Ser and Thr. The humanized antibody or fragment thereof according to claim 34, which is replaced by a more selected amino acid, especially Ser. Tyr at Kabat position 87 in the framework region of the light chain variable region of human or primate origin shown in SEQ ID NO: 12 by an amino acid selected from the group consisting of Phe, Leu, Val, Ile, and Ala. The humanized antibody or fragment thereof according to claim 34, which is particularly replaced by Leu and Phe, in particular by Phe. Trp at Kabat position 47 in the framework region of the heavy chain variable region of human or primate origin shown in SEQ ID NO: 15 was selected from the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe. Amino acids, especially Leu and Ile, and Arg at Kabat position 94 in the framework region of the heavy chain variable region of human or primate origin set forth in SEQ ID NO: 15, consisting of Ser and Thr. Tyr at Kabat position 87 in the framework region of the light chain variable region of human or primate origin shown in SEQ ID NO: 12, replaced by more selected amino acids, especially Ser, in Phe, Leu, Val, Ile, The humanized antibody or fragment thereof according to claim 34, which is replaced by an amino acid selected from the group consisting of and Ala, particularly by Leu and Phe, especially by Phe. Trp at Kabat position 47 in the framework region of the heavy chain variable region of human or primate origin shown in SEQ ID NO: 15 was selected from the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe. 35. The humanized antibody or fragment thereof, which is replaced by amino acids, especially Leu and Ile, especially Leu. Claim: Arg at Kabat position 94 in the framework region of the heavy chain variable region of human or primate origin set forth in SEQ ID NO: 15 is replaced by an amino acid selected from the group consisting of Ser and Thr, especially by Ser. 36. The humanized antibody or fragment thereof. Claim 37, claim 37, where the Trp at Kabat position 47 in the framework region of the heavy chain variable region of human or primate origin shown in SEQ ID NO: 15 is replaced by an amino acid selected from the group consisting of Leu and Ile, especially Leu. The humanized antibody or fragment thereof described. The humanized antibody or fragment thereof according to claim 38, wherein the Tyr at Kabat position 87 in the framework region of the light chain variable region of human or primate origin shown in SEQ ID NO: 12 is replaced by Phe. SEQ ID NO: 15: Trp at Kabat position 47 in the framework region of the heavy chain variable region of human or primate origin shown in 15 is replaced with an amino acid selected from the group consisting of Leu and Ile, especially Leu, and SEQ ID NO: The Arg at Kabat position 94 in the framework region of the heavy chain variable region of human or primate origin shown in 15 is replaced by Ser, and the light chain variable region of human or primate origin shown in SEQ ID NO: 12 The humanized antibody or fragment thereof according to claim 39, wherein the Tyr at Kabat position 87 in the framework region is replaced by Phe. One of claims 1-44, wherein the heavy chain variable region (HCVR) has an amino acid sequence that is 90%, particularly 95%, more particularly 98% identical to the sequences set forth in SEQ ID NOs: 15 and 16, respectively. The chimeric antibody or fragment thereof described or a humanized antibody or fragment thereof. Any one of claims 1-44, wherein the light chain variable region (LCVR) has an amino acid sequence that is 90%, particularly 95%, more particularly 98% identical to the sequences set forth in SEQ ID NOs: 12 and 13, respectively. The described chimeric antibody or fragment thereof or humanized antibody or fragment thereof. Amino acids in which at least two, especially three, of the CDR regions of a heavy chain variable region (HCVR) are 90%, particularly 95%, and more particularly 98% identical to the corresponding CDR regions set forth in SEQ ID NOs: 1-3. The humanized antibody or fragment thereof according to any one of claims 1 to 44, which has a sequence. Amino acids in which at least two, especially three, of the light chain variable region (LCVR) CDR regions are 90%, particularly 95%, and more particularly 98% identical to the corresponding CDR regions set forth in SEQ ID NOs: 4-6. The humanized antibody or fragment thereof according to any one of claims 1 to 44, which has a sequence. A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof, which comprises the light chain variable region of SEQ ID NO: 13. The chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to claim 49, further comprising the light chain constant region of SEQ ID NO: 14. A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof, comprising a light chain variable region of SEQ ID NO: 13 and a light chain constant region of SEQ ID NO: 14. A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof, which comprises a heavy chain variable region of SEQ ID NO: 16. The chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to claim 52, further comprising the heavy chain constant region of SEQ ID NO: 17. A chimeric antibody or fragment thereof or a humanized antibody or fragment thereof, comprising a heavy chain variable region of SEQ ID NO: 16 and a heavy chain constant region of SEQ ID NO: 17. The chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to any one of claims 50, 51, 53, and 54, wherein the N-terminal Lys of the heavy chain constant region has been removed. The chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to any one of the above claims, wherein the antibody is an _IgG4 isotype containing IgG1, IgG2, IgG3, or mutated IgG4, particularly an IgG4 isotype. Any of the above claims, wherein the antibody is bi-effective in that it exhibits both aggregation inhibitory and deaggregation properties, especially in combination with a high degree of conformational sensitivity. The chimeric antibody or fragment thereof according to the above item, or the humanized antibody or fragment thereof. Β-Amyloid monomer peptides such as, for example, Aβ monomeric peptides 1-39; 1-40, 1-41 or 1-42, with soluble amyloid peptides of amyloid monomers and / or polymers. And / or a polymer containing the Aβ monomer unit of the plurality of Aβ polymers containing a soluble β-amyloid peptide, particularly Aβ _1-42 monomer and / or the Aβ _1-42 monomer unit of the Aβ 1-42 monomer. By co-incubation with a soluble amyloid peptide, the antibody inhibits the aggregation of the Aβ monomer to the fibrils or filaments of the polymer polymer and the amyloid monomer peptide, in particular the Aβ monomer peptide 1-. 39; fibrils of preformed high molecular weight polymer amyloids formed by aggregation of β-amyloid monomeric peptides such as 1-40, 1-41, or 1-42, especially Aβ _1-42 monomeric peptides. Alternatively, by co-incubation with the filament, the antibody can further disaggregate the fibrils or filaments of the preformed polymer.
The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to any one of the above claims. The chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to any one of the above claims, which substantially binds to Aβ aggregated in a mammalian brain, particularly a human brain. The chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to any one of the above claims, which reduces the Aβ plaque load in a mammalian brain, particularly a human brain. A nucleic acid molecule comprising the nucleotide sequence encoding the chimeric antibody or fragment thereof according to any one of claims 1 to 60 or the humanized antibody or fragment thereof. Nucleic acid molecule comprising a nucleotide sequence encoding an antibody variable region comprising SEQ ID NOs: 2 and 3 representing complementarity determining regions (CDRs) 2 and 3 of heavy chain variable regions (HCVR). A nucleic acid molecule comprising a nucleotide sequence encoding an antibody variable region comprising SEQ ID NO: 4, representing complementarity determining regions (CDRs) 1 of the light chain variable region (LCVR). 62. The nucleic acid molecule of claim 62, comprising the nucleotide sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 19. 13. The nucleic acid molecule of claim 63, comprising the nucleotide sequence set forth in SEQ ID NO: 20. Nucleic acid molecule comprising a nucleotide sequence encoding the light chain variable region of SEQ ID NO: 22. A nucleic acid molecule comprising a nucleotide sequence encoding a light chain variable region of SEQ ID NO: 22 and a light chain constant region of SEQ ID NO: 23. SEQ ID NO:: Nucleic acid molecule containing a nucleotide sequence encoding a heavy chain variable region of 25. A nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable region of SEQ ID NO: 25 and a heavy chain constant region of SEQ ID NO: 26. An expression vector comprising the nucleotide sequence according to any one of claims 60 to 69. A cell comprising the expression vector according to any one of claims 60 to 68, or the expression vector according to claim 70. A composition comprising the antibody according to any one of claims 1 to 60, comprising a therapeutically effective amount of any functionally equivalent antibody or functional portion thereof. 