PATENT DOCUMENT

Abstract:
The present invention pertains to the prevention, treatment and diagnosis of neurodegenerative diseases, in particular Alzheimer&#39;s disease, and other similar disease. More specifically to high affinity antibodies selective for amyloid beta protein (Aβ) in its protofibril conformation and of IgG class and IgG1 or IgG4 subclass or combinations thereof or mutations thereof, retaining high Fc receptor binding and low C1(C1q) binding, effective in clearance of Aβ protofibrils and with reduce risk of inflammation.

Full Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is the U.S. National Stage of International Application No. PCT/SE2007/000292, filed Mar. 23, 2007, which claims the benefit of SE 0600662-1, filed Mar. 23, 2006, and SE 0602591-0, filed Nov. 30, 2006. 
     BACKGROUND 
     Field of Invention 
     This invention pertains to the prevention, treatment and diagnosis of neurodegenerative diseases, in particular Alzheimer&#39;s disease, and other similar disease. More precisely, to high affinity 10 −7  M, preferably 10 −8  M, even less than 10 −9 M or less than 10 −10  M or 10 −11  M antibodies, selective for amyloid beta protein (Aβ) in its protofibril conformation and of IgG class and IgG1 or IgG4 subclass or combinations thereof or mutations thereof, retaining high Fc receptor binding and low C1(C1q) binding, effective in clearance of Aβ protofibrils and with reduce risk of inflammation. 
     Alzheimer&#39;s disease (AD) is a progressive and irreversible neurodegenerative disorder causing cognitive, memory and behavioural impairments. It is the most common cause of dementia in the elderly population affecting roughly 5% of the population above 65 years and 20% above 80 years of age. AD is characterized by an insidious onset and progressive deterioration in multiple cognitive functions. The neuropathology involves both extracellular and intracellular argyrophillic proteineous deposits. The extracellular deposits, referred to as neuritic plaques, mainly consist of amyloid beta protein (Aβ) surrounded by dystrophic neurites (swollen, distorted neuronal processes). Aβ within these extracellular deposits are fibrillar in its character with a β-pleated sheet structure. Aβ in these deposits can be stained with certain dyes, e.g. Congo Red, and display a fibrillar ultra structure. These characteristics, adopted by Aβ in its fibrillar structure in neuritic plaques, are the definition of the generic term amyloid. The classic intracellular AD pathologic lesion is the neurofibrillary tangle (NFT) which consists of filamentous structures called paired helical filaments (PHFs), composed of twisted strands of hyperphosphorylated microtubule-associated protein tau. Frequent neuritic plaques and neurofibrillary tangle deposits in the brain are diagnostic criteria for AD, as carried out post mortem. AD brains also display macroscopic brain atrophy, nerve cell loss, local inflammation (microgliosis and astrocytosis) and often cerebral amyloid angiopathy (CAA) in cerebral vessel walls. 
     Two forms of Aβ peptides, Aβ40 and Aβ42, are the dominant species in AD neuritic plaques while Aβ40 is the prominent species in cerebrovascular amyloid associated with AD. Enzymatic activities allow Aβ to be continuously formed from a larger protein called the amyloid precursor protein (APP) in both healthy and AD afflicted subjects in all cells of the body. Two major APP processing events through β- and γ-secretase activities enables Aβ production, while a third enzyme called α-secretase, prevents Aβ generation by cleavage inside the Aβ sequence (Selkoe, 1994; Ester 2001; U.S. Pat. No. 5,604,102). The Aβ42 is a fortytwo amino acid long peptide, i.e. two amino acids longer at the C-terminus, as compared to Aβ40. Aβ42 is more hydrophobic, and does more easily aggregate into larger structures of Aβ peptides (Jarret 1993) such as Aβ dimers, Aβ trimers, Aβ tetramers, Aβ oligomers, Aβ protofibrils or Aβ fibrils. Aβ fibrils are hydrophobic and insoluble, while the other structures are all less hydrophobic and soluble. All these higher molecular structures of Aβ peptides are individually defined based on their biophysical and structural appearance e.g. in electron microscopy, and their biochemical characteristics e.g. by analysis with size-exclusion chromatography/western blot. These Aβ peptides, particularly Aβ42, will gradually assemble into a various higher molecular structures of Aβ during the life span. AD, which is a strongly age-dependent disorder, will occur earlier in life if this assembly process occurs more rapidly. This is the core of the “amyloid cascade hypothesis” of AD which claims that APP processing, the Aβ42 levels and their assembly into higher molecular structures is a central cause of AD. All other neuropathology of AD brain and the symptoms of AD such as dementia are somehow caused by Aβ or assembled forms thereof. 
     Aβ can exist in different lengths i.e. 1-39, 1-40, 1-42 and 1-43 and fragments sizes i.e. 1-28 and 25-35. Truncations might occur at the N-terminus of the peptide. All these peptides can aggregate and form soluble intermediates and insoluble fibrils, each molecular form having a unique structural conformation and biophysical property. Monomeric Aβ1-42 for example, is a 42 amino acid long soluble and non toxic peptide, that is suggested to be involved in normal synapse functions. Under certain conditions, the Aβ1-42 can aggregate into dimers, trimers, tetramers, pentamers up to 12-mer and higher oligomeric forms, all with its distinct physicochemical property such as molecular size, EM structure and AFM (atomic force microscopy) molecular shape. An example of a higher molecular weight soluble oligomeric Aβ form is the protofibril (Walsh 1997), which has an apparent molecular weight &gt;100 kDa and a curvelinear structure of 4-11 nm in diameter and &lt;200 nm in length. It has recently been demonstrated that soluble oligomeric Aβ peptides such as Aβ protofibrils impair long-term potentiation (LTP) a measure of synaptic plasticity that is thought to reflect memory formation in the hippocampus (Walsh 2002). Furthermore, oligomeric Arctic Aβ peptides display much more profound inhibitory effect than wtAβ on LTP in the brain, likely due to their strong propensity to form Aβ protofibrils (Klyubin 2003). 
     There are also other soluble oligomeric forms described in the literature that are distinctly different from protofibrils. One such oligomeric form is ADDL (Amyloid Derived Diffusible Ligand) (Lambert 1998). AFM analysis of ADDL revealed predominantly small globular species of 4.7-6.2 nm along the z-axis with molecular weights of 17-42 kDa (Stine 1996). Another form is called ASPD (Amyloidspheroids) (Hoshi 2003). ASPD are spherical oligomers of Aβ1-40. Toxicity studies showed that spherical ASPD&gt;10 nm were more toxic than lower molecular forms (Hoshi 2003). This idea has gained support from recent discovery of the Arctic (E693) APP mutation, which causes early-onset AD (US 2002/0162129 A1; Nilsberth et al., 2001). The mutation is located inside the Aβ peptide sequence. Mutation carriers will thereby generate variants of Aβ peptides e.g. Arctic Aβ40 and Arctic Aβ42. Both Arctic Aβ40 and Arctic Aβ42 will much more easily assemble into higher molecular structures i.e. protofibrils. Thus, the pathogenic mechanism of the Arctic mutation suggests that the soluble higher molecular protofibrils are causing AD and contains a specific unique epitope i.e. “the AD disease epitope”. 
     In the Alzheimer&#39;s disease (AD) brain, extracellular amyloid plaques are typically found in parenchyma and vessel walls. The plaques are composed of amyloid (Aβ38-43 amino acid long hydrophobic and self-aggregating peptides, which gradually polymerize prior to plaque deposition. The soluble Aβ oligomeric species have been proposed to be better disease correlates than the amyloid plaques themselves (McLean et al., 1999; Näslund et al., 2000). Among these pre-fibrillar intermediate Aβ species, oligomeric forms have been shown to elicit adverse biological effects both in vitro and in vivo (Walsh et al., 2002) and may thus play a central role in disease pathogenesis. Several oligomeric Aβ species of various molecular sizes are known. Importantly, the conformation of monomeric, oligomeric and fibrillar forms of Aβ are different and can be targeted by conformational selective antibodies. The identity of the main Aβ pathogen is unclear, although some evidence suggests high-molecular weight Aβ oligomers to be especially neurotoxic (Hoshi et al., 2003). 
     Pathogenic mutations in the amyloid precursor protein (APP) gene, causing early onset AD have been described. One of them, the Swedish APP mutation (Mullan et al., 1992), causes increased levels of Aβ. The other the Arctic APP mutation (E693G) located within the Aβ domain, was found to enhance the formation of protofibrils, large Aβ oligomers, suggesting these Aβ intermediates to be particularly pathogenic ((US 2002/0162129 A1; Nilsberth et al, 2001). The identification of the Arctic APP mutation and the elucidation of toxic effects for Aβ protofibrils have increased the focus on Aβ oligomers in AD pathogenesis. 
     Active immunization as a therapeutic strategy for Alzheimer&#39;s disease was first reported by (Schenk et al. 1999). The target for the immunization strategy was the fibrillar form of Aβ found in Alzheimer plaques. A recent clinical phase I/II trial of active Aβ vaccination using fibrillized Aβ as a vaccine (AN-1792) had to be halted because of the development of meningoencephalitis in a small number of patients (Bayer et al., 2005). The side effects seen in this study were likely caused by anti-Aβ antibodies reacting against fibrillar amyloid in vessel walls. The fibrillary amyloid in CAA is in close proximity to the blood-brain-barrier (BBB) and the antigen-antibody reaction could thus generate damage to the BBB leading to infiltration of T-lymphocytes into the CNS (Pfeifer et al., 2002; Racke et al., 2005). Moreover, only a minority of the participating patients displayed an immune response to the Aβ vaccine. Although the study ended prematurely, it seems to imply that active Aβ immunization may be beneficial only to a subset of AD patients. 
     Monoclonal antibodies selective for human Aβ protofibrils have been described (US 2002/0162129 A1). The method to generate highly pure and stable human Aβ protofibrils, involves the use synthetic Aβ42 peptides with the Arctic mutation (Glu22Gly). The mutation facilities immunization and hybridoma screening for Aβ protofibril selective antibodies. Importantly, these antibodies bind both wild-type Aβ protofibrils and Aβ-Arc protofibrils (PCT/SE 2005/000993). 
     Antibodies that are selective towards other conformations of Aβ such as Aβ fibrils (O&#39;Nuallain 2002), micellar Aβ (Kayed 2003), ADDL (Lambert 2001), have been described. However, non of these are Aβ protofibril selective. 
     SUMMARY OF THE INVENTION 
     The present invention pertains to improved antibodies i.e. high affinity (less than 10 −7  M) Aβ protofibril selective antibodies of class IgG and subclass IgG1 or IgG4 or combination thereof or mutations thereof, with reduced risk of inflammation, for improved prevention, treatment and diagnosis of Alzheimer&#39;s disease, Downs syndrome or other neurodegenerative disorders. Said antibodies have been developed by classical hybridoma techniques and antibody engineering. 
     The invention discloses the consensus amino acid sequence of the CDR1-3 regions on the VL and VH chains from antibodies that selectively bind oligomeric Aβ forms, i.e. Aβ protofibrils constituting the “Alzheimer disease epitope”, combined with modifications of the Fc region to reduce complement factor C1q binding, reducing the risk for complement activation and inflammation. 
     The constant region of an antibody has many important functions notably binding Fc-receptors and complement factor C1q. The latter function has been inactivated to avoid inflammatory reactions. 
     In summary, this type of high affinity protofibril selective antibodies have the following distinct advantages as compared to other known immunotherapeutic treatment modalities:
     1) targets disease causing Aβ protofibrils with high affinity   2) reduces the risk for inflammatory side-effects i.e. meningioencephalitis, by low or no binding to complement factor C1q   3) high affinity antibody reduces the clinical dose needed for an effective treatment   4) provides a modality of accurate dosing   5) less binding to Aβ fibrils in the blood vessel wall i.e. CAA, reducing the risk for inflammatory side-effects.   6) Less antibody is bound in the periphery, thus more will cross the blood brain barrier and be available for binding and elimination of Aβ oligomeric forms in the brain.   

