HK1176075B - Antibody glycosylation in the variable region - Google Patents

Description

Glycosylation of antibody variable regions

The patent application of the invention is a divisional application of the patent application with the international application number of PCT/EP2006/011914, the international application date of 11/12/2006, the application number of 200680046307.9 entering the Chinese national stage and the invention name of "glycosylation of antibody variable regions".

Technical Field

The invention relates to a purified antibody molecule preparation, characterized in that it comprises a heavy chain variable region (V)_H) Wherein at least one antigen binding site comprises a glycosylated asparagine (Asn). More specifically, purified antibody molecules are provided that specifically recognize the β -A4 peptide/A β 4 and are present in the heavy chain variable region (V)_H) Comprising glycosylation. The invention specifically relates to mixtures of antibodies comprising one or two glycosylated antigen binding sites with a glycosylated asparagine (Asn) in the variable region of the heavy chain, i.e. the variable region of the heavy chain (V)_H) A mixture of antibody isotypes containing a glycosylated Asn. Compositions or antibody preparations comprising particular glycosylated antibody isoforms are also disclosed. Also provided are pharmaceutical and diagnostic uses of these antibodies. The antibody isoforms may be used, for example, in the pharmaceutical intervention of amyloid formation or amyloid plaque formation and/or in diagnostic uses thereof.

Background

Approximately 70% of all cases of dementia result from alzheimer's disease, which is associated with selective impairment of brain regions and neural circuits that are critical for cognition. Alzheimer's disease is characterized by neurofibrillary tangles, particularly occurring in hippocampal pyramidal neurons and numerous amyloid plaques, which contain a dense core of most starch deposits and a mild halo (defused halos).

Extracellular neuritic plaques contain a number of dominant fibrous peptides, known as "beta amyloid," "a-beta," "a β 4," "β -a4," or "a β"; see Selkoe (1994), Ann. Rev. cell biol.10, 373-403, Koo (1999), PNAS 96 Vol., 9989-9990, U.S. Pat. No. 4,666,829 or Glenner (1984), BBRC 12, 1131. This beta amyloid protein is derived from "Alzheimer's precursor protein/beta-amyloid precursor protein" (APP). APP is an integral membrane glycoprotein (see Sisodia (1992), PNAS, Vol. 89, p. 6075) and is endoproteinase within the A.beta.sequence by a cytoplasmic membrane protease alpha-secretase (see Sisodia (1992), supra). Moreover, other secretase activities, in particular β -secretase and γ -secretase activities, cause extracellular release of amyloid- β (a β), a β comprises amino acid 39(a β 39), amino acid 40(a β 40), amino acid 42(a β 42) or amino acid 43(a β 43); see Sinha (1999), PNAS 96, 11094-; price (1998), Science 282, 1078-1083; WO 00/72880 or Hardy (1997), TINS 20, 154.

It is noteworthy that a β has several naturally occurring forms, of which the human forms refer to a β 39, a β 40, a β 41, a β 42 and a β 43 mentioned above. The most predominant form, a β 42, has the following amino acid sequence (from the N-terminus): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 3). In A β 41, A β 40, A β 39, the C-terminal amino acids A, IA and VIA were lost, respectively. In the A β 43 form, the C-terminus of the above sequence (SEQ ID NO: 3) also contains a threonine residue.

The time required for a β 40 fibril nucleation was significantly longer than for a β 42 fibril nucleation; see Koo citation and Harper (1997), Ann. Rev. biochem.66, 385-407, supra. More commonly, A.beta.42 is associated with neuritic plaques, which are thought to be more prone to forming fibers in vitro, as described by Wagner (1999), J.Clin.Invest.104, 1239-1332. Jarrett (1993), Cell 93, 1055-1058 suggested that A β 42 acts as a "seed" in the nucleation-dependent multimerization of ordered non-crystalline A β peptides.

Modified APP processing and/or production of extracellular plaques containing proteinaceous deposits is known not only from alzheimer's pathology, but also from subjects suffering from other neurological and/or neurodegenerative diseases. These diseases include, in particular, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis of the Dutch type, Parkinson's disease, ALS (amyotrophic lateral sclerosis), Curie's disease, HIV-related dementia and motor neuropathy.

To date, only limited medical intervention regimens for amyloid-related diseases have been described. For example, cholinesterase inhibitors such as galantamine, rivastigmine or donepezil have been discussed as being beneficial only in patients with mild to moderate alzheimer's disease. However, adverse events due to cholinergic action of such drugs have also been reported. While such cholinergic-enhancing treatments do provide benefits for certain symptoms, most treated patients fail to produce a satisfactory therapeutic response. It is estimated that significant cognitive improvement occurs in only about 5% of treated patients, and there is little evidence that this treatment can significantly alter the progression of this progressive disease. Therefore, there is an urgent clinical need for more effective treatments, particularly those that can prevent or delay the progression of the disease.

Also, NMDA-receptor antagonists, such as memantine, have recently become common. However, adverse events due to their pharmacological activity have also been reported. Furthermore, such treatments with NMDA-receptor antagonists are only considered symptomatic treatments and not treatments to ameliorate the disease.

Methods of immunomodulation are also proposed for the treatment of amyloid-related diseases. WO99/27944 discloses conjugates comprising a portion of an a β peptide and a carrier molecule, wherein the carrier molecule should enhance the immune response. WO 00/72880 mentions another active immunization method in which the a β fragment is also used to induce an immune response.

Passive immunization methods using conventional anti-a β antibodies have also been proposed in WO99/27944 or WO 01/62801, and WO 02/46237, WO 02/088306 and WO 02/088307 describe specific humanized antibodies against a certain portion of a β. WO 00/77178 describes antibodies that bind to the transition states adopted by β -amyloid in hydrolysis. WO03/070760 discloses antibody molecules that recognize two discrete amino acid sequences on the a β peptide.

WO 03/016466 describes a humanized anti-A β antibody which has been modified to avoid glycosylation of its heavy chain, since Wallick (1988) J.Exp.Med.168, 1099-1109 have assumed glycosylation in the variable region of the antibody.

Disclosure of Invention

The technical problem underlying the present invention is to provide effective means and methods for the medical management of amyloid diseases, in particular means and methods for reducing unwanted amyloid plaques in patients in need of (corresponding) medical intervention.

First, the present invention provides a purified antibody molecule characterized by a heavy chain variable region (V)_H) Wherein at least one antigen binding site comprises a glycosylated asparagine (Asn). The purified antibodies or antibody compositions of the invention provided herein are particularly targeted to a β and/or a β fragments. The purified antibody molecules provided herein, in particular the antibody compositions or antibody preparations of the invention, may be used for the preparation of a pharmaceutical or diagnostic composition for the treatment, amelioration, prevention of amyloidosis and/or amyloid plaque formation related diseases. Examples of such diseases are Alzheimer's disease.

In the present invention, it has surprisingly been found that purified antibody molecules comprising N-linked glycosylation of at least one antigen binding site in the heavy chain variable region are particularly useful for reducing starch spotting. Furthermore, the glycosylated antibodies or antibody compositions provided in the context of the present invention were found to be particularly useful and shown to be able to effectively cross the blood brain barrier/blood brain boundary by effective plaque binding.

In sharp contrast to the knowledge of the prior art, WO 03/016466 discloses antibodies specifically engineered to lack N-glycosylation sites in the heavy chain and indicates that glycosylation in the variable region has a negative effect on antibody affinity. The prior art indicates that the anti-a β antibodies in deglycosylated form of the heavy chain variable CDR2 region have significantly higher affinity for a β peptides synthesized and purified in vitro.

The present invention therefore relates to improved, purified antibody molecules or antibody preparations, in particular anti-a β 4/a β peptide (β amyloid) and very effective in vivo. The improvement of the antibody molecule/antibody preparation of the present invention resides in the provision of a purified antibody molecule comprising N-glycosylation in at least one heavy chain variable region, such as the CDR2 region of said heavy chain variable region. As previously mentioned, this is in contrast to the prior art such as WO 03/016466, WO 03/016466 which states that such N-glycosylation must be avoided in a β antibodies.

Examples of antibody molecules of the invention are immunoglobulin molecules, such as IgG molecules. IgG is characterized by comprising two heavy chains and two light chains (as shown in figure 14), and this class of molecules comprises two antigen binding sites. The antigen binding site comprises a "variable region" consisting of a heavy chain (V)_H) Certain moiety and light chain (V)_L) A certain part. Antigen-binding site consisting of V_HAnd V_LThe domains are formed side by side. General information on antibody molecules or immunoglobulin molecules can also be found in common textbooks, such as "cellular and molecular immunology" by Abbas, w.b. sounders Company (w.b. sendo) (2003).

In one aspect, for example, in providing an immunoglobulin molecule identified in the present invention, the corresponding heavy chain variable region (V) of an antigen binding site is described_H) An antibody comprising a glycosylated asparagine (Asn). The antibodies are hereinafter referred to as "mono-glycosylated antibodies"; see fig. 14.

In another aspect, corresponding heavy chain variable regions (V) of two antigen binding sites are provided_H) Comprising glycosylated asparagine (Asn) in the immunoglobulin molecule. The antibody molecule is hereinafter referred to as "doubly glycosylated antibody", see fig. 14.

Heavy chain variable region (V) of antigen binding site_H) The aglycosylated asparagine (Asn) -free immunoglobulin is hereinafter referred to as an "aglycosylated antibody”。

The mono-, di-and non-glycosylated antibodies may comprise the same amino acid sequence or different amino acid sequences. Thus, the term "antibody" includes antibody molecules, particularly recombinantly produced antibody molecules such as immunoglobulins. However, as discussed below, the term "antibody molecule" also encompasses known isotypes and modifications of immunoglobulins, such as single chain antibodies or single chain Fv fragments (scAB/scFv) or bispecific antibody constructs, characterized by comprising at least one glycosylated V as defined herein_HAnd (4) a zone. A specific example of such isoforms or modifications may be V_H-V_LOr V_L-V_HForm of an sc (single chain) antibody, wherein said V_HComprising glycosylation as described herein. Also contemplated are bispecific scFVs, in the form of V_H-V_L-V_H-V_L、V_L-V_H-V_H-V_L、V_H-V_L-V_L-V_HAnd the CDR-2 region thereof comprises glycosylation as described herein.

In the context of the present invention, the term "ANTIBODY" (ANTIBODY) is used in capital letters for the sake of clarity. However, the lowercase term "antibody" (antibody) is also used in the context of the present application. "ANTIBODY"/"ANTIBODIES"/"ANTIBODY" and "ANTIBODIES" are used interchangeably.

The mono-and di-glycosylated antibodies are referred to above as "glycosylated antibody isotypes". Characterised by the variable region of the heavy chain (V)_H) A purified antibody molecule in which at least one antigen-binding site comprises a glycosylated asparagine (Asn) is a mono-glycosylated antibody that contains no or little di-glycosylated or non-glycosylated antibodies, i.e., is a "purified mono-glycosylated antibody". In the context of the present invention, a doubly glycosylated antibody contains no or very little mono-or non-glycosylated antibody, i.e. is a "purified doubly glycosylated antibody".

The term "free or containing little" means completely free of other (glycosylated) isoforms or at a concentration of other (glycosylated) isoforms of at most 10%, such as at most 5%, such as at most 4%, such as at most 3%, such as at most 2%, such as at most 1%, such as at most 0.5%, such as at most 0.3%, such as at most 0.2%. The following and the accompanying examples will further provide information in this regard.

In the context of the present invention, the term "mono-glycosylated antibody" relates to one (V) in a single antibody molecule (e.g.an immunoglobulin, such as an IgG, such as IgG1)_H) Antibody molecules containing N-glycosylation in the region. For example, the "monoglycosylated form" comprises glycosylation in one of the variable regions of the heavy chain, as defined below at the asparagine "Asn 52" position. The "mono-glycosylated IgG1 form or mono-glycosylated isotype" may also comprise (as shown herein) glycosylation at a very conserved glycosylation site in the Fc-portion, such as asparagine Asn306 in the non-variable Fc-portion.

The term "bis-glycosylated antibody" as intended by the present invention comprises two heavy chain variable regions (V) as defined herein_H) Glycosylation of (a). Again, both of the heavy chain variable regions of this "disaccharified form" contain glycosylation, specifically at asparagine Asn52 as shown below and in the accompanying examples. This "doubly glycosylated IgG1 form or doubly glycosylated isotype" may also comprise (as shown herein) glycosylation at glycosylation sites that are well conserved in the non-variable/constant Fc-portion, such as in particular position 306 of an immunoglobulin. Figure 14 shows the corresponding antibody molecule.

In the context of the present invention, variable regions, such as the heavy chain variable region (two (V)_H) Region) is referred to as an "unglycosylated form" and its heavy chain variable region is not glycosylated. However, this "unglycosylated form" may still comprise glycosylation in the asparagine (Asn)306 of the antibody constant region (C-region), such as the Fc-portion, which is generally well conserved as glycosylation sites, in particular the non-variable/constant Fc-portion as defined herein, see SEQ ID NO: 6.

heavy chain variable region (V)_H) The glycosylated asparagine (Asn) of (A) can be located in the complementarity determining region 2(CDR2 region).The heavy chain variable region (V)_H) The glycosylated asparagine (Asn) of (a) may be located at position 52 of the variable region as shown below and in SEQ ID No.2 (or in SEQ ID NO: position 52 of 6, also comprising the Fc-portion of the antibody heavy chain described herein).

The antibody isotype may also contain other glycosylation in the constant/non-variable portion of the antibody molecule, such as the Fc-portion of IgG 1. Glycosylation of the Fc-portion is associated with a very conserved glycosylation (site) characterized by the position of Asn306 in the heavy chain, see the following sequence:

QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV

SAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

RGKGNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG

GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES

NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPGK(SEQ ID NO：6)

the sequence is also described below, with the CDRs, CH-regions, heavy chain regions, and two N-glycosylation sites (N52 and N306) indicated:

QVELVESGGGLVQPGGSLRLSCAASWVRQAPGKGLEWV

S

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

R

WGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

TVPSSSLGTQTYICNVNHKPSNTKVDKKV

SEQ ID NO：6)

CDR1、2、3

underlining：CH1

Italic: hinge region

Marking off：CH2

CH3

Bold N: n-linked glycosylation sites

The IgG-Fc region of the antibodies of the invention may be a homodimer consisting of: interchain disulfide hinge region, glycosylated C_H2 domain, C_H2N-linked oligosaccharide carried on asparagine 306(Asn-306) and non-covalently paired C_H3 domain. The oligosaccharide glycosylated at Asn-306 is of the complex biantennary type and may comprise a core heptaglycan structure with a variable outer arm sugar.

Oligosaccharides influence or determine the structure and function of Fc (Jefferis (1998) Immunol Rev.163, 50-76). Jefferis (2002) Immunol Lett.82(1-2), 57-65 and Krapp (2003) J Mol biol.325(5), 979-89 discuss effector function, numbering/effector ligand interactions for specific IgG-Fc. This conserved Fc-position Asn-306 corresponds to the "Asn-297" of the Kabat-system (Kabat (1991) protein Sequence of Immunological Interest (Sequence of Proteins of Immunological Interest), 5 th edition, national institute of Health, Public Health services (Public Health Service, national institutes of Health, Bethesda MD).

An exemplary heavy chain may be encoded by the following sequence:

caggtggaattggtggaaagcggcggcggcctggtgcaaccgggcggcagcctgcgtctgagctgcgcggcc

tccggatttacctttagcagctatgcgatgagctgggtgcgccaagcccctgggaagggtctcgagtgggtgagc

gctattaatgcttctggtactcgtacttattatgctgattctgttaagggtcgttttaccatttcacgtgataattcgaaaa

acaccctgtatctgcaaatgaacagcctgcgtgcggaagatacggccgtgtattattgcgcgcgtggtaagggta

atactcataagccttatggttatgttcgttattttgatgtttggggccaaggcaccctggtgacggttagctcagcctc

caccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggct

gcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgca

caccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttg

ggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagccca

gatatcgtgcgatatcgtgcaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggg

ggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc

gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcata

atgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgc

accaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaa

aaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctg

accaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggaga

gcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctac

agcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctct

gcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga(SEQ ID NO：5)。

the heavy chain may also comprise (especially during recombinant production) other sequences, such as a "leader sequence".

A corresponding example is encoded by the following sequence:

atgaaacacctgtggttcttcctcctgctggtggcagctcccagatgggtcctgtcc (rear joint)

caggtggaattggtggaaagcggcggcggcctggtgcaaccgggcggcagcctgcgtctgagctgcgcggcc

tccggatttacctttagcagctatgcgatgagctgggtgcgccaagcccctgggaagggtctcgagtgggtgagc

gctattaatgcttctggtactcgtacttattatgctgattctgttaagggtcgttttaccatttcacgtgataattcgaaaa

acaccctgtatctgcaaatgaacagcctgcgtgcggaagatacggccgtgtattattgcgcgcgtggtaagggta

atactcataagccttatggttatgttcgttattttgatgtttggggccaaggcaccctggtgacggttagctcagcctc

caccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggct

gcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgca

caccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttg

ggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagccca

aatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctctt

ccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagc

cacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgc

gggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatgg

caaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaa

gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagc

ctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggaga

acaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggaca

agagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcag

aagagcctctccctgtctccgggtaaatga(SEQ ID NO：25)

The corresponding amino acid sequence is:

MKHLWFFLLLVAAPRWVLS (rear joint)

QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA

INASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGK

GNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：26)

The above sequences also comprise a "signal peptide" which is cleaved enzymatically in the secretory pathway by a host signal peptidase in the production of the antibody molecule of the invention in a host cell, such as a CHO cell.

Alternatively, the heavy chain may be encoded by a nucleic acid sequence optimized for recombinant production, as shown in the following sequence:

1gagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtga

51 ttcatggaga aatagagaga ctgagtgtga gtgaacatga gtgagaaaaa

101 ctggatttgt gtggcatttt ctgataacgg tgtccttctg tttgcaggtg

151 tccagtgt

is connected to

ca ggtggagctg gtggagtctg ggggaggcct ggtccagcct

201 ggggggtccc tgagactctc ctgtgcagcg tctggattca ccttcagtag

251 ctatgccatg agctgggtcc gccaggctcc aggcaagggg ctcgagtggg

301 tgtccgccat aaacgccagc ggtacccgca cctactatgc agactccgtg

351 aagggccgat tcaccatctc cagagacaat tccaagaaca cgctgtatct

401 gcaaatgaac agcctgagag ccgaggacac ggctgtgtat tactgtgcga

451 gaggcaaggg gaacacccac aagccctacg gctacgtacg ctactttgac

501 gtgtggggcc aaggaaccct ggtcaccgtc tcctcaggtg agtcctcaca

551 acctctctcc tgcggccgca gcttgaagtc tgaggcagaa tcttgtccag

601 ggtctatcgg actcttgtga gaattagggg ctgacagttg atggtgacaa

651 tttcagggtc agtgactgtc tggtttctct gaggtgagac tggaatatag

701 gtcaccttga agactaaaga ggggtccagg ggcttttctg cacaggcagg

751 gaacagaatg tggaacaatg acttgaatgg ttgattcttg tgtgacacca

801 agaattggca taatgtctga gttgcccaag ggtgatctta gctagactct

851 ggggtttttg tcgggtacag aggaaaaacc cactattgtg attactatgc

901 tatggactac tggggtcaag gaacctcagt caccgtctcc tcaggtaaga

951 atggcctctc caggtcttta tttttaacct ttgttatgga gttttctgag

1001 cattgcagac taatcttgga tatttgccct gagggagccg gctgagagaa

1051 gttgggaaat aaatctgtct agggatctca gagcctttag gacagattat

1101 ctccacatct ttgaaaaact aagaatctgt gtgatggtgt tggtggagtc

1151 cctggatgat gggataggga ctttggaggc tcatttgagg gagatgctaa

1201 aacaatccta tggctggagg gatagttggg gctgtagttg gagattttca

1251 gtttttagaa tgaagtatta gctgcaatac ttcaaggacc acctctgtga

1301 caaccatttt atacagtatc caggcatagg gacaaaaagt ggagtggggc

1351 actttcttta gatttgtgag gaatgttcca cactagattg tttaaaactt

1401 catttgttgg aaggagctgt cttagtgatt gagtcaaggg agaaaggcat

1451 ctagcctcgg tctcaaaagg gtagttgctg tctagagagg tctggtggag

1501 cctgcaaaag tccagctttc aaaggaacac agaagtatgt gtatggaata

1551 ttagaagatg ttgcttttac tcttaagttg gttcctagga aaaatagtta

1601 aatactgtga ctttaaaatg tgagagggtt ttcaagtact cattttttta

1651 aatgtccaaa atttttgtca atcaatttga ggtcttgttt gtgtagaact

1701 gacattactt aaagtttaac cgaggaatgg gagtgaggct ctctcatacc

1751 ctattcagaa ctgactttta acaataataa attaagttta aaatattttt

1801 aaatgaattg agcaatgttg agttgagtca agatggccga tcagaaccgg

1851 aacacctgca gcagctggca ggaagcaggt catgtggcaa ggctatttgg

1901 ggaagggaaa ataaaaccac taggtaaact tgtagctgtg gtttgaagaa

1951 gtggttttga aacactctgt ccagccccac caaaccgaaa gtccaggctg

2001 agcaaaacac cacctgggta atttgcattt ctaaaataag ttgaggattc

2051 agccgaaact ggagaggtcc tcttttaact tattgagttc aaccttttaa

2101 ttttagcttg agtagttcta gtttccccaa acttaagttt atcgacttct

2151 aaaatgtatt tagaattcga gctcggtaca gctttctggg gcaggccagg

2201 cctgaccttg gctttggggc agggaggggg ctaaggtgag gcaggtggcg

2251 ccagcaggtg cacacccaat gcccatgagc ccagacactg gacgctgaac

2301 ctcgcggaca gttaagaacc caggggcctc tgcgcctggg cccagctctg

2351 tcccacaccg cggtcacatg gcaccacctc tcttgcagcc tccaccaagg

2401 gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc

2451 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac

2501 ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac accttcccgg

2551 ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg

2601 ccctccagca gcttgggcac ccagacctac atctgcaacg tgaatcacaa

2651 gcccagcaac accaaggtgg acaagaaagt tggtgagagg ccagcacagg

2701 gagggagggt gtctgctgga agccaggctc agcgctcctg cctggacgca

2751 tcccggctat gcagccccag tccagggcag caaggcaggc cccgtctgcc

2801 tcttcacccg gagcctctgc ccgccccact catgctcagg gagagggtct

2851 tctggctttt tcccaggctc tgggcaggca caggctaggt gcccctaacc

2901 caggccctgc acacaaaggg gcaggtgctg ggctcagacc tgccaagagc

2951 catatccggg aggaccctgc ccctgaccta agcccacccc aaaggccaaa

3001 ctctccactc cctcagctcg gacaccttct ctcctcccag attccagtaa

3051 ctcccaatct tctctctgca gagcccaaat cttgtgacaa aactcacaca

3101 tgcccaccgt gcccaggtaa gccagcccag gcctcgccct ccagctcaag

3151 gcgggacagg tgccctagag tagcctgcat ccagggacag gccccagccg

3201 ggtgctgaca cgtccacctc catctcttcc tcagcacctg aactcctggg

3251 gggaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga

3301 tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa

3351 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa

3401 tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg

3451 tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac

3501 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat

3551 ctccaaagcc aaaggtggga cccgtggggt gcgagggcca catggacaga

3601 ggccggctcg gcccaccctc tgccctgaga gtgaccgctg taccaacctc

3651 tgtccctaca gggcagcccc gagaaccaca ggtgtacacc ctgcccccat

3701 cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa

3751 ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc

3801 ggagaacaac tacaagacca cgcctcccgt gctggactcc gacggctcct

3851 tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg

3901 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac

3951 gcagaagagc ctctccctgt ccccgggcaa atga(SEQ ID NO：23)

SEQ ID NO: 23 "alternative" protein sequences comprise the same coding sequence as the first alternative, but in some slightly different genomic compositions comprise other introns and slightly different "leader sequence"/"signal sequence". The "leader sequence" may also comprise (other) introns as indicated above. The corresponding exon/intron structures in the sequences shown herein can be easily deduced by the skilled person by routine methods.