72. The composition of claim 72, which is a pharmaceutical composition optionally further comprising a pharmaceutically acceptable carrier. The composition according to any one of claims 72 to 73, which comprises a therapeutically effective amount of the antibody. The chimeric antibody or fragment thereof or humanized antibody or fragment thereof according to any one of claims 1 to 60, which comprises a therapeutically effective amount of any functionally equivalent antibody or functional portion thereof, and optionally further. A mixture comprising a bioactive substance and / or a pharmaceutically acceptable carrier and / or a diluent and / or an excipient. The mixture according to claim 75, wherein the further bioactive substance is a compound used in the treatment of amyloid or amyloid-like proteins associated with amyloid or amyloid-like proteins, such as the Aβ protein involved in Alzheimer's disease, and amyloidosis. DNA repair of compounds of the invention and optionally pharmaceutically acceptable carriers and / or diluents and / or excipients, such as compounds against oxidative stress, anti-aplastic compounds, metal chelating agents, pyrenzepine and metabolites. Inhibitors, 3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS), α-secretase activators, β- and γ-secretase inhibitors, tau proteins, nerves Transmitter, β-sheet breaker, attractant of cellular components that remove / deplete amyloid β, inhibitor of N-terminal cleavage type amyloid β including pyroglutamine-oxidized amyloid β3-42, anti-inflammatory molecule, Or at least selected from the group consisting of cholineresterase inhibitors (ChEI) such as taclin, rivastigmin, donepezil, and / or galantamine, M1 agonists, and other drugs including any amyloid or tau modifiers, as well as nutritional supplements. The mixture according to claim 76, which comprises one compound. The mixture according to claim 77, wherein the compound is a cholinesterase inhibitor (ChEI). The mixture according to any one of claims 75 to 77, wherein the compound is a compound selected from the group consisting of tacrine, rivastigmine, donepezil, galantamine, niacin, and memantine. The mixture according to any one of claims 75 to 79, comprising a therapeutically effective amount of an antibody and / or a bioactive substance. Alzheimer's disease (AD), Levy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); neurological disorders such as Guam Parkinson-dementia complex; as well as progressive nuclear paralysis, multiple sclerosis Diseases; Kreuzfeld Jacob's disease, Parkinson's disease, HIV-related dementia, ALS (muscle atrophic lateral sclerosis), adult-onset diabetes; senile heart amyloidosis; endocrine tumors, and other diseases including luteal degeneration. A group associated with amyloid plaque formation, including, but not limited to, secondary amyloidosis and age-related amyloidosis, including, but not limited to, other diseases based on or associated with amyloid-like protein. A chimeric antibody or fragment thereof, or a humanized antibody or fragment thereof, and / or any one of claims 1 to 60 for preparing a drug for treating or alleviating the effects of amyloidosis, which is a disease and disorder of the disease. And / or the pharmaceutical composition according to any one of claims 72 to 74, or the mixture according to any one of claims 75 to 80. The use according to claim 81, wherein the amyloid-related disease or condition is Alzheimer's disease. 17. The use of claim 81, wherein the amyloid-related condition is characterized by a loss of cognitive memory capacity in animals, particularly mammals or humans. The use according to claim 81, wherein treatment of an animal, particularly a mammal or a human, suffering from an amyloid-related condition characterized by loss of cognitive memory capacity results in an increase in cognitive memory capacity. 