     One aspect of the invention is the discovery of the antibody consensus amino acid sequence of the CDR regions that bind human wild type Aβ protofibrils (Example 1). This discovery defines the binding sites (CDR regions) that confer high affinity and high selectivity for wild-type human Aβ protofibrils for use as therapeutics or diagnostics. The basic structure of an immunoglobulin (IgG) molecule comprises two identical light chains and two identical heavy chains linked together by disulphide bridges ( FIG. 1 ). The light chain, which is either lambda or kappa, has a variable region (VL) and a constant region (CL) of approximately 110 amino acid residues each. The heavy chain has a variable region (VH) of about 110 amino acid residues, but a much larger constant region (CH) of 300-400 amino acid residues, comprising CHγ1, CHγ2 and CHγ3 regions or domains. 
     The constant region (Fc) activates the complement system and binds to a Fc receptor on macrophages, microglia and neutrophiles, which ingest and destroys infecting microorganisms or foreign/non-self antigens. This function is particular important since it is part of the therapeutic principle of the antibody, i.e. Fc receptor mediated microglial phagocytosis and clearance of Aβ protofibrils. Other antibody mediated clearance mechanisms are also operating, i.e. anti-aggregation properties of Aβ antibodies and clearance of Aβ protofibrils in the periphery, according to the sink hypothesis. The variable region of the heavy and light chains contains 3 hyper variable regions called complementary determining regions or CDRs. The CDR regions are short stretches of about 13-23 amino acid long, located in the VL and VH regions. The six CDRs regions on one “arm” of the antibody forms the “pocket” that binds the antigen.  FIG. 1  shows the basic structure of an IgG immunoglobulin and its subdomains. 
     Another aspect of the invention pertains to protofibril selective antibodies of high affinity. Affinities in the range of 10 −7  M preferably 10 −8  M, even less than 10 −9  M, less than 10 −10  M, or less than 10 −11  M for protofibrils are described (Example 2). These antibodies have the advantage that they can be administered at lower doses compared to antibodies with affinities in the 10 −6  M range. This has significant clinical advantage in that these high affinity antibodies, which are administered by injection, can be given subcutaneously since only a low amount of the antibody is needed to achieve efficacy. Administration modalities are not limited to subcutaneous injections. Furthermore, the lower doses needed for efficacy will reduce cost of goods for production of the antibody. 
     Another aspect of the invention is that the antibodies are of IgG class, suitable for therapeutic use since it can pass over the blood brain barrier. Clearance of Aβ protofibrils in the brain parenchyma is achieved by Fc receptor mediated phagocytosis by microglia cells. Other anti-Aβ clearance mechanisms are likely to operate as well. This clearance of soluble Aβ protofibrils is a central mechanism of the treatment. Aβ protofibrils are considered highly neurotoxic, initiating and driving the disease process. Clearance of Aβ protofibrils in the brain is of significant clinical value. In addition to clearance of Aβ protofibrils, other Aβ oligomeric forms including Aβ fibrils, will be reduced indirectly via removal of Aβ protofibrils since different Aβ aggregated forms, i.e. dimers, trimers, tetramers and higher oligomeric forms including protofibrils and fibrils, are in equilibrium. Example of reduction of plaques, which contain Aβ fibrils, is shown in a Alzheimer transgenic mouse model (APPswe) after 72 hour treatment with a high affinity protofibril selective antibody (mAb 158) (Example 3). Hence, clearance of Aβ protofibrils by said antibody will also have the advantage to indirectly reduce other Aβ aggregated or oligomeric forms. 
     Yet another aspect of the invention is a high affinity human Aβ protofibril selective antibody of subclass IgG1, which has a high affinity for human FcγRI receptors present on microglial cells in the brain. A high affinity antibody will lead to efficient clearance of Aβ protofibrils which will be of significant therapeutic value. Hence, the antibodies will exhibit clearance of Aβ protofibrils, both in CNS and periphery as compared to other immunotherapeutic strategies such as active vaccination or monoclonal antibody treatments with other monoclonal antibodies of IgG1 subclass targeting other Aβ forms. Importantly, the treatment will be efficient early in the disease process when toxic soluble Aβ spices such as Aβ protofibrils are present at elevated levels but also later in the disease process. Elevated levels of oligomeric Aβ forms have been described in a transgenic mouse model exhibiting the Swedish and Arctic mutations APP swearc (Lord A. et al. 2006). 
     Yet another aspect of the invention is that the high affinity Aβ protofibril selective antibodies can reduce or inhibit Aβ aggregation thereby reducing levels of soluble oligomeric AP forms in the brain. 
     Yet, another aspect of the invention is that the high affinity Aβ protofibril selective antibodies can bind oligomeric forms of Aβ, i.e. Aβ protofibrils outside CNS as well, thereby shifting the equilibrium of said Aβ forms over the blood brain barrier in such a way as to lower CNS levels of said Aβ forms (drainage). 
     As discussed above, the Elan clinical study using an Aβ vaccine (AN-1792) selective for Aβ fibrils to treat Alzheimer patients resulted in a side-effect, i.e. meningioencephalitis, in 6% of the cases. The strategy to target Aβ fibrils, that are the core of amyloid plaques present in the brain parenchyma but importantly also in the blood vessel walls, resulted in severe side-effects. The side-effects was most likely caused by the binding of the antibodies to CAA (Cerebral Amyloid Angiopathy) in the blood vessel walls of the brain, starting an inflammatory process. This significant clinical problem is avoided by the improved high affinity protofibril selective antibodies with reduced complement activation activity. These antibodies will retain high clearance efficacy of Aβ protofibrils reduced risk of side-effects, i.e. meningioencephalitis. 
     Another aspect of the invention is that the high affinity protofibril selective antibodies have low Aβ fibril binding (See example 2), reducing the risk for side effects, by less binding to Aβ fibrils present in CAA. 
     Yet another aspect of the invention is that the high affinity Aβ protofibril selective IgG antibodies are engineered to reduce complement factor C1q binding to the CH2 domain of IgG1 and reduce complement activation and risk of inflammation. This modification can be done in several different ways. One way is to make a chimeric antibody where the CHγ2 domain of the IgG1 constant region has been deleted and exchanged for the corresponding domain from IgG4 or part of the domain that confers C1q binding. It is well established that IgG4 does not bind C1q and hence does not activate the complement cascade. To achieve this the constant region of the heavy chain (CH) is engineered is such a way as to combine the high affinity Fc-receptor domain (CHγ3) on IgG1 with the IgG4 domain (CHγ2) which has no binding for the complement factor C1q. This new antibody containing the chimeric constant heavy chain (IgG1 :CHγ1, CHγ2:IgG4, CHγ3:IgG1) will have the important properties of both efficient clearance of Aβ protofibrils through Fc-receptor mediated phagocytosis and reduced risk for side-effects, i.e inflammation such as meningioencephalitis. 
     Yet another way of reducing the risk of inflammation is to alter the oligosaccharides structure of the antibody which will reduce complement factor C1q binding and complement activation. 30 different structures of the complex biantennary oligosaccharides at Asn-297 in human IgG1 has been described. The absence of CH2 associated carbohydrates is believed to cause a conformational change in the “hinge” region of the antibody, reducing interaction efficacies with effector molecules and loss of complement activation function and C1q binding. 
     The modification of a high affinity human Aβ protofibril selective antibody by site-directed mutagenesis of Asn-297 to any other amino acid will generate an antibody of retained Fc-receptor binding with less C1q binding and hence reduced risk of inflammation in particular at the blood brain barrier. An alternative to modify the glycosylation on the antibody is to expressing the antibody in a cell type where the enzyme N-acteylglucosaminyl-transferase I has been inactivated. This will yield an antibody with altered carbohydrate structure at Asn-297. A structure of Man 5 GlcNAc 2 , but not limited to this structure, is formed. This carbohydrate modification will reduce complement factor C1q binding and inhibit inflammation (Wright at al. 1998). Alternatively, glycosylated protofibril selective antibodies can be achieved by culturing cells expressing antibodies in the presence of tunicamycin, which inhibits glycosylation. These antibodies will have altered complement activating activity as well as altered Fc-receptor function (Leatherbarrow el al. 1985). Screening of clones expressing antibodies with low complement activation and high Fc-receptor binding will generate protofibril selective antibodies that exhibit high Fc-mediated clearance of Aβ protofibrils and low C1q binding. 
     Yet another aspect of the invention is a high affinity human Aβ protofibril selective antibody, of IgG1 subclass, where the complement factor C1q binding site has been modified, i.e. Pro331&gt;Ser331 (Xu et al. 1994), in such a way as to reduce or inhibit binding of complement factor C1q, for the treatment or prevention of AD. The proline residue at position 331 in human IgG1 can also be changed to a threonine or glycine or any other polar amino acid. This modification can be achieved by standard molecular biology techniques such as site-directed mutagenesis or DNA deletions. 
     Yet another aspect of the invention is the use of high affinity human Aβ protofibril selective IgG antibodies to specifically determine protofibril levels in human tissues, in particular in cerebrospinal fluid, blood, urine or saliva as a diagnostic tool or biomarker for Alzheimer&#39;s disease. Levels of human Aβ protofibrils in CSF or blood are likely to be different as compared to a matched elderly control group not having Alzheimer&#39;s disease. A person who is developing Alzheimer&#39;s disease is likely to have increased levels of Aβ protofibril levels in CSF or blood. Hence, by determination of Aβ protofibril levels in CSF or blood an early diagnosis of the disease can be made. This is possible to achieve with the new high affinity Aβ protofibril selective antibodies in combination with a sandwich ELISA method (Example 2A), where Aβ protofibrils have been determined down to 10 pM level. Interference of other Aβ forms such as Aβ fibrils, Aβ monomers and Aβ fragments (1-16; 17-40) in the assay, is 10% or less. 
     The invention further pertains to the use of a high affinity protofibril specific antibodies for determinations of Aβ protofibrils in human and animal tissues, for example, cerebrospinal fluid, blood, serum, urine and brain tissue but not limited to these tissues, providing for a possible diagnostic method for Alzheimer&#39;s disease. Suitable methods for assaying Aβ protofibrils in these tissues as well as in cell cultures using an anti-Aβ protofibril antibody are immunoassays such as ELISA, RIA, Western blotting or dot blotting. The method would be suitable to follow treatment efficacy (protofibril reduction) in clinical trials and suitable as a diagnostic test for Alzheimer&#39;s disease or Down&#39;s syndrome. 
     Since Aβ protofibrils levels are very low in CSF and blood, a high affinity Aβ protofibril selective antibody is needed in a diagnostic test based on an ELISA method, to be able to measure low levels of Aβ protofibrils. Other supersensitive methods such as proximity ligation (Example 4) (Gullberg 2004) or similar amplification systems or Biacore or similar techniques, can be used to increase sensitivity. The proximity ligation technique is based on the discovery that different antibodies, raised against different epitopes on an analyte (in this case a protein), may bind near each other on said analyte. If said different antibodies are conjugated to oligonucleotides, the distance between said oligonucleotides will be short enough for a connector oligonucleotide, with the aid of ligation components, to form a bridge between the oligonucleotides. Amplification components are also added, upon which RT-PCR may be performed. By this principle, an amplifiable DNA sequence, reflecting the identity and amount of the target protein, is generated. This technique makes it possible to obtain an enhanced signal response and thus to detect lower concentrations of analyte. 
     The present inventors surprisingly discovered that a modified proximity ligation technique may also be used with their Aβ protofibril-specific antibodies, to detect low concentrations of larger Aβ peptide structures, i.e. Aβ protofibrils but not Aβ monomers. They discovered that the Aβ peptides, in the protofibril conformation, exhibits a structure (repetitive units) that makes it possible for two antibodies, according to the present invention, to bind sufficienttly near each other on the protofibril. If said antibodies are conjugated to oligonucleotides, said oligonucleotides may be bridged using a connector oligonucleotide. PCR is performed using amplification components. By this principle, an amplifiable DNA sequence, reflecting the identity and amount of the target protofibril, is generated (see  FIG. 4 ). 
     Proximity ligation or a version of the technique called “rolling circle”, is a highly sensitive technique and particularly well suited for detection of polymeric structures with repeated sequences, such as Aβ protofibrils to be used for diagnosis of Alzheimer&#39;s disease and other neurodegenerative disorders. 
     The invention further pertains to the use of high affinity protofibril specific antibodies in imaging for detection, localization and quantitation of Aβ protofibrils in human and animal tissues. The antibody could be label with a radioactive ligand such as I 131 , C 14 , H 3  or Gallium 68 , but not limited to these radioisotopes, for detection purposes. The method will be suitable as a diagnostic tool for Alzheimer&#39;s disease or Down&#39;s syndrome. 
     Yet another aspect of the invention is to make the antibody spices specific for use in veterinary medicine. The diagnostic methods outlined are also suitable for veterinary use. 
     Another aspect of the invention is the humanization of said antibodies to avoid side-effect, i.e. to avoid an immunoresponse against said antibodies in humans when used as a therapeutic or diagnostic agent. 
     Yet another aspect is a formulation of the antibody in a physiological buffer, for example PBS but not limited to PBS, suitable for administration to humans and animals. The antibody product can be freeze dried for better stability. The freeze dried formulation can contain an excipient such as manitol but not limited to manitol to stabilize the product after freeze drying. 
     The antibody product can contain an antibacterial agent. 
     The antibodies or fragments according to the inventions may exhibit amino acid deletions, substitutions and insertions within said CDR regions and/or its framework. Inserted or substituted amino acids may also be amino acid derivatives, with the proviso that the affinity and specificity of the antibody is still intact. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the basic structure of an IgG immunoglobulin and its subdomains. 
         FIG. 2A  shows the characterization of a high affinity protofibril selective monoclonal antibody as measured by sandwich ELISA. 
         FIG. 2B  shows the characterization of a high affinity protofibril selective monoclonal antibody as measured by competitive ELISA. 
         FIG. 3  shows the therapeutic efficacy of a high affinity protofibril selective antibody in transgenic mouse model (APP Swe ). 
         FIG. 4  shows human Aβ protofibrils measured at pM levels by proximity ligation technique. 
         FIG. 5A  shows the reactivity of mAb158 with Aβ protofibril, Aβ fibril, medin fibril, iset amyloid polypeptide (IAPP) fibril, and α-synuclein fibril in a dot blot assay. 
         FIG. 5B  shows the reactivity of 6E10 (Aβ), pAb179 (medin), pAbA110 (IAPP), and mAb211 (α-synuclein) with Aβ protofibril, Aβ fibril, medin fibril, IAPP fibril, and α-synuclein fibril in a dot blot assay. 
         FIG. 5C  shows immunoprecipitation data from HEK-cell culture media (mock, APP Swe  and APP Arc-Swe ) and mouse brain homogenates (non-transgenic, APP Swe , and APP Arc-Swe ). mAb158 and 6E10 were used for immunoprecipitation and 6E19 was used as the detecting antibody. 
         FIG. 6A  shows sandwich ELISA data using mAb158 as both the capturing and the detecting antibody. 
         FIG. 6B  shows ELISA data validating the conformation specificity of mAb158. 
         FIG. 6C  shows data from a mAB158 ELISA and a 6E10-6E10 sandwich ELISA performed by adding an increasing excess of Aβ1-16 to a fixed concentration of Aβ protofibrils. 
         FIG. 7A  shows the Aβ protofibril concentration in APP Arc-Swe  HEK-cell culture media and APP Swe  HEK-cell culture media. 
         FIG. 7B  shows the Aβ rotofibril levels in APP Arc-Swe  and APP Swe  transgenic mouse brains. 
         FIG. 8  shows the Aβ protofibril levels in APP Swe-Arc  transgenic mouse brain TBS extracts after four months treatment with either mAb158 or placebo. 
         FIG. 9  shows the total Aβ levels in  APP   Swe-Arc  transgenic mouse brain formic acid extracts after four months. 
         FIG. 10  shows the vector map of pKN100. 
         FIG. 11  shows the vector map of pG1D200. 
         FIG. 12  shows Aβ monomer binding by chimeric and mouse 158 antibodies. 
         FIG. 13  shows competition of monomeric and protofibrillar Aβ for binding to chimeric 158 or mouse 158 antibody. 
         FIG. 14  shows competition of monomeric and protofibrillar Aβ for binding to chimeric 158, mouse 158 antibody and humanized 158 antibody (BAN2401). 
     