The exemplary antibodies described herein also comprise a light chain that may comprise or have the following amino acid sequence:

DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY

GASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQ

GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC(SEQ ID NO：8)

it may be encoded by the following nucleic acid sequences:

gatatcgtgctgacccagagcccggcgaccctgagcctgtctccgggcgaacgtgcgaccctgagctgcagag

cgagccagagcgtgagcagcagctatctggcgtggtaccagcagaaaccaggtcaagcaccgcgtctattaatt

tatggcgcgagcagccgtgcaactggggtcccggcgcgttttagcggctctggatccggcacggattttaccctg

accattagcagcctggaacctgaagactttgcgacttattattgccttcagatttataatatgcctattacctttggcca

gggtacgaaagttgaaattaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttga

aatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtgga

taacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctc

agcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagg

gcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO：7)。

the "light chain" of the exemplary antibodies described herein also encompasses "leader sequences" that are particularly useful in the production of the technology.

The corresponding sequence may be (or may be comprised in, for example, a vector system) the following:

atggtgttgcagacccaggtcttcatttctctgttgctctggatctctggtgcctacggg (rear joint)

gatatcgtgctgacccagagcccggcgaccctgagcctgtctccgggcgaacgtgcgaccctgagctgcagagcga

gccagagcgtgagcagcagctatctggcgtggtaccagcagaaaccaggtcaagcaccgcgtctattaatttatggcg

cgagcagccgtgcaactggggtcccggcgcgttttagcggctctggatccggcacggattttaccctgaccattagcag

cctggaacctgaagactttgcgacttattattgccttcagatttataatatgcctattacctttggccagggtacgaaagttga

aattaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttg

tgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactc

ccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagc

agactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaa

caggggagagtgttag(SEQ ID NO：27)

The sequence encodes the following amino acid sequence:

MVLQTQVFISLLLWISGAYG (rear joint)

DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY

GASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQ

GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC(SEQ ID NO：28)

Alternatively, the light chain may be encoded by a nucleic acid sequence optimized for recombinant production, as shown in the following sequence:

1gacatga gggtcctcgc tcagctcctg gggctcctgc tgctctgttt

51 cccaggtaag gatggagaac actagcagtt tactcagccc agggtgctca

101 gtactgcttt actattcagg gaaattctct tacaacatga ttaattgtgt

151 ggacatttgt ttttatgttt ccaatctcag gcgccagatg t

is connected to

gatatcgtg

201 ttgacgcagt ctccagccac cctgtctttg tctccagggg aaagagccac

251 cctctcctgc cgggccagtc agagtgttag cagcagctac ttagcctggt

301 accagcagaa acctggccag gcgcccaggc tcctcatcta tggcgcatcc

351 agcagggcca ctggcgtgcc agccaggttc agtggcagtg ggtctgggac

401 agacttcact ctcaccatca gcagcctgga gcctgaagat ttcgcgacct

451 attactgtct gcagatttac aacatgccta tcacgttcgg ccaagggacc

501 aaggtggaaa tcaaacgtga gtagaattta aactttgcgg ccgcctagac

551 gtttaagtgg gagatttgga ggggatgagg aatgaaggaa cttcaggata

601 gaaaagggct gaagtcaagt tcagctccta aaatggatgt gggagcaaac

651 tttgaagata aactgaatga cccagaggat gaaacagcgc agatcaaaga

701 ggggcctgga gctctgagaa gagaaggaga ctcatccgtg ttgagtttcc

751 acaagtactg tcttgagttt tgcaataaaa gtgggatagc agagttgagt

801 gagccgtagg ctgagttctc tcttttgtct cctaagtttt tatgactaca

851 aaaatcagta gtatgtcctg aaataatcat taagctgttt gaaagtatga

901 ctgcttgcca tgtagatacc atgtcttgct gaatgatcag aagaggtgtg

951 actcttattc taaaatttgt cacaaaatgt caaaatgaga gactctgtag

1001 gaacgagtcc ttgacagaca gctcaagggg tttttttcct ttgtctcatt

1051 tctacatgaa agtaaatttg aaatgatctt ttttattata agagtagaaa

1101 tacagttggg tttgaactat atgttttaat ggccacggtt ttgtaagaca

1151 tttggtcctt tgttttccca gttattactc gattgtaatt ttatatcgcc

1201 agcaatggac tgaaacggtc cgcaacctct tctttacaac tgggtgacct

1251 cgcggctgtg ccagccattt ggcgttcacc ctgccgctaa gggccatgtg

1301 aacccccgcg gtagcatccc ttgctccgcg tggaccactt tcctgaggca

1351 cagtgatagg aacagagcca ctaatctgaa gagaacagag atgtgacaga

1401 ctacactaat gtgagaaaaa caaggaaagg gtgacttatt ggagatttca

1451 gaaataaaat gcatttatta ttatattccc ttattttaat tttctattag

1501 ggaattagaa agggcataaa ctgctttatc cagtgttata ttaaaagctt

1551 aatgtatata atcttttaga ggtaaaatct acagccagca aaagtcatgg

1601 taaatattct ttgactgaac tctcactaaa ctcctctaaa ttatatgtca

1651 tattaactgg ttaaattaat ataaatttgt gacatgacct taactggtta

1701 ggtaggatat ttttcttcat gcaaaaatat gactaataat aatttagcac

1751 aaaaatattt cccaatactt taattctgtg atagaaaaat gtttaactca

1801 gctactataa tcccataatt ttgaaaacta tttattagct tttgtgtttg

1851 acccttccct agccaaaggc aactatttaa ggacccttta aaactcttga

1901 aactacttta gagtcattaa gttatttaac cacttttaat tactttaaaa

1951 tgatgtcaat tcccttttaa ctattaattt attttaaggg gggaaaggct

2001 gctcataatt ctattgtttt tcttggtaaa gaactctcag ttttcgtttt

2051 tactacctct gtcacccaag agttggcatc tcaacagagg ggactttccg

2101 agaggccatc tggcagttgc ttaagatcag aagtgaagtc tgccagttcc

2151 tcccaggcag gtggcccaga ttacagttga cctgttctgg tgtggctaaa

2201 aattgtccca tgtggttaca aaccattaga ccagggtctg atgaattgct

2251 cagaatattt ctggacaccc aaatacagac cctggcttaa ggccctgtcc

2301 atacagtagg tttagcttgg ctacaccaaa ggaagccata cagaggctaa

2351 tatcagagta ttcttggaag agacaggaga aaatgaaagc cagtttctgc

2401 tcttacctta tgtgcttgtg ttcagactcc caaacatcag gagtgtcaga

2451 taaactggtc tgaatctctg tctgaagcat ggaactgaaa agaatgtagt

2501 ttcagggaag aaaggcaata gaaggaagcc tgagaatacg gatcaattct

2551 aaactctgag ggggtcggat gacgtggcca ttctttgcct aaagcattga

2601 gtttactgca aggtcagaaa agcatgcaaa gccctcagaa tggctgcaaa

2651 gagctccaac aaaacaattt agaactttat taaggaatag ggggaagcta

2701 ggaagaaact caaaacatca agattttaaa tacgcttctt ggtctccttg

2751 ctataattat ctgggataag catgctgttt tctgtctgtc cctaacatgc

2801 cctgtgatta tccgcaaaca acacacccaa gggcagaact ttgttactta

2851 aacaccatcc tgtttgcttc tttcctcagg aactgtggct gcaccatctg

2901 tcttcatctt cccgccatct gatgagcagt tgaaatctgg aactgcctct

2951 gttgtgtgcc tgctgaataa cttctatccc agagaggcca aagtacagtg

3001 gaaggtggat aacgccctcc aatcgggtaa ctcccaggag agtgtcacag

3051 agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg

3101 agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca

3151 tcagggcctg agctcgcccg tcacaaagag cttcaacagg ggagagtgtt

3201 ag(SEQ ID NO：24)

The exemplary light chain "sequences" described above likewise have slightly different genomic structures. "alternative sequences" comprise different and/or additional introns. Accordingly, mutatis mutandis to the description of the "heavy chain".

In the context of the present invention, the term "antibody molecule" relates to an intact immunoglobulin molecule, such as IgM, IgD, IgE, IgA or IgG, such as IgG1, IgG2, IgG2b, IgG3 or IgG4, as well as to a part of such an immunoglobulin molecule, such as a Fab-fragment, a Fab '-fragment, a F (ab) 2-fragment, a chimeric F (ab)2 or chimeric Fab' fragment, a chimeric Fab-fragment or an isolated V_H-or CDR-regions (separating said isolated V)_HOr CDR-regions are integrated or engineered into the corresponding "framework regions"). The term "antibody molecule" also includes diabodies and molecules comprising an antibody Fc domain as a carrier linked to at least one antigen binding moiety/peptide such as the peptibodies described in WO 00/24782. Thus, the term "heavy chain variable region (V) in the present context_H) "is not limited to the variable region of a whole immunoglobulin, but also relates to the variable region of the heavy chain (V)_H) Such as the CDRs, or CDRs 1, 2 and/or 3, alone or in combination, or the corresponding "framework" of the variable regions. Thus, an antibody molecule of the invention may be an antibody construct which may contain a glycosylated heavy chain given variable region (V) as an antigen binding site_H) Or at least one CDR. The heavy chain variable region (V) of the antibody constructs of the invention is made as described herein_H) Of said corresponding part ofGlycosylation, for example, comprises a glycosylated asparagine (Asn) at the antigen binding site. Heavy chain variable region (V)_H) Examples of "isolated moieties" are SEQ ID NOs: 12 (or encoded by the nucleic acid sequence shown in SEQ ID NO: 11).

Furthermore, the term "antibody molecule" relates to modified and/or altered antibody molecules like chimeric, humanized or fully humanized antibodies.

The "fully humanized antibody" molecule was also identified and described as a "fully human" antibody. All such antibodies can be produced by methods known in the art. For example, recombinant antibody molecules can be generated by phage display techniques using in vitro maturation as described by Knappik (2000) J Mol biol.296(1), 57-86 and Rauchenberger (2003), J Biol chem.278(40), 38194-205 using fully human immunoglobulin gamma and subclass-1 framework (IgG 1).

The term antibody relates to e.g. IgG molecules and IgG1 as described in the accompanying examples. The term also relates to modified or altered monoclonal or polyclonal antibodies, as well as to antibodies produced recombinantly or synthetically. The term also relates to whole antibodies and antibody fragments/portions thereof, such as isolated light and heavy chains, Fab/c, Fv, Fab 'and F (ab')₂. The term "antibody molecule" also includes antibody derivatives, bifunctional antibodies and antibody constructs, such as single chain fvs (scfv) or antibody-fusion proteins. It is likewise conceivable to include glycosylation V_HCatalytic and/or proteolytic antibodies of a domain, glycosylated V as defined herein_H-a CDR. The term "antibody molecule" also relates to recombinantly produced antibody molecules/antibody constructs which may comprise in addition to one specificity (e.g., anti-a β/a β) also other or more specificities. Such constructs may include, but are not limited to, "bispecific" or "trispecific" constructs. Additional details of the term "antibody molecule" of the invention are provided below.

As indicated above and with respect to at least one antigen binding site, a single chain antibody, chimeric antibody, CDR-grafted antibody, bivalent antibody-construct, antibody fusion protein, cross-cloned antibody or synthetic antibody comprising a glycosylation as defined herein in at least one of the glycosylated heavy chain variable regions described herein. For example, when producing a single chain antibody, the "heavy chain variable region" described herein is not limited to the heavy chain itself, but also relates to the corresponding portion derived from a whole antibody heavy chain, e.g., a whole immunoglobulin, e.g., an IgG. This portion may be used alone or together with a corresponding frame portion as a corresponding CDR. Moreover, genetic variants of immunoglobulin genes are also contemplated herein in the context. For example, a genetic variant of immunoglobulin heavy chain subclass 1(IgG1) may comprise a G1m (17) or G1m (3) allotypic marker in the CH1 domain, or a G1m (1) or G1m (non-1) allotypic marker in the CH3 domain. IgG1 of the Gm (17) (z) and Gm (1) (a) allotypes is preferably used herein. The antibody molecules of the invention also comprise modified or mutated antibodies, such as mutant iggs with enhanced or impaired Fc-receptor binding or complement binding. In one embodiment, the antibodies provided herein are fully humanized or "fully human" antibodies.

Thus, the antibodies of the invention also include cross-cloned antibodies, i.e., antibodies comprising different antibody regions (e.g., CDR-regions) from one or more parent or affinity-optimized antibodies as described herein. These cross-cloned antibodies may be antibodies in several different frameworks, such as an IgG-framework, such as a (human) IgG1-, IgG2 a-or IgG2 b-framework. For example, the antibody framework is that of a mammal, such as a human. The overall structure of the domains of the light and heavy chains is identical, each containing four framework regions, the sequences of which are relatively conserved and linked by three hypervariable domains (termed complementarity determining regions) (CDR 1-3).

As used herein, "human framework region" refers to a framework region that is substantially identical (about 85% or more, typically 90-95% or more) to a naturally occurring human immunoglobulin framework region. The framework regions of the antibody (e.g., the combination of the framework regions of the constituent light and heavy chains) are used to position the CDRs. The CDRs are primarily responsible for binding to an epitope of antigen. It is noteworthy that not only may the cross-cloned antibodies described herein be present in the preferred (human) antibody framework, antibody molecules comprising the CDRs of the antibodies described herein may likewise be introduced into an immunoglobulin framework. Examples of framework regions include IgG1, IgG2a, and IgG2 b. Most preferred are human frameworks and human IgG1 frameworks, as shown in SEQ id no: 6, antibody heavy chain shown.

In one embodiment, the heavy chain variable region of an antibody isotype can comprise those comprising the following amino acids

GFTFSSYAMS(SEQ ID NO：10)

The CDR1 may be encoded by the following nucleic acid sequence:

ggatttacctttagcagctatgcgatgagc(SEQ ID NO：9)

the antibody isotypes may comprise the following in the heavy chain variable region

AINASGTRTYYADSVKG(SEQ ID NO：12)

(N: N-linked glycosylation site of complete heavy chain Asn-52)

The above-mentionedMay be encoded by the following nucleic acid sequences:

gctattaatgcttctggtactcgtacttattatgctgattctgttaagggt(SEQ ID NO：11)

according to the invention N-glycosylation occurs in the CDR2 region at the corresponding Asn52 position in the variable region of the heavy chain (V)_H) Consisting of SEQ ID NO: 1 and has the sequence of SEQ ID NO: 2.

Furthermore, the heavy chain variable region of an antibody isotype may comprise a sequence comprising the following amino acids

GKGNTHKPYGYVRYFDV(SEQ ID NO：14)

The above-mentionedMay be encoded by the following nucleic acid sequences:

ggtaagggtaatactcataagccttatggttatgttcgttattttgatgtt(SEQ ID NO：13)

antibody isotypes can comprise light (L) chains, characterized by the following CDRs:

RASQSVSSSYLA(SEQ ID NO：16)

agagcgagccagagcgtgagcagcagctatctggcg(SEQ ID NO：15)

GASSRAT(SEQ ID NO：18)

ggcgcgagcagccgtgcaact(SEQ ID NO：17)

LQIYNMPI(SEQ ID NO：20)

cttcagatttataatatgcctatt(SEQ ID NO：19)

the antibody heavy chain amino acid Sequence of an antibody isotype, such as the well-conserved glycosylation site Asn306 in the non-variable Fc-portion (corresponding to "Asn 297" in the Kabat-system (Kabat (1991) protein Sequence of Immunological Interest in immunology), 5 th edition, Besserda, Md.: National Center for Biotechnology Information, National Library of Medicine) may contain other potential glycosylation sites (as known in the art to contain the Asn-X-Ser/Thr motif), which is or includes the Sequence encoded by SEQ ID NO. 5 provided above, i.e., SEQ ID NO: 6.

In one embodiment of the invention, the antibody isotype is characterized by a heavy chain variable region (V) of at least one antigen binding site_H) Comprising a glycosylated asparagine (Asn), said V_HEncoded by the following sequence:

(a) comprises the amino acid sequence shown as SEQ ID NO: 1 by the nucleotide sequence shown in the specification:

CAGGTGGAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGC

AGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGC

GATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAG

CGCTATTAATGCTTCTGGTACTCGTACTTATTATGCTGATTCTGTTAAGG

GTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAA

ATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTG

GTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTT

TGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA(SEQ ID NO：1)；

(b) encodes a polypeptide comprising SEQ ID NO: 2:

QVELVESGGGLVQPGGSLRLSCAASGFTFS SYAMSWVRQAPGKGLEWV

SAIASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

RGKGNTHKPYGYVRYFDVWGQGTLVTVSS (SEQ ID NO: 2; bold face)Asn, at position 52 of the variable region of the heavy chain, as defined herein);

(c) a nucleic acid molecule which hybridizes to the nucleic acid molecule (a) or (b) and encodes a polypeptide capable of binding to the following β -a4 peptide/a β 4 amino acid sequence or a fragment thereof comprising at least 15 amino acids:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO：3)；

(d) hybridizes with a nucleic acid molecule (a) or (b) and encodes a polypeptide capable of hybridizing with at least two regions of the following beta-a 4 peptide/Α β 4 amino acid sequence or with a sequence SEQ ID NO: 3 at least two regions of a fragment:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO：3)。

wherein the two regions of the β -A4 peptide A β 4 or the fragment thereof comprise amino acids 3-6 and 18-26 of SEQ ID No. 3; or

(e) A nucleic acid sequence which is degenerate to a nucleic acid sequence as defined in any one of (a) to (d).

The person skilled in the art is aware that the term "nucleic acid molecule hybridizing to a nucleic acid molecule (a) or (b) and encoding a polypeptide capable of binding to at least two regions of the β -a4 peptide/a β 4" as used herein relates to the coding strand of a double stranded nucleic acid molecule, wherein the non-coding strand hybridizes to the nucleic acid molecule (a) and (b) as described above.

As described above, purified antibody molecules containing Asn-glycosylation as defined herein can be identified and expressed as antibody molecules comprising a variable region containing glycosylation in the heavy chain selected from the group consisting of:

(a) SEQ ID NO: 5. 23 or 25, or a light chain polypeptide encoded by a nucleic acid molecule as set forth in seq id no;

(b) comprises the amino acid sequence shown in SEQ ID NO: 6 or 26;

(c) a heavy chain polypeptide encoded by a nucleic acid molecule capable of hybridising to nucleic acid molecule (a) and encoding a polypeptide capable of binding to the β -a4 peptide/a β 4 peptide represented by the amino acid sequence β -a4 or a fragment thereof comprising at least 15 amino acids

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 3); or

(d) A heavy chain polypeptide encoded by a nucleic acid molecule capable of hybridizing to nucleic acid molecule (a) and encoding a polypeptide capable of binding to at least two regions of the beta-a 4 peptide/a beta 4 or at least two regions of the fragment SEQ ID No.3 comprising at least 15 amino acids as shown in the amino acid sequence

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA(SEQID NO：3)；

Wherein the two regions of the β -A4 peptide A β 4 or the fragment thereof comprise amino acids 3-6 and 18-26.

The antibodies identified above (e.g., the exemplary antibodies herein) can also comprise an L-chain having the following amino acid sequence:

DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGA

SSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKV

EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ

SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT

KSFNRGEC(SEQ ID NO：22)

or an L chain encoded by, for example, the following nucleic acid sequences:

gatatcgtgctgacccagagcccggcgaccctgagcctgtctccgggcgaacgtgcgaccctgagctgcagagcga

gccagagcgtgagcagcagctatctggcgtggtaccagcagaaaccaggtcaagcaccgcgtctattaatttatggcg

cgagcagccgtgcaactggggtcccggcgcgttttagcggctctggatccggcacggattttaccctgaccattagcag

cctggaacctgaagactttgcgacttattattgccttcagatttataatatgcctattacctttggccagggtacgaaagttga

aattaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttg

tgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactc

ccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagc

agactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaa

caggggagagtgttag(SEQ ID NO：21)

as described above, a purified antibody molecule having a heavy chain containing Asn-glycosylation as defined herein may further comprise a light chain selected from the group comprising:

(a) SEQ ID NO: 7. 21, 24 or 27, or a light chain polypeptide encoded by a nucleic acid molecule as set forth in seq id no;

(b) has the sequence shown in SEQ ID NO: 8. 22 or 28 a;

(c) a light chain polypeptide encoded by a nucleic acid molecule which is capable of hybridising to nucleic acid molecule (a) and which encodes a polypeptide which is capable of binding to the β -a4 peptide/a β 4 represented by the amino acid sequence:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 3); or

(d) A light chain polypeptide encoded by a nucleic acid molecule which hybridizes with nucleic acid molecule (a) and encodes a polypeptide which binds to at least two regions of the β -a4 peptide/a β 4 or at least two regions of the fragment SEQ ID No.3 comprising at least 15 amino acids as shown in the amino acid sequence

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA(SEQID NO：3)；

The term "hybridize" as used herein in reference to a nucleic acid sequence/DNA sequence may relate to hybridization under stringent or non-stringent conditions. Non-stringent conditions are preferred, unless further indicated. The hybridization conditions can be determined according to established experimental methods, for example: sambrook, Russell molecular cloning, A Laboratory Manual, Cold Spring harbor Laboratory, New York (Cold Spring harbor Laboratory, N.Y.) (2001); ausubel, New compiled Molecular Biology protocols (Current protocols in Molecular Biology), Green publishing Association and Weiley Interscience, N.Y. (1989)), or Higgins and Hames (eds), "Nucleic acid hybridization, practice methods" (Nucleic acid hybridization, a practical approach), IRL Oxford Press, Washington's region (IRL Press Oxford, Washington DC) (1985). The conditions are set as is well within the skill of the art and can be determined by experimental methods described in the art. Thus, detection of only specifically hybridizing sequences typically requires stringent hybridization and washing conditions, such as 0.1XSSC, 0.1% SDS, 65 ℃. Non-stringent hybridization conditions for detecting homologous or non-exact complementary sequences can be set at 6XSSC, 1% SDS, 65 ℃. It is well known that the length of the probe and the composition of the nucleic acid to be detected constitute further parameters of the hybridization conditions. Note that: the above conditions can be altered by adding and/or replacing alternative blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Deng's (Denhardt's) reagent, BLOTTO, heparin, denatured salmon sperm DNA and all commercial preparations. Due to compatibility issues, it may be necessary to modify the hybridization conditions described above by adding specific blocking reagents. Hybrid nucleic acid molecules also include fragments of the above molecules. Such a fragment may represent a nucleic acid sequence encoding a non-functional antibody molecule as defined herein or a non-functional fragment or CDR thereof, and is at least 12 nucleotides, preferably at least 15, more preferably at least 18, more preferably at least 21, more preferably at least 30, more preferably at least 40, most preferably at least 60 in length. Furthermore, nucleic acid molecules which hybridize to any of the above-described nucleic acid molecules also include complementary fragments, derivatives, and allelic variants of these molecules. In addition, a hybridization complex refers to a complex formed by two nucleic acid sequences relying on complementary hydrogen bonding of G, C and A, T bases; base stacking interactions can further stabilize these hydrogen bonds. The hydrogen bonding of two complementary nucleic acid sequences is in an antiparallel conformation. Hybridization complexes can be formed in solution (e.g., Cot or Rot assays) or between a nucleic acid sequence in one solution and another nucleic acid sequence immobilized to a solid support (e.g., a membrane, filter, chip, pin, or slide such as a fixed cell slide). The term complementary or complementarity refers to the natural binding of polynucleotides by base pairing under permissive salt and temperature conditions. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". The complementarity of two single stranded molecules may be "partial" complementarity where only a portion of the nucleic acids are bound, or complete complementarity where all complementarity between the single stranded molecules is complete. The degree of complementarity between nucleic acid strands significantly affects the efficiency and length of hybridization between nucleic acid strands. This is particularly important when performing amplification reactions that rely on binding between nucleic acid strands.