17. The use of claim 81, wherein treatment of an animal, particularly a mammal or human, suffering from an amyloid-related condition characterized by loss of cognitive memory capacity results in a complete recovery of cognitive memory capacity. Alzheimer's disease (AD), Levy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guam Parkinson-neurological disorders such as dementia complex; as well as progressive nuclear paralysis, multiple Sclerosis; Kreuzfeld Jacob's disease, Parkinson's disease, HIV-related dementia, ALS (muscle atrophic lateral sclerosis), adult-onset diabetes; senile heart amyloidosis; endocrine tumors, and other diseases including luteal degeneration, etc. A group of diseases associated with amyloid plaque formation, including, but not limited to, secondary amyloidosis and age-related amyloidosis, including, but not limited to, other diseases based on or associated with amyloid-like protein. The pharmaceutical composition according to any one of claims 72 to 74 or any of claims 75 to 80 for use in a method for preventing, treating, or alleviating the effects of amyloidosis, which is a disorder. A method for preparing the mixture according to the above item, which comprises the step of formulating the antibody of the present invention in a pharmaceutically acceptable form. 86. The method of claim 86, wherein the antibody is included in the composition in a therapeutically effective amount. 86. The method of claim 86, wherein the amyloid-related disease or condition is Alzheimer's disease. 86. The method of claim 86, wherein the amyloid-related condition is characterized by a loss of cognitive memory capacity in an animal, particularly a mammal or human. 46. The method of claim 86, wherein treatment of an animal, particularly a mammal or human, suffering from an amyloid-related condition characterized by loss of cognitive memory capacity results in an increase in cognitive memory capacity. 86. The method of claim 86, wherein the amyloid-related condition impairs cognitive memory capacity in animals, particularly mammals or humans. Treatment of animals, especially mammals or humans, suffering from amyloid-related conditions characterized by loss of cognitive memory capacity reverses cognitive memory capacity and, in particular, complete recovery of cognitive memory capacity. Bringing, the method of claim 86. Alzheimer's disease (AD), Levy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guam Parkinson-neurological disorders such as dementia complex; as well as progressive nuclear paralysis, multiple Sclerosis; Kreuzfeld Jacob's disease, Parkinson's disease, HIV-related dementia, ALS (muscle atrophic lateral sclerosis), adult-onset diabetes; senile heart amyloidosis; endocrine tumors, and other diseases including luteal degeneration, etc. A group of diseases associated with amyloid plaque formation, including, but not limited to, secondary amyloidosis and age-related amyloidosis, including, but not limited to, other diseases based on or associated with amyloid-like protein. The effects of amyloidosis, which is a disorder, are affected by the chimeric antibody or fragment thereof according to any one of claims 1 to 60 or a humanized antibody or fragment thereof, and / or a functional portion thereof, and / or claims 72 to 74. The pharmaceutical composition according to any one of the above, or the mixture according to any one of claims 75 to 80, is prevented or treated by administration to an animal or human affected by such a disorder. A method comprising the step of administering the antibody in a therapeutically effective amount, which is a method for alleviating or alleviating the disease. 93. The method of claim 93, wherein the amyloid-related disease or condition is Alzheimer's disease. 39. The method of claim 93, wherein the amyloid-related condition is characterized by a loss of cognitive memory capacity in an animal, particularly a mammal or human. 39. The method of claim 93, wherein treatment of an animal, particularly a mammal or human, suffering from an amyloid-related condition characterized by loss of cognitive memory capacity results in increased retention of cognitive memory capacity. 39. The method of claim 93, wherein the amyloid-related condition impairs cognitive memory capacity in animals, particularly mammals or humans. 39. The method of claim 93, wherein treatment of an animal, particularly a mammal or human, suffering from an amyloid-related condition characterized by loss of cognitive memory capacity results in a complete recovery of cognitive memory capacity. A method of diagnosing an amyloid-related disease or condition in a patient.
(A) A step of contacting a sample suspected to contain amyloid protein or a specific body part or body part with the antibody according to any one of claims 1 to 60;
(B) Step of binding the antibody to an amyloid protein;
A method comprising (c) detecting an antibody bound to the protein; and (d) correlating the presence or absence of antibody binding with the presence or absence of an amyloid protein in a sample or specific body part or site. A method of determining the degree of amyloid formative burden in tissues and / or body fluids.