    
    
     DESCRIPTION 
     The following examples are provided for illustration and are not intended to limit the invention to these specific examples. 
     EXAMPLE 1 
     Human wild-type Aβ protofibril selective monoclonal antibodies were cloned and sequenced. The amino acid sequence of the variable heavy chain region (VH) and the variable light chain region (VL) are shown in Table 1. The positions of the CDR regions 1-3 are underlined and shown as well in Table 2 and 3. The amino acid sequences of the CDR regions form the structural basis for binding human wild-type Aβ protofibrils constituting the “Alzheimer disease epitope”. 
     The amino acid sequence of the CDR regions 1-3 of the VL and VH chains for a high affinity protofibril specific antibody BA9/158 is shown in Table 1, 2 and 3. Sequencing data of other protofibril selective antibodies (BA2, BA3, BA4 and BA7) provide alternative amino acids sequences of the CDR regions but not limited to these. The combined amino acid sequences of the CDR1-3 regions of the VH and VL chains create the molecular “pocket” which binds human Aβ wild-type protofibrils with high affinity and specificity. This “pocket” forms the structural basis of the “Alzheimer&#39;s disease epitope”. Variations in the CDR amino acid sequence length are observed in both the VH chain and the VL is compatible binding to human Aβ protofibrils (Table 2 and 3). A shorter CDR region provides a more restricted three dimensional structure of the binding pocket of the antibody, whereas a longer is more flexible. 
     We claim the CDR sequences as shown in Tables 1, 2 and 3 as well as amino acid sequences in the “mouse framework” regions of the VH and VL chains, i.e. outside the CDR regions as well as the human VL and VH framework regions for protofibril specific antibodies as shown in Table 4 and 5, but not limited to those. 
     The amino acid sequence of the framework region of VL and VH regions 1-3 of the VL and VH chains from a high affinity protofibril specific antibody BA9/158 is shown in Table 4 and 5. 
     Other amino acid substitution in the CDR regions than what is shown in Table 1, 2 and 3 are compatible with high affinity and high specificity binding to human wild-type Aβ protofibrils. Where a polar amino acid is present in a particular position in a CDR region that particular amino acid can be substituted by another polar amino acid, with retained or improved high affinity and specificity binding to Aβ protofibrils. Likewise, if a non-polar or negatively or positively charged amino acids is present at a certain position, that amino acid can be substituted for by a similar amino acid from the same group. 
     Also, a particular amino acid or amino acids are exchanged in any position in the CDR regions by functional equivalents that confers a similar function and structure to the antibody. 
     EXAMPLE 2 
     Characterization of an High-affinity Human Aβ Wild-type Profibril Selective Monoclonal Antibody by ELISA 
     Example 2 shows a high affinity protofibril selective antibody that cross-reacts a 200-1000-fold less with Aβ monomers and less than 40-fold with Aβ fibrils, as measured by a sandwich ELISA ( FIG. 2A ). From competitive ELISA experiments, the antibody has a strong affinity for human Aβ42 wild-type protofibrils, but only very weak affinity for the N-terminal part of the Aβ peptide and Aβ monomers. No binding was observed to the C-terminal fragment of Aβ ( FIG. 2B ). Furthermore, the antibody does not cross-react with other types of amyloids, like medin or transthyretin. Furthermore the antibody does not recognize human APP, the abundant precursor of Aβ. 
     In  FIG. 2A  a sandwich ELISA is shown. Antibody 158 was coated in the wells and different Aβ forms subsequently added to the well in increasing concentrations. Measurement of bound Aβ forms was made by adding biotinylated mAb 158 and HRP labelled Streptavidine. Colour development was measured according to the procedure recommended by the manufacturer. 
     In  FIG. 2B  a competitive ELISA is shown. An ELISA plate was coated with human Aβ protofibrils. Antibody 158 was subsequently incubated with increasing amounts of different Aβ forms (competition). The incubation mix was added to the microtiter plate wells and free antibody was allowed to bind to immobilized protofibrils in the wells. Bound 158 antibody was measured by a second antibody using standard procedures. 
     EXAMPLE 3 
     The efficacy of high affinity Aβ protofibril selective antibody was determined in an Alzheimer transgenic mouse model (APPswe) by an acute intracranial injection. Transgenic mice used for efficacy evaluation express human APP, with the Swedish mutation (APP Swe ). In this paradigm, antibodies are injected directly into plaque-rich regions of the brain parenchyma and effects on neuropathology are assessed after 72 hours (Wilcock et al., 2003). Other studies have shown that the direct application of anti-Aβ antibodies results in a rapid clearance of amyloid deposits in vivo (Bacskai et al, 2001; Brendza et al., 2005). The injection of high affinity Aβ protofibril selective antibody leads to a significant plaque reduction in the APP Swe  mouse model ( FIG. 3 ). 
     In  FIG. 3  the therapeutic efficacy of a high affinity protofibril selective antibody in transgenic mouse model (APPswe) was tested. A: A 14 months old APPSwe transgenic mouse was intracranially injected with PBS and B: high affinity protofibril selective antibody (158) at 1 μg/μl and examined 72 hours following injection. Marked clearance of Aβ burden is noticeable in the subiculum close to the injection site (B; arrow) as compared to the control side (A; arrow). 
     EXAMPLE 4 
     Proximity ligation in combination with high affinity protofibril selective antibody for measurement of Aβ protofibrils. Human wild-type Aβ protofibrils were detected down to 10 pM-range whereas the Aβ monomer preparation were not detected at all. The combination of the hypersensitive proximity ligation method and a high affinity antibody is particularly advantageous since it provides a system to determine only oligomeric forms of the analyte, which is particularly suitable when diagnosing Alzheimer&#39;s disease and other protein “aggregation” diseases such as prion disease, Creutzfelt-Jacob, amyloidosis and Parkinson&#39;s disease. 
     In  FIG. 4  human Aβ protofibrils are measured at pM levels by the proximity ligation technique. Proximity ligation assay: Method description (from Gullberg et al., 2004): Step 1, incubation of sample with proximity probe pair (≈1 h); step 2, addition of all components required for ligation and detection by quantitative PCR (≈5 min ligation time). A high affinity protofibril selective monoclonal antibody was used in the assay; step 3, quantitative PCR (≈2 h). Synthetic Aβ monomer and Aβ protofibril preparations were diluted and tested for their reactivity in proximity ligation assay described above. 
     EXAMPLE 5 
     mAb 158 does not Recognize a Generic Amyloid Epitope. 
     Previously reported Aβ conformation dependent antibodies have been shown to bind oligomers and fibrils of other amyloidogenic proteins, suggesting a common epitope present on all amyloid aggregates. Due to technical difficulties in generating protofibrils from other amyloidogenic proteins than Aβ, mAb158 was instead tested against different amyloid fibrils. The dot blot assay was used for these experiments since inhibition ELISA, where the antibody-antigen reactions take place in solution, is not suitable for insoluble antigens like fibrils. The dot blot assay is however not suitable for evaluation of antibody specificity for various Aβ forms, i.e. for measuring differences in selectivity for profibrils and fibrils. Fibrils of medin, islet amyloid polypeptide (IAPP) and α-synuclein were immobilized on a nitrocellulose membrane to maintain their native conformations. mAb158 did not exhibit reactivity with any amyloid other the Aβ fibril ( FIG. 5A ). The binding of mAb 158 to Aβ fibrils suggests that part of the Aβ protofibril epitope is present also in the Aβ fibril structure. As positive controls the antibodies 6E10 (Aβ), pAb179 (medin), pAbA110 (IAPP) and mAb211 (α-synuclein) were used ( FIG. 5B ). Representative blots from repeated experiments (n=3). 
     mAb158 does not Bind APP 
     Levels of APP and soluble APP fragments commonly exceed the levels of Aβ in biological samples such as CSF and brain homogenate, and therefore an Aβ-antibody&#39;s cross-reactivity to APP could inhibit a treatment by binding to APP, resulting in less free antibody for binding and elimination of Aβ protofibrils and/or Aβ oligomers. Also, it could disturb measurements of Aβ protofibrils in biological samples by a sandwich ELISA assay of Aβ. To elucidate whether mAb158 binds to native APP, immunoprecipitation experiments were performed. HEK-cell culture media (mock, APP Swe  and APP Arc-Swe ) and mouse brain homogenates (non-transgenic, APP Swe  and APP Arc-Swe ) were immunoprecipitated with mAb158 or 6E10, followed by a denaturing Western blot with 6E10 as detecting antibody ( FIG. 5C ). As seen in  FIG. 5C , mAb158 did not immunoprecipitate αAPPs from cell culture media or full length APP from mouse brain homogenates, whereas, as expected, 6E10 did. The synthetic Aβ protofibrils used as control were immunoprecipitated equally well by both antibodies ( FIG. 5C ). Representative blots from repeated experiments (n=3). 
     EXAMPLE 6 
     Establishment of an Aβ protofibril specific sandwich ELISA. To enable measurements of Aβ protofibrils in biological samples a sandwich ELISA with mAb158 as both capturing and detecting antibody was established. This assay measures Aβ protofibrils with a detection limit of 1 pM and with a linear range up to 250 pM ( FIG. 6A , lines indicate linear regression of the standard curves). Due to uncertainties concerning the size of the Aβ protofibrils used in the standard curve, the concentration 1 pM is based on the molecular weight of one Aβ monomer (4514 g/mol), Though, since the molecular weight of a protofibril has been estimated to be at least 100 kDa, the limit of detection calculated as molar Aβ protofibrils could be as low as 50 fM. A standard curve of AβArc protofibrils gave a lower signal than wild type Aβ protofibrils, possibly due to differences in Aβ protofibril size ( FIGS. 6A ,  6 B). Titrated synthetic LMW-Aβ (Low Molecular Weight Aβ). By the term “Low Molecular Weight Aβ”, it is meant monomers, dimers and trimers of Aβ having a molecular weight of approximately 4-12 kDa. Aβ protofibrils and Aβ1-16 were used to validate the conformation specificity of the ELISA ( FIG. 6B ), where the hydrophilic Aβ1-16 peptide was used since it is not expected to aggregate. An ELISA composed of two identical antibodies requires at least a dimer of a protein to produce a signal and as predicted, Aβ1-16 was not detected with the mAb 158 sandwich-ELISA even at μM-concentrations ( FIG. 6B ). When pre-treating the LMW-Aβ and Aβ protofibrils with 70% formic acid (FA), known to dissociate aggregated Aβ into monomers, the sandwich ELISA the signal was lost (data not shown). Hence, the detection of LMW-Aβ at high nM concentrations ( FIG. 6B ) is probably due to a small aggregate content of the peptide preparation. 
     A large excess of monomeric Aβ, holoAPP and APP-fragments, naturally occurring in biological samples, could interfere with the Aβ protofibril analysis by occupying binding sites of the capture antibody coat, thus inhibiting the protofibrils from binding. This problem was investigated by adding an increasing excess of Aβ1-16 to a fixed concentration of Aβ protofibrils (50 pM, expressed as monomer units) and analyzing it with both the mAb158 ELISA and a 6E10-6E10 sandwich ELISA ( FIG. 6C ). A 500 000-fold molar excess of Aβ1-16, as compared to Aβ protofibrils, did not disturb the measurements with the mAb158 sandwich ELISA, as expected since Aβ1-16 binds poorly to the capture antibody. In contrast, a 500 fold excess of Aβ1-16 was enough to decrease the signal in the 6E10-6E10 ELISA, where Aβ1-16 binds with high affinity to the capture antibody ( FIG. 6C ). Moreover, when synthetic Aβ protofibrils was added to mock HEK cell culture media or non-transgenic mouse brain homogenates, 90% of the signal was recovered (data not shown). 
     EXAMPLE 7 
     Measurement of Aβ Protofibrils in Biological Samples. 
     The presence of Aβ protofibrils in cell and mouse models carrying the Arctic mutation have been suggested, though until now there has been no method for direct assaying of Aβ protofibrils in biological samples. The mAb158 sandwich ELISA therefore provides the first opportunity to measure Aβ protofibril levels in such cell and mouse models and to compare them to models without this intra-Aβ mutation. Samples from cells and mice carrying only the Swedish mutation were compared to the wild type Aβ protofibril standard curve, whereas samples from cells and mice expressing Aβ with the Arctic mutation were compared to AβArc protofibril standard curve ( FIG. 6A ). To ensure that all Aβ measured in this assay was in a soluble state, and to exclude any possible interference from Aβ fibrils, all samples were centrifuged for 5 min at 17 900×g before analysis. Groups of cell media from transiently transfected APP Swe  and APP Arc-Swe  HEK-cells were analyzed and compared to mock HEK-cell culture media. Aβ protofibril levels were calculated from the standard curves ( FIG. 6A ) as the mean value of triplicates and were then normalized to APP levels to compensate for differences in transfection levels (according to Stenh et al.). The Aβ protofibril concentration in APP Arc-Swe  HEK-cell culture media was 28 pM (±2), significantly higher (p&lt;0.0001) than the 8.2 pM (±0.3) seen in APP Swe  ( FIG. 7A ). No Aβ protofibrils could be detected in mock media. Levels of Aβ protofibrils were also measured in brains from 10 months old APP Arc-Swe  and APP Swe  transgenic mice with both plaques and intraneuronal Aβ pathology (according to Lord et al.). Brains were homogenized in TBS and centrifuged prior to analysis in order to recover the soluble Aβ fraction. Similar to the analysis using cell culture media, Aβ protofibril levels differed significantly (p=0.005) between the groups, with 397 pM (±59) in APP ArcSwe  and 108 pM (±14) in APP Swe  transgenic mouse brains ( FIG. 7B ). 
     In the above-mentioned figures ( FIGS. 6 and 7 ) the number of samples were; mock cells (n=3) and transiently transfected with APP Swe  (n=8) and APP Arc-Swe  (n=11). Levels of Aβ protofibrils in APP Arc-Swe  media were approximately 9 fold higher than in APP Swe  media, whereas mock media gave no signal (A). Measurements of Aβ protofibril levels in the TBS-soluble fraction of non-transgenic mouse brain homogenates (n=6) were compared to transgenic mice (APP Swe , n=3, and APP Arc-Swe , n=6) (B). Similar to the cell culture media, Aβ protofibril levels of APP Arc-Swe  mice were 7 fold higher than in APP Swe  mice. Error bars show ±SEM. 
     EXAMPLE 8 
     mAb158 Significantly Lowers Aβ Protofibrils and total Aβ in APPswearc transgenic mice After i.p. Administration 
     mAb158 (12 mg/kg) was injected i.p. once weekly for 18 weeks in 9-10 months old APPswearc mice. After the study, brains were isolated and homogenised in TBS and subsequently centrifuged to sediment insoluble material. The insoluble material was solubilised in formic acid. Hence, two fractions were obtained from mouse brains i.e. a TBS fraction and a formic acid fraction. Aβ protofibril levels in the TBS fractions were determined by an ELISA. A significant reduction of Aβ protofibrils was found in the mAb158 treatment group compared to the placebo group ( FIG. 8 ).  FIG. 8  shows the Aβ protofibril levels in APPswearc transgenic mouse brain TBS extracts after 4 months treatment with either mAb158 or placebo. 
     Total Aβ in the formic acid fraction was determined by an ELISA (the formic acid was used to solubilise all Aβ forms, in order to make all Aβ forms detectable). A significant reduction of total Aβ was observed in the treatment group compared to the placebo group ( FIG. 9 ).  FIG. 9  shows the total Aβ levels in APPswearc transgenic mouse brain formic acid extracts after 4 months treatment with either mAb158 or placebo. 
     EXAMPLES 9-11 
                                       Abbreviations                                A   Adenine       Ab protocol   AERES biomedical protocol       BHK   baby hamster kidney       bp   base pairs       C   Centrigrade       C   Cytosine       CHO   Chinese Hamster Ovary       CMF   Calcium and Magnesium Free       COS 7   African green monkey kidney fibroblast cell line       dhfr   Dihydrofolate-reductase       DMEM   Dulbecco&#39;s Modified Eagles Medium       DMSO   Dimethyl sulphoxide       DNA   Deoxyribonucleic acid       ELISA   Enzyme linked immuno-adsorbent assay       FCS   Foetal Calf Serum       g   grams       G   Guanine       hr   hour       HRP   Horseradish peroxidase       IgG   Immunoglobulin       K   G or T (IUPAC convention)       LSAP   Large Soluble Amyloid Product       mAb   monoclonal antibody       sec   second       min   minute       M   A or C (IUPAC convention)       MTX   Methotrexate       NIMR   National Institute for Medical Research (UK)       nm   nanometer       OD   optical density       PBS   Phosphate Buffered Saline       PCR   Polymerase chain reaction       R   A or G (IUPAC convention)       RT   Room Temperature       S   C or G (IUPAC convention)       T   Thymine       UV   Ultra Violet       V   variable       V   A or C or G (IUPAC convention)       VH   Immunoglobulin heavy chain variable region       VK   Immunoglobulin kappa light chain variable region       W   A or T (IUPAC convention)       Y   C or T (IUPAC convention)                    
Materials
 