The term "hybridizing sequence" preferably refers to a sequence which has at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, particularly preferably at least 80%, especially more preferably at least 90%, especially more preferably at least 95% and most preferably at least 97% sequence identity to the above-mentioned nucleic acid sequence encoding the antibody molecule. Furthermore, the term "hybridizing sequence" refers to a sequence encoding an antibody molecule which has at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, especially preferably at least 80%, especially more preferably at least 90%, especially more preferably at least 95% and most preferably at least 97% identity to the amino acid sequence of the antibody molecule described herein above.

According to the present invention, the term "identical" or "percent identity" when two or more nucleic acid or amino acid sequences are present, means that the two or more sequences or subsequences are the same, or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 65% identical, preferably 70-95% identical, more preferably at least 95% identical), when compared and aligned for maximum correspondence over the window of comparison or designated region, using sequence comparison algorithms well known in the art, or by manual alignment or visual inspection. For example, two sequences are considered substantially identical when the sequence identity is 60% to 95% or greater. This definition also applies to the complementary strand of the test sequence. Preferably, the identity exists over a region of at least about 15 to 25 amino acids or nucleotides in length, more preferably over a region of at least about 50 to 100 amino acids or nucleotides in length. Those skilled in the art know how to judge the percent identity between sequences, for example using the algorithms based on the CLUSTALW computer program (Thompson Nucl. acids Res.2(1994), 4673-.

Although the FASTDB algorithm generally does not consider non-matching deletions or additions, i.e., gaps, within sequences, in the calculation, errors can be corrected manually to avoid overestimating identity%. CLUSTALW, however, does not account for sequence gaps in identity calculations. The BLAST and BLAST 2.0 algorithms (Altschul, Nucl. acids Res.25(1997), 3389-. The nucleic acid sequence BLASTN program used default word size (W) of 11, expect (E) of 10, M =5, N =4, and performed double strand comparisons. For amino acid sequences, the BLASTP program uses a default word size (W) of 3 and an expectation (E) of 10. BLOSUM62 scoring matrix (Henikoff proc. natl. acad. sci., USA, 89(1989), 10915) used alignment (B) of 50, expectation (E) of 10, M =5, N =4 and double strand comparisons were performed.

Furthermore, the invention also relates to nucleic acid molecules whose sequence is degenerate as compared with the above-described hybrid molecules. The term "degeneracy due to the genetic code" as used herein means that the same amino acid can be encoded by different nucleotide sequences due to redundancy in the genetic code.

To test whether amino acid residues or nucleotide residues in a given antibody sequence correspond to those of SEQ id no: 1.5, 23 and 25, by artificial means or by using computer program means, such as the means mentioned below in connection with the definition of "hybridization" and the degree of homology.

For example, Local sequence alignments can be searched using BLAST 2.0(Altschul (1997), supra; Altschul (1993), supra; Altschul (1990), supra) which represents the Basic Local Alignment search tool (Basic Local Alignment search BLAST). BLAST as discussed above is capable of aligning nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignment, BLAST is particularly useful in determining exact pairings or identifying similar sequences. The basic unit of BLAST algorithm output is the High-scoring Segment Pair (HSP). HSPs are composed of two sequence segments of arbitrary equal length, the alignment of which is the local maximum, the alignment score reaching or exceeding a user-set threshold or cut-off score. The BLAST method is to look for HSPs between query and database sequences, evaluate the statistical significance of any pair found, and report only those pairs that meet the significance threshold selected by the user. Parameter E establishes a domain of statistical significance for the reported database sequence pair. E can be interpreted as the upper limit of the frequency of occurrence of the desired HSP (or set of HSPs) in the context of the entire database search. The program outputs a database sequence reporting any pairings that satisfy E.

Similar computer techniques using BLAST (Altschul (1997) supra; Altschul (1993) supra and Altschul (1990) supra) are used to search nucleotide databases, such as GenBank or EMBL, for the same or related molecules. This assay is significantly faster than membrane-based multiplex hybridization. Further, the sensitivity of the computer search may be altered to determine whether any particular pairing is classified as accurate or similar. The basis for the search is the product score, which is defined as follows:

% sequence identity x% maximum BLAST score

100

The degree of similarity between the two sequences and the length of the sequence pair are taken into account. For example, when the product score is 40, the pairing is accurate with 1-2% error; if 70, the pairing is exact. Molecules with product scores between 15-40 were selected to identify similar molecules, although lower scores are likely to identify related molecules. Examples of programs known in the art that are capable of generating sequence alignments are the CLUSTALW computer program (Thompson, Nucl. acids Res.2(1994), 4673-.

In one embodiment, the invention provides glycosylated antibody isotypes wherein the V is located_HAsn glycosylation of a region is selected from:

(a) a biantennary complex type coreless fucosylated saccharide structure;

(b) a double-antenna heterozygote sugar structure;

(c) a sugar structure of the biantennary oligomannose type; and

(d) the dual antenna configuration of any of the configurations provided in fig. 5 or 27.

In one embodiment of the antibody of the invention, the corresponding sugar structure does not comprise core fucosylation.

The corresponding N-glycosylation can consist mainly of biantennary complex type coreless fucosylated sugar structures (more than or equal to 75%; mainly 80-90%), and up to more than 80% antennary high sialylation. The minority sugar structures belong to the biantennary heterozygote and the oligomannose type (. ltoreq.25%), respectively, see also FIGS. 5 and 27. The glycosylation structure of the variable region is not cleaved by the N-glycosidase F (amino acid polypeptide) from the protein (amino acid polypeptide).

In one embodiment, the predominant complex biantennary sugar structures are further identified by the following method

-contains one or two sialic acids linked to one or the other or both antennae. Sialic acid is an N-acetylneuraminic acid type, most likely bound to a terminal β 1,4 linked galactose with an α -2,3 linkage.

Lack of core fucosylation, i.e. a fucose residue linked to the most core N-acetylglucosamine by an α -1 → 6 linkage at the reducing end of the sugar chain.

In one embodiment, the hybrid sugar structure is further identified by:

complex antennae (lactosamine units attached to the core sugar structure (GlcNAc-Gal)) are contained as one arm of the bi-antenna structure. The arm contains mainly N-acetyl neuraminic acid linked to a terminal β -1,4 linked galactose.

Contain 1-3 other mannose subunits linked to the core sugar structure as other antennae. Lack of core fucosylation, i.e. a fucose residue linked to the most core N-acetylglucosamine by an α -1 → 6 linkage at the reducing end of the sugar chain.

In one embodiment, the oligomannose-type sugar structure is further identified by the following method

-4 (Man4 → GlcNAc2), 5 (Man5 → GlcNAc2) or 6 (Man6 → GlcNAc2) mannose subunits within the intact sugar structure, i.e. there are 3 branching mannose subunits in a typical N-linked core sugar structure.

Lack of core fucosylation, i.e. a fucose residue linked to the most core N-acetylglucosamine by an α -1 → 6 linkage at the reducing end of the sugar chain.

In another embodiment of the invention, a composition is provided comprising a heavy chain variable region (V) characterized by_H) An antibody molecule comprising a glycosylated asparagine (Asn) at one antigen binding site of (A), and is characterized in the variable region of the heavy chain (V)_H) The two antigen binding sites of (a) comprise glycosylated asparagine (Asn), i.e. a composition comprising a mono-glycosylated antibody and a di-glycosylated antibody, hereinafter referred to as an antibody composition. The term antibody composition is also contemplated to comprise a peptide containing at least one glycosylation V as described herein_HRegion or at least one of said V_HA composition of molecules of glycosylated CDRs of a region, wherein the molecules may be immunoglobulins or immunoglobulin isotypes and modifications as described above. For example, the composition may also comprise single chain antibodies (scFvs) or contain glycosylated V_HBispecific molecules from which CDR regions are derived.

Additional definitions of antibody compositions of the invention are provided below.

The antibody composition contains no or only very small amounts of "V_HAglycosylated "antibody molecules, i.e. variable regions, in particular heavy chain variable regions (V)_H) An antibody that does not contain glycosylation as defined herein.

In the context of the present invention, especially in the antibody mixtures provided herein, the term "free or only containing a very small amount of non-glycosylated antibody molecules" means that the antibody composition comprises less than 10%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1%, such as less than 0.5% or less of the non-glycosylated isoforms described herein.

Thus, in one embodiment, the invention provides an antibody preparation comprising antibody molecules that are mono-and/or di-glycosylated (glycosylation is in the heavy chain variable region) but do not contain variable regions that are not glycosylated.

Again, the term "antibody molecule without variable region aglycosylation" relates to an antibody preparation/antibody mixture/antibody library wherein the content of the aglycosylated isoforms described herein is at most 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, e.g. at most 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%.

In one embodiment, the invention provides compositions of antibody isotypes that do not contain more than 0.5% of the variable regions that are unglycosylated (e.g., the heavy chain variable regions are unglycosylated).

As indicated above, in embodiments of the invention, a mixture of mono-and di-glycosylated antibodies, such as immunoglobulins, is provided, said mixture being free of antibody molecules whose variable regions are not glycosylated. In the present invention, variable regions such as two heavy chain variable regions (two (V)_H) -region) antibodies that do not contain such post-translational modifications are referred to as "unglycosylated forms" which do not contain glycosylation in the variable region of the heavy chain. However, this "unglycosylated form" may still comprise glycosylation of the antibody constant region (C-region), e.g. the highly conserved glycosylation site most commonly located in the Fc-portion, in particular the presentAsparagine (Asn)306 in a non-variable/constant Fc-moiety as defined herein.

Glycosylated antibody isoforms alone or mixtures of mono-and di-glycosylated isoforms are very useful and advantageous therapeutic antibody preparations in the treatment of Alzheimer's Disease (AD) and other amyloid related diseases such as down syndrome, hereditary cerebral hemorrhage with amyloidosis of the dutch type, parkinson's disease, ALS (amyotrophic lateral sclerosis), creutzfeldt-jakob disease, HIV-related dementia and motor neuropathy. The individual glycosylated antibody isoforms or mixtures of mono-and di-glycosylated isoforms are also unique diagnostic tools.

The two glycosylated isoforms described herein have improved and highly efficient brain penetration in vivo. An AD-associated amyloidosis mouse model PS2APP mouse shows potent brain penetration and specific binding to beta amyloid plaques.

Moreover, in vitro immunohistochemical staining detected improved specificity for human beta-starch plaques and significantly reduced non-specific adhesion. As described in the accompanying examples, the minimum effective concentration of human beta-amyloid plaque uniformity staining (dependent staining) was determined to be 10 ng/ml.

As described in the accompanying examples, the isolation and characterization of differently glycosylated antibodies, such as immunoglobulins, revealed that glycosylation of the heavy chain variable region has a surprising effect on antigen binding a β peptide, diagnostic value, pharmacological properties and functional activity. Purified antibody molecules can be subjected to MS-analysis, binding studies to soluble A.beta. (Biacore) and epitope mapping (Pepspot analysis), dissociation of aggregated A.beta.and microscopy analysis of bound beta.amyloid plaques in vitro and in vivo.

In one embodiment of the invention, the purified antibody or antibody composition is capable of specifically recognizing the β -a4 peptide/a β 4.

As described herein, in a particular embodiment, the purified antibody or antibody composition relates to an antibody or antibody composition capable of specifically recognizing both regions (N-terminal region and central/middle portion) of a β/a β 4.

The term "specifically recognisable" as used herein refers to antibody molecules which are capable of specifically interacting with and/or binding to at least two amino acids per one of the two regions of β -a4 as defined herein. The term relates to the specificity of the antibody molecule, i.e. to its ability to distinguish a specific region of the β -a4 peptide as defined herein from another unrelated region of the β -a4 peptide or another non-APP-related protein/peptide/(unrelated) test peptide. Thus, specificity can be detected by methods known in the art and disclosed herein. Such methods include, but are not limited to, Western blotting, ELISA-, RIA-, ECL-, IRMA-detection, and peptide scanning. Such methods also include detecting K_DValues as described in the accompanying examples. Peptide scanning (pepspot matrix) is often used to delineate a linear epitope map of polypeptide antigens. Peptides are overlapped with each other on activated cellulose to continuously synthesize the primary sequence of the polypeptide. The recognition of a particular peptide by an antibody to be tested for its ability to detect or recognize a particular antigen/epitope can be scored by conventional chromogenic methods (secondary antibodies containing enzymes or dyes such as horseradish peroxidase or 4-chloronaphthol or hydrogen peroxide), chemiluminescent reactions, or similar methods known in the art. When using a chemiluminescent reaction or a fluorescent secondary antibody, the reaction can be quantified. When an antibody reacts with a certain set of overlapping peptides, the minimum amino acid sequence necessary for the reaction can be deduced; see the illustrative examples provided by the present invention.

The same experiment revealed two distant clusters of reactive peptides, indicating recognition of a non-continuous, i.e.conformational, epitope in the antigenic polypeptide (Geysen (1986), mol. Immunol.23, 709-715).

In addition to the pepspot assay, a standard ELISA assay can be performed. As shown in the appended examples, small hexapeptides can be conjugated to proteins and coated onto immunoassay plates to react with test antibodies. Scoring can be done by standard color development (e.g., horseradish peroxidase and secondary antibody as well as tetramethyl benzidine and hydrogen peroxide). Responses in some wells were scored by optical density (e.g., 450 nm). The background (= negative reaction) may typically be 0.1OD, and the positive reaction typically is 1 OD. This means that the difference (ratio) of positive/negative reactions is more than 10-fold. The accompanying examples will give more details. Furthermore, quantitative methods for detecting the specificity and the ability to "specifically recognize" two regions of the herein defined β -a4 peptide will be given below.

The term "two regions of the β -A4 peptide" relates to two regions which are related to the central/medial epitope of the N-terminal amino acids 3-6 and amino acids 18-24 of SEQ ID No.3(β -A4 peptide). As described in the accompanying examples, the disaccharified antibody isoform A specifically provided and exemplified herein (see accompanying examples) detects two portions of the A.beta.molecule, the first portion comprising the N-terminal amino acids 1-10 and the second portion comprising the central/middle portion of A.beta.amino acids 17-26 (as shown in SEQ ID No. 3). Thus, in the antibody mixtures provided herein comprising the herein described monoglycosylated and bisglycosylated antibody isotypes, the two regions may also be somewhat relaxed to comprise amino acids 1-10 (or-11 or-12) or shorter portions thereof and amino acids 17-26 (or amino acids 16-27) or shorter portions between amino acids 17-26, such as amino acids 19-26 or 20-26. The term "β -a4 peptide" in the context of the present invention relates to a β 39, a β 41, a β 43 as described above, in particular to a β 40 and a β 42. Attached SEQ ID NO: 3 also describes a β 42. It is to be noted that the term "two regions of the β -a4 peptide" also relates to "epitopes" and/or "antigenic determinants" comprising two regions of the β -a4 peptide or parts thereof as defined herein. According to the invention, two regions of the β -a4 peptide are separated by at least one amino acid, such as at least two, three, four, five and six amino acids, in the primary structure of the β -a4 peptide (at the amino acid sequence level). As shown herein and as noted in the appended examples, the antibody/antibody molecule of the present invention detects/acts on and/or binds to two regions of the β -a4 peptide as defined herein, wherein the two regions are separated (at the level of the primary structure of the amino acid sequence) by at least one amino acid, wherein the sequence separating the two regions/"epitope" may comprise more than seven, eight, ten or even about fourteen amino acids.

The term "two regions of the β -a4 peptide" also relates to conformational or discontinuous epitopes consisting of said two regions or parts thereof; see Geysen (1986), supra. In the context of the present invention, conformational epitopes are defined by two or more discrete amino acid sequences spaced apart in the primary sequence but concentrated at the surface when the polypeptide is folded as a native protein (Sela, (1969) Science 166, 1365 and Laver, (1990) Cel, 161, 553-6). It is contemplated that the antibody molecules of the present invention specifically bind to and interact with conformational epitopes consisting of or comprising two regions of β -a4 as described herein or portions thereof as disclosed below. The "antibody molecule" of the invention is believed to have dual specificity for (a) the amino acid arm containing amino acids 1-11 (or portions thereof) of β -A4(SEQ ID No.3) and (b) the amino acid arm containing amino acids 16-27 (or portions thereof) of β -A4(SEQ ID No.3) simultaneously and independently. Fragments or portions of these arms comprise at least two, and in most cases at least three amino acids.

Antibody molecules, such as immunoglobulins, can be expressed in three systems: a) transiently transfected human embryonic kidney cells (HEK 293 EBNA, Invitrogen), b) transiently transfected chinese hamster ovary Cells (CHO) and c) stably transfected CHO cell lines (CHO K1 and CHO K1SV, longza Biologics) containing epstein-barr virus nuclear antigen. Three different antibody molecules (either unglycosylated, mono-glycosylated or di-glycosylated) can be separated using specific purification steps, including protein a purification, cation exchange chromatography, and size exclusion column separation as described in detail below.

In one embodiment of the invention, the antibody molecule is recombinantly produced in, e.g., CHO cells or HEK293 cells, preferably CHO cells. In a specific embodiment, the glycosylation pattern identified above is obtained after expression in CHO cells. CHO cells are well known in the art and include CHO cells used in the experimental section, such as CHO K1 or CHO K1SV cells. A commonly used HEK293 cell is HEK293 EBNA.

The glycosylated antibody of the invention is expressed recombinantly in eukaryotic expression systems, in particular in CHO cells, as described in the examples. However, other expression cells, i.e., eukaryotic cells, are also contemplated. Eukaryotic cells include, for example, fungal or animal cells. Examples of suitable fungal cells are yeast cells, such as Saccharomyces, Saccharomyces cerevisiae. Suitable animal cells are, for example, insect cells, vertebrate cells, for example mammalian cells, such as NSO, MDCK, U2-OSHela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, C33A. Human cell lines are also contemplated. These host cells, such as CHO cells, provide post-translational modifications to the antibody molecules of the invention, including removal of leader or signal peptide sequences, folding and assembly of the H (heavy) and L (light) chains, and most importantly glycosylation at the correct site in the molecule, i.e. glycosylation in the heavy chain variable region. Such signal peptides or leader sequences are cleaved by the signal peptidase of the host during secretion in recombinant production processes, as in CHO cells. Other suitable cell lines known in the art are available from cell line stores, such as the American Type Culture Collection (ATCC). It is also contemplated that primary cells/cell cultures may be used as host cells in accordance with the present invention. The cells are specifically derived from insects (such as fruit flies or blattaria) or mammals (such as humans, pigs, mice or rats). The host cell may also comprise cells from and/or derived from a cell line, such as a neuroblastoma cell line.

Thus, the antibody molecules of the invention are produced by recombinant expression systems. One example of such a system (as described above) is a mammalian expression system using Chinese Hamster Ovary (CHO) cells. These systems can be used with the Glutamine Synthetase (GS) system (WO 87/04462; WO 89/01036; Bebbington, 1992, Biotechnology (New York), 10, 169-75). The system involves transfecting CHO cells with a gene encoding GS enzyme and the desired antibody gene. CHO cells were selected using glutamine-free medium culture and inhibited the GS enzyme with methionine iminosulfone (MSX). To survive, the cell will increase GS enzyme expression with concomitant expression of mAb.

Another possible expression system is the CHO dhfr system, in which CHO cells are deficient in dihydrofolate reductase (dhfr-) and rely on thymidine and hypoxanthine for growth. The parental (parental) CHO dhfr-cell line was transfected with antibodies and dhfr gene to enable selection of CHO cell transformants of the dhfr + phenotype. Selection was performed in the absence of thymidine and hypoxanthine. Expression of antibody genes can be elevated using Methotrexate (MTX). The drug is a direct inhibitor of the dhfr enzyme, so that amplified dhfr gene copy number and antibody gene resistant colonies sufficient to survive under these conditions are isolated.

Purified antibody molecules, such as immunoglobulins, may be prepared by a method comprising the steps of:

(a) recombinantly expressing a heterologous nucleic acid molecule encoding an antibody molecule as described above in a mammalian cell, such as a CHO or HEK293 cell; and

(b) purifying the recombinantly expressed antibody molecule by a method comprising the following steps

(b1) Purifying a protein A column;

(b2) ion exchange column purification, such as cation exchange chromatography; and, optionally

(b3) And (5) purifying by using a size exclusion column.

The purification protocol may also include other steps, such as other concentration steps, such as diafiltration, or an analytical step, such as the use of an analytical column. The method/step may further comprise a virus inactivation step and/or a virus removal step, such as filtration/nanofiltration. It is also contemplated that it may be possible to repeat certain steps (e.g., two step ion exchange chromatography may be performed) or to omit certain steps (e.g., size exclusion column chromatography).

Protein a is a group of specific ligands that are capable of binding to the Fc region of most IgG1 isotypes. It is synthesized by and can be isolated from certain strains of Staphylococcus aureus (Staphylococcus aureus) and coupled to chromatographic beads. Several types of gel formulations are commercially available.

An example of a protein A column that can be used is a MabSelect (trade Mark) column. Ideally, the column is equilibrated with 25mM Tris/HCl, 25mM NaCl, 5mM EDTA and the cell culture supernatant is applied to the column. The column was washed with 1M Tris/HCl pH 7.2 and the antibody was eluted with 100mM acetic acid pH 3.2.

Cation exchange chromatography using positively charged groups in the stationary phase and a sample in the mobile phase

And (4) interaction. When using a weak cation exchange column (e.g. CM Toyopearl)) Then, the following chromatography steps were carried out: after pre-equilibration with 100mM acetic acid pH 4, the protein A eluate is loaded onto the column, washed with 100mM acetic acid pH 4, and the antibodies are eluted with 250mM sodium acetate (pH 7.8-8.5) and 500mM sodium acetate (pH 7.8-8.5) and fractionated. In the first step, a mixture of the doubly glycosylated isoform component and the singly glycosylated isoform component is eluted normally, and in the second step, the unglycosylated isoform component is eluted normally.