(A) Steps to obtain a sample representative of the tissue and / or body fluid under investigation;
(B) A step of inspecting the sample for the presence of an amyloid protein using the antibody according to any one of claims 1 to 60;
A method comprising (c) determining the amount of antibody bound to the protein; and (d) calculating the plaque load in the tissue and / or body fluid. A test kit for detecting and diagnosing amyloid-related diseases and conditions, which comprises the antibody according to any one of claims 1 to 60. A container containing one or more antibodies according to the present invention, and an immunological complex formed by binding to the amyloid protein so that the presence or absence of the immunological complex correlates with the presence or absence of the amyloid protein. 10. The test kit of claim 101, comprising an instruction manual for using the antibody for the purpose of detecting the formation of a scientific complex. A framework region of human or primate origin, and at least one CDR with the amino acid sequence of SEQ ID NO: 4 representing CDR1 of the light chain variable region (LCVR).
Light chain variable region (LCVR), including. At least one amino acid sequence selected from the group consisting of a framework region derived from humans or primates, and a sequence number consisting of SEQ ID NO: 2 representing CDR2 and SEQ ID NO: 3 representing CDR3 in the heavy chain variable region (HCVR). Two CDRs
Heavy chain variable region (HCVR), including. A sequence consisting of a heavy chain framework region derived from humans or primates, and a heavy chain variable region (HCVR) consisting of SEQ ID NO: 1, representing CDR1, SEQ ID NO: 2, representing CDR2, and SEQ ID NO: 3 representing CDR3. At least two CDRs with amino acid sequences selected from the group
Heavy chain variable region (HCVR), including. A sequence consisting of a light chain framework region of human or primate origin, and a light chain variable region (LCVR) of SEQ ID NO: 4, representing CDR1, SEQ ID NO: 5, representing CDR2, and SEQ ID NO: 6 representing CDR3. At least two CDRs with amino acid sequences selected from the group
Light chain variable region (LCVR), including. Heavy chain framework regions of human or primate origin, and in particular in the order shown above, represent CDR1 of the heavy chain variable region (HCVR): 1, SEQ ID NO: 2, and CDR3. SEQ ID NO: CDR with amino acid sequence of
Heavy chain variable region (HCVR), including. Incorporated human or primate-derived light chain framework regions and light chain variable regions (LCVRs), SEQ ID NO: 4 for CDR1, SEQ ID NO: 5 for CDR2, and SEQ ID NO: 6 for CDR3. CDRs having the amino acid sequences of
Light chain variable region (LCVR), including. SEQ ID NO: 12 light chain variable region (LCVR). Provided is a humanized antibody or fragment thereof comprising the light chain variable region (LCVR) of SEQ ID NO: 12. Light chain variable region (LCVR) containing the signal sequence shown in SEQ ID NO: 13. SEQ ID NO: A humanized antibody or fragment thereof comprising a complete light chain variable region (LCVR) comprising the signal sequence set forth in SEQ ID NO: 13. A humanized antibody or fragment thereof comprising the light chain variable region (LCVR) of SEQ ID NO: 12 and the light chain constant region of SEQ ID NO: 14. A humanized antibody or fragment thereof comprising the complete light chain variable region (LCVR) of SEQ ID NO: 13 and the light chain constant region of SEQ ID NO: 14. SEQ ID NO: 15 heavy chain variable region (HCVR). A humanized antibody or fragment thereof comprising the heavy chain variable region (HCVR) of SEQ ID NO: 15. Heavy chain variable region (HCVR) containing the signal sequence shown in SEQ ID NO: 16. SEQ ID NO: A humanized antibody or fragment thereof comprising a complete heavy chain variable region (HCVR) comprising the signal sequence set forth in SEQ ID NO: 16. A humanized antibody or fragment thereof comprising a heavy chain variable region (HCVR) of SEQ ID NO: 15 and a heavy chain constant region of SEQ ID NO: 17. A humanized antibody or fragment thereof comprising a heavy chain variable region (HCVR) of SEQ ID NO: 16 and a heavy chain constant region of SEQ ID NO: 17. The chimeric antibody or fragment thereof according to any one of the above claims, which substantially binds to the Aβ monomer but preferably does not show any significant cross-reactivity with amyloid precursor protein (APP). Or a humanized antibody or fragment thereof. At least about 1 × 10 ^-7 M to at least about 1 × 10 ^-12 M, especially at least about 1 × 10 ^-8 M to at least about 1 × 10 ^-11 M, more particularly at least about 1 × 10 ^-8 M. To Aβ monomers with a binding affinity represented by KD in the range of at least about 1 × 10 ^-10 _M , and more particularly at least about 1 × 10 ^-8 M to at least about 5 × 10 ^-8 M. The chimeric antibody or fragment thereof or humanized antibody or fragment thereof according to claim 122, which binds but preferably exhibits no significant cross-reactivity with amyloid precursor protein (APP). The chimera according to any one of the above claims, which substantially binds to a fiber, fibril, or filament of Aβ, but preferably exhibits no significant cross-reactivity with amyloid precursor protein (APP). Antibodies or fragments thereof or humanized antibodies or fragments thereof. At least about 1 × 10 ^-7 M to at least about 1 × 10 ^-12 M, especially at least about 1 × 10 ^-8 M to at least about 1 × 10 ^-11 M, and more particularly at least about 1 × 10 ^-9 M. Aβ fibers, fibrils with binding affinity represented by KD in the range of at least about 1 × 10 ^-10 _M , and more particularly at least about 2 × 10 ^-9 M to at least about 8 × 10 ^-9 M. , Or a fragment thereof or a humanized antibody or fragment thereof according to claim 123, which binds to a filament, but preferably shows no significant cross-reactivity with amyloid precursor protein (APP). The chimeric antibody according to any one of the above claims, which substantially binds to the soluble polymer amyloid β, but preferably shows no significant cross-reactivity with amyloid precursor protein (APP). Fragment or humanized antibody or fragment thereof. Substantially binds to Aβ monomers and / or fibers, fibrils, or filaments of Aβ, and / or soluble polymer amyloid β, but is preferably markedly cross-reactive with amyloid precursor protein (APP). The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to any one of the above claims, which does not indicate any of the above. The chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to claim 126, wherein the binding affinity increases in ascending order. Claimed to exhibit a binding affinity for Aβ fibers, fibrils, or filaments that is at least 10-fold, particularly at least 15-fold, more particularly at least 20-fold, and particularly at least 25-fold higher than the binding affinity for Aβ monomers. 127 The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to the above. The said claim, which substantially binds to aggregated Aβ, including Aβ plaques in mammalian, especially human brain, but preferably shows no significant cross-reactivity with amyloid precursor protein (APP). The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to any one of the above. Substantially binding to soluble fibers containing Aβ monomers in mammalian, especially human brain, but preferably showing no significant cross-reactivity with amyloid precursor protein (APP), said. The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to any one of the claims. By inducing a conformational transformation, the fibers are deaggregated to soluble polymer and monomeric forms and bound to deaggregated and solubilized Aβ forms in tissues and / or body fluids, especially in the brain. And any one of the above claims, wherein by stabilizing the Aβ form, the equilibrium between the soluble Aβ and the aggregated Aβ can be transferred to the soluble form. Chimera antibody or fragment thereof or humanized antibody or fragment thereof. β- to α-helix and / or random coil conformations, especially random coil conformations, and even more particularly to random coil conformations at a given location in the molecule, especially in the environment of Tyr 10 and Val 12 for Aβ proteins. Antibodies or fragments thereof capable of inducing transfer of sheet conformation, with increased random coil conformation at the expense of β-sheet conformation, and fibrils or filaments of preformed high molecular weight polymer amyloid. The chimeric antibody or fragment thereof according to claim 131 or a humanized antibody or fragment thereof, which results in improved solubilization. The reduction in β-sheet conformation is at least 30%, especially at least 35%, and more particularly at least compared to the fibrils or filaments (controls) of each preformed amyloid polymer incubated in buffer. The chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to claim 132, which reaches 40% or more. Substantially binds to soluble Aβ protein species that are polymeric and less aggregated, including monomeric forms, thus modifying the peripheral removal and catabolism of these Aβ protein species and their toxic effects. The chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to claim 131, which is to be lowered. At least one light chain or fragment thereof or at least one heavy chain incorporating at least one, particularly two, and more particularly three CDR regions obtained from a mouse donor antibody, particularly from the mouse antibody ACI-01-Ab7C2. Antibodies containing the fragments or fragments thereof that have at least 5-fold, particularly at least 8-fold, more particularly at least 10-fold, especially at least 15-fold higher affinity for Aβ antigens than mouse donor antibodies. The humanized antibody or fragment thereof according to any one of the above claims. The use according to any one of claims 83-85, wherein the humanized antibody or fragment thereof does not necessarily bind to aggregated amyloid β in the brain. The method of any one of claims 89-91, wherein the humanized antibody or fragment thereof does not necessarily bind to aggregated amyloid β in the brain. 17. The use of claim 81, wherein treatment of an animal, particularly a mammal or human, suffering from an amyloid-related condition characterized by loss of cognitive memory capacity results in retention of cognitive memory capacity. 46. The method of claim 86, wherein treatment of an animal, particularly a mammal or human, suffering from an amyloid-related condition characterized by loss of cognitive memory capacity results in retention of cognitive memory capacity. The chimeric antibody or fragment thereof or humanized antibody thereof according to any one of the above claims, which can shift the equilibrium between Aβ in a soluble state and Aβ in an aggregated state to its soluble form. piece. By deaggregating the fibers to soluble polymer and monomeric forms, the equilibrium between the soluble Aβ and the aggregated Aβ can be transferred to that soluble form. The chimeric antibody or fragment thereof or the humanized antibody or fragment thereof according to any one of the following items. It does not substantially bind to Aβ in human tissues and / or body fluids, especially in the brain, but induces conformational transformations to disaggregate the fibers into soluble polymer and monomeric forms and tissue. And / or the equilibrium between the soluble and aggregated Aβ by binding to and stabilizing the deaggregated and solubilized Aβ morphology in body fluids, especially in the brain. The chimeric antibody or fragment thereof or a humanized antibody or fragment thereof according to any one of the above claims, which is capable of transferring the antibody to its soluble form. The humanization according to any one of the above claims, which exhibits a binding affinity that is at least 5-fold, 10-fold, particularly at least 15-fold, more particularly at least 20-fold, and particularly at least 100-fold higher than the binding affinity of a mouse antibody. Antibodies or fragments thereof. The humanized antibody or fragment thereof according to any one of the above claims, wherein the affinity, specificity, and stability of the antibody or fragment thereof are modified by changes in the glycosylation profile. An antibody or variant thereof comprising the variable regions listed in SEQ ID NO: 27. A cell line expressing the antibody according to claim 145. SEQ ID NO: An antibody gene or variant thereof comprising the variable region listed in SEQ ID NO: 29. A cell line expressing the antibody according to claim 147. A method for deaggregating preformed β-amyloid fibrils, comprising the step of interacting the hC2 antibody with preformed β-amyloid fibrils. The humanized antibody or fragment thereof according to any one of the above claims, which protects neurons from Aβ-induced degradation. A method for preventing Aβ-induced neuronal degradation, comprising treating a neuron with an effective amount of the humanized antibody or fragment thereof according to any one of the above claims. Use of the humanized antibody or fragment thereof according to any one of the above claims for the preparation of a pharmaceutical agent for preventing neuronal degeneration due to exposure to Aβ oligomer.

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