     
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 Equipment 
               
             
          
           
               
                 Equipment 
                 UK Supplier 
                 Catalog Number 
               
               
                   
               
               
                 DNA thermal cycler:  
                 Perkin Elmer 
                 N801-0177 
               
               
                 GeneAmp 9600 
                   
                   
               
               
                 A designated tissue culture  
                 Walker Safety Cabinets 
                 N/a 
               
               
                 laboratory containing a class  
                 Ltd. 
                   
               
               
                 II microbiological safety  
                   
                   
               
               
                 cabinet fitted with a UV-lamp 
                   
                   
               
               
                 Innova ® bench top  
                 New Brunswick Scientific 
                 4000 
               
               
                 incubator shaker 
                   
                   
               
               
                 Bench top centrifuge 
                 Fisher Scientific 
                 CEK-126-010N 
               
               
                 CO2-gassed 37° incubator 
                 RossLab plc 
                 HSO-501TVBB 
               
               
                 Microbiological incubator 
                 Kendro/Heraeus 
                 B6060 
               
               
                 Electroporator Model:  
                 Bio-Rad Laboratories Ltd. 
                 341BR-3092 
               
               
                 Gene Pulser II 
                   
                   
               
               
                 ELISA reader: Microplater  
                 Bio-Rad Laboratories Ltd. 
                 3550 
               
               
                 Reader 3550 
                   
                   
               
               
                 Microplate Manager ® 2.2  
                 Bio-Rad Laboratories Ltd. 
                 N/a 
               
               
                 data analysis software  
                   
                   
               
               
                 package for Macintosh  
                   
                   
               
               
                 computer 
                   
                   
               
               
                 96-Well GeneAmp PCR  
                 ABI 
                 N8050200 
               
               
                 System 9700 
                   
                   
               
               
                 ABI PRISM 310 Genetic  
                 Applied Biosystems 
                 310-00-100/120 
               
               
                 Analyzer 
                   
                   
               
               
                 T100 surface plasmon  
                 Biacore 
                   
               
               
                 resonance detector 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 Plastic consumables 
               
             
          
           
               
                   
                   
                 Catalog  
               
               
                 Article 
                 UK Supplier 
                 Number 
               
               
                   
               
               
                 175 cm2 tissue culture flask 
                 Sarstedt Ltd 
                 83.1812.002 
               
               
                 25 cm2 tissue culture flask 
                 Corning Costar 
                 3056 
               
               
                 30 ml universal container 
                 Sterilin 
                 128C 
               
               
                 75 cm2 tissue culture flask 
                 Sarstedt Ltd 
                 83.1813.002 
               
               
                 Electroporation cuvettes 
                 Bio-Rad Laboratories Ltd. 
                 165-2088 
               
               
                 ELISA plates: Nunc MaxiSorp 
                 Invitrogen Life  
                 43945A 
               
               
                   
                 Technologies 
                   
               
               
                 GeneAmp ™ PCR reaction  
                 Perkin Elmer 
                 N801-0180 
               
               
                 tubes 
                   
                   
               
               
                 Glasstic ® disposable  
                 Bio-stat Diagnostic 
                 887144 
               
               
                 cell-counting slide 
                   
                   
               
               
                 Nunc inoculating needles 
                 Life Technologies 
                 254399 
               
               
                 tissue culture petri 100 × 20  
                 Helena Biosciences 
                 93100 
               
               
                 mm, multi-vent 
                   
                   
               
               
                 tissue culture plate: 6-well + lid 
                 Corning 
                 C3516 
               
               
                 tissue culture plate: 24-well + lid 
                 Corning 
                 C3526 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
           
               
                   
               
               
                 Immunology and molecular biology reagents 
               
             
          
           
               
                 Article 
                 UK Supplier 
                 Catalog No. 
                 Lot No. 
               
               
                   
               
               
                 1st strand synthesis kit 
                 Amersham  
                 27-9261-01 
                 3375313 
               
               
                   
                 Biosciences 
                   
                   
               
               
                 Advantage ®-HF 2 PCR Kit 
                 Clontech 
                 639123 
                 6040151 
               
               
                 Agarose (UltraPure ™) 
                 Invitrogen 
                 15510-027 
                 3019491 
               
               
                 Albumin bovine (BSA) 
                 Calbiochem 
                 126575 
                 B65755 
               
               
                 Ampicillin 
                 Sigma 
                 A-9518 
                 63H0992 
               
               
                 Apa I 
                 Promega 
                 R636 
                 16007003 
               
               
                 Themoprime + DNA  
                 Abgene 
                 AB0301 
                 014/0103/11 
               
               
                 Polymerase 
                   
                   
                 019/0607/13 
               
               
                   
                   
                   
                 020/1808/13 
               
               
                 Bam HI 
                 Promega 
                 R602 
                 15851606 
               
               
                 BigDye ® Terminator v3.0  
                 ABI 
                 4390242 
                 0605143 
               
               
                 Cycle Sequencing Ready  
                   
                   
                 0608154 
               
               
                 Reaction Kit 
                   
                   
                   
               
               
                 Ethidium Bromide (10 mg/ml) 
                 Sigma 
                 E-1510 
                 43H9414 
               
               
                 Goat anti-human IgG (Fc  
                 Stratech  
                 109-005-098 
                 68215 
               
               
                 fragment specific) antibody 
                 Scientific 
                   
                   
               
               
                 Goat anti-human kappa chain 
                 Sigma 
                 A7164 
                 032K9157 
               
               
                 horseradish peroxidase  
                   
                   
                   
               
               
                 conjugate 
                   
                   
                   
               
               
                 Hind III 
                 Promega 
                 R604 
                 16834803 
               
               
                 Human IgG1/kappa antibody. 
                 The Binding  
                 BP078 
                 223729 
               
               
                   
                 Site 
                   
                   
               
               
                 K-Blue HRP substrate 
                 SkyBio 
                 308176 
                 060823 
               
               
                 Oligonucleotides 
                 Sigma 
                 n.a. 
                   