When using a strong cation exchange column (such as SP Toyopearl 650), the antibody is eluted with a salt step: after pre-equilibration with 50mM acetic acid pH 5.0, the protein a eluate was loaded onto the column and the first step elution was performed at pH 4 using 50mM acetic acid and 210mM sodium chloride. The second elution step used 50mM acetic acid and 350mM sodium chloride. The mixture of the doubly glycosylated isoform component and the singly glycosylated isoform component is eluted normally by the first salt step and the unglycosylated isoform is eluted normally by the second salt step.

In addition, it can also be obtained from strong cation exchange columns (such as SP-) Antibody elution by salt gradient: after pre-equilibration, column loading and column washing at pH 4.5, a salt gradient from 50mM MES pH 5.8-to 50mM MES/1M sodium chloride pH 5.8 was used. Typically, the components of the doubly glycosylated isoform, the mono-glycosylated isoform and the non-glycosylated isoform are eluted separately. The di-glycosylated isoform component and the mono-glycosylated isoform component may then be collected to form a product library and/or a desired antibody mixture.

Further purification of the mixture of di-and mono-glycosylated antibody molecules, such as immunoglobulins, may use size exclusion chromatography. Examples of useful pillarsIs SuperdexAnd (3) a column. Examples of running buffers include histidine/sodium chloride, such as 10mM histidine/125 mM sodium chloride/pH 6 and Phosphate Buffered Saline (PBS).

Flow-through mode anion exchange chromatography followed by a concentration/diafiltration step is another purification step. QAre examples of anion exchange step resins. For example, the SP chromatography eluate was diluted three-fold with 37.5mM Tris/HCl pH 7.9 and passed through a 25mM Tris/83 mM sodium acetate pre-equilibrated Q-Sepharose column. Collecting the effluent and adjusting the pH to 5.5 using, for example, Hydrosart 30And (5) performing membrane ultrafiltration concentration. The concentrate was then diafiltered with 10 volumes of 20mM histidine/HCl pH 5.5.

The purification scheme may also comprise a further step (c) of analytical chromatography, for example using a Mono-SHR5/5 column. However, other steps such as diafiltration are also contemplated to concentrate the antibody molecules.

In one embodiment of the invention, there is provided a composition, antibody preparation or antibody library comprising an antibody molecule as described herein or prepared by a method as described above. In this embodiment of the invention, the composition comprises a mono-or di-glycosylated antibody. In another embodiment, the composition comprises a mono-or di-glycosylated (in the heavy chain variable region) antibody and the composition is derived from an antibody molecule lacking glycosylation in the variable region. In the context of this embodiment, the term "antibody library" relates to a mixture of mono-or di-glycosylated (in the heavy chain variable region) antibodies, which can be separated separately and mixed into one mixture. An antibody mixture or antibody library provided herein can comprise 50% mono-glycosylated antibodies and 50% di-glycosylated antibodies (as defined herein). However, ratios of 30/70 to 70/30 are also contemplated. However, it will be apparent to those skilled in the art that other ratios can be used in the antibody mixture of the invention. For example, 10/90 or 90/10, 20/80 or 80/20, and 40/60 or 60/40 may also be used in the context of the present invention. As described in the examples, particularly useful ratios of antibody mixtures herein comprising di-glycosylated and mono-glycosylated antibodies as defined herein are from 40/60-to 45/55.

The compositions provided herein are particularly useful in diagnostic or pharmaceutical compositions.

Accordingly, the present invention provides a diagnostic or pharmaceutical composition comprising:

(a) the above antibody molecule comprising an antigen binding site with a glycosylated Asn;

(b) the above antibody molecule comprising two antigen binding sites with glycosylated Asn; or, most preferably

(c) A mixture of antibody molecules (a) and (b).

The mixture provided herein comprising one antibody molecule with an antigen-binding site at a glycosylated Asn and two antibody molecules with an antigen-binding site at a glycosylated Asn lacks the aglycosylated isotype involved in the variable region of the heavy chain. As indicated above, the term "isotype lacking aglycosylation (referring to the heavy chain variable region)" relates to a mixture/antibody repertoire/antibody preparation in which less than 5%, 4%, 3%, 2%, 1%, or even less than 0.5% of the antibody species are aglycosylated in the heavy chain variable region. As described in the examples, the mixture/antibody repertoire/antibody preparation may contain little (less than 0.5%) of the aglycosylated isoform. The percentage and/or number of a given glycosylated isoform (as described herein, glycosylation of the heavy chain variable region, see fig. 14) in a given antibody composition is readily determined using methods known in the art, including, but not limited to, mass spectrometry, SDS-PAGE analysis, ion exchange, HPLL, ELISA, and the like.

As shown in the appended examples, specific and sensitive immunological modification of real Alzheimer's disease beta-amyloid plaques by the antibodies of the present invention was demonstrated by immunohistochemical staining experiments using frozen sections of brain tissue from AD patients. Human anti-A β antibodies from A β -immunized patients have also been shown to stain brain sections effectively for β -amyloid plaques (Hock, 2002, Nature Medicine, 8, 1270-. In addition, immunological modifications have been shown in transgenic animal models loaded with human β -amyloid plaques (Richards, 2003, j. neuroscience, 23, 8989-9003). This plaque binding has been shown to lead to its clearance and ultimately to amelioration of disease-related symptoms in similar animal models, and involvement of Fc-dependent processes is discussed (Bard, 2000, Nature Medicine, 6, 916-. Furthermore, efficient binding of anti-A β antibodies to β amyloid plaques has been reported to be associated with slowing of disease progression (Hock, 2002, Nature Medicine, 8, 1270-. This finding, as well as autopsy analysis of human brain tissue, suggests that phagocytosis of microglia is involved in the mechanism of plaque clearance in humans (Nicoll, 2003, Nature Medicine, 9, 448-. The antibody of the invention or a specific antibody comprised in the pharmaceutical composition is therefore human IgG1, IgG1 being primarily responsible for FcR dependent processes in humans. The effective immune modification of beta amyloid plaques by the antibodies/mixtures of the invention indicates that the drug is effective in eliciting passive immunity in humans to clear the beta amyloid plaques present and prevent the formation of beta amyloid plaques.

Furthermore, the antibody preferably can cross the blood brain barrier to reach its destination. For macromolecules such as human IgG, this process is significantly reduced, so that only about 0.1-0.2% plasma antibody concentration in CSF can be achieved. The mechanism of plaque clearance is also a controversial topic in which the peripheral effects of A.beta.peptides may be involved (Dodart, 2002, Nature Neuroscience, 5: 452-. Thus, the resulting therapeutic antibodies or corresponding mixtures of mono-and di-glycosylation (heavy chain variable regions) of the invention are also of value for in vitro depolymerization of A β polymers without the involvement of Fc-dependent processes and binding to soluble A β monomers and oligomers in CSF, since neutralizing soluble monomeric A β peptides or oligomeric A β peptides (e.g., aggregation intermediates) can also contribute to overall amyloidosis reduction (Du, 2003, Brain, 126: 1-5).

The compositions of the present invention may be administered in solid or liquid form, and may be in the form of a powder, tablet, solution or aerosol, and the like. The composition may comprise one or more antibodies/antibody molecules of the invention, most preferably a mixture of mono-and di-glycosylated antibodies as provided herein.

The pharmaceutical composition preferably optionally comprises a pharmaceutically acceptable carrier and/or diluent. The pharmaceutical compositions disclosed herein are particularly useful for treating neurological and/or neurodegenerative diseases. Such diseases include, but are not limited to, Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS), hereditary cerebral hemorrhage with amyloidosis of the Dutch type, Down's syndrome, HIV-dementia, Parkinson's disease, and aging-related neuronal disorders. The pharmaceutical compositions of the present invention are contemplated to be useful as potent inhibitors of plaque formation or potent stimulators of plaque depolymerization. Accordingly, the present invention provides pharmaceutical compositions comprising the compounds of the present invention for the treatment of amyloidogenic diseases/disorders. The term "amyloidogenic disease/disorder" includes any disease associated with or caused by the formation or deposition of amyloid fibrils and/or pathological APP proteolysis. Examples of amyloidogenic diseases include, but are not limited to, Alzheimer's Disease (AD), down's syndrome, dementia associated with Lewy body formation, parkinson's disease dementia, mild cognitive impairment, cerebral amyloid angiopathy, and vascular dementia. Different amyloidogenic diseases are defined by or characterized by the properties of the polypeptide component of amyloid deposits. For example, amyloid beta is characterized by amyloid deposits found in subjects with alzheimer's disease.

Examples of suitable pharmaceutical carriers, excipients, and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, disinfectant solutions, and the like. Compositions comprising such carriers may be formulated using known conventional methods. Suitable carriers may comprise any material that retains high affinity binding for a β when combined with an anti-a β specific binding agent or antibody and does not react with the subject's immune system, including excipients, surfactants, enhancers, and the like; see Remington's Pharmaceutical Sciences (1980)16 th edition, Osol, A. These pharmaceutical compositions may be administered to a subject at a suitable dosage. Suitable compositions can be administered by different means, such as parenteral, subcutaneous, intraperitoneal, topical, intrabronchial, intrapulmonary and intranasal administration, and, if desired for topical treatment, intralesional administration. Parenteral administration includes intraperitoneal, intramuscular, intradermal, subcutaneous, intravenous or intraarterial administration. Particularly preferred modes of administration are injection and/or delivery to a site in a cerebral artery or directly to brain tissue. The compositions of the invention may also be administered directly to a target site, such as by biolistic delivery to an external or internal target site, such as the brain.

The antibodies of the desired purity are mixed with any physiologically acceptable carrier, excipient, stabilizer, surfactant, buffer, and/or tonicity agent to prepare a pharmaceutical composition comprising the glycosylated antibodies described herein. Acceptable carriers, excipients, and/or stabilizers are nontoxic at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides, and other sugars; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucamine (so-called "meglumine"), galactosamine and neuraminic acid; and/or a non-ionic surfactant, such as tween, Brij pluronic, Triton-X or polyethylene glycol (PEG).

The pharmaceutical composition may be in liquid form, lyophilized form, or reconstituted liquid form in lyophilized form, wherein the lyophilized formulation is reconstituted with a sterile solution prior to administration. The standard procedure for reconstitution of a lyophilized composition is to add back a volume of purified water (usually the same amount as that removed in lyophilization), however solutions containing an antibacterial agent may also be used to produce pharmaceutical compositions for parenteral administration; see Chen (1992) Drug Dev IndPharm 18, 1311-54.

Exemplary antibody concentrations in pharmaceutical compositions can range from about 1mg/mL to 200mg/mL or from about 50mg/mL to 200mg/mL, or from about 150mg/mL to 200 mg/mL. For the sake of clarity, it is emphasized that the concentrations indicated herein are concentrations in liquids or in liquids that are precisely reconstituted from solid form.

Aqueous formulations of the antibodies can be prepared in a pH-buffer, such as a pH range of about 4.0-7.0, or about 5.0-6.0, or about 5.5. Examples of suitable buffers having a pH within this range include phosphate, histidine, citrate, succinate, acetate buffers and other organic acid buffers. The buffer concentration may be about 1mM to 100mM, or about 5mM to 50mM, depending on the buffer and the desired formulation tonicity.

The antibody formulation may comprise a tonicity agent to adjust the tonicity of the formulation. Exemplary tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars and combinations thereof. Isotonic aqueous formulations are preferred, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" means that the solution has the same tonicity as other solutions, such as physiological saline solution and serum. The tonicity agent may be used in an amount of about 5mM to 350mM, especially 105mM to 305 nM.

Surfactants may also be added to the antibody formulation to reduce aggregation of the formulation antibodies and/or to minimize particle formation in the formulation and/or to reduce adsorption. Illustrative examples of the surfactant include polyoxyethylene sorbitan fatty acid ester (Tween), polyoxyethylene alkyl ether (Brij), alkylphenylpolyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymerSubstances (poloxamers, pluronics) and Sodium Dodecyl Sulfate (SDS). A preferred polyoxyethylene sorbitan fatty acid ester is polysorbate 20 (trade mark Tween 20)^TM) And polysorbate 80 (trade mark Tween 80)^TM). The preferred polyethylene-polypropylene copolymer is available under the trademark PluronicF68 or Poloxamer 188^TMThe substance of (1). Preferred polyoxyethylene alkyl ethers are those sold under the trade name Brij^TMThe substance of (1). An exemplary concentration range for the surfactant may be about 0.001% -1% w/v.

Lyoprotectants (lyoprotectants) may also be added to protect labile active ingredients (e.g., proteins) against the conditions of instability during lyophilization. For example, known lyoprotectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol, and glycerol), and amino acids (including alanine, glycine, and glutamic acid). The lyoprotectant is typically used in an amount of about 10mM-500 nM.

In one embodiment, the formulation comprises the above-described substances (i.e., glycosylated antibody, surfactant, buffer, stabilizer, and/or tonicity agent), substantially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof. In another embodiment, preservatives may be included in the formulation, such as in a concentration range of about 0.001-2% (w/v).

In one embodiment, an antibody formulation of the invention is a liquid or lyophilized formulation suitable for parenteral administration, which may comprise:

-about 1-200mg/mL of a glycosylated antibody or antibody composition described herein;

-from about 0.001% to about 1% of at least one surfactant;

-about 1-100mM buffer;

-optionally about 10-500mM stabilizer and/or about 5-305mM tonicity agent;

-a pH of about 4.0-7.0.

In a preferred embodiment, the parenteral formulation of the invention is a liquid or lyophilized formulation comprising:

-about 1-200mg/mL of the glycosylated antibody or antibody composition described herein,

-0.04% Tween 20w/v,

-20mM L-histidine,

-250mM of sucrose, and (c) in the presence of sucrose,

-pH 5.5。

in a more preferred embodiment, the parenteral formulation of the invention may also include a lyophilized formulation comprising:

-15mg/mL of a glycosylated antibody or antibody composition described herein,

-0.04% Tween 20w/v,

-20mM L-histidine,

-250mM of sucrose, and (c) in the presence of sucrose,

-pH 5.5；

or

-75mg/mL of a glycosylated antibody or antibody composition as described herein,

-0.04% Tween 20w/v,

-20mM L-histidine,

-250mM of sucrose, and (c) in the presence of sucrose,

-pH 5.5；

or

-75mg/mL of a glycosylated antibody or antibody composition as described herein,

-0.02% Tween 20w/v,

-20mM L-histidine,

-250mM of sucrose, and (c) in the presence of sucrose,

-pH 5.5；

or

-75mg/mL of a glycosylated antibody or antibody composition as described herein,

-0.04% Tween 20w/v,

-20mM L-histidine,

-250mM of trehalose and, optionally, a pharmaceutically acceptable carrier,

-pH 5.5；

or

-75mg/mL of a glycosylated antibody or antibody composition as described herein,

-0.02% Tween 20w/v,

-20mM L-histidine,

-250mM of trehalose and, optionally, a pharmaceutically acceptable carrier,

-pH 5.5

in another more preferred embodiment, the parenteral formulation of the invention may also include a liquid formulation comprising:

7.5mg/mL of a glycosylated antibody or antibody composition described herein,

-0.022% Tween 20w/v,

-120mM L-histidine,

-250125 mM of sucrose (sucrose content: C),

-pH 5.5；

or

37.5mg/mL of a glycosylated antibody or antibody composition described herein,

-0.02% Tween 20w/v,

-10mM L-histidine,

-125mM of sucrose, with a concentration of sucrose,

-pH 5.5；

or

37.5mg/mL of a glycosylated antibody or antibody composition described herein,

-0.01% Tween 20w/v,

-10mM L-histidine,

-125mM of sucrose, with a concentration of sucrose,

-pH 5.5；

or

37.5mg/mL of a glycosylated antibody or antibody composition described herein,

-0.02% Tween 20w/v,

-10mM L-histidine,

-125mM of trehalose and, optionally, glucose,

-pH 5.5；

or

37.5mg/mL of a glycosylated antibody or antibody composition described herein,

-0.01% Tween 20w/v,

-10mM L-histidine,

-125mM of trehalose and, optionally, glucose,

-pH 5.5；

or

-75mg/mL of a glycosylated antibody or antibody composition as described herein,

-0.02% Tween 20w/v,

-20mM L-histidine,

-250mM of trehalose and, optionally, a pharmaceutically acceptable carrier,

-pH 5.5；

or

-75mg/mL of a glycosylated antibody or antibody composition as described herein,

-0.02% Tween 20w/v,

-20mM L-histidine,

-250mM of mannitol,

-pH 5.5；

or

-75mg/mL of a glycosylated antibody or antibody composition as described herein,

-0.02% Tween 20w/v,

-20mM L-histidine,

-140mM of sodium chloride,

-pH 5.5；

or

-150mg/mL of a glycosylated antibody or antibody composition as described herein,

-0.02% Tween 20w/v,

-20mM L-histidine,

-250mM of trehalose and, optionally, a pharmaceutically acceptable carrier,

-pH 5.5。

or

-150mg/mL of a glycosylated antibody or antibody composition as described herein,

-0.02% Tween 20w/v,

-20mM L-histidine,

-250mM of mannitol,

-pH 5.5。

or

-150mg/mL of a glycosylated antibody or antibody composition as described herein,

-0.02% Tween 20w/v,

-20mM L-histidine,

-140mM of sodium chloride,

-pH 5.5。

or

-10mg/mL of an antibody to A β,

-0.01% Tween 20w/v,

-20mM L-histidine,

-140mM of sodium chloride,

-pH 5.5。

in one embodiment, the pharmaceutical composition of the present invention is a liquid formulation comprising:

-10mg/mL of an antibody to A β,

-0.01% Tween 20w/v,

-20mM L-histidine,

-140mM of sodium chloride,

-pH 5.5。

in another embodiment, the pharmaceutical composition of the present invention is a lyophilized formulation comprising:

-75mg/mL of an antibody to A β,

-0.04% Tween 20w/v,

-20mM L-histidine,

-250mM of sucrose, and (c) in the presence of sucrose,

-pH 5.5。

the term "glycosylated antibody as described herein" in the context of an exemplary formulation may include mono-glycosylated antibodies as described herein, di-glycosylated antibodies as described herein, and mixtures thereof.

The attending physician and clinical factors determine the dosage regimen. As is well known in the medical arts, the dosing regimen for any one patient depends on a number of factors, including; patient size, body surface area, age, the particular compound administered, sex, time and mode of administration, general health, and other drugs administered concurrently. The amount of proteinaceous pharmaceutically active substance administered is 1ng-20mg/kg body weight per dose, e.g. 0.1mg-10mg/kg body weight, 0.5mg-5mg/kg body weight; however, dosages below or above this exemplary range are also contemplated, particularly in view of the above factors. If the regimen is a continuous infusion, the amount administered per minute should be in the range of 1 μ g to 10mg/kg body weight.

The pharmaceutical compositions described herein may be formulated as short-acting, immediate-release, long-acting, or sustained-release formulations. Thus, the pharmaceutical composition may be adapted for sustained or controlled release.

Sustained release formulations are prepared using methods well known in the art. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of the sustained-release base include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers, and poly-D- (-) -3-hydroxybutyric acid. Possible loss of biological activity and changes in immunogenicity of antibodies contained in sustained release formulations can be prevented by the use of suitable additives, control of moisture content, development of specific polymer matrix compositions.

The process is monitored for periodic assessments. The composition, i.e., the mono-or di-glycosylated antibody of the present invention or a mixture thereof, may be administered locally or systemically. Notably, peripherally administered antibodies can enter the central nervous system, see Bard (2000), Nature Med.6, 916-. Parenteral formulations include aqueous and non-aqueous sterile solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose solution, dextrose and sodium chloride, lactose-modified ringer's injection, or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose solution), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the present invention may also comprise other substances depending on the intended use of the pharmaceutical composition. The substance can be a drug acting on central nervous system, such as neuroprotective factor, cholinesterase inhibitor, M1 muscarinic receptor agonist, hormone, antioxidant, inflammation inhibitor, etc. It is especially preferred that other substances such as neurotransmitters and/or neurotransmitter replacement molecules, vitamin E or alpha lipoic acid are included in the pharmaceutical composition.

Those skilled in the art, particularly but not limited to biochemists, biologists, chemists, pharmacists and groups of such professionals, will readily be able to produce such pharmaceutical compositions. It is also clear to the skilled medical person, e.g. the attending physician, how to administer such pharmaceutical compositions to patients in need of treatment with the pharmaceutical compositions described herein. Such administration may comprise systemic administration, such as by infusion and/or injection. However, it is also contemplated that the compounds and/or compound mixtures of the present invention are administered directly to the brain. For example, the compound or compound mixture or compound formulation can be administered directly to the brain via intraventricular or intrathecal injection, preferably by slow infusion to reduce the effect on the brain parenchyma. Slow intracerebral implantation may also be used. Methods of using gene therapy, such as implantation of recombinant cells producing the antibodies of the invention, are also contemplated. These "recombinant cells" should be capable of providing variable region glycosylation as defined herein/some portion of an antibody described herein, in particular an anti-a β antibody of the invention. However, as indicated above, the advantage of the antibody/antibody compositions of the invention is that they are able to cross the blood brain barrier and bind to starch plaques. The pharmaceutical compositions of the invention described below are useful in the treatment of all disease types heretofore unknown or related to or dependent on pathological APP aggregation or pathological APP processing. They are particularly useful in the treatment of alzheimer's disease or other diseases where extracellular beta amyloid deposits appear to play a role. They are preferably used in humans, but the methods, uses, and compositions described herein may also be used for animal therapy.

In a preferred embodiment of the present invention, the above-described composition of the present invention is a diagnostic composition, optionally further comprising a suitable detection means. The diagnostic composition comprises a compound of the invention as described above, i.e., at least one of the glycosylated antibodies described herein.

The diagnostic composition may comprise a compound of the invention, in particular a glycosylated antibody molecule of the invention, in soluble form/in liquid phase, but it is also contemplated that the compound is bound/attached and/or attached to a solid support.

A solid support may be used in conjunction with the diagnostic compositions described herein, or a compound of the invention may be directly bound to the solid support. Such supports are well known in the art and include commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction vessels, plastic tubing, and the like. The compounds of the invention, and in particular the antibodies of the invention, may be bound to a number of different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamides, agarose, and magnetite. For the purposes of the present invention, the carrier characteristic may be soluble or insoluble. Suitable labels and methods of labeling are identified above and described further below. Methods suitable for immobilizing/curing the compounds of the present invention are well known and include, but are not limited to, ionic, hydrophobic, covalent interactions, and the like.

It is especially preferred to use the diagnostic composition of the invention for the detection and/or quantification of APP and/or APP-processed products, such as beta amyloid, or for the detection and/or quantification of pathologically and/or (genetically) modified APP cleavage sites.

As shown in the accompanying examples, the glycosylated antibody molecules of the present invention are particularly useful as diagnostic reagents for the detection of authentic human amyloid plaques in brain slices of Alzheimer's disease patients by direct immunofluorescence.

Preferably, the compounds of the invention used in the diagnostic composition are detectably labeled. The biomolecules are labeled using several techniques known to those skilled in the art and within the scope of the present invention. Several different labels and methods of labeling are known to those of ordinary skill in the art. Examples of the types of labels that can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds.

Common labels include fluorescent dyes (e.g., fluorescein, rhodamine, Texas Red, etc.), enzymes (e.g., horseradish peroxidase,. beta. -galactosidase, alkaline phosphatase), radioisotopes (e.g., beta. -galactosidase, alkaline phosphatase)³²P or¹²⁵I) Biotin, digoxigenin, colloidal metals, chemical or bioluminescent compounds (such as dioxetane, luminoamine or acridine)) And the like. Labeling processes, such as covalent coupling of enzymatic or biotin groups, iodination, phosphorylation, biotinylation, and the like are well known in the art.

Detection methods include, but are not limited to: autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, and the like. Common detection methods include radioisotope or non-radioisotope methods. These methods include Western blotting, overlay (radioimmunoassay) assay, RIA (radioimmunoassay) and IRMA (immunoradiometric immunoassay), EIA (enzyme immunoassay), ELISA (enzyme linked immunosorbent assay), FIA (fluorescence immunoassay) and CLIA (chemiluminescent immunoassay).

Furthermore, the present invention provides the use of a glycosylated antibody molecule according to the invention, or an antibody molecule produced by a method according to the invention, or a mixture of mono-and di-glycosylated antibodies as provided herein for the preparation of a medicament or diagnostic composition for the prevention, treatment and/or diagnosis of a disease associated with amyloid formation and/or amyloid plaque formation. Further preferred is the use of a compound described herein, in particular an antibody molecule of the invention, for the prevention and/or treatment of neurological diseases associated with altered or abnormal APP processing and/or amyloid formation. Antibody molecules, such as (engineered) immunoglobulin formats, such as antibodies in an IgG framework, in particular an IgG1 framework, or chimeric antibody formats (especially fully humanized antibodies or whole antibodies), bispecific antibodies, single chain fv (scFv), or bispecific scFv and the like are used for the preparation of the pharmaceutical compositions described herein. However, the antibody molecules and mixtures provided herein may also be used for diagnostics, as described in the appended examples, since the antibody molecules of the invention are capable of specifically acting on or detecting a β 4 and/or amyloid deposits/plaques.

Thus, the inventive use of the compounds of the invention is for the preparation of a pharmaceutical composition for the treatment of a neurological disease requiring improvement, for example by breaking down β -amyloid plaques, clearing amyloid (plaques) or passive immunization against β -amyloid plaque formation. As shown in the accompanying examples, the antibody molecules of the invention are particularly useful in preventing disaggregation of a β aggregates and formed amyloid aggregates. Thus, the glycosylated antibodies or the mixture of mono-and di-glycosylated antibodies of the present invention described herein can be used to reduce amyloid deposits/plaques, clear amyloid plaque/plaque precursors and protect neurons. The antibody molecules of the invention are particularly contemplated for use in preventing the formation of starch plaques in vivo and for clearing existing starch plaques/deposits in vivo. Furthermore, the antibody molecules or mixtures of the invention can be used to generate passive immunity against A β peptides and A β aggregates, i.e., β amyloid plaques. The medical use of the antibodies of the invention comprising an Fc-moiety can achieve the goal of clearing A β 4/A β 4 deposits. The Fc-portion of the antibody is particularly useful in Fc-receptor mediated immune responses, such as the attraction of macrophages (phagocytes and/or microglia) and/or helper cells. In mediating Fc-part related immune responses, the antibody molecule of the invention is preferably located in the (human) IgG1 framework. As discussed herein, preferably the subject to be treated with an antibody molecule or antibody cocktail of the invention is a human subject. It is also contemplated that other frameworks, such as IgG2 a-or IgG2 b-frameworks, may be used in the antibody molecules of the invention. Immunoglobulin frameworks in the form of IgG2a and IgG2b are especially contemplated in mice, e.g. in scientific applications of the antibody molecules of the invention, such as testing in transgenic mice expressing (human) wild-type or mutant APP, APP-fragment and/or Α β 4.

Examples of such diseases associated with amyloidogenesis and/or amyloid plaque formation include, but are not limited to, dementia, Alzheimer's disease, motor neuropathy, Parkinson's disease, ALS (amyotrophic lateral sclerosis), scrapie, HIV-associated dementia, and Guillain-Barre disease, hereditary cerebral hemorrhage with amyloidosis of the Dutch type, Down's syndrome, and neuronal diseases associated with aging. The antibody molecules of the invention and the compositions provided herein may also be used to ameliorate and/or prevent inflammatory processes associated with amyloid formation and/or amyloid plaque formation.

Accordingly, the present invention also provides a method of treating, preventing and/or delaying a neurological and/or neurodegenerative disease comprising the step of administering to a subject suffering from said neurological and/or neurodegenerative disease and/or administering to a subject susceptible to a neurological and/or neurodegenerative disease an effective amount of an anti-a β antibody molecule or a mixture of mono-and disaccharides provided herein of the invention and/or a composition as defined above. The treatment provided herein may include administration of the compounds/compositions of the present invention alone or in a co-therapeutic form, i.e., in combination with other drugs and pharmaceuticals. In a particularly preferred embodiment of the present invention, there is provided a method of treating, preventing and/or delaying neuropathy and/or neurodegenerative disease comprising the step of administering to a patient in need of corresponding medical intervention a mixture of antibodies comprising mono-and di-glycosylated anti-a β antibodies as provided herein.

The term "treatment" as used herein contemplates administration of mono-or di-glycosylated antibodies (or mixtures thereof) as described herein to a patient in need thereof. The patient may be a human patient, in one embodiment a human, suffering from or susceptible to a disease associated with pathological APP processing. Thus, the term "treatment" as used herein includes prophylactic and therapeutic administration of a compound or mixture of compounds provided herein.

One disease that can be treated with the compounds and compositions provided herein is alzheimer's disease. Patients with Alzheimer's disease may be diagnosed according to the national institute of neurologic and language disorders, stroke/Alzheimer's disease and related diseases diagnostic criteria (NINCDS/ADRDA criteria) (Mckhan et al, 1984).

The compounds and/or compositions provided herein may also be used in the context of "co-therapy", e.g., when treating APP-related diseases, such as alzheimer's disease. In such cases, co-treatment with other approved drugs such as memantine, donepezil, rivastigmine tartrate or galantamine is contemplated.

In a further embodiment, the present invention provides a kit comprising at least one glycosylated antibody molecule as defined herein or a mixture of the inventive mono-and/or di-glycosylation methods provided herein. Conveniently, the kits of the invention also optionally include buffers, stock solutions and/or other reagents and materials required for medical, scientific or diagnostic experiments or purposes. Furthermore, a portion of the kit of the present invention may be packaged in a vial or bottle alone, or in combination in a container or multi-container unit.

The kits of the invention are suitable for performing the methods of the invention and may be used in several applications cited herein, such as for use as diagnostic kits, research tools or medical tools. Furthermore, the kits of the invention may comprise detection means suitable for scientific, medical and/or diagnostic purposes. The kits are preferably produced according to standard procedures known to those skilled in the art.

Drawings

FIG. 1: plasmid map of heavy and light chain sequence insertion sites

FIG. 2: analytical chromatography legend

FIG. 3: CMT column chromatograms described herein. The di-and mono-glycosylated isoforms elute in peak 1 and the non-glycosylated isoforms elute in peak 2.

FIG. 4: complete IgG ESI-MS analysis of antibody A isotype. The molecular weight of the main peak is expressed in Da. A: unglycosylated antibody a; b: a mono-glycosylated antibody a; c: bisglycosylated antibody A

FIG. 5: scheme for the derivation of the N-glycosylation pattern of antibodies. Structures that are only partially glycosylated are indicated in parentheses. A: a type of complex; b: heterozygote types; c: an oligomannose type; GlcNAc = N-acetyl-glucosamine, Man = mannose; gal = galactose; fuc = fucose; NeuAc = N-acetylneuraminic acid.

FIG. 6: MS and HPAEC-PAD analysis of deduced sugar structure of antibody a Asn 306. Structures that are only partially glycosylated are indicated in parentheses. GlcNAc = N-acetyl-glucosamine, Man = mannose; gal = galactose; fuc = fucose; NeuAc = N-acetylneuraminic acid

FIG. 7: antibody a isotype binds to immobilized fibrillar Α β 40 (Biacore sensor chip). Antibody concentration was 60 nM. Binding curves for all isotype mixtures prior to purification are shown.

FIG. 8: pepspot assay epitope mapping of antibody a compositions. A) Pepspot signals of the individual overlapping decapeptide spots are shown. B) Densitometric analysis of signal intensity of overlapping decapeptide spots alone.

FIG. 9: depolymerization experiments. Antibody a compositions and antibody a isotypes induce release of biotinylated a β from aggregated a β.

FIG. 10: antibody a compositions comprising antibody a isoforms capture soluble a β from human cerebrospinal fluid (CSF). Cerebrospinal fluid samples from 4 alzheimer patients were analyzed in 2 pools on average. Two immunoprecipitations were performed per pool, followed by Western blot analysis, and quantitation of captured a β was performed using Western blot optical density. The highest A β value in a given series of Western blots was taken as 100%.

FIG. 11: the antibody A is used for carrying out indirect immunofluorescence staining on the human starch plaques in vitro. The antibody A concentration of 10ng/ml is used for carrying out highly sensitive and specific detection after the real isolated human beta starch plaques are stained. Using (a) an antibody a composition; (B) doubly glycosylated antibody a; (C) a mono-glycosylated antibody a; and (D) a goat anti-human (H + L) -Cy3 antibody of unglycosylated antibody a detects bound antibody a. Scale =80 μm.

FIG. 12: confocal microscopy revealed in vivo immunological modification of PS2APP transgenic mouse plaques containing glycosylated antibody a isoform. Immune modification revealed in vivo binding of antibody a isotype 3 days after a single administration of 1mg of antibody a isotype to mice. Representative pictures of antibody a isotype profiles are shown, including the bis (a), mono (B), and unglycosylated (C) antibody a isotypes. Scale =80 μm.

FIG. 13: binding assay of anti-a β antibodies to cell surface APP. Human APP-transfected HEK293 cells and non-transfected control cells were analyzed for binding to antibodies by flow cytometry.

FIG. 14: schematic representation of antibody a non-glycosylated, mono-glycosylated and di-glycosylated antibody molecules (immunoglobulins).

FIG. 15: antibody compositions (comprising mono-and di-glycosylated antibody a), di-and mono-glycosylated antibody a isoforms (20mg/kg per week, i.v.) or vehicle treatment for 5 months, total plaque surface area (a), total plaque number (B) and plaque number and size distribution (C) in the thalamic region.

FIG. 16: antibody compositions (comprising mono-and di-glycosylated antibody a), di-and mono-glycosylated antibody a isotypes (20mg/kg i.v. per week) or vehicle treatment total spot surface area (a), total spot number (B) and spot number and size distribution (C) of cortex and callus regions after 5 months of treatment.

FIG. 17: antibody compositions (comprising mono-and di-glycosylated antibody a), di-and mono-glycosylated antibody a isoforms (20mg/kg i.v. weekly) or vehicle treatment total spot surface area (a), total spot number (B) and spot number and size distribution (C) in hippocampal area after 5 months of treatment.

FIG. 18: antibody compositions (comprising mono-and di-glycosylated antibody a), di-and mono-glycosylated antibody a isoforms (20mg/kg i.v. weekly) or vehicle treatment total plaque surface area (a), total plaque number (B) and plaque number and size distribution (C) in the lower foot area after 5 months of treatment.

FIG. 19: fluorescence intensity of immunostaining antibody a composition bound to β -amyloid plaques was measured after i.v. administration of 0.1mg/kg to PS2APP mice 1, 2 and 4 times every two weeks. Analysis was performed 2 weeks after the last injection.

FIG. 20: fluorescence intensity of immunostaining antibody a composition bound to β -amyloid plaques was measured after i.v. administration of 0.15mg/kg to PS2APP mice 1 and 3 times per month. Analysis was performed 2 weeks after the last injection.

FIG. 21: fluorescence intensity of immunostaining antibody a composition bound to β -amyloid plaques was measured after two weeks of 1 for 4 injections giving 0.05, 0.1 and 0.30mg/kg to PS2APP mice, indicating the dose-dependence of amyloid plaque binding. Analysis was performed 2 weeks after the last injection.

FIG. 22: fluorescence intensity of immunostaining antibody a composition bound to beta starch plaque was measured after 3 injections of 0.075, 0.15 and 0.45mg/kg given to PS2APP mice for 1 month, indicating starch plaque binding as a dose-related. Analysis was performed 2 weeks after the last injection.

FIG. 23: human AD brain sections were incubated with antibody A composition at the indicated concentrations and live, differentiated human primary macrophages (80 ten thousand cells/ml) for 40 hours, and the sections were stained for A.beta.using an anti-A.beta.murine monoclonal antibody (BAP-2). The results show a reduction in amyloid loading, indicating antigen-dependent cellular phagocytosis of beta amyloid plaques by the antibody a composition. Scale =300 μm.

FIG. 24: after incubation of human AD brain sections with 80 ten thousand cells/ml, the antibody a composition reacted to the dose of beta-amyloid plaques in the sections. (A) Total spot area is shown and (B) is staining intensity.

FIG. 25: fluorescence micrographs of P388D1 cells incubated with 0, 0.1, 1, and 10. mu.g/ml antibody A composition (A-D, respectively).

FIG. 26 dose response (expressed as relative fluorescence units, RFU) of antibody A compositions was quantitated using A.beta.conjugated fluorescent beads and P3881D1 cells. Two independent experiments showed that the potency range of the antibody a composition was quite large.

FIG. 27 is a schematic view showing: tables showing different carbohydrate structures of antibody A in the heavy chain constant region (Asn 306; column 2 above) and the heavy chain variable region (Asn 52; columns 3, 4).

Detailed Description

Examples

The invention is illustrated by the following non-limiting examples.

Example 1: generation of antibody A by cloning

According to the present invention, the IgG1 molecule was produced by a common cloning technique. Using heavy chain variable regions (V)_H) To identify the coding sequence and expressed amino acid sequence of antibody a. The following DNA sequences encode the corresponding heavy chain examples:

caggtggaattggtggaaagcggcggcggcctggtgcaaccgggcggcagcctgcgtctgagctgcgcggcc

tccggatttacctttagcagctatgcgatgagctgggtgcgccaagcccctgggaagggtctcgagtgggtgagc

gctattaatgcttctggtactcgtacttattatgctgattctgttaagggtcgttttaccatttcacgtgataattcgaaaa

acaccctgtatctgcaaatgaacagcctgcgtgcggaagatacggccgtgtattattgcgcgcgtggtaagggta

atactcataagccttatggttatgttcgttattttgatgtttggggccaaggcaccctggtgacggttagctcagcctc

caccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggct

gcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgca

caccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttg

ggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagccca

gatatcgtgcgatatcgtgcaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggg

ggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc

gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcata

atgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgc

accaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaa

aaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctg

accaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggaga

gcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctac

agcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctct

gcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga(SEQ ID NO：5)。

it encodes the following immunoglobulin H chain:

QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV

SAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

RGKGNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG

GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES

NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPGK(SEQ ID NO：6)

sequences comprising additional "leader sequences" as shown below may also encode the same heavy chain:

atgaaacacctgtggttcttcctcctgctggtggcagctcccagatgggtcctgtcc

caggtggaattggtggaaagcggcggcggcctggtgcaaccgggcggcagcctgcgtctgagctgcgcggcctccg

gatttacctttagcagctatgcgatgagctgggtgcgccaagcccctgggaagggtctcgagtgggtgagcgctattaat

gcttctggtactcgtacttattatgctgattctgttaagggtcgttttaccatttcacgtgataattcgaaaaacaccctgtatct

gcaaatgaacagcctgcgtgcggaagatacggccgtgtattattgcgcgcgtggtaagggtaatactcataagccttatg

gttatgttcgttattttgatgtttggggccaaggcaccctggtgacggttagctcagcctccaccaagggtccatcggtctt

ccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccg

aaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcag

gactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatca

caagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcc

cagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggac

ccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgt

ggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcacc

gtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcga

gaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctg

accaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaat

gggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctc

accgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactac

acgcagaagagcctctccctgtctccgggtaaatga(SEQ ID NO：25)

the corresponding amino acid sequence is

MKHLWFFLLLVAAPRWVLS

QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA

INASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGK

GNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：26)

Similarly, the following nucleotide sequence encodes the light chain of antibody a:

gatatcgtgctgacccagagcccggcgaccctgagcctgtctccgggcgaacgtgcgaccctgagctgcagag

cgagccagagcgtgagcagcagctatctggcgtggtaccagcagaaaccaggtcaagcaccgcgtctattaatt

tatggcgcgagcagccgtgcaactggggtcccggcgcgttttagcggctctggatccggcacggattttaccctg

accattagcagcctggaacctgaagactttgcgacttattattgccttcagatttataatatgcctattacctttggcca

gggtacgaaagttgaaattaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttga

aatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtgga

taacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctc

agcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagg

gcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO：7)

and encodes the following amino acid sequence (L chain):

DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY

GASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQ

GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC(SEQ ID NO：8)

"leader sequences" may likewise be used, the corresponding sequences being

atggtgttgcagacccaggtcttcatttctctgttgctctggatctctggtgcctacggg

gatatcgtgctgacccagagcccggcgaccctgagcctgtctccgggcgaacgtgcgaccctgagctgcagagcga

gccagagcgtgagcagcagctatctggcgtggtaccagcagaaaccaggtcaagcaccgcgtctattaatttatggcg

cgagcagccgtgcaactggggtcccggcgcgttttagcggctctggatccggcacggattttaccctgaccattagcag

cctggaacctgaagactttgcgacttattattgccttcagatttataatatgcctattacctttggccagggtacgaaagttga

aattaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttg

tgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactc

ccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagc

agactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaa

caggggagagtgttag(SEQ ID NO：27)

The sequence encodes the following amino acid sequence

MVLQTQVFISLLLWISGAYG

DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY

GASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQ

GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC(SEQ ID NO：28)

The above sequences are distinguished from MAB31 described in WO 03/070760.

However, the heavy and light chains of exemplary antibody a may also be encoded by the following sequences:

a) heavy chain

atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgattcatggagaaatagagagactgagtgt

gagtgaacatgagtgagaaaaactggatttgtgtggcattttctgataacggtgtccttctgtttgcaggtgtccagt

gtcaggtggagctggtggagtctgggggaggcctggtccagcctggggggtccctgagactctcctgtgcagc

gtctggattcaccttcagtagctatgccatgagctgggtccgccaggctccaggcaaggggctcgagtgggtgtc

cgccataaacgccagcggtacccgcacctactatgcagactccgtgaagggccgattcaccatctccagagaca

attccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagag

gcaaggggaacacccacaagccctacggctacgtacgctactttgacgtgtggggccaaggaaccctggtcac

cgtctcctcaggtgagtcctcacaacctctctcctgcggccgcagcttgaagtctgaggcagaatcttgtccaggg

tctatcggactcttgtgagaattaggggctgacagttgatggtgacaatttcagggtcagtgactgtctggtttctctg

aggtgagactggaatataggtcaccttgaagactaaagaggggtccaggggcttttctgcacaggcagggaaca

gaatgtggaacaatgacttgaatggttgattcttgtgtgacaccaagaattggcataatgtctgagttgcccaaggg

tgatcttagctagactctggggtttttgtcgggtacagaggaaaaacccactattgtgattactatgctatggactact

ggggtcaaggaacctcagtcaccgtctcctcaggtaagaatggcctctccaggtctttatttttaacctttgttatgga

gttttctgagcattgcagactaatcttggatatttgccctgagggagccggctgagagaagttgggaaataaatctg

tctagggatctcagagcctttaggacagattatctccacatctttgaaaaactaagaatctgtgtgatggtgttggtg

gagtccctggatgatgggatagggactttggaggctcatttgagggagatgctaaaacaatcctatggctggagg

gatagttggggctgtagttggagattttcagtttttagaatgaagtattagctgcaatacttcaaggaccacctctgtg

acaaccattttatacagtatccaggcatagggacaaaaagtggagtggggcactttctttagatttgtgaggaatgtt

ccacactagattgtttaaaacttcatttgttggaaggagctgtcttagtgattgagtcaagggagaaaggcatctagc

ctcggtctcaaaagggtagttgctgtctagagaggtctggtggagcctgcaaaagtccagctttcaaaggaacac

agaagtatgtgtatggaatattagaagatgttgcttttactcttaagttggttcctaggaaaaatagttaaatactgtga

ctttaaaatgtgagagggttttcaagtactcatttttttaaatgtccaaaatttttgtcaatcaatttgaggtcttgtttgtgt

agaactgacattacttaaagtttaaccgaggaatgggagtgaggctctctcataccctattcagaactgacttttaac

aataataaattaagtttaaaatatttttaaatgaattgagcaatgttgagttgagtcaagatggccgatcagaaccgg

aacacctgcagcagctggcaggaagcaggtcatgtggcaaggctatttggggaagggaaaataaaaccactag

gtaaacttgtagctgtggtttgaagaagtggttttgaaacactctgtccagccccaccaaaccgaaagtccaggct

gagcaaaacaccacctgggtaatttgcatttctaaaataagttgaggattcagccgaaactggagaggtcctctttt

aacttattgagttcaaccttttaattttagcttgagtagttctagtttccccaaacttaagtttatcgacttctaaaatgtat

ttagaattcgagctcggtacagctttctggggcaggccaggcctgaccttggctttggggcagggagggggcta

aggtgaggcaggtggcgccagcaggtgcacacccaatgcccatgagcccagacactggacgctgaacctcgc

ggacagttaagaacccaggggcctctgcgcctgggcccagctctgtcccacaccgcggtcacatggcaccacc

tctcttgcagcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcaca

gcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctga

ccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgc

cctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaa

gaaagttggtgagaggccagcacagggagggagggtgtctgctggaagccaggctcagcgctcctgcctgga

cgcatcccggctatgcagccccagtccagggcagcaaggcaggccccgtctgcctcttcacccggagcctctg

cccgccccactcatgctcagggagagggtcttctggctttttcccaggctctgggcaggcacaggctaggtgccc

ctaacccaggccctgcacacaaaggggcaggtgctgggctcagacctgccaagagccatatccgggaggacc

ctgcccctgacctaagcccaccccaaaggccaaactctccactccctcagctcggacaccttctctcctcccagat

tccagtaactcccaatcttctctctgcagagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccaggta

agccagcccaggcctcgccctccagctcaaggcgggacaggtgccctagagtagcctgcatccagggacagg

ccccagccgggtgctgacacgtccacctccatctcttcctcagcacctgaactcctggggggaccgtcagtcttcc

tcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtga

gccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagcc

gcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaat

ggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagcca

aaggtgggacccgtggggtgcgagggccacatggacagaggccggctcggcccaccctctgccctgagagtg

accgctgtaccaacctctgtccctacagggcagccccgagaaccacaggtgtacaccctgcccccatcccggga

tgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagt

gggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttctt

cctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatg

aggctctgcacaaccactacacgcagaagagcctctccctgtccccgggcaaatga(SEQ ID NO：23)

b) Light chain

atggacatgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtaaggatggagaacactagc

agtttactcagcccagggtgctcagtactgctttactattcagggaaattctcttacaacatgattaattgtgtggacat

ttgtttttatgtttccaatctcaggcgccagatgtgatatcgtgttgacgcagtctccagccaccctgtctttgtctcca

ggggaaagagccaccctctcctgccgggccagtcagagtgttagcagcagctacttagcctggtaccagcaga

aacctggccaggcgcccaggctcctcatctatggcgcatccagcagggccactggcgtgccagccaggttcag

tggcagtgggtctgggacagacttcactctcaccatcagcagcctggagcctgaagatttcgcgacctattactgt

ctgcagatttacaacatgcctatcacgttcggccaagggaccaaggtggaaatcaaacgtgagtagaatttaaact

ttgcggccgcctagacgtttaagtgggagatttggaggggatgaggaatgaaggaacttcaggatagaaaaggg

ctgaagtcaagttcagctcctaaaatggatgtgggagcaaactttgaagataaactgaatgacccagaggatgaa

acagcgcagatcaaagaggggcctggagctctgagaagagaaggagactcatccgtgttgagtttccacaagta

ctgtcttgagttttgcaataaaagtgggatagcagagttgagtgagccgtaggctgagttctctcttttgtctcctaag

tttttatgactacaaaaatcagtagtatgtcctgaaataatcattaagctgtttgaaagtatgactgcttgccatgtaga

taccatgtcttgctgaatgatcagaagaggtgtgactcttattctaaaatttgtcacaaaatgtcaaaatgagagactc

tgtaggaacgagtccttgacagacagctcaaggggtttttttcctttgtctcatttctacatgaaagtaaatttgaaatg

atcttttttattataagagtagaaatacagttgggtttgaactatatgttttaatggccacggttttgtaagacatttggtc

ctttgttttcccagttattactcgattgtaattttatatcgccagcaatggactgaaacggtccgcaacctcttctttaca

actgggtgacctcgcggctgtgccagccatttggcgttcaccctgccgctaagggccatgtgaacccccgcggt

agcatcccttgctccgcgtggaccactttcctgaggcacagtgataggaacagagccactaatctgaagagaaca

gagatgtgacagactacactaatgtgagaaaaacaaggaaagggtgacttattggagatttcagaaataaaatgc

atttattattatattcccttattttaattttctattagggaattagaaagggcataaactgctttatccagtgttatattaaaa

gcttaatgtatataatcttttagaggtaaaatctacagccagcaaaagtcatggtaaatattctttgactgaactctcac

taaactcctctaaattatatgtcatattaactggttaaattaatataaatttgtgacatgaccttaactggttaggtagga

tatttttcttcatgcaaaaatatgactaataataatttagcacaaaaatatttcccaatactttaattctgtgatagaaaaa

tgtttaactcagctactataatcccataattttgaaaactatttattagcttttgtgtttgacccttccctagccaaaggca

actatttaaggaccctttaaaactcttgaaactactttagagtcattaagttatttaaccacttttaattactttaaaatgat

gtcaattcccttttaactattaatttattttaaggggggaaaggctgctcataattctattgtttttcttggtaaagaactct

cagttttcgtttttactacctctgtcacccaagagttggcatctcaacagaggggactttccgagaggccatctggca

gttgcttaagatcagaagtgaagtctgccagttcctcccaggcaggtggcccagattacagttgacctgttctggtg

tggctaaaaattgtcccatgtggttacaaaccattagaccagggtctgatgaattgctcagaatatttctggacaccc

aaatacagaccctggcttaaggccctgtccatacagtaggtttagcttggctacaccaaaggaagccatacagag

gctaatatcagagtattcttggaagagacaggagaaaatgaaagccagtttctgctcttaccttatgtgcttgtgttca

gactcccaaacatcaggagtgtcagataaactggtctgaatctctgtctgaagcatggaactgaaaagaatgtagt

ttcagggaagaaaggcaatagaaggaagcctgagaatacggatcaattctaaactctgagggggtcggatgacg

tggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaa

gagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattt

taaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtga

ttatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcaggaactgt

ggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgct

gaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccagg

agagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcag

actacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttc

aacaggggagagtgttag(SEQ ID NO：24)