               
               
                 PBS Tablets 
                 Sigma 
                 P4417 
                 11K8204 
               
               
                 QIAGEN Plasmid Maxi Kit  
                 Qiagen 
                 12162 
                 124114870 
               
               
                 (25) 
                   
                   
                   
               
               
                 QIAprep Spin Miniprep Kit 
                 Qiagen 
                 27106 
                 124117906 
               
               
                 QIAquick gel purification kit 
                 Qiagen 
                 28704 
                 11549740 
               
               
                 QIAquick PCR purification kit 
                 Qiagen 
                 28106 
                 G10.1.12 
               
               
                 Red Stop Solution (For K Blue) 
                 SkyBio Ltd, 
                 301475 
                 060104 
               
               
                   
                 Qiagen 
                 74106 
                 10916587 
               
               
                 Shrimp alkaline phosphatase 
                 USB 
                 70092Y 
                 107635 
               
               
                 Subcloning Efficiency ™  
                 Invitrogen 
                 44 0098 
                 1164658 
               
               
                 DH5α ™ Chemically  
                   
                   
                   
               
               
                 Competent  E. coli   
                   
                   
                   
               
               
                 T4 DNA Ligase 
                 Promega 
                 M1801 
                 167080 
               
               
                 TMB One-Step substrate for  
                 SkyBio Ltd, 
                 KB176 
                   
               
               
                 HRP 
                   
                   
                   
               
               
                 TOPO-TA Cloning ® kit 
                 Invitrogen 
                 45-0641 
                 1350772 
               
               
                 X-Gal 
                 Sigma 
                 B-9146 
                 20965701 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 Solutions from National Institute of Medical Research 
               
             
          
           
               
                 Solution name: 
                 Components 
                 Amount 
               
               
                   
               
             
          
           
               
                 PBS ‘A’ Dulbeccos (Ca &amp; 
                 NaCl 
                 8 
                 g 
               
               
                 Mg Free) 
                 KCl 
                 0.2 
                 g 
               
               
                   
                 Na 2 HPO 4   
                 1.15 
                 g 
               
               
                   
                 KH 2 PO 4   
                 0.2 
                 g 
               
               
                   
                 water 
                 1 
                 L 
               
               
                 LB 
                 Bacto Tryptone 
                 10 
                 g 
               
               
                   
                 Yeast Extract 
                 5 
                 g 
               
               
                   
                 NaCl 
                 10 
                 g 
               
               
                   
                 water 
                 1 
                 L 
               
               
                 LB agar 
                 LB 
                 1 
                 L 
               
               
                   
                 Agar (Difco) 
                 15 
                 g 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Culture Reagents 
               
             
          
           
               
                   
                 UK  
                 Catalog 
                 Lot 
                 Expiry 
               
               
                 Article 
                 Supplier 
                 Number 
                 Numbers 
                 date 
               
               
                   
               
               
                 DMEM (1X)  
                 Invitrogen 
                 41966-047 
                 9206 
                 July 2007 
               
               
                 Dulbecco&#39;s Modified 
                   
                   
                   
                   
               
               
                 Eagle Medium (High  
                   
                   
                   
                   
               
               
                 glucose) with  
                   
                   
                   
                   
               
               
                 GlutaMAX ™ I,  
                   
                   
                   
                   
               
               
                 4500 mg/L D- 
                   
                   
                   
                   
               
               
                 Glucose, Sodium  
                   
                   
                   
                   
               
               
                 Puruvate 
                   
                   
                   
                   
               
               
                 DMSO (Dimethyl  
                 Sigma 
                 D2650 
                 125K2409 
                 December  
               
               
                 sulfoxide) 
                   
                   
                   
                 2007 
               
               
                 Penicillin &amp;  
                 Invitrogen 
                 15070-063 
                 1298401 
                   
               
               
                 Streptomycin 
                   
                   
                   
                   
               
               
                 Serum: Fetal Clone I 
                 Perbio 
                 SH30080 
                 AMM17779 
                 December  
               
               
                   
                 Science 
                   
                   
                 2007 
               
               
                 SOC 
                 Invitrogen 
                 15544-034 
                 1306051 
                   
               
               
                 Trypan Blue 
                 Sigma 
                 T8154 
                 19H2388 
                   
               
               
                 Trypsin-EDTA  
                 Sigma 
                 T4049 
                 48K2342 
                 April 2008 
               
               
                 solution, cell culture  
                   
                   
                   
                   
               
               
                 tested, 0.25% 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 9 
     DNA Sequence of 158 Antibody 
     9.1—RNA Preparation 
     Snap-frozen cell pellets of the mouse hybridoma 158, (labelled vials 060824#158 5×10 6  cells) were received by TAG on Oct. 3, 2006. These cells were stored frozen until processing using the Qiagen RNeasy midi kit to isolate RNA following the manufacturers protocol. 
     9.2—1 st  Strand Synthesis 
     About 5 micrograms of 158 RNA was subjected to reverse transcription to produce 158 cDNA using the Amersham Biosciences 1st strand synthesis kit following the manufacturers protocol—This was repeated to generate 3 independent cDNA products (rounds 1, 2 and 3) in order to obviate DNA mutations due to the RT reaction. 
     9.3 Cloning of the 158 Immunoglobulin cDNA 
     Hybridoma 158 cDNA was amplified by PCR in 23 separate reactions. Immunoglobulin kappa chain variable region (VK) cDNA was amplified using 11 VK primers (MKV1-11) in combination with the kappa constant region primer MKC (Table 6). Similarly, immunoglobulin heavy chain variable region (VH) cDNA was amplified by PCR using 12 different VH primers (MHV1-12) in combination with a mix of the four IgG constant region primers (MHCG1/2a/2b/3: Table 7 ). 
     The result of the initial set of IgH PCR reactions was the single amplification product using MHV5 primer. None of the other 11 primer pairs gave a PCR product. The product of the PCR reaction primed by the oligonucleotide primers: MHV5+(MHCG1/2a/2b/3 mixture) was ligated into the pCR2.1®-TOPO® vector using the TOPO-TA cloning® kit. The result of the initial set of IgK PCR reactions was two single amplification products using primers MKV1 and MKV2 with MKC. The other 9 primer pairs generated no product. The products of the PCR reaction primed by the oligonucleotide primers: MKV1 or MKV2+MKC were ligated into the pCR2.1®-TOPO® vector using the TOPO-TA cloning® kit.  E.coli  TOP10 bacteria transformed with the ligated vector were cloned on LB/ampicillin/X-gal agar plates, by picking onto agar grid and into PCR screening mixture. The cloned plasmid inserts were screened by PCR amplification. The PCR products were gel electrophoresed and clones producing the correct-sized PCR amplification product (500 bp approx) were identified. Overnight cultures (5 ml) of each clone were processed using the QIAprep Spin Miniprep Kit Protocol, to produce DNA plasmid minipreps. 
     9.4—cDNA Sequence Determination 
     The complete cycle of RT-PCR, cloning, and DNA sequence analysis was repeated to obtain three completely independent sets of sequence information for each immunoglobulin chain. Plasmid clones from each independent set of RT-PCR reactions were sequenced in both directions using the 1212 and 1233 primers (Table 10). Plasmids were sequenced using the BigDye® Terminator v3.0 Cycle Sequencing Ready Reaction Kit (ABI), cycled on a GeneAmp9600 PCR machine and analysed on an ABI 310 capillary sequencer. 
     9.5—158 VK DNA Sequence 
     Sequences of VK clones generated using PCR primers MKV2 and MKC on 1 st  strand cDNAs rounds 1 and 2, were identical to a sterile kappa transcript originating from the myeloma fusion partner such as MOPC-21, SP2 and Ag8. This is a sterile transcript The consensus sequence (158 VK) of VK clones generated using PCR primers MKV1 and MKC on 1 st  strand cDNAs rounds 1-3 is shown in Table 11. This is a functional rearrangement. Table 11 shows some differences from the sequence shown in Tables 1, 4 and 5. These differences are in the FW1 region where the PCR primer was located. The mouse VK leader sequence most identical to the fragment of leader in 158 VK, not encoded by our primers, was K5.1# (Table 12). The prediction for the signal peptide to cleave correctly the #K5.1 signal sequence was done by a prediction program. Most likely predicted cleavage site was correctly between amino acid residue 19 and 20. (Table 13). The chimeric 158VK protein and DNA sequence is shown in Table 14. 
     9.6—158 VH DNA Sequence 
     The consensus sequence (158 VH) of VH clones generated using PCR primers MHV5 and MHCG1/2a/2b/3 mixture on 1 st  strand cDNAs rounds 1-3 is shown in Table 15. As with 158 VK, there are some differences from the FW1 sequence shown in Tables 1, 4 and 5. The most identical mouse VH leader sequence to the fragment of leader, not encoded by our primers, was NL-1 (Table 16). 
     EXAMPLE 10 
     Construction of Chimeric Expression Vectors 
     Construction of chimeric expression vectors entails adding a suitable leader sequence to VH and VK, preceded by a Hin dIII restriction site and a Kozak sequence. The Kozak sequence (Table 8) ensures efficient translation of the variable region sequence. It defines the correct AUG codon from which a ribosome can commence translation, and the most critical base is the adenine at position-3, upstream of the AUG start. The leader sequence is selected as the most similar mouse leader sequence in the Kabat database. These additions are encoded within the forward primers (Table 9). Furthermore, the construction of the chimeric expression vectors entails introducing a 5′ fragment of the human γ1 constant region, up to a natural Apa I restriction site, contiguous with the 3′ end of the J region of 158. The CH is encoded in the expression vector downstream of the inserted VH sequence but lacks the V-C intron. For the light chain, the natural splice donor site (Table 8) and a Bam HI site is added downstream of the V region. The splice donor sequence facilitates splicing out the kappa V:C intron which is necessary for in-frame attachment of the VK to the constant region. The mouse VH and VK genes were analysed to identify any unwanted splice donor sites, splice acceptor sites, Kozak sequences and for the presence of any extra sub-cloning restriction sites which would later interfere with the subcloning and/or expression of functional whole antibody. In this case none were found. 
     10.1—Expression Vectors 
     Plasmid DNA preparations of the expression vectors pKN100, and pG1D200 were purified using Qiagen Maxi kits following the manufacturers protocol. Plasmid DNA Purification using QIAGEN Plasmid Midi and Maxi Kits, from 500 ml cultures of TOP10 bacteria transfected with either vector. The vector maps are shown in  FIGS. 10 and 11 . 
     10.2—The Light Chain Chimerisation Primers 
     The mouse leader sequence K5.1# was incorporated into the design of the chimeric 158 VK. Primers were designed to generate a PCR product containing this complete leader, and 158 VK, with terminal restriction sites Hind III and Bam HI for cloning into the pKN100 expression vector (Table 9). The forward primer 158vl introduces a Hind III restriction site; a Kozak site and the K5.1# leader sequence. The back primer 158vlrev introduces: a splice donor site and a Bam HI restriction site. 
     10.3—The Heavy Chain Chimerisation Primers 
     The leader sequence NL-1 was incorporated into the design of the chimeric 158 VH. Primers were designed to generate a PCR product containing this leader, and the 158 VH region, with terminal restriction sites Hin dIII and Apa I for cloning into the pG1D200 expression vector. These are shown in Table 9. The forward primer, 158vh, introduces a Hin dIII restriction site; a Kozak translation initiation site and the NL-1 leader sequence. The back primer, 158vhrev, introduces the 5′ end of the γ1 C region and a natural Apa I restriction site. The signal peptide cleavage site prediction for K5.1 leader sequence of VK is shown in Table 17. 
     10.4—Generation of the Chimeric 158 VH Construct: pG1D200158VH 
     The 158 VH DNA fragment was amplified with primers: 158vh and 158vhrev (Table 9). The 450 bp (approx) PCR product was T-A ligated into the vector pCR2.1 and used to transform chemically competent TOP10 bacteria. Clones were selected by appropriate insert size and sequenced using the 1212 primer (Table 10). The correct expression insert was subcloned into pG1D200 expression vector and the correct subclone was selected by DNA sequencing using primer BDSH61R (Table 10). This clone was grown in 200 ml culture to produce plasmid DNA using the Qiagen Maxi Kit using the manufacturers protocol. The chimeric 158VH protein and DNA sequence is shown in Table 18. 
     10.5—Generation of the Chimeric 158 VK Construct: pKN100158VK 
     The 158 VK DNA fragment was amplified with primers 158vl and 158vlrev (Table 9). The 450 bp (approx) PCR product was T-A ligated into vector pCR2.1 and used to transform chemically competent TOP10 bacteria. Clones were selected by insert size and sequenced using the 1212 primer (Table 10). The correct clone was subcloned into pKN100 expression vector. The correct subclone was selected by screening for insert size and DNA sequencing using primer Hu-K2 (Table 10). This clone was grown in 200 ml culture to produce plasmid DNA using the Qiagen Maxi Kit using the manufacturers protocol. 
     EXAMPLE 11 
     Production and Binding Properties of Chimeric 158 Antibody 
     11.1—COS 7 Cell Transformation and Cell Culture 
     One vial of COS 7 cells was thawed and grown in DMEM supplemented with 10% Fetal clone I serum and antibiotics. One week later, cells (0.8 ml at 10 7 /ml) were electroporated with pG1D200158VH plus pKN100158VK (10 μg DNA each). The cells were grown in 8 ml of growth medium in petri dishes for 3 days. 
     11.2—Chimeric Antibody Production 
     A sandwich ELISA was used to measure antibody concentrations in the COS 7 supernatants. Chimeric 158 VH×158 VK antibody was expressed at 0.3 μg/ml and subsequently at 3.7 μg/ml (Table 19) in transiently co-transfected COS cell conditioned media. 
     11.3—Chimeric Antibody Activity 
     Two ELISAs was used to analyse the antigen binding of chimeric 158. Using the 3.7 μg/ml chimeric antibody conditioned medium, binding to Aβ monomer was measured by a direct ELISA protocol ( FIG. 12 ) and compared to the mouse 158 IgG. Secondly, a competition ELISA was done using either monomer or protofibril mixed in the fluid phase with antibody, which subsequently bound to Aβ monomer in the solid phase ( FIG. 13 ). These showed that the chimeric 158 antibody binds to amyloid Aβ monomer and protofibril similarly to the original 158 mouse antibody. 
     Comment 
     Later sequencing has shown that the mouse antibody sequence data, as shown in Tables 1 and 4 contain errors in both VH and VK chains at the 5′ end. We suggest that this is due to the use of primers located within the V region. In later sequencing, primers located within the leader sequences, which cannot introduce mutations within the V regions, were used. The later sequencing showed sequence differences (see Tables 15 and 11). Said differences are however not located within the CDR regions. 
     The chimeric antibody binds amyloid Aβ monomer and protofibrils as shown by the direct binding ELISA and the competition ELISA respectively. This evidence confirms that the combination of 158 VH and 158 VK chains encodes the anti-LSAP antibody 158 and indicates that these sequences are suitable for the humanisation procedure to generate a humanised 158 antibody. 
     EXAMPLE 12 
     Humanised Antibody Design and Discussion 
     