Example 1.1 vector construction

The antibody A sequence is derived from secondary maturation after primary screening of a synthetic phage display library MorphoSys HuCAL library. The DNA of antibody A was originally provided in the vector pMorph of MorphoSys, Germany, corresponding to the Fab expression vector shown in FIG. 2 in WO03/070760, appendix p 6/43. In the construction of the vectors for the purposes of the present invention, the encoding vectors pEE6.1 and pEE14.4 (both from Longza Biopsis) were used to obtain a construct containing both strands in the same vector, see FIG. 1; see WO 87/04462 or WO 89/01036. Cloning was performed using the following protocol:

the Ig kappa chain was PCR isolated from the vector MS-Roche # 7.9H 7_ Ig kappa chain (as described in WO 03/070760) using primer ACGTAAGCTTGCCGCCACCATGGTGTTGCAG (sense strand; HindIII; SEQ ID NO.29) and primer ACGTGAATTCCTAACACTCTCCCCTGTT (antisense strand, EcoRI; SEQ ID NO.30), inserted into pCR 2.1 Topo TA, and the inserts were fully sequenced. The pCR Topo 2.1 was digested with HinDIII/EcoR to remove the Ig kappa chain insert and ligated into the vector pEE14.4 as a HindIII/EcoRI insert.

The Ig γ 1 heavy chain was PCR cloned from the vector pMorph MS-Roche #7.9. H7-IgG 1 using primer ACGTAAGCTTGCCGCCACCATGAAACACCTG (sense strand, HindIII; SEQ ID NO: 31) and primer ACGTGAATTCTCATTTACCCGGAGACAG (antisense strand, EcoRI; SEQ ID NO: 32), inserted into pCR 2.1 Topo TA, and the inserts were completely sequenced. pCR Topo 2.1 was digested with HindIII/EcoRI to remove the Ig γ 1 heavy chain insert, which was ligated into the vector pEE 6.4 as a HindIII/EcoRI insert. NotI/SalI digestion of pEE 6.4 IgG1 removed the heavy chain expression cassette and the isolated fragment was inserted into SalI/NotI digested pEE 14.4. kappa. to yield the final two-gene construct pEE14.4 mAb-31.

Example 1.2: transfection of CHO cells and expression of antibody A

Transfection was performed according to standard experimental methods. The host cell line CHO K1 was derived from the Lonza Working Cell Bank (WCB) #028-W2(Lonza, 2002, 1-179), and the host cell line CHO K1SV was derived from the Lonza Master Cell Bank (MCB) #269-M (Lonza, 2003, 1-87).

Adherent CHO K1 cells derived from WCB #028-W2 were transfected with the vector pEE14.4 MAb31 containing the heavy and kappa light chain genes using lipofection (Fugene, Roche diagnostics). Transfection isolates were selected in the presence of DMEM, GS supplement (both from JRH Biotechnology (JRH Biosciences)), 10% dialysed FCS (PAA Laboratories, CoS # R0-CEP 2001-083-Rev 00) and 50. mu.M methionine sulphonimide (MSX from Sigma). After 2 weeks, colonies were picked and transferred to 96-well plates and antibody production was detected by ELISA. The highest expression of antibody a was cloned by limiting serial dilution of the 4 colonies to obtain single cell derived cultures, and 82 clones were derived and expanded after one week.

One of the clones was selected with the highest specific yield of 48 pg/cell/day in the adherent state. Further subcloning was performed by limiting dilution to obtain good antibody producers with high stability (Pu, (1998) Mol Biotechnol, 10, 17-25). In addition, the vector pEE14.4 MAb31 was transfected into suspension variant CHO K1SV cells (from MCB #269-M) of CHO K1 cells using electroporation. Transfectants were selected as described above, and the resulting clones were single cell cloned by limiting dilution to give several high producing clones of antibody A.

EXAMPLE 1.3 Adaptation of antibody A expressing clones to suspension culture

The best growth properties of the CHO K1 clone were tested in DHI medium containing different protein hydrolysates: the cells were finally adapted to DHI medium with/without glutamine (Invitrogen) which was a mixture of DMEM, Ham's F12 and IMDM mixed at a ratio of 1:1:2(v: v: v), respectively (Schlaeger and Schumpp, 1992, J Immunol Methods, 146, 111-20) with the following changes: soy and rice hydrolysates: 0.2% soy Hypep 1510 and 0.2% rice Hypep 5603 (Kerry biosciences), 0.03% pluronic F68 (Invitrogen), 25 μ M MSX (Sigma) and 5% dialytic FCS (PAA laboratories). The FCS concentration was gradually decreased until the cells grew exponentially in serum-free DHI medium. Primary seed cell banks of several recombinant cell clones were cryopreserved with serum-free DHI medium.

The CHO K1SV clone was adapted to grow in suspension in chemically defined CD-CHO medium (Gibbco-Invitrogen) containing 25. mu.M MSX using a two-step procedure (Lonza). Cell banks were generated in CD-CHO. Optionally, any other serum-free, protein-free medium for CHO cell growth can be used in suspension culture and as a basis for antibody expression.

Example 2: production of antibody A

Production of antibody A (by Fed-batch fermentation)

CHO clones were prepared by fermentation from shake-or spinner-cultured stock cultures using the following method:

each clone of a cryopreserved tube was thawed separately with each medium containing 25 μ MMSX in 100ml shake flasks or spinner flasks of nominal volume 50-75 ml.

Mixing the raw materials in a ratio of 1: the 5-ratio serial flask was used to expand cells and to obtain a 500ml volume of storage culture in a shake flask or spinner flask. Cells for fermentation inoculation can be derived from these storage cultures up to 90 days after thawing. In 2L shake flasks or spinner flasks, a seed train (seed train) consisted of 2x1000ml steps, followed by inoculation in another 10L fermentor. Alternatively, the 10L fermentor may itself be used as a fed-batch fermentor vessel or used to inoculate 100L through a fed-batch fermentor. The medium contained MSX as a selective condition until the MSX was removed when the 10L fermentor was inoculated.

And (3) fermentation process:

day 0: from 3-4x10⁵Starting with/ml cells (from seed culture 1:4-1:5 flasks).

Day 2-3: at the beginning of the feed, the cell density should be higher than 1.5X10⁶Per milliliter.

Feeding: 2% continuous or one-shot feed per day.

Throughout the fermentation, the antibody a isotype composition was monitored using ion exchange chromatography (see below).

Day 14-18: when cell viability begins to decline (50%) and reaches the desired titer, cell supernatants are collected by centrifugation and/or filtration and sterile filtration and further processed as described in the next section.

Fermentation is carried out according to standard procedures, see for example Werner, (1993), Arzneimitelforschung, 43, 1242-9 or Rendall, (2003), the eighteenth ESACT meeting (Proceedings of the18th ESACT meeting), 5.11-14.2003, 1, 701- "704).

Example 3: purification of antibody A

The purification process is based on three chromatographic steps and one diafiltration step: protein a affinity chromatography, cation exchange chromatography, anion exchange chromatography and diafiltration using a 100kD membrane. Gel type and column size were 1lMabSelect (GE Healthcare, Art.17-5199, column diameter 9CM, bed length 18+/-2CM), 0.4l CM-Toyopearl 650M (TosoBioscience, Art.007972, Small ion Capacity =85 micro equivalents/ml, diameter 5.0CM, bed length 20+/-2CM), 1.3l Q-Sepharose FF (GE Healthcare, Art.17-0510-04), diameter 9CM, bed length 20+/-2 CM. The column was run at room temperature. The fractions were stored at 2-8 ℃. Detection was performed at 280 nm. The using area is 0.1m²Concentrated and diafiltered using a Biomax 100 ultrafiltration module (Milibo group, Art. P2B100A01).

Protein a chromatography

The following solutions were prepared using purified water:

solution a (equilibration buffer): 25mM Tris, 25mM NaCl, 5mM EDTA, adjusted to pH 7.1+/-0.1 with HCl

Solution B (wash buffer 1): 100mM acetic acid, adjusted to pH 4.5+/-0.1 with NaOH

Solution C (elution buffer): 100mM acetic acid, adjusted to pH 3.2+/-0.1 with NaOH

Solution D (wash buffer 2): 100mM acetic acid, 75mM NaCl, pH 3+/-0.1

Solution E (regeneration buffer): 2M guanidine hydrochloride, 100mM Tris, adjusted to pH7.5+/-0.1 with HCl

Solution F (storage buffer): 200mM benzyl alcohol, 100mM acetic acid, adjusted to pH 5.0+/-0.1 with NaOH

The column was first equilibrated with 3 bed volumes of solution a.

The clarified cell culture supernatant was then used to pack a column (45l, 386mg/l antibody)

Washed with 5 bed volumes of solution a,

washed with 3 bed volumes of solution B,

eluting with 3.5 bed volumes of solution C, and collecting the eluate,

washing with 3 column volumes of solution D, and

regeneration is carried out with 2 column volumes of solution E,

equilibrated with 3 bed volumes of buffer A,

and washed with bed volume buffer F for storage.

All chromatography steps used a linear flow rate of 100 cm/h.

The column loading was 17.4g antibody/l Mabselect gel, the yield of the total isotype mixture was 96%.

Inactivation of viruses

The following solutions were prepared using purified water:

solution G (conditioning solution): 2M sodium acetate

The pH of the protein A eluate is adjusted to pH3.5-3.7 by adding concentrated acetic acid or 2M sodium acetate (solution G). Stirring for 15 minutes and then adjusting to pH 4+/-0.1 by addition of 2M sodium acetate (solution G).

Cation exchange chromatography

The following solutions were prepared using purified water:

solution H (equilibration buffer): 100mM acetic acid, adjusted to pH 4.0+/-0.1 with NaOH

Solution I (elution buffer 1): 250mM sodium acetate, without pH adjustment, pH 7.8-8.5

Solution J (elution buffer 2): 500mM sodium acetate, pH 7.8-8.5 without pH adjustment

Solution K (regeneration solution): 0.5M sodium hydroxide

Solution L (storage buffer): 0.01M sodium hydroxide

The column was first regenerated with 2 bed volumes of solution K and then equilibrated with 5 bed volumes of solution H.

The column was then aliquoted with protein a eluent and washed with 1 bed volume of solution H.

Followed by elution with 6 bed volumes of solution I. In this step, the di-glycosylated and mono-glycosylated isoforms are eluted. In the next step, the non-glycosylated isoforms are eluted with 3 bed volumes of solution J.

After regenerating the column with two bed volumes of solution K, the column was stored in this buffer for 24 hours, followed by a further 2 bed volumes of solution K wash. Wash with 3 bed volumes of solution L for storage.

Figure 3 shows a chromatographic legend.

The chromatographic fractions were analyzed by analytical IEX as described below.

All chromatography steps used a linear flow rate of 100 cm/h.

The column loading was 14.3g antibody/l CM Toyopearl 650M, the yield of mixture of di-and mono-glycosylated isoforms was 79% and the yield of the non-glycosylated isoform was 6.2%.

Flow through chromatography with Q-Sepharose FF

The following solutions were prepared using purified water:

solution M (dilution buffer): 37.5mM Tris, adjusted to pH 7.9+/-0.1 with acetic acid

Solution N (conditioning solution): 2M Tris

Solution O (equilibration buffer): 83mM sodium acetate, 25mM Tris, pH7.5+/-0.1

Solution P (regeneration buffer 1): 0.5M NaOH/1M NaCl

Solution Q (regeneration buffer 2): 0.2M acetic acid/1M NaCl

Solution R (storage buffer): 0.01M NaOH

First, with a solution M1: 3 the eluate from the CMT column (acidic) is diluted and adjusted to pH7.5 with solution N.

The column was first equilibrated with 2 bed volumes of solution O, then the diluted CMT column eluate was passed through the column and the effluent collected. The product on the column was washed off with solution O until the absorbance at 280nm was below 0.1 (the effluent was collected).

The column was regenerated with 1.5 bed volumes of solution P and after 1 hour of storage the column was regenerated with 1.5 bed volumes of solution P. The column was then regenerated with 2 bed volumes of solution Q, washed with 3 bed volumes of solution R and stored.

All chromatography steps used a linear flow rate of 100 cm/h.

The column loading was 3.5g antibody/l Q Sepharose FF, and the yield of mixture of di-and mono-glycosylated isoforms was 91%.

Diafiltration (Perfect Inflation)

The following solutions were prepared using purified water:

solution S (diafiltration buffer): 20mM histidine, adjusted to pH 5.5 with HCl

The filter holder Pellicon 2 (Mitigo group Co.) was equipped with 1 model Biomax 100 (Mitigo group Co., area =0.1 m)²And art. p2b 100a01). WATSON-MARLOW 501U pump fitted with silicone tubing was used for pumping. The system was rinsed with buffer O and then at 4-11 deg.C for 1 hour3.8 liters (1.1g antibody/l) of QS chromatography effluent (adjusted to pH 5.5 with concentrated acetic acid) were concentrated to 250-300 ml. Followed by diafiltration (V = constant) (4-11 °) with 3l buffer S (approximately 10 volumes). Finally, the product was sterilized by filtration using a Millipac 20 filter (Mitigo group Co.). The yield of the ultrafiltration/diafiltration step was 91%. The product concentration was 15 mg/ml. The product was frozen at-70 ℃.

Analytical IEX method for analyzing components

Column: Mono-S HR5/5 (GE healthcare company, Art.17-0547-01)

Buffer 1:50 mM morpholinoethanesulfonic acid, adjusted to pH 5.8 with sodium hydroxide

Buffer 2: 50mM morpholinoethanesulfonic acid, 1M NaCl, pH 5.8 adjusted with sodium hydroxide

Flow rate: 1ml/min

And (3) detection: 280nm

Sample loading amount: 36-72 mu

Gradient:

figure 2 gives an exemplary chromatogram:

yield of

Example 4: identification of antibody A isoforms by SDS-PAGE

SDS-PAGE analysis was performed according to standard experimental methods using 4-12% NuPage gradient Bis-Tris gels (Invitrogen) and MARK12 (Invitrogen) as controls. 1-3 μ g protein A purified fermentation supernatant (Prod 01, 02, 03) or spinner culture supernatant (all other lanes) was loaded per well. When analyzed under reducing conditions, a single band of peak 1 (doubly glycosylated antibody a), two bands of peak 2 (singly glycosylated antibody a), and a single band of peak 3 (unglycosylated antibody a) were obtained in the heavy chain molecular weight range. The molecular weights of the two bands of peak 2 correspond to the molecular weights of peak 1 and peak 2, respectively.

Similar results were obtained using several expression systems, such as: transient transfection of HEK293 EBNA cells, transient transfection of CHO cells and stable transfection of CHO cells.

Example 5: mass Spectrometry (MS) analysis identified antibody a isotype.

The complete antibody mass spectrum of all antibody a isotypes was determined using electrospray ionization mass spectrometry a (ESI-MS).

For this purpose, a sample of antibody a was prepared under non-reducing conditions. Samples were desalted into 2% formic acid and 40% acetonitrile by G25 gel filtration and then analyzed by ESI-MS on a Q-Tof2 or LCT-mass spectrometer from Waters, Inc. (Waters).

The difference between the molecular weight of the unglycosylated antibody A obtained by molecular weight separation and that of the monoglycosylated antibody A was 1623. The molecular weight of the unglycosylated antibody A, predicted from the amino acid sequence, is 145,987Da, which is consistent with an experimentally determined molecular weight of 145,979 Da. Similarly, the difference between the molecular weights of the mono-and di-glycosylated antibody isotypes is 1624Da as shown in FIG. 4. The observed molecular weight differences are consistent with the type of N-glycosylation described in further detail below.

Example 6: asn-52 glycosylation structure of antibody A

Asn52 is a part of the heavy chain variable part sequence aaa-aaa-Asn-Ala-Ser-aaa-aaa, which corresponds to the N-glycosylation consensus sequence Asn-aaa-Ser/Thr. N-linked glycosylation of Asn52 was confirmed by tryptic peptide mapping and mass spectrometry evaluation of Asn 52-containing peptides HC/T4 for antibody a isotype. In the tryptic peptide map of unglycosylated antibody A, only the peptide corresponding in mass to the unglycosylated HC/T4 peptide appeared, indicating that Asn52 is unglycosylated, whereas in the mono-and di-glycosylated antibody A, the peptide corresponding in mass to HC/T4 containing the N-linked sugar structure was detected.

To further confirm glycosylation of the consensus sequence in the heavy chain proteolytic peptide HC/T4, the glycosylated HC/T4 peptide was isolated from the peptide map of the glycosylated antibody a isoform and MALDI-mass spectrometry was performed before and after N-glycosidase F incubation. Before the action of N-glycosidase F, the mass corresponding to HC/T4 peptide comprising the N-linked carbohydrate structure was obtained. However, the mass of HC/T4 peptide acted on by N-glycosidase F corresponds to the expected mass of the desired unglycosylated HC/T4+1Da, as expected when N-glycosidase F removes the sugar chain from asparagine (Asn-Asp switch).

The presence of N-acetylneuraminic acid in the sugar structure attached to Asn52 further indicates the presence of N-linked complexes and hybrid type sugar structures. Thus, glycosylated antibody a isoform was treated with N-glycosidase F alone (to remove the N-sugar at Asn306 but not Asn 52) or in combination with ceramidase and analyzed after HC and LC separation by denaturation, reduction and desalting. The HC masses for both methods differed by approximately 291Da or 582Da, corresponding to 1 or 2 sialic acids. This indicates that the complex and/or hybrid type of N-linked sugar is attached to Asn 52.

Asn-52 glycosylation (N-glycosylation) consists mainly of coreless fucosylated biantennary complex-type sugar structures (. gtoreq.75%; mainly 80-90%) and is highly sialylated, so that up to 80% of complex-type antennary contains N-acetylneuraminic acid. The minority sugar structures belong to the biantennary heterozygote and the oligomannose type (. ltoreq.25%), respectively (FIG. 5 or FIG. 27). The commonality of all Asn52 glycosylation structures is that they are not cleaved from intact antibody a by N-glycosidase F.

Example 7: asn306 glycosylation structure of antibody A

As indicated above, antibody A contains antibody-type glycosylation of asparagine 306(Asn306) attached to the Fc-portion of the Heavy Chain (HC), which consists of a complex biantennary oligosaccharide chain. It is well known that antibodies comprise different isoforms of such complex biantennary oligosaccharide chains, differing in the degree of terminal galactosylation, sialylation and the degree of core fucosylation. Furthermore, it is known that the degree of lack of core fucosylation in the Fc sugar chain structure is important for the in vivo potency of antibodies, and it is widely accepted that the degree of core fucosylation modulates the effector functions of antibodies.

Typical changes in antibodies for the Fc sugar chain attached to Asn306 were found for antibody A bodies (Router (1997), glycoconjugate 14(2), 201-; 207; Raju (2003), bioprocess International, 44-52) to be associated with terminal galactosylation and core fucosylation.

The heterogeneity of the degree of terminal galactosylation (G0: G1: G2 structure) was detected to be about 35-40% G0-structure, about 45% G1-structure and about 15-20% G2-structure (see FIG. 6 or the schematic structure of FIG. 27).

The content of Fc-sugar structures lacking core fucosylation, i.e. lacking a fucose unit linked to the innermost N-acetylglucosamine of the core sugar structure, may be important for antibodies, since the presence or absence of a fucose unit may modulate the binding of the antibody to Fc-receptors of effector cells, thereby affecting the activity of these cells.

The relative content of the sugar chain isoform lacking core fucosylation at Asn306 in antibody a was determined using two different methods:

A) mass spectrum of fully glycosylated HC:

antibody A was denatured with 6M guanidine hydrochloride and 250mM TCEP and reduced to Light Chain (LC) and glycosylated HC. The reduced sample was desalted into 2% formic acid and 40% acetonitrile and used for ESI-MS analysis on a Watts Q-Tof2 or LCT-HC mass spectrometer. From the obtained m/z spectra, the relative content of each oligosaccharide isoform was calculated from the peak heights of glycosylated HC of the individual oligosaccharide isoforms containing the selected single m/z state. For the calculation of the relative content of sugar structures lacking core fucosylation, the peak height of the G0 structure lacking core fucose (G0-Fuc) correlates with the sum of G0+ (G0-Fuc).

The individual carbohydrate structures were distinguished by differences in mass of glycosylated HC and HC, and the oligosaccharide structures of HC were removed by incubation with N-glycosidase F prior to MS-analysis in control experiments.

B) Chromatographic analysis of released oligosaccharides by HPEAC-PAD:

a sample of antibody a was incubated with N-glycosidase F in sodium phosphate buffer, pH 7.2, in order to release the oligosaccharide chain at Asn306 (under the conditions used, the sugar structure of Asn52 was not released from the intact non-denatured antibody). The released sugar chains were separated from the antibody A protein by centrifugation and analyzed in a BioLC system using a Carbo Pac PA200 column from Dionex, using a gradient of sodium acetate with strong basicity (pH 13). The column used is capable of separating nonfucosylated oligosaccharide chains from fucosylated oligosaccharide chains. By comparing the sample residence time for analysis on a Carbo Pac PA200 column with the residence time of a suitable oligosaccharide standard and by determining the molar mass of the peaks separated and collected by MALDI mass spectrometry, each peak obtained can be assigned to a corresponding carbohydrate structure. To calculate the relative content of the core fucose-deficient structures, the area% of all the core fucose-deficient structures was summed.

Analysis of several batches of the mixture of di-, mono-glycosylated antibody a isoforms and purified antibody a isoforms revealed a content of non-fucosylated Asn 306-linked oligosaccharide chains in the range of-14% -27% (MS assay) and 6% -26% (HPAEC-PAD assay), respectively.

Example 8: determination of the KD values of antibody A compositions and isotypes (e.g., unglycosylated, mono-or di-glycosylated antibodies of the invention) bound to A β 1-40 and A β 1-42 fibers using Surface Plasmon Resonance (SPR) in vitro

Surface Plasmon Resonance (SPR) on-line measurement of binding of antibody a to Α β fibers, the affinity of molecular interactions was determined as follows: measurements were performed using Biacore2000 and Biacore3000 instruments. Synthetic peptide was incubated at a concentration of 200. mu.g/ml for three days at 37 ℃ in 10mM sodium acetate buffer (pH 4.0) to produce A.beta.1-40 and A.beta.1-42 fibers in vitro. Electron microscopy confirmed the fibrous structure of both peptides, with Α β 1-40 showing predominantly shorter fibers (<1 micron) and Α β 1-42 showing predominantly longer fibers (>1 micron). It is hypothesized that these fibers are closer to aggregated a β peptide in human AD brain than amorphous aggregates and unclear mixtures with no structural precipitates. Fibers were diluted 1:10 and coupled directly to CM5(BIA application Handbook, AB edition, Biacore AB, uppsala, 1998) as described in the manufacturer's instruction manual.

The coupling process comprises an activation step in which the surface is contacted with an aqueous mixture of N-hydroxysuccinimide and 1-ethyl-1- (3-diaminopropyl) -carbodiimide hydrochloride to convert carboxyl groups of the surface to chemically reactive succinimide ester groups, and an immobilization step in which the activated surface is contacted with fibres dissolved in 10mM acetic buffer (pH 4.5) to a surface loading of about 1 picogram/mM and 200-350 resonance units (1 resonance unit corresponds to a surface loading of about 1 picogram/mM)²). Then, the surface loading fiber is contacted with an antibody solution with the concentration range of 200nM C0.15 nM. Fig. 7 shows typical time-dependent reaction curves (= sensorgrams) for the monitored bound phase (during contact with buffer) and dissociated phase (during subsequent contact with buffer).

The following table shows the K binding of A.beta.1-40 and A.beta.1-42 fibers to antibody A isotype_DThe value is obtained. Briefly, K is calculated using Scatchard type analysis_DValue, the assay uses a concentration-dependent equilibrium binding reaction. These equilibrium binding constants can be obtained in two ways.

Since the binding process is very slow at low antibody concentrations, the contact time interval to reach equilibrium is very long (fig. 7). However, such contact time intervals can be achieved on a Biacore instrument and Scatchard analysis can be performed on experimental equilibrium reactions.

Equilibrium binding data can also be obtained by extrapolating the shorter time-dependent binding curve indefinitely. These theoretically obtained equilibrium binding levels can be used again for determining K_DThe value is obtained.

Independent of the manner in which the equilibrium sensor response curves are determined, Scatchard plots may be acquired independently. Higher (bivalent) and lower (monovalent) affinity interactions of antibody a isoforms derived from the second affinity maturation cycle can be derived from Scatchard plots. These two affinities represent lower and higher Ks in the ranges shown in the table below_DThe value:

the table above shows the K for low affinity complexes (monovalent) and high affinity complexes (divalent) formed by the interaction of antibody A isotype and A β 1-40 fibers as detected by surface plasmon resonance_DThe value is obtained. K is given as the extrapolated equilibrium reaction result (labeled "extrapolated") and the experimentally determined equilibrium reaction result (labeled "experiment")_DThe value is obtained. The extrapolated values are measured at least 6 times and the standard deviation is given. K based on experimental and extrapolated equilibrium sensor responses_DEqual within the limits given by the standard deviation.

Example 9: epitope mapping of antibody A compositions and isotypes (e.g., unglycosylated, mono-glycosylated or di-glycosylated antibodies of the invention) by Pepspot decapeptide analysis

Epitopes (antigenic determinants) can be linear or conformational. The bi-epitopic specificity described herein is the reactivity of an antibody with two non-continuous linear peptides.

Epitope mapping methods for defining specific epitope recognition are based on ELISA techniques using 6-peptide conjugates to coat microplates or on pepspot technology. The latter technique allows the detection and quantification of antibodies using known experimental methods of Western blotting of proteins onto PVDF membranes.

Epitope mapping techniques are designed to specifically detect linear epitopes, but these techniques are not capable of mapping spatially more complex epitopes such as non-continuous or conformational epitopes. Techniques such as domain scanning and combinatorial peptide matrices that can be applied to conformational or discontinuous epitope mapping require long peptides (domains) up to 36 amino acids in length or peptide combinations each consisting of 12 amino acids.

The applied technology is therefore considered to be linear epitope specific and does not include discrete or discontinuous interspersed conformational epitopes.

In summary, the presented data indicate that two regions within a β peptides as defined herein resemble independent linear epitopes, recognized simultaneously according to the unique bi-epitope specificity of the antibodies studied for one hexameric or decameric a β peptide.

The following amino acid sequence comprising A.beta.1-42 was divided into 43 overlapping decapeptides containing 1 amino acid frameshift. The numbers refer to the essential amino acids from the sequence of a β 1-40 that must be present in order for the antibody to optimally bind the decapeptide.

ISEVKM¹DAEF RHDSGYEVHH QKLVFFAEDV GSNKGAIIGLMVGGVVI⁴²ATV IV (SEQ ID NO: 4). Thus, DAEF RHDSGYEVHHQKLVFFAEDV GSNKGAIIGL MVGGVVIA (SEQ ID NO: 3) represents amino acids 1-42 of the A β 4/β -A4 peptide.

43 decapeptides synthesized by the supplier (Jerini BioTools, Berlin) were N-terminally acetylated and C-terminally covalently linked to a cellulose sheet ("pepspot"). The cellulose discs were incubated for 2 hours on a shaking platform with monoclonal antibodies (1. mu.g/ml) in blocking buffer (50mM Tris-HCl, 140mM NaCl, 5mM NaEDTA, 0.05% NP40 (Fruka), 0.25% gelatin (Sigma), 1% bovine serum albumin fragment V (Sigma), pH 7.4). The fiber discs were washed three times with TBS (10mM Tris.HCl, 150mM NaCl, pH 7.5) on a shaking platform for three minutes each. The fiber sheet was then pressed against a filter paper, wetted with cathode buffer (25mM Tris base, 40mM 6-aminocaproic acid, 0.01% SDS, 20% methanol), transferred to a semi-dry blotting rack with the peptide side facing an equal size PVDF membrane (Biorad)).

The semi-dry blotting rack contained freshly wetted filter paper slightly larger than the peptide sheet (Whatman No. 3):

three sheets of paper wetted with cathode buffer

Peptide tablet

Methanol-wetted PVDF membrane

Anode buffer 1(30mM Tris base, 20% methanol) moistened three sheets of paper

Anode buffer 2(0.3mM Tris base, 20% methanol) moistened three sheets of paper

The current density at the cathode and the anode is 0.8mA/cm²Down-transfer for 1 hour, a time sufficient to completely elute the antibody from the cellulose sheet and transfer to a PVDF membrane. The PVDF membrane was immersed in the blocking buffer for 10 minutes. Fluorescein IRdyne 800-labeled goat anti-human IgG (H + L) (Lokran, cat. 609-132-123) was added to the Ordoxey (Odyssey) blocking buffer (Li-Cor) at a dilution of 1:10000 and further diluted with PBS and 0.05% Tween 201: 1. The membrane was incubated on a shaking platform for 1 hour. Wash with TBST (TBS containing 0.005% tween 20) for 3x10 minutes. The membrane was dried and scanned for 800nm fluorescence using a long wavelength fluorescence scanner (Odyssey), as shown in figure 8.

The PVDF membrane was labeled with a needle punch so as to accurately label the antibody reaction spots. The epitope of the antibody under investigation was defined as the smallest amino acid sequence in the reactive peptide. The fluorescence intensity of each spot was integrated and recorded as Relative Fluorescence Units (RFU). For comparison, two mouse monoclonal antibodies were analyzed in the same manner, with BAP-1 corresponding to antibody 6E10(Kim (1998)) specific for the N-terminal domain and BAP-44 corresponding to antibody 4G8(Kim (1998)) specific for the middle domain, except that anti-mouse Ig was used instead of anti-human Ig in the assay.

Notably, affinity maturation of monovalent Fab fragments and conversion to full-length IgG1 antibodies generally resulted in broadening of epitope recognition sequences as shown by pepspot and ELISA assays. This may involve either the recruitment of more contact points in the antigen binding region of the antibody due to affinity maturation, or the stronger binding to the minimal epitope so that weak effects on adjacent amino acids are detectable. The latter may be the case when probing A.beta.derived peptides with full-length IgG antibodies. As shown in the table below, the recognition sequences for the N-terminal and middle epitopes were extended by three amino acids when comparing the parent Fab with the corresponding fully mature IgG. However, it is noteworthy that the decapeptide is modified for covalent attachment at the C-terminus of an amino acid that is not readily accessible to full-length antibodies due to steric hindrance. If in this case the last C-terminal amino acid does not contribute significantly to the epitope recognition sequence, and the C-terminal decrease of the smallest recognition sequence by one amino acid must be taken into account in the pepspot analysis used in the present invention.

The table above relates to pepspot analysis of full-length IgG antibody binding to decapeptide on cellulose discs. The numbers refer to the positions of the amino acids in the sequence of A.beta.1-40, which must be present in order to bind the decapeptide to an antibody. Further extensions of the epitope are indicated in parentheses to indicate the flanking amino acids required to achieve maximal binding.

Example 10: disaggregation experiments using antibody a isotypes (i.e., the unglycosylated, mono-glycosylated, or di-glycosylated antibodies of the invention) to induce release of biotinylated a β from aggregated a β.

The experimental settings for detecting the potential of antibody a isotype to induce disaggregation of aggregated a β are as follows:

biotinylated A.beta.1-40 was first incorporated into preformed A.beta.1-40/A.beta.1-42 fibers prior to antibody A isotype action. The release of biotinylated a β was detected using streptavidin-POD conjugate experiments as described below.

After incubation in aqueous buffer for several days, the synthetic a β spontaneously aggregates and forms a fibrous structure similar to the starch deposits seen in the brains of alzheimer's patients. The following in vitro experiments are suitable for detecting incorporation or release of biotinylated A.beta.into preformed A.beta.aggregates, and for analyzing A.beta.neutralization of anti-A.beta.antibodies and other A.beta.binding proteins such as albumin (Bohrmann (1999) J.biol.chem.274, 15990-15995). Antibody A isotype-induced aggregated A.beta.disaggregation was measured by release of incorporated biotinylated A.beta.1-40.

The experimental steps are as follows:

NUNC Maxisorb microtiter plates (MTPs) were coated with a 1:1 mixture of A.beta.1-40 and A.beta.1-42 (each 2. mu.M, 100. mu.l per well) at 37 ℃ for 3 days. Under this condition, the pore surfaces adsorb and solidify highly aggregated and fibrillated a β. The coating was removed and the plate was dried at room temperature for 2-4 hours. The dried plates can be stored at-20 ℃. For incorporation of biotinylated A.beta.the solution was dissolved in a solution containing 0.05% NaN at 37 deg.C₃20nM biotinylated A β 1-40 incubation of TBS coated plates overnight (200 μ l/well). The plate was washed 3 times with 300. mu.l/well T-PBS and added with 0.05% NaN₃TBS serially diluted antibodies and incubated at 37 ℃ for 3 hours. The plate was washed and analyzed for the presence of biotinylated A.beta.1-40. After washing 3 times with 300. mu.l T-PBS, streptavidin-POD conjugate (100. mu.l/well) diluted 1% BSA in T-PBS 1:1000 (Roche Molecular Biochemicals) was added and incubated at room temperature for 2 hours, the plate was washed 3 times with 300. mu. l T-PBS, 100. mu.l/well freshly prepared Tetramethylbenzidine (TMB) solution was added [ preparation of TMB solution: 10ml 30mM citric acid pH 4.1 (adjusted with KOH) +0.5ml TMB (12mg TMB in 1ml acetone +9ml methanol) +0.01ml 35% H₂O₂]. Add 100. mu.l/well 1N H₂SO₄The reaction was stopped and the absorbance at 450nm was read on a microtiter plate reader.

As shown in fig. 9, antibody a isotype-induced disaggregation of aggregated a β was measured using the release of incorporated biotinylated a β 1-40. Antibody A isotype and mouse monoclonal antibody BAP-1 were similar in activity (FIG. 9), but it was evident that the release of biotinylated A β from the solidified A β stack by the BAP-2, BAP-17 and 4G8 antibodies was less potent (data not shown). BAP-1 can be clearly distinguished from glycosylated antibody A by the property of reacting with full-length APP on the cell surface. Antibodies with such properties, such as BAP-1, would not be useful for therapeutic applications because of their potential to induce an autoimmune response. Interestingly, although BAP-2 is specific for exposure to amino acid residues 4-6 of aggregated A.beta.in this experiment BAP-2 activity was significantly lower, suggesting that not all N-terminal specific antibodies have an equivalent ability to release A.beta.from preformed aggregates. BAP-17 (C-terminal specificity) and 4G8 (amino acid residues 16-24 specificity) were relatively less effective in this experiment because of the property of these two epitopes to be cryptic in aggregating A.beta.. BSA at the concentrations used was not effective for aggregating a β.

Compared to the bis-glycosylated isoform, the mono-glycosylated isoform has a higher capacity to disaggregate aggregated a β peptide in vitro, and may be the same in vivo.

Example 11: antibody A compositions and methods comprising isotypes (mono-and di-glycosylated antibodies of the invention) for capturing soluble A β from human cerebrospinal fluid (CSF)

The ability to capture soluble a β from human CSF was determined using Immunoprecipitation (IP) and semi-quantitative Western Blot (WB) analysis.

The experimental steps are as follows:

human CSF was immunoprecipitated according to the following protocol:

70 μ l human CSF

20 μ l incubation buffer (50mM Tris, 140mM NaCl, 5mM EDTA, 0.05% NP-40, 1% BSA, 0.25% gelatin, 0.25% milk powder, pH 7.2)

10μlAntibody A from mother liquor (1000-10. mu.g/ml)

100μl

The solution was left at 4 ℃ for 1 hour. Mu.l of protein G agarose beads (Amersham Biosciences) # 17-0618-01; PBS wash, 50% slurry) were added and incubated for 2 hours at 4 ℃ on a rotator. After centrifugation at 500g for 3 minutes at 4 ℃ the supernatant was removed and 200. mu.l PBS was added to the beads, transferred to a Millipore filter tube 0.45 μm (Millipore # UFC3OHVNB) and centrifuged at 500g for 3 minutes at 4 ℃. A further 200. mu.l PBS was added to the beads, vortexed and centrifuged at 2000g for 3 minutes at 4 ℃. Mu.l of DTT-containing 1xNuPage sample buffer was added, left at 70 ℃ for 10 minutes and centrifuged at 2000g at 4 ℃ for 3 minutes.

In SDS-PAGE, 18. mu.l of protein G eluate were applied to NuPage gel 10% Bis-Tris gel, while A.beta._1-42(Bakem corporation (Bachem)) was added directly to the sample buffer as an internal standard and electrophoresed in a MES buffer system.

The gel was transferred to a Hybond C extra membrane (Norwegian Kex semi-dry system). Dry film at room temperature for 3 minutes. The film was transferred to pre-warmed PBS and heated in a 600W microwave oven for 3 minutes. Blocking was performed with SuperBlock solution (Pierce) for 1 hour, followed by blocking with T-PBS containing 5% milk powder (Burley) for 1 hour (PBS containing 0.1% Tween 20).

anti-Abeta antibody W02 antibody (1:1500-1:2000 diluted, Gentak, zurich, Switzerland) was incubated overnight on a rotator at 4 ℃ and then washed three times with T-PBS for 5 minutes each, followed by incubation with anti-mouse IgG-HRP (Dako) diluted 1:5000 in T-PBS for 2 hours at room temperature. Washed three more times with T-PBS for 5 minutes each, followed by incubation with LumiLight Plus for 5 minutes at room temperature. Western blots were digitized and analyzed for optical density using the Alpha Innotech digital camera system.

As shown in figure 10, immunoprecipitation and Western blot experiments showed that antibody a compositions (comprising mono-and di-glycosylated antibody a isoforms) bind efficiently to soluble a β in human CSF. Notably, in this experiment, the mono-glycosylated antibody a was able to capture soluble a β more efficiently than the di-glycosylated antibody a (fig. 10).

Example 12: antibody a compositions and isotypes (e.g., unglycosylated, mono-glycosylated or di-glycosylated antibodies of the invention) are immunostained in vitro on human starch plaques.

The ability of glycosylated antibody a isoform to stain authentic human beta-amyloid plaques obtained from brain sections from patients with severe alzheimer's disease was examined by immunohistochemical analysis using indirect immunofluorescence. Shows a specific and sensitive staining for authentic human beta-amyloid plaques.

The indirect immunofluorescent marker is derived from a frozen section of the non-fixed tissue of the thalamus obtained at autopsy from a patient positive for Alzheimer's disease diagnosis. Bound antibody A isotype was detected using a two-step incubation sequence as shown by affinity purified goat anti-human (GAH) IgG (H + L) coupled to Cy3 (# 109-. The control group included an irrelevant human IgG1 antibody (sigma) and a secondary antibody alone, both of which produced a negative result.

All types of beta-amyloid plaques were detected sensitively and specifically and revealed consistently when the concentration of antibody A was 10ng/ml (FIG. 11).

When the concentration of the glycosylated antibody A isoform is as high as 1 mu g/ml, the real human beta-amyloid plaques can be specifically and sensitively stained.

Background staining was observed at a concentration of 10 μ g/ml, most notably with the aglycosylated antibody a isotype. The unglycosylated isoforms have considerable non-specific viscosity on the contacted slide surface and on almost all tissue components by in vitro tissue sectioning. This appears to be non-specific binding due to ionic and/or hydrophobic interactions.

Example 13: modification of beta amyloid plaques with antibody A in a mouse model of Alzheimer's disease

To investigate the ability of glycosylated antibody a isoform to immunologically modify beta amyloid plaques in vivo, a single-dose study was performed in PS2APP double transgenic mice (Richards (2003), j. neuroscience, 23, 8989-9003). The glycosylated antibody a isoform was administered at a dose of 1 mg/mouse, and 3 days later the animals were perfused with phosphate buffered saline, and the brains were frozen on dry ice and prepared for frozen sections.

Both glycosylated isoforms show improved and highly efficient brain penetration in vivo (compared to the unglycosylated form). Effective brain penetration and specific binding to β amyloid plaques are shown in PS2APP mice, a mouse model of AD-associated amyloidosis.

The presence of beta-amyloid plaque binding antibodies was assessed using non-fixed frozen sections using either indirect immunofluorescent single labeling with goat anti-human IgG (H + L) coupled to Cy3 (# 109-.

Bound antibody a was detected by immunofluorescence staining. The sections were adhered to pre-cooled slides, hydrated in PBS and treated with-20 deg.C pre-cooled acetone for 2 minutes. Washed twice with PBS for 2 minutes each time. Blocking of non-specific binding sites was performed with PBS containing 1% BSA or by incubation in UltraV block (LabVision) for 5 minutes, PBS washed, incubated in a strong blocking solution containing 10% normal sheep serum (biogenet) for 20 minutes. After washing in PBS containing 10% normal sheep serum, the slides were incubated with affinity purified goat anti-human (GAH) IgG (H + L) (#109-165-003, batch 49353, Jackson Immunol at 15. mu.g/ml) coupled to Cy3 for 1 hour at room temperature. The starch plaques were counterstained by incubation with Alexa 488-conjugated mouse monoclonal antibody, BAP-2, at a concentration of 0.5. mu.g/ml for 1 hour at room temperature. At 4mM CuSO₄To quench the autofluorescence of lipofuscin. The coverslips were rinsed with double distilled water and washed twice with 500. mu.l PBS/slide, and the coverslips were embedded with fluorescent coverslips (Dako S3023).

Confocal laser microscopy was used for imaging and co-localized quantitative analysis of the pictures was performed using IMARIS and COLOCALIZATION software (bitplan, switzerland).

Three days after a single administration of 1 mg/mouse, glycosylated antibody a isoform was found to cross the blood brain barrier and efficiently immune-modify/bind all β -amyloid plaques in vivo. Fig. 12 shows a representative picture. A clear comparison is that no unglycosylated form was detected at the starch spots.

Example 14: study of antibody A isoform binding to Amyloid Precursor Protein (APP) expressed on the surface of HEK293 cells

Flow cytometry is well known in the art. The relative fluorescence units measured by flow cytometry (as FL1-H) indicate the binding of each antibody to the cell surface. Fluorescence changes in APP-transfected HEK293 compared to untransfected HEK293 indicate adverse reactions with the cell surface. For example, anti-N-terminal domain antibodies BAP-1 and BAP-2 significantly altered FL-1 signaling of HEK293/APP (FIG. 13, solid line, right panel) compared to untransfected HEK293 cells (FIG. 13, dashed line, right panel). Similarly, the changes caused by the BAP-44 antibody (intermediate A.beta.epitope specificity) are similar in size. In contrast, all antibody a isotypes (fig. 13 left panel) (N-terminal and intermediate a- β epitope specificity) showed no significant fluorescence change. Untransfected HEK293 cells showed higher background fluorescence than APP transfected cells due to different cell size and surface properties. The FACScan instrument was used in conjunction with the Cellquest Pro software package, both products of Becton Dickinson (Becton Dickinson).

Antibody a isotype was non-reactive to cell surface APP (figure 13).

Example 15: morphological analysis of Abeta amyloid plaque deposition in mouse Alzheimer's disease model

The ability of antibody a composition or antibody a isoform to reduce amyloidosis in vivo in different regions of the PS2APP mouse brain (thalamus, cortex, hippocampus and inferior crura) receiving 5 months treatment with the antibody a composition or antibody a isoform was investigated using quantitative computer-assisted image analysis.