       
         
               
             
               
               
             
           
               
                   
               
               
                 Abbreviations and definitions 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 158 
                 mouse monoclonal anti-LSAP ™ antibody 158 
               
               
                 158 VH 
                 VH of mouse 158 antibody 
               
               
                 158 VK 
                 VK of mouse 158 antibody 
               
               
                 158RKAss 
                 Humanised version of 158 VK  
               
               
                   
                 retaining cryptic splice sites 
               
               
                 158RKA 
                 Humanised version of 158 VK with  
               
               
                   
                 cryptic splice sites removed 
               
               
                 158RHAss  
                 Humanised version of 158 VH retaining  
               
               
                   
                 cryptic splice sites 
               
               
                 158RHA 
                 Humanised version of 158 VH with  
               
               
                   
                 cryptic splice sites removed 
               
               
                 A 
                 Adenine 
               
               
                 bp 
                 base pairs 
               
               
                 C 
                 Cytosine 
               
               
                 CDR 
                 Complementarity determining region  
               
               
                   
                 in the immunoglobulin variable regions, 
               
               
                   
                 defined using the Kabat numbering system 
               
               
                 D-gene 
                 Diversity gene 
               
               
                 DNA 
                 Deoxyribonucleic acid 
               
               
                 FW 
                 Framework region: the immunoglobulin  
               
               
                   
                 variable regions excluding the CDR 
               
               
                   
                 regions 
               
               
                 G 
                 Guanine 
               
               
                 IgG 
                 Immunoglobulin G 
               
               
                 J-gene 
                 Joining gene 
               
               
                 Kabat 
                 an immunoglobulin alignment and numbering  
               
               
                   
                 system pioneered by Elvin A 
               
               
                   
                 Kabat 
               
               
                 mAb 
                 monoclonal antibody 
               
               
                 MRCT 
                 Medical Research Council Technology 
               
               
                 T 
                 Thymine 
               
               
                 VCI 
                 Framework residue classified as vernier  
               
               
                   
                 or canonical or VH-VL interface 
               
               
                 V-gene 
                 The gene segment that is rearranged together  
               
               
                   
                 with a J (and D for VH) gene to 
               
               
                   
                 generate a complete VH or VK 
               
               
                 V region  
                 The segment of IgG chains which is  
               
               
                   
                 variable in sequence between different 
               
               
                   
                 antibodies. It extends to Kabat residue 109  
               
               
                   
                 in the light chain and 113 in the 
               
               
                   
                 heavy chain. 
               
               
                 VH 
                 Immunoglobulin heavy chain variable region 
               
               
                 VK 
                 Immunoglobulin kappa light chain variable region 
               
               
                   
               
             
          
         
       
     
                                       Equipment            Hardware &amp; software   Origin               SGW02 computer   Silicon Graphics       PC computer   Hewlett Packard       SR 7.6   Steve Searle, Wellcome Trust           Sanger Institute, Cambridge.       Lasergene 6.0   DNAstar Inc       Modeler 9.0   Accelrys Ltd.       SignalP   Center for Biol. Sequence Analysis,           Technical University of Denmark website       BlastP   NCBI website                    
12.1—Human V Gene Databases
 
     The protein sequences of human and mouse immunoglobulins from the International Immunogenetics Database 2006 and the Kabat Database Release 5 of Sequences of Proteins of Immunological Interest (last update 17 Nov. 1999) were used to compile a database of immunoglobulin protein sequences in Kabat alignment. Our database contains 9322 human VH and 2689 human VK sequences. The sequence analysis program, SR 7.6, was used to query the human VH and VK databases with 158 VH and 158 VK protein sequences (Table 20). 
     12.2—Selection of a Human Framework for 158RHA 
     12.2.1—Comparison of 158 VH with Human VH Sequences 
     Human VH sequences with highest identity to 158 VH at Vernier (Foote, J. and G. Winter. 1992. Antibody framework residues affecting the conformation of the hypervariable loops.  J Mol. Biol.  224:487-499.), Canonical (Morea, V., A. M. Lesk, and A. Tramontano. 2000. Antibody modeling: implications for engineering and design.  Methods  20:267-279.) and VH-VL Interface (Chothia, C., J. Novotny, R. Bruccoleri, and M. Karplus. 1985. Domain association in immunoglobulin molecules. The packing of variable domains.  J Mol. Biol.  186:651-663.) (VCI) residues, located within the V-region framework (FW), are shown in Table 21. The number of VCI residues (VCI score) and FW residues (FW score) identical to 158 are also shown. All these VH sequences share identical VCI residues, and CDR lengths, as shown in Table 22. AJ556669 has an unusual Pro74 not seen in the other human sequences in this dataset, leading us to discount it in the initial analysis. Pro74 is, however, present in the 158VH sequence, so AJ556669 could be considered as an alternative FW for humanisation, if the VH construct based on AF062243 does not bind antigen. The alignment of these sequences (Table 23) highlights their differences. AF062243 uniquely within this dataset has the conservative change T(82a)S and the conservation of F79. The other features of AF062243 are the conservative changes D1E, K19R, A23S, T77S, S118T. All other FW changes were common to all the frameworks in Table 23. AF062243 was selected as the framework on which to base 158RHA. 
     12.3—Generation of 158RHA 
     The design of 158RHA is simply the grafting of CDR 1, 2 and 3 from 158 VH into the acceptor FW of AF062243. The human germline V-gene most identical to AF062243 is VH M99649 (VH3-07), (Table 24) from which the leader peptide was extracted (Table 25). The SignalP algorithm (Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.  Protein Eng  10:1-6.) predicted that it would cut appropriately with signal peptidase (Table 26). Table 27 shows the scheme of grafting 158 VH CDR 1, 2 and 3 into the AF062243 FW, to generate 158RHA protein sequence. Table 28 shows the generation of the DNA sequence 158RHAss from the natural DNA sequences of 158 VH and AF062243. Analysis of the 158RHAss DNA sequence predicted the presence of splice donor sites, the prediction scores of which are shown in Table 29. Non-coding mutations were introduced to inactivate these predicted splice sites, as shown in Table 30 to generate the final 158RHA DNA sequence (Table 31). 
     12.4—Selection of a Human Framework for 158RKA 
     12.4.1—Comparison of 158 VK with Human VK Sequences 
     The human VK sequences with highest identity to 158 VK at VCI residues are shown in Table 32 together with the number of VCI residues (VCI score) and FW residues (FW score) identical to 158 VK. Eleven sequences have all VCI residues identical to 158 VK. Table 33 shows that all these sequences have CDR lengths identical to 158 VK. Table 34 highlights their differences, showing that K45 is retained in AB064054 only, which also retains 185. The G100P change is unremarkable because P100 is common, having an incidence of 15% in our human VK database. The two substitutions: T7S and K74R, are conservative, and all other substitutions are common to all the sequences in Table 34. For these reasons AB064054 was selected to generate 158RKA. 
     12.5—Generation of 158RKA 
     The design of 158RKA is the simple grafting of the CDRs 1, 2 and 3 from 158 VK into the acceptor FW of human AB064054. The nearest germline V-gene to AB064054 is A19 (Table 35), from which the leader peptide was extracted (Table 36). The SignalP algorithm predicted appropriate cutting (Table 37) of this leader peptide. Table 38 shows the generation of the protein sequence of 158RKA by intercalation of the 158 VK CDRs into the FW of AB064054. Table 39 shows the generation of the DNA sequence of 158RKAss from the natural DNA sequence of 158 VK and AB064054. Analysis of the 158RKAss predicted the presence of splice donor sites, the scores of which are shown in Table 40. Non-coding mutations (41) were introduced to inactivate these sites and generate the final 158RKA DNA construct (Table 42). 
     12.6 Humanized Antibody (BAN2401) Binding Activity 
     The 158RKA and 158RHA genes were inserted into an expression vector containing the IgG1 constant region. This construct was expressed in COS cells to generate the humanized 158 antibody. The humanized 158 antibody was tested for binding activity and specificity in a competitive ELISA. The humanised antibody exhibited identical binding properties as to mAb158 and the 158 chimeric antibody (see  FIG. 14 .) 
     12.7 Additional Mutations in the 158RHA and 158RKA Chains. 
     By comparing mouse germline V genes VH AAK71612 to 158 VH a single somatic mutation A60G in the CDR2 was identified. Furthermore, the molecular model of antibody 158 which contains three VH FW residues within 5 Å of CDR residues which are unconserved in 158RHA. These substitutions are D1E, P74A and T82S (Table 43). Similarly, there are two VK FW residues within 5 Å of CDR residues which is unconserved in 158RKA. This substitution is L3V and G100P (Table 44). Introduction of back mutations at positions VH-1, VH-74, VH-82, VK-3 and VK-100 into 158RHA and 158RKA, in humanised versions 158RHB, 158RHC, 158RHD, 158RKB and 158RKC are shown in Table 43 and 44. 
     REFERENCES 
     
         
         Bacskai et al., Nat. Med. 7:369-372, 2001. 
         Bard et al., Nat. Med. 6:916-919, 2000. 
         Bayer et al., Neurology 64:94-101, 2005. 
         Brendza et al., J. Clin. Invest. 115:428-33, 2005. 
         Chen et al., Nature, 408:975-9, 2000. 
         Chothia, C. et al,  J Mol. Biol.,  186:651-663, 1985. 
         Ester W. P. Science 293, 1449-1459, 2001. 
         Gullberg et al., Proc. Natl Acad Sci, 101:8420-4, 2004. 
         Foote, J. et al.,  J Mol. Biol.,  224:487-499, 1992. 
         Hoshi et al. Proc. Natl Acad. Sci, 100:6370-6375, 2003. 
         Jarret J. T. , Biochemistry, 32, 4693-4697,1993. 
         Leatherbarrow R. J. et al., Mol. Immunol. 22, 407, 1985. 
         Lord et al., Neurobiol. Aging, 27:67-77, 2006. 
         McLean et al., Ann. Neurol. 46:860-866, 1999. 
         Morea, V. et al.,  Methods  20:267-279, 2000. 
         Mullan et al., Nat Genet. 1:345-347, 1992. 
         Nielsen, H. et al.  Protein Eng  10: 1-6, 1997. 
         Nilsberth et al., Nat Neurosci. 4:887-893, 2001. 
         Näslund et al., JAMA, 283:1571-1577, 2000. 
         Pfeifer et al., Science 298:1379, 2002. 
         Racke et al., J. Neurosci 25 :629-36, 2005. 
         Schenk D. et al. Nature, 400, 173-177, 1999. 
         Stenh et al., Ann. Neurol. 58:147-50, 2005. 
         Walsh D. M. et al., 272, 22364-22372, 1997 
         Walsh D. M. et al., Nature, 416, 535-9, 2002. 
         Wilcock et al., J. Neurosci., 23:3745-51, 2003. 
         Wright A. et al., J. of Immunology, 3393-3402, 1998. 
         Xu Y. et al. J. Biol. Chem. 269, 3469-3474, 1994. 
       
    
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Amino acid sequence of variable regions of the heavy  
               
               
                 chain (VH) and light chain (VL/Vκ) from six different monoclonal  
               
               
                 antibodies specific for human wild-type Aβ protofibrils. (SEQ ID NOS: 13-24) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 (SEQ ID 
                 VH-BA1: 
                 EVKLVESGGGLVQPGGSRKLSCAASGFT FSSFGMR VVRQAPEKGLEWVA YISSGSSTIYYADTVK GRFT 
               
               
                 NO: 13) 
                 X731 
                 ISRDNPKNPLPLQKTSLRSEDTAMYYCA RYGNYAM-------DYW GQGTSVTVSS 
               
               
                   
               
               
                 (SEQ ID 
                 VH-BA2: 
                 EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRFT 
               
               
                 NO: 14) 
                 X736 
                 ISRDNAKNTLYLQKSTSLRSEDAMYYCARNYGSRRYF-----DVWGASTSYTVSS 
               
               
                   
               
               
                 (SEQ ID 
                 VH-BA3: 
                 QVHLQQSGPELVKPGASVEMSCKASGYTFTSYVNHWVKQKPGQGLEWIGYINPYNDSTKYNEKFKGKAT 
               
               
                 NO: 15) 
                 X745 
                 LTSDKSSSTAYKELSSLTSEDSAVYYCARRVSPLTSYAM---DYWGQGTSVTVSS 
               
               
                   
               
               
                 (SEQ ID 
                 VH-BA7: 
                 QVQLKESGPGLVAPPQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALMS-RLS 
               
               
                 NO: 16) 
                 X746 
                 ISKDNSKSQVPLKHNSLQTDDTAMYYCARGRYDGKTRFA----YWGDGTLVTVSS 
               
               
                   
               
               
                 (SEQ ID 
                 VH-BA4: 
                 EVKLMESGGGLVQPGGSRKLSCAASGFTFSGFGMHEVRQAPEKGLEWVAYISSGSSTTYYADYVKGRFT 
               
               
                 NO: 17) 
                 X748 
                 ISRDNPKNTLPLQMTSLRSEDTAMYYCARGDSF---------DYWGDGTTLTVSS 
               
               
                   
               
               
                 (SEQ ID 
                 VH-BA9: 
                 EVQRVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTIYYGDTVKGRFT 
               
               
                 NO: 18) 
                 X758 
                 ISRDNPKNTLPLQKTSLRSEDTAMYYCAREGGYYYGRSYYTMDYEGQSTSVTVSS 
               
               
                   
               
               
                 (SEQ ID 
                 Vκ-BA1: 
                 DVVMFQFFLSLPVSLGDQASISC RSSQSIYSSNGNYYLS- WYLQKPGQSPKLLIY KVSNRFS GVPDRFS 
               
               
                 NO: 19) 
                 X731 
                 GSGSGTDFTLKISRVEAEDLGVYYC FQGSNVP PFFGGGTKLEIK 
               
               
                   
               
               
                 (SEQ ID 
                 Vκ-BA2: 
                 DIVMTQAFKFLLVSAGDEVTTTCKASQSVSNDVA------WYQQKPGQSPKLLIYYASNRYTGVPDRFT 
               
               
                 NO: 20) 
                 X736 
                 GSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPFFFGSGTKLEIK 
               
               
                   
               
               
                 (SEQ ID 
                 Vκ-BA3: 
                 DIVMTQAPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYAASTRESGVPDRFT 
               
               
                 NO: 21) 
                 X745 
                 GSGSGTDPTLTISSVQAEDLAVYYCKQSYNL-WTFGGGTKLRIK 
               
               
                   
               
               
                 (SEQ ID 
                 Vκ-BA7: 
                 ENVLTQSPAINSASPGEKVTNECRASSSVSSSYLH-----WYQQKSGASPKLWIYSTSNLASGVPARFS 
               
               
                 NO: 22) 
                 X746 
                 GSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTPGAGTKLELK 
               
               
                   
               
               
                 (SEQ ID 
                 Vκ-BA4: 
                 DIVMTQAPLSLPVSLEDQASISCRSSQSLVRSNGNFYLH-WYLQKPGQSLKLLKYKVSNRFSGVPDRPSV 
               
               
                 NO: 23) 
                 X748 
                 GSGSGTDFTLKISREAEDLSVYFCSQSTAVPLTFGAGTKLELK 
               
               
                   
               
               
                 (SEQ ID 
                 Vκ-BA9: 
                 DFVKTQAPLSLPVSLGDQASISCRSSQSIVHSNGNTYL-SHYLQKPGQSPKLLIYKVSNRFSGVPDRFSG 
               
               
                 NO: 24) 
                 X758 
                 SGSGTDPTLKISRVEAEDLGIYYCFQGSHVPPTFGGGYKLEIK 
               
               
                   
               
               
                 *Position of the various CDR regions (1-3) are underlined in VL and VH. The boundaries of the CDR regions (1-3) are shown in Table 3 and Table 4. Antibody BA9, also named 158 in the patent application., is an example of a high affinity protofibril specific antibody according to the invention. 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Amino acid sequences of CDR1-2 regions from VH 
               
               
                 chain from a protofibril selective antibody and 
               
               
                 amino acid substitutions that are compatible with 
               
               
                 high affinity binding to human wildtype Aβ proto- 
               
               
                 fibrils. (SEQ ID NOS: 25-32) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 VH chain CDR-1 region 
                   
               
               
                 AASGFTFSSFGMHWVR (SEQ ID NO: 25) 
                 Antibody 158 
               
               
                 ---------YA-S--- (SEQ ID NO: 26) 
                 Substitutions* 
               
               
                   
               
               
                 VH chain CDR-2 region 
                   
               
               
                 WVAYISSGSSTIYYGDTVKGRFT 
                 Antibody 158 
               
               
                 (SEQ ID NO: 27) 
                   
               
               
                 --------------A-------- 
                 Substitutions* 
               
               
                 (SEQ ID NO: 28) 
                   
               
               
                 ---T----G-YT--P-S------ 
                 Substitutions* 
               
               
                 (SEQ ID NO: 29) 
                   
               
               
                   
               
               
                 VH chain CDR-3 region 
                   
               
               
                 CAREG-GYYYGRSYY-TMDYWGQ 
                 Antibody 158 
               
               
                 (SEQ ID NO: 30) 
                   
               
               
                 CARYGxxxxxNYxxxxAMDYWGQ 
                 Substitutions 
               
               
                 (SEQ ID NO: 31) 
                 and deletions* 
               
               
                 CARNYxxxxGSRRxxxYFDVWGA 
                 Substitutions 
               
               
                 (SEQ ID NO: 32) 
                 and deletions* 
               
               
                   
               
               
                 *The amino acid substitutions (other amino acid than in antibody 158) are shown with one amino acid letter code. Deletions are shown with (x). 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Amino acid sequences of CDR 1-3 regions from V/L 
               
               
                 chain from a protofibril selective antibody and 
               
               
                 amino acid substitutions that are compatible with 
               
               
                 high affinity binding to human wildtype Aβ proto- 
               
               
                 fibrils (SEQ ID NOS: 33-38) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 VL chain CDR-1 region 
                   
               
               
                 ISCRSSQSIVHSNGNTYLEWYL 
                 Antibody 158 
               
               
                 (SEQ ID NO: 33) 
                   
               
               
                 ITCKASQSVxxSNDxxxVAWYQ 
                 Substitutions and 
               
               
                 (SEQ ID NO: 34) 
                 deletions* 
               
               
                   
               
               
                 VL chain CDR-2 region 
                   
               
               
                 LIYKVSNRFSGVP (SEQ ID NO: 35) 
                 Antibody 158 
               
               
                 ---YA---YT--- (SEQ ID NO: 36) 
                 Substitutions* 
               
               
                   
               
               
                 VL chain CDR-3 region 
                   
               
               
                 YYCFQGSHVPPTFGG (SEQ ID NO: 37) 
                 Antibody 158 
               
               
                 -F-Q-DYSS-F---S (SEQ ID NO: 38) 
                 Substitutions* 
               
               
                   
               
               
                 *The amino acid substitutions (other amino acid than in antibody 158) are shown with one amino acid letter code. Deletions are shown with (x). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                 Amino acid sequence of mouse framework regions of the mouse and human variable light 
               
               
                 chain (VL) region from protofibril specific antibodies (SEQ ID NOS: 39-47) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Mouse framework* VL regions 
                   
                   
               
               
                 Divmtqaplslpvslgdqasiscwylqkpgpspklliygvpdrfsgsgsgtdftlkisrveaedlgiyyc 
                 antibody 158 
                 (SEQ ID NO: 39) 
               
               
                   
               
               
                 ...................................................................... 
                 BA9_VL_fr123 
                 (SEQ ID NO: 40) 
               
               
                   
               
               
                 .v....t...........................................................v... 
                 BA1_VL_fr123 
                 (SEQ ID NO: 41) 
               
               
                   
               
               
                 ........kf.l..a..rvt.t...q..................t...y.....ft..t.q....av.f. 
                 BA2_VL_fr123 
                 (SEQ ID NO: 42) 
               
               
                   
               
               
                 Human framework VL regions 
                   
                   
               
               
                 ......t......tp.ep...............q..............................v.v... 
                 VKII-3-1-(1)-O11 
                 (SEQ ID NO: 43) 
               
               
                   
               
               
                 ......s......tp.ep...............q..............................v.v... 
                 VKII-4-1-(1)-A19 
                 (SEQ ID NO: 44) 
               
               
                   
               
               
                 ......t....s.tp.qp...............q..............................v.v... 
                 VKII-4-1-(1)-A18 
                 (SEQ ID NO: 45) 
               
               
                   
               
               
                 ......t....s.tp.qp.............p.q..............................v.v... 
                 VKII-4-1-(1)-A2 
                 (SEQ ID NO: 46) 
               
               
                   
               
               
                 .v....s......t..qp......fq.r.....rr.............................v.v... 
                 VKII-4-1-(1)-A17 
                 (SEQ ID NO: 47) 
               
               
                   
               
               
                 *Framework region is the region outside the CDR regions. The CDR regions has been deleted for clarity. 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 Amino acid sequence of mouse and human framework regions of the mouse and human 
               
               
                 variable light heavy (VH) region from protofibril specific antibodies (SEQ ID NOS: 48-56) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Mouse framework* VH regions 
                   
                   
               
               
                 Evklmesggglvqpggsrklscaaswvrqapekglewvarftisrdnpkntlflqmtslrsedtamyycar 
                 antibody 158 
                 (SEQ ID  
               
               
                   
                   
                 NO: 48) 
               
               
                   
               
               
                 ....................................................................... 
                 BA9_VH_fr123 
                 (SEQ ID 
               
               
                   
                   
                 NO: 49) 
               
               
                   
               
               
                 ....v.................................................................. 
                 BA1_VH_fr123 
                 (SEQ ID 
               
               
                   
                   
                 NO: 50) 
               
               
                   
               
               
                 ....v.......k....l...........t...r.............a....y...s.............. 
                 BA2_VH_fr123 
                 (SEQ ID 
               
               
                   
                   
                 NO: 51) 
               
               
                   
               
               
                 Human framework VH regions 
                   
                   
               
               
                 ..q.v............lr............g...............a..s.y...n...a....v..... 
                 VH3-7_fr123 
                 (SEQ ID 
               
               
                   
                   
                 NO: 52) 
               
               
                   
               
               
                 ..q.v......i.....lr............g......s........s....y...n...a....v..... 
                 VH3-53_fr123 
                 (SEQ ID 
               
               
                   
                   
                 NO: 53) 
               
               
                   
               
               
                 ..q.l............lr............g......s........s....y...n...a....v....k 
                 VH3-23_fr123 
                 (SEQ ID 
               
               
                   
                   
                 NO: 54) 
               
               
                   
               
               
                 ..q.v............lr............g......s........a..s.y...n...d....v..... 
                 VH3-48_fr123 
                 (SEQ ID 
               
               
                   
                   
                 NO: 55) 
               
               
                   
               
               
                 ..q.v............lr............g...v..s........a....y...n...a....v..... 
                 VH3-74_fr123 
                 (SEQ ID 
               
               
                   
                   
                 NO: 56) 
               
               
                   
               
               
                 *Framework region is the region outside the CDR regions. The CDR regions has been deleted for clarity. 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 PCR primers for cloning mouse VK 
               
               
                 (SEQ ID NOS: 57-68) 
               
             
          
           
               
                 Name 
                 Sequence (5′ → 3′) 
                   
               
               
                   
               
               
                 MKV1 
                 ATGAAGTTGVVTGTTAGGCTGTTGGTGCTG 
                 (SEQ ID NO: 57) 
               
               
                   
               
               
                 MKV2 
                 ATGGAGWCAGACACACTCCTGYTATGGGTG 
                 (SEQ ID NO: 58) 
               
               
                   
               
               
                 MKV3 
                 ATGAGTGTGCTCACTCAGGTCCTGGSGTTG 
                 (SEQ ID NO: 59) 
               
               
                   
               
               
                 MKV4 
                 ATGAGGRCCCCTGCTCAGWTTYTTGGMWTC 
                 (SEQ ID NO: 60) 
               
               
                   
                 TTG 
                   
               
               
                   
               
               
                 MKV5 
                 ATGGATTTWAGGTGCAGATTWTCAGCTTC 
                 (SEQ ID NO: 61) 
               
               
                   
               
               
                 MKV6 
                 ATGAGGTKCKKTGKTSAGSTSCTGRGG 
                 (SEQ ID NO: 62) 
               
               
                   
               
               
                 MKV7 
                 ATGGGCWTCAAGATGGAGTCACAKWYYCW 
                 (SEQ ID NO: 63) 
               
               
                   
                 GG 
                   
               
               
                   
               
               
                 MKV8 
                 ATGTGGGGAYCTKTTTYCMMTTTTTCAAT 
                 (SEQ ID NO: 64) 
               
               
                   
                 TG 
                   
               
               
                   
               
               
                 MKV9 
                 ATGGTRTCCWCASCTCAGTTCCTTG 
                 (SEQ ID NO: 65) 
               
               
                   
               
               
                 MKV10 
                 ATGTATATATGTTTGTTGTCTATTTCT 
                 (SEQ ID NO: 66) 
               
               
                   
               
               
                 MKV11 
                 ATGGAAGCCCCAGCTCAGCTTCTCTTCC 
                 (SEQ ID NO: 67) 
               
               
                   
               
               
                 MKC 
                 ACTGGATGGTGGGAAGATGG 
                 (SEQ ID NO: 68) 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 PCR primers for cloning mouse heavy VH 
               
               
                 (SEQ ID NOS: 69-84) 
               
             
          
           
               
                 Name 
                 Sequence(5′ → 3′) 
                   
               
               
                   
               
               
                 MHV1 
                 ATGAAATGCAGCTGGGGCATSTTCTTC 
                 (SEQ ID NO: 69) 
               
               
                   
               
               
                 MHV2 
                 ATGGGATGGAGCTRTATCATSYTCTT 
                 (SEQ ID NO: 70) 
               
               
                   
               
               
                 MHV3 
                 ATGAAGWTGTGGTTAAACTGGGTTTTT 
                 (SEQ ID NO: 71) 
               
               
                   
               
               