Thus, male PS2APP transgenic mice were injected intravenously with the antibody a composition or antibody a isotype and vector. 75 PS2APP mice, 5-6 months old, were divided into 5 groups (A-E), 15 mice each. Starting on day 0, each mouse received 0.1mL of vehicle (0mg/kg), or antibody a formulation (20mg/kg) by tail vein bolus injection. A. B, C, D and group E PS2APP mice received a vector (histidine buffered saline), an antibody A composition containing mono-and di-glycosylated antibody A but no non-glycosylated antibody A, a di-glycosylated antibody A, a mono-glycosylated antibody A and a non-glycosylated antibody A, respectively.

Injection of anti-CD-4 antibody (hybridoma clone GK 1.5 from ATCC) induced immune tolerance to administration of human anti-A β antibody. Monitoring of drug-resistant antibodies indicated that the antibody-treated animals developed only moderate immune responses after 16 weeks of treatment and that the detectable antibodies had low affinity or were produced only in small amounts (data not shown).

Mice were sacrificed 5 months after treatment. Unfixed brains were subjected to sagittal sectioning, including thalamus, hippocampal structures, and cortical regions. Each hemisphere was prepared as 50 slices as follows: starting from flank 1.92, 5 sections of 5X10 μm and 5X20 μm were cut out in succession. The tissue used was 750 μm in total, with no gaps in the serial sections. The series of slices thus ends at approximately 1.20 of the side (Paxinos and Franklin, 2003). For morphological quantification, one per ten sections was used.

The staining of the starchy plaque deposits in sections was carried out using the bisglycosylated antibody A isoform at a concentration of 5. mu.g/ml. Staining of a β with mouse monoclonal antibody (BAP-2) conjugated to Alexa-488 fluorophore (5 μ g/ml) was comparable, although significant intracellular and background staining of neurons interfered with the following routine processing of pictures. Affinity purified Cy 3-coupled goat anti-human (GAH) IgG (H + L) (# 109-. After washing twice with 500. mu.l PBS/slide, the slide was embedded with a fluorescent coverslipper (S3023, Daco).

Images were acquired using a GenePix personal 4100A microarray scanner (axon instruments, now Molecular Devices, ca, usa). Beta starch plaque load and number were measured by computer-aided image analysis using an unbiased morphometric method using two parameters, the percentage of beta starch plaque coverage area and the number of beta starch plaques. Plaque load and number were quantified using MCID M7 erite software (Image Research Inc), san kaeselin, Ontario, Canada. And enhancing the scanning image by using a detail extraction filter and a target emphasis filter. The resulting image is binarized and the threshold is adjusted according to the staining intensity. Artifacts, vessels and edge effects are marked on the original reference image. A region of interest is delineated on the reference image. The areas of these regions, the spot occupancy area, and the number of spots were measured in the binary pictures for final quantification. A single pixel is ignored. Calculations were performed using general spreadsheet software (microsoft Excel, redmond, washington). Spot sizes were divided into 11 groups ranging from<100 to>1000μm². Statistical evaluation was performed using a two-tailed variance t-test.

For comparison and statistical evaluation, a study was initiated with a group (15 animals) of untreated 6-month old PS2APP mice to determine the amyloidosis (β amyloidosis) baseline. Results with significant levels are shown in FIGS. 15-18 (. p.ltoreq.0.05;. p.ltoreq.0.01;. p.ltoreq.0.001).

The reduction of amyloid plaques was particularly pronounced in the thalamic region (fig. 15). The average reduction of the total beta amyloid plaque surface area in the antibody treatment group was determined: antibody a composition 64%, doubly glycosylated antibody a 70%, singly glycosylated antibody a 81% and unglycosylated antibody a 44%. Average reduction in total beta-amyloid plaque number: antibody a composition 70%, doubly glycosylated antibody a 78%, singly glycosylated antibody a 82% and unglycosylated antibody a 36%. Notably, the significance of the unglycosylated antibody was low, with considerable variation observed.

FIG. 16 shows the reduction of neocortical areas and callose amyloid plaques. Mean reduction in total beta amyloid plaque surface area in the tested antibody treatment groups: antibody a composition 19%, doubly glycosylated antibody a 27%, singly glycosylated antibody a 30% and unglycosylated antibody a 10%. Average reduction in total beta-amyloid plaque number: antibody a composition 40%, doubly glycosylated antibody a 46%, singly glycosylated antibody a 42% and unglycosylated antibody a 11%.

Figure 17 shows the reduction of whole hippocampal starch plaques. Mean reduction in total beta amyloid plaque surface area in the tested antibody treatment groups: antibody a composition 12%, doubly glycosylated antibody a 24%, singly glycosylated antibody a 24% and unglycosylated antibody A6%. Average reduction in total beta-amyloid plaque number: antibody a composition 36%, doubly glycosylated antibody a 46%, singly glycosylated antibody a 37% and unglycosylated antibody A3%.

Figure 18 shows the reduction of subcerebral amyloid plaques in the area of high susceptibility to amyloid. Mean reduction in total beta amyloid plaque surface area in the tested antibody treatment groups: antibody a composition 2%, doubly glycosylated antibody a12%, singly glycosylated antibody A5% and unglycosylated antibody A1%. Average reduction in total beta-amyloid plaque number: antibody a composition 22%, doubly glycosylated antibody a 36%, singly glycosylated antibody a 13% and unglycosylated antibody A1%. Antibody a compositions and the main N-glycosylated isoforms (doubly glycosylated antibody a and mono-glycosylated antibody a) were comparable in potency to reduce beta starch plaque load and plaque number. The reduction in plaque load was most pronounced and was statistically significant in the low or moderate amyloidosis regions.

In summary, the reduction in the number of beta amyloid plaques was found to be statistically significant in all measured brain regions following treatment with the antibody a composition and the two Asn 52-glycosylation containing antibody a isoforms. In contrast, it was found that there was little effect on the number of thalamic beta amyloid plaques, and that treatment with the unglycosylated isoform of antibody a had no significant effect on the number of beta amyloid plaques in other areas of the study brain, where the unglycosylated isoform was excluded from the antibody a composition after purification as detailed herein.

We also investigated the plaque removal efficacy related to plaque size. In general, human anti-A β antibodies have been found to significantly clear small β -amyloid plaques in subjects. This phenomenon was observed in all brain regions (fig. 15C, 16C, 17C and 18C). In contrast, there was little or no significant trend observed with the unglycosylated isotype of antibody a.

Comparative analysis of antibody a and the major Asn 52-glycosylated isoform showed comparable capacity to reduce plaque load, whereas the non-glycosylated isoform had no significant effect on reducing plaque load.

Example 16: in vivo pharmacokinetics of antibody a compositions binding to beta amyloid plaques.

Two dosing frequencies were compared to investigate the binding kinetics of the antibody a composition, which contained both mono-and di-glycosylated antibody a, without unglycosylated antibody a, as defined above.

Thus, the PS2APP transgenic male mouse antibody A composition was administered by tail vein injection four times at 0.05, 0.1, and 0.3mg/kg every two weeks or three times at 0.075, 0.15, and 0.45mg/kg every one month. For comparison, 0.1mg/kg was administered once and twice every two weeks, 0.15mg/kg was administered twice every one month, and all mice were sacrificed two weeks after the last administration. Unfixed PS2APP brain tissue (including thalamus, hippocampal structures, and cortical regions) was prepared for sagittal sectioning at lateral-1.92 to 1.2mm according to Paxinos and Franklin. Brain sections of 40 μm were made in a cryo-apparatus.

Bound antibody a composition antibodies were detected using immunofluorescence ex vivo immunostaining. Thus, the brain was sectioned and incubated with a detection antibody-affinity purified Cy 3-coupled goat anti-human (GAH) IgG (H + L) (#109-165-003, batch 49353, Jackson immuno Research) (15. mu.g/ml) for 1 hour at room temperature. A mouse anti-A.beta.monoclonal antibody BAP-2 (0.5. mu.g/ml) conjugated to an Alexa488 fluorophore was used for 1 hour incubation at room temperature to counterstain the beta-starch plaques.

Images of the cortical occipital lobe near the cerebellum were recorded using a lycra (Leica) TCS SP2 AOBS confocal laser scanning microscope as described above. Computer-assisted image processing was performed using IMARIS software (Bitplane, switzerland). The spot image of the lower dose (except for the two highest doses 0.3 and 0.45 mg/kg) was first selected using the cropping function of the software, since different gain settings were required to obtain a linear signal record for the highest dose group. After setting the threshold (T) to the reading of the bound GAH-Cy3 at the β -amyloid plaque site, voxels (voxels) were selected using the SURPASS (SURPASS) function. The thresholds were set at 19 and 12 for the lower and higher dose groups, respectively. As a control specific for beta amyloid plaques, images of the double-labeled GAH-Cy3 stained plaques were compared to images of Alexa 488-conjugated mouse monoclonal BAP 2-stained plaques and recorded on different channels.

Descriptive statistics were performed on the quantitative description of all images using IMARIS MeasurementPro software module. The mean voxel fluorescence intensity (MVI) values were determined by selecting either the low dose group beta-starch plaques or the high dose group image total signal. The baseline mvi (b) is caused by machine noise, tissue scatter signals and lipofuscin autofluorescence. To correct for background, the average signal intensity away from the beta starch spot area was measured to determine B and B was subtracted from all the assay images MVI (MVI-B = S). Signal intensities (S) similar to the mean intensity of plaques were obtained from 3-4 images taken from each brain slice of each mouse in each dose group. For comparability, signal intensities were normalized to reference samples obtained from earlier studies. We used PS2APP mouse brain sections after a single administration of 0.25mg/kg as reference. One week after dosing was used as the measurement endpoint.

All measured intensity values were normalized to the mean intensity at beta starch plaques measured one week after using a single administration of 0.25mg/kg antibody a composition (see table below). After immunostaining and averaging the signal intensities of 3 animals per dose group, a normalized value of the average relative fluorescence intensity of the immunopositive beta-starch plaques was obtained using CLSM. Plaques without antibody a derived from the antibody a composition were observed only in the low dose group, likely to be lost during sectioning due to limited or partial occupation of the plaque surface by antibody a derived from the antibody a composition. Therefore, only immune positive plaques were included in the comparative analysis.

The following table shows the average relative fluorescence intensity for each dose group following multiple intravenous bolus injections of the antibody a composition in PS2APP transgenic mice:

¹the experimental data represent intensity values normalized to data obtained after 1 week with a single dose of 0.25 mg/kg.

FIG. 19 shows the relationship between antibody A composition binding and the number of consecutive biweekly administrations of 0.1 mg/kg. After two administrations, although the degree of immunostaining differed greatly and did not reach significance, the average intensity appeared to increase. After 4 injections, the immunostaining of beta-amyloid plaques was more uniform, but the average intensity only slightly increased. Overall, the data from the bi-weekly dosing clearly show that the trend of increased plaque binding correlates with the number of doses administered.

FIG. 20 shows the relationship between antibody A composition binding and the number of consecutive monthly administrations of 0.15 mg/kg. Interestingly, the levels obtained after two and three administrations were comparable. This is not an expected result and may indicate that early effects are initiated which lead to time-dependent differences in clearance mechanisms (e.g. delayed microglial activation).

Figures 21, 22 show the relationship between the binding potency of the antibody a composition and the administered dose. The dose relationship is clearly shown by bi-weekly administrations of 0.05, 0.1 and 0.3mg/kg (FIG. 21) and monthly administrations of 0.075, 0.15 and 0.45mg/kg (FIG. 22). There is evidence that the response is nonlinear, and other factors such as time delay in microglial activation may also affect the observed nonlinearity.

The following conclusions are therefore drawn: antibody a compositions bound to mouse a β plaques with dose correlation, indicating that multiple administrations had an additive effect.

Example 17: antigen-dependent endocytosis assay.

To detect the endocytic effect mediated by the antibody a composition, authentic a β plaques from AD brain sections were preincubated with different concentrations of the antibody a composition (antibody a composition comprising mono-and di-glycosylated antibody a but no non-glycosylated antibody a as defined above) and contacted with live human primary monocytes.

Non-fixed human AD brain tissue sections of the occipital cortical region were prepared from severe AD cases (Braak stage IV). Sections were rehydrated in PBS for 5 minutes before live cells were added. Antibody a composition antibody at the indicated concentration in PBS was incubated for 1 hour. After PBS wash, live cells were added. The culture density was 0.8 and 1.5X10 in RPMI 1640 (Gibbs #61870-⁶Pre-stimulated human primary monocytes in ml for 2-4 days. Methods for preparing pre-stimulated human primary monocytes are well known in the art, e.g., using stimulating factors such as macrophage colony stimulating factor (M-CSF).

After incubation, the medium was carefully removed and chemically fixed in PBS containing 2% formaldehyde for 10 minutes to preserve the sections. The residual amyloid plaque load was stained by incubating the mouse monoclonal antibody BAP-2 conjugated to Alexa488 (molecular probes): A-20181, monoclonal antibody labeling kit) at a concentration of 10mg/ml for 1 hour at room temperature.

The amount of plaque removal was determined by measuring the residual a β plaques of the immunofluorescent stain. Images were recorded using a lycra TCSSP2 AOBS confocal laser scanning microscope. An optical slice was recorded using a488 nm excitation wavelength, pinhole setting 4, HCX PL FL 20x/0.40 correction objective, but in one experiment, HCPL fluorotar 10x/0.30 objective, pinhole setting 3 was used. Instrument settings were kept constant in all images to facilitate comparison of relevant quantitative results. Specifically, the laser power, gain and offset are adjusted to bring the observed signal intensity within the dynamic monitoring range. For each antibody a composition concentration, gray matter regions were recorded at similar locations in successive sections in order to minimize fluctuations in plaque load that might be caused by anatomical differences. Potential competitive binding of antibody a composition and detection antibody BAP-2 was determined in the absence of cells at all concentrations of antibody a composition. An irrelevant human IgG1 (sarotake (Serotec), PHP010) antibody was used as another control. Image analysis was performed using IMARIS software (blamplan, switzerland). An iso-surface of BAP-2 positive pixels representing a BAP-2 target bound to a plaque is created by setting an intensity threshold. The surface area and total fluorescence intensity values were calculated using the "iso-surface function" of the surfasspro software module. Data are expressed as mean stained area and total stained intensity values obtained from 5 gray matter regions of a brain slice. Instrument noise forms the signal baseline and tissue scatter signals are found to be negligible and therefore do not need to be subtracted from the total intensity signal.

As shown in fig. 23, a reduction in a β plaque staining indicates enhanced phagocytosis of a β plaques in brain sections from human AD, thus demonstrating the qualitative effect of antibody a compositions. Immunohistochemistry revealed a significant reduction in the stained a β plaques after 40 hours of preincubation with 100ng/ml of antibody a composition. This effect was very pronounced at antibody A composition concentrations of 1 and 5 mg/ml. At 5mg/ml, endocytosis clearly cleared a β plaques in large amounts, leaving only a few large a β plaques. Figure 24 shows quantitative measurements based on immunoreactive signals expressed as area and intensity in the same experiment.

Alternatively, antibody a composition-mediated endocytosis was measured using Α β -conjugated fluorescent polystyrene beads. Thus, fluorescent beads (3mm, Fluoresbrite carbon YG, Porsense Inc. (Polysciences Inc.)) were coupled to A β. Briefly, beads were washed twice by suspension in coupling buffer (50mM MES buffer, pH 5.2, 1% DMSO) and centrifugation. The pellet (about 10. mu.l) was suspended in 200ul of coupling buffer, and 20. mu.l of 20% EDC solution (ethyl-diaminopropyl-carbodiimide, Pierce) was added to the coupling buffer for activation. The coupling reaction was initiated by the immediate addition of 20 μ g A β (1-40) or A β (1-42) (prepared with 0.1% ammonium hydroxide, Bakem corporation (Bachem)). After overnight incubation, the beads were washed three times with 0.5ml 10mM Tris.HCl pH8.0 and 0.5ml storage buffer (10mM Tris.HCl pH8.0, 0.05% BSA, 0.05% NaN3), respectively. The 1% suspension was stored at 4 ℃ until use. Fluoresbrite Carboxy NYO (Red fluorescent) beads coupled to all D-amino acids Abeta (1-40) (prepared with 0.1% ammonium hydroxide in the dark, Bakeka corporation) were used as negative controls.

Murine monocytes/macrophages (cell line P388D1) were grown to approximately 50% confluence in C24 clear tissue culture plates or C96 black microtiter plates. The medium was IMEM containing 5% FBS, glutamine and antibiotics. To block non-specific scavenger receptors, 10ml fucoidan (10 mg/ml aqueous solution, Frouka) was added to a200 ml culture volume and incubated for 2 hours. The serially diluted antibody a composition was added and pre-incubated for 30 minutes. Fluorescent Α β bead suspension (20 μ l) was added and incubated for 3 hours for endocytosis. Adherent cells were washed vigorously once and twice with ice cold EDTA and PBS, respectively, to remove adherent material from the cell surface. The remaining beads were monitored visually using a Zeiss (Zeiss) Axiovert 405 or quantified by a microplate fluorometer (Fluoroscan, laboratory systems) using a 444nm (excitation light) and 485nm (emission light) filter set.

Figure 25 shows the qualitative effect of antibody a composition on endocytosis of conjugated fluorescent beads of synthetic a β aggregates in P388D1 cells. Figure 26 shows the results of quantitative determination of the dose response of antibody a compositions. Two independent experiments reveal EC₅₀The range is 30-200ng/ml, and the range of MEC is 10-60 ng/ml. Incubation stoichiometry differences, i.e., bead/cell ratios, may account for the observed differences. Monovalent antibodies interact with a limited antigen resulting in a concentration>A decrease in bead endocytosis was observed at 200 ng/ml.

It was therefore concluded that the antibody a composition was effective in a dose-related manner in inducing endocytosis of a β plaques in AD brain tissue sections.

Claims (24)

1. A method for preparing an antibody molecule contained in a composition, said antibody molecule being capable of specifically recognizing β -a4 peptide/a β 4, said method comprising the steps of:

(a) recombinantly expressing a heterologous nucleic acid molecule encoding an antibody molecule in cultured mammalian cells;

(b) purifying the recombinantly expressed antibody molecule using a method comprising the steps of:

(b1) purifying a protein A column;

(b2) purifying by an ion exchange column; and

(b3) purifying by a size exclusion column; and

the antibody molecules contained in the composition are mono-or di-glycosylated antibody molecules, or the antibody molecules contained in the composition are a mixture of the mono-and di-glycosylated antibody molecules,

wherein the antibody molecule comprises in the heavy chain variable region the amino acid sequence of SEQ ID NO: 10, CDR1 shown in SEQ ID NO: 12, and the CDR2 shown in SEQ ID NO: 14, comprising in the light chain variable region the CDR3 of SEQ ID NO: 16, CDR1 shown in SEQ ID NO: 18 and the CDR2 shown in SEQ ID NO: 20 CDR3 shown in fig. 20; and

wherein the mono-glycosylated antibody molecule is variable in the heavy chain (V)_H) Containing a glycosylated asparagine (Asn) in the heavy chain variable region (V) at two antigen binding sites_H) Containing a glycosylated asparagine (Asn) in which the variable region of the heavy chain (V)_H) The glycosylated asparagine (Asn) in SEQ ID NO: 12 in CDR 2;

less than 4% of the antibody molecules in the composition have a heavy chain variable region (V) of the antigen binding site_H) Without glycosylated asparagine (Asn).

2. The method of claim 1, wherein the ion exchange column purification comprises cation exchange chromatography.

3. The method of claim 1, further comprising an additional step (c): analytical chromatography and/or a further concentration step.

4. The method of claim 1, wherein the β -a4 peptide/a β 4 is selected from the group consisting of:

(a) SEQ ID NO: 3; and

(b) SEQ ID NO: 3, said fragment comprising a fragment of at least 15 amino acids of SEQ ID NO: 3, amino acid residues 3 to 6 and amino acid residues 18 to 26.

5. The method of claim 1, wherein the variable region of the heavy chain (V) of at least one antigen binding site of the antibody molecule_H) Comprising a glycosylated asparagine (Asn), said V_HEncoded by the following sequence:

(a) consisting of SEQ ID NO: 1 of the sequence set forth in seq id no:

CAGGTGGAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCTATTAATGCTTCTGGTACTCGTACTTATTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA；

(b) encoding the amino acid sequence of SEQ ID NO: 2:

QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSS (SEQ ID NO: 2); or

(c) A nucleic acid sequence which is degenerate to a nucleic acid sequence of any one of (a) to (b).

6. The method of claim 1, wherein the variable region comprising a glycosylated asparagine (Asn) is comprised in a heavy chain selected from the group consisting of:

(a) SEQ ID NO: 5. 23 or 25, or a light chain polypeptide encoded by a nucleic acid molecule as set forth in seq id no; or

(b) Consisting of SEQ ID NO: 6 or 26.

7. The method of claim 1, wherein said locus at V_HAsn glycosylation of a region is selected from:

(a) a biantennary complex-type sugar structure;

(b) double-antenna heterozygote sugar structure;

(c) a sugar structure of the biantennary oligomannose type; and

(d) the bi-antennary sugar structures of any of the structures provided in figures 5 or 27.

8. The method of claim 7, wherein said sugar structures do not comprise core fucosylation.

9. The method of claim 1, wherein the antibody molecule is recombinantly produced.

10. The method of claim 9, wherein the antibody molecule is produced in CHO cells.

11. The method of claim 10, wherein the CHO cell is CHO K1 or CHO K1 SV.

12. The method of claim 1, wherein the composition is a diagnostic or pharmaceutical composition.

13. A composition comprising antibody molecules produced by the method of any one of claims 1 to 12.

14. Use of a composition according to claim 13 for the preparation of a medicament for the prevention, amelioration and/or treatment of a disease associated with amyloidogenesis and/or amyloid plaque formation.

15. Use of a composition according to claim 13 for the preparation of a diagnostic kit for the detection of a disease associated with amyloidogenesis and/or amyloid plaque formation.

16. Use of a composition according to claim 13 for the manufacture of a medicament for decomposing a β -amyloid plaque.

17. Use of a composition according to claim 13 for the preparation of a pharmaceutical composition for passive immunization against beta amyloid plaque formation.

18. Use of a composition according to claim 13 for the preparation of a pharmaceutical composition for the prophylactic treatment of a disease associated with amyloidogenesis and/or amyloid plaque formation.

19. The use of claim 18, wherein pre-existing beta starch plaques or beta starch plaque aggregation intermediates are to be reduced.

20. Use of a composition according to claim 13 for the manufacture of a diagnostic kit for diagnosing a disease associated with amyloidogenesis and/or amyloid plaque formation in a patient or for diagnosing a susceptibility of a patient to suffering from a disease associated with amyloidogenesis and/or amyloid plaque formation.

21. The use according to any one of claims 14, 15, 18-20, wherein the disease is dementia, motor neuropathy, down's syndrome, creutzfeldt-jakob disease, hereditary cerebral hemorrhage with amyloidosis of the dutch-type, parkinson's disease, or an aging-related neuronal disease.

22. The use according to claim 21, wherein the dementia is dementia associated with Lewy body formation or dementia associated with HIV.

23. The use of claim 21, wherein the disease is alzheimer's disease or amyotrophic lateral sclerosis.

24. A kit comprising the composition of claim 13 or an antibody produced by the method of any one of claims 1-12.