                 MHV4 
                 ATGRACTTTGGGYTCAGCTTGRTTT 
                 (SEQ ID NO: 72) 
               
               
                   
               
               
                 MHV5 
                 ATGGACTCCAGGCTCAATTTAGTTTTC 
                 (SEQ ID NO: 73) 
               
               
                   
                 CTT 
                   
               
               
                   
               
               
                 MHV6 
                 ATGGCTGTCYTRGSGCTRCTCTTCTGC 
                 (SEQ ID NO: 74) 
               
               
                   
               
               
                 MHV7 
                 ATGGRATGGAGCKGGRTCTTTMTCTT 
                 (SEQ ID NO: 75) 
               
               
                   
               
               
                 MHV8 
                 ATGAGAGTGCTGATTCTTTTGTG 
                 (SEQ ID NO: 76) 
               
               
                   
               
               
                 MHV9 
                 ATGGMTTGGGTGTGGAMCTTGCTATTC 
                 (SEQ ID NO: 77) 
               
               
                   
                 CTG 
                   
               
               
                   
               
               
                 MHV10 
                 ATGGGCAGACTTACATTCTCATTCCTG 
                 (SEQ ID NO: 78) 
               
               
                   
               
               
                 MHV11 
                 ATGGATTTTGGGCTGATTTTTTTTATTG 
                 (SEQ ID NO: 79) 
               
               
                   
               
               
                 MHV12 
                 ATGATGGTGTTAAGTCTTCTGTACCTG 
                 (SEQ ID NO: 80) 
               
               
                   
               
               
                 MHCG1 
                 CAGTGGATAGACAGATGGGGG 
                 (SEQ ID NO: 81) 
               
               
                   
               
               
                 MHCG2a 
                 CAGTGGATAGACCGATGGGGC 
                 (SEQ ID NO: 82) 
               
               
                   
               
               
                 MHCG2b 
                 CAGTGGATAGACTGATGGGGG 
                 (SEQ ID NO: 83) 
               
               
                   
               
               
                 MHCG3 
                 CAAGGGATAGACAGATGGGGC 
                 (SEQ ID NO: 84) 
               
               
                   
               
               
                 Legend: Wobble bases are defined in Abbreviations (Section 2). 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Sequences important for efficient expression of 
               
               
                 immunoglobulin in mammalian cells 
               
               
                 (SEQ ID NOS: 85-88) 
               
             
          
           
               
                 Name 
                 Consensus DNA Sequence (5′ → 3′) 
               
               
                   
               
               
                 Kozak transla- 
                 G C C G C C  R  C C −1    A   +1    U G G   
               
               
                 tion initiation 
                 (SEQ ID NO: 85) 
               
               
                 site 
                   
               
               
                   
               
               
                 Kappa light 
                   A C ::  G T  R A G T 
               
               
                 chain splice 
                 (SEQ ID NO: 86) 
               
               
                 donor site 
                   
               
               
                   
               
               
                 Heavy chain 
                 M A G ::  G T  R A G T 
               
               
                 splice donor 
                 (SEQ ID NO: 87) 
               
               
                 site 
                   
               
               
                   
               
               
                 Immunoglobulin 
                 Y Y Y Y Y Y Y Y Y Y Y N C  A G  :: G 
               
               
                 splice acceptor 
                 (SEQ ID NO: 88) 
               
               
                 site 
               
               
                   
               
               
                 Legend: Bases shown in  bold  are considered to be invariant within each consensus sequence. Splice sites are defined by the symbol “::”. Wobble bases are defined in Abbreviations (see Examples 9-11). 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Oligonucleotide primers used to generate 
               
               
                 chimeric 158 (SEQ ID NOS: 89-92) 
               
             
          
           
               
                 Oligonucletotide name 
                 Sequence (5′ → 3′) 
               
               
                   
               
               
                 158 vh (SEQ ID NO: 89) 
                   AAGCTT   GCCGCCACC ATGGACTCCAGGCTC 
               
               
                   
               
               
                 158 vhrev 
                   GGGCCC TTGGTGGAGGCTGAGGAGACGGTGACTGAGG 
               
               
                 (SEQ ID NO: 90) 
                   
               
               
                   
               
               
                 158 vl (SEQ ID NO: 91) 
                   AAGCTT   GCCGCCACC ATGAAGTTGCCTGTTAGG 
               
               
                   
               
               
                 158 vlrev (SEQ ID NO: 92) 
                   GGATCC ACTCACGTTTGATTTCCAGCTTGG 
               
               
                   
               
               
                 Legend: Restriction sites are  underlined . Kozak sequences are in  bold type . 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Oligonucleotide primers used for sequencing 
               
               
                 (SEQ ID NOS: 93-96) 
               
             
          
           
               
                 Oligonucletotide name 
                 Sequence (5′→3′) 
               
               
                   
               
               
                 1212 (17mer) 
                 GTTTTCCCAGTCACGAC 
               
               
                 (SEQ ID NO: 93) 
                   
               
               
                   
               
               
                 1233 (24mer) 
                 AGCGGATAACAATTTCACACAGGA 
               
               
                 (SEQ ID NO: 94) 
                   
               
               
                   
               
               
                 Hu-K2 (17mer) 
                 CTCATCAGATGGCGGGA 
               
               
                 (SEQ ID NO: 95) 
                   
               
               
                   
               
               
                 BDSH61R (SEQ ID NO: 96) 
                 CGCTGCTGAGGGAGTAGAGTC 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 11 
               
               
                   
               
               
                 DNA sequence of 158 VK, primer MKV1 and the VK sequence derived using primers located within the V 
               
               
                 region (SEQ ID NOS: 97-99) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 (SEQ ID NO: 97) 
                   
               
               
                   1 
                 ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTG 
                 158 VK 
               
               
                   
                 (SEQ ID NO: 98) 
                   
               
               
                   1 
                 .........VV................... 
                 MKV1 
               
               
                   
                 (SEQ ID NO: 99) 
                   
               
               
                   1 
                 ---------------------------------------------------------...A..G..........GG.............. 
                 *** VK 
               
               
                   
               
               
                  91 
                 CCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTAC 
                 158 VK 
               
               
                  34 
                 .......................................................................................... 
                 *** VK 
               
               
                   
               
               
                 181 
                 CTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGA 
                 158 VK 
               
               
                 124 
                 .......................................................................................... 
                 *** VK 
               
               
                   
               
               
                 271 
                 TCAGGGACAGATTTCACACTGAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCTCCG 
                 158 VK 
               
               
                 214 
                 .......................................................................................... 
                 *** VK 
               
               
                   
               
               
                 361 
                 ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTG 
                 158 VK 
               
               
                 304 
                 ................................. 
                 *** VK 
               
               
                   
               
               
                 Legend: Residues identical to 158 VK are indicated by a dot. 
               
               
                 ***Sequencing using primers located within the V region. 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
           
               
                 TABLE 12 
               
               
                   
               
               
                 Chimeric VK leader sequence selection - K5.1# leader 
               
               
                 selection for the chimeric VK (SEQ ID NOS: 100-102) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 158 VK 
                 MKLPVRLLVLMFWIPASSS (SEQ ID NO: 100) 
               
               
                   
               
               
                 K5.1#Protein 
                 MKLPVRLLVLMFWIPASSS (SEQ ID NO: 101) 
               
               
                   
               
               
                 K5.1#DNA 
                 ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCT 
               
               
                   
                 GCTTCCAGCAGT (SEQ ID NO: 102) 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                 SignalP result 6 for K5.1# leader (SEQ ID NO: 103) 
               
             
          
           
               
                 &gt;Sequence 
                 length = 40 
               
             
          
           
               
                 # Measure  
                 Position 
                 Value 
                 Cut off  
                 signal peptide? 
               
               
                   
               
             
          
           
               
                 max. 
                 C 
                 20 
                 0.970 
                 0.32 
                 YES 
               
               
                 max. 
                 Y 
                 20 
                 0.890 
                 0.33 
                 YES 
               
               
                 max. 
                 S 
                 13 
                 0.989 
                 0.87 
                 YES 
               
               
                 mean 
                 S 
                 1-19 
                 0.954 
                 0.48 
                 YES 
               
               
                   
                 D 
                 1-19 
                 0.922 
                 0.43 
                 YES 
               
               
                   
               
               
                 # Highest probability for cleavage is between amino acid residue 19 and 20 (SSS-DV) 
               
             
          
         
       
     
     
       
         
               
             
               
             
               
               
             
           
               
                 TABLE 14 
               
               
                   
               
               
                 Protein and DNA sequence of chimeric 158 VK construct (SEQ ID NOS: 104-105) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
             
          
           
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 15 
               
               
                   
               
               
                 DNA sequence of 158 VH, primer MHV5 and the sequence derived using primers 
               
               
                 located within the V region (SEQ ID NOS: 106-108) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 (SEQ ID NO: 106) 
                   
               
               
                 1 
                 ATGGACTCCAGGCTCAATTTAGTTTTCCTTGTCCTTATTTTAAAAGGTGTCCAGTGTG 
                 158 VH 
               
               
                   
                 ATGTGCAGCTGGTGGAGTCTGGGGGAGGCTTA 
                   
               
               
                   
               
               
                   
                 (SEQ ID NO: 107) 
                   
               
               
                 1 
                 --------------------------------------------------------- 
                 *** VH 
               
               
                   
                 ..G...A.....A....A............... 
                   
               
               
                   
               
               
                   
                 (SEQ ID NO: 108) 
                   
               
               
                 1 
                 .............................. 
                 MHV5 
               
               
                   
               
               
                 91 
                 GTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTA 
                 158 VH 
               
               
                   
                 GCTTTGGAATGCACTGGGTTCGTCAGGCTCCA 
                   
               
               
                   
               
               
                 34 
                 .......................................................... 
                 *** VH 
               
               
                   
                 ................................ 
                   
               
               
                   
               
               
                 181 
                 GAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTAGTACCATCTACTATG 
                 158 VH 
               
               
                   
                 GAGACACAGTGAAGGGCCGATTCACCATCTCC 
                   
               
               
                   
               
               
                 124 
                 .......................................................... 
                 *** VH 
               
               
                   
                 ................................ 
                   
               
               
                   
               
               
                 271 
                 AGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACA 
                 158 VH 
               
               
                   
                 CGGCCATGTATTACTGTGCAAGAGAGGGGGGA 
                   
               
               
                   
               
               
                 214 
                 .......................................................... 
                 *** VH 
               
               
                   
                 ................................ 
                   
               
               
                   
               
               
                 361 
                 TATTACTACGGTAGGAGTTACTATACTATGGACTACTGGGGTCAAGGAACCTCAGTCAC 
                 158 VH 
               
               
                   
                 CGTCTCCTCAGCCAAAACAACAGCCCCA 
                   
               
               
                   
               
               
                 304 
                 ........................................................... 
                 *** VH 
               
               
                   
                 ............................ 
               
               
                   
               
               
                 Legend: Residues identical to 158 VH are indicated by a dot. 
               
               
                 ***Sequencing using primers located within the V region. 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
           
               
                 TABLE 16 
               
               
                   
               
               
                 Chimeric VH leader selection- 
               
               
                 NL-1 VH leader sequence (SEQ ID NO: 109-111) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 159 VH leader 
                 MDSRLNLVFLVLILKGVQC (SEQ ID NO: 109) 
               
               
                   
               
               
                 NL-1 protein 
                 MDSRLNLVFLVLILKGVQC (SEQ ID NO: 110) 
               
               
                   
               
               
                 NL-1 DNA 
                 ATGGACTCCAGGCTCAATTTAGTTTTCCTTGTCCTTA 
               
               
                   
                 TTTTAAAAGGTGTCCAGTGT (SEQ ID NO: 111) 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 17 
               
             
             
               
                   
               
               
                 SignalP result 6 for NL-1 VH leader sequence (SEQ ID NO: 112) 
               
             
          
           
               
                 # Measure  
                 Position 
                 Value 
                 Cut off  
                 signal peptide? 
               
               
                   
               
             
          
           
               
                 max. 
                 C 
                 20 
                 0.775 
                 0.32 
                 YES 
               
               
                 max. 
                 Y 
                 20 
                 0.795 
                 0.33 
                 YES 
               
               
                 max. 
                 S 
                 13 
                 0.953 
                 0.87 
                 YES 
               
               
                 mean 
                 S 
                 1-19 
                 0.866 
                 0.48 
                 YES 
               
               
                   
                 D 
                 1-19 
                 0.830 
                 0.43 
                 YES 
               
               
                   
               
               
                 # Highest probability for cleavage is between amino acid residue 19 and 20 (VQC-DV) 19 and 20: VQC-DV 
               
             
          
         
       
     
     
       
         
               
             
               
             
               
               
             
           
               
                 TABLE 18 
               
               
                   
               
               
                 Protein and DNA sequence of chimeric 158 VH (SEQ ID NOS: 113-114) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
             
          
           
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 19 
               
             
             
               
                   
               
               
                 Expression of chimeric 158 antibody in COS cells 
               
             
          
           
               
                   
                 Expression  
                 Antibody  
               
               
                 Number of 
                 Vector Constructs  
                 Concentration 
               
               
                 Co-transfections 
                 Co-Transfected 
                 (ng/ml) 
               
               
                   
               
             
          
           
               
                 2 pooled 
                 pG1D200158 and pKN100158 
                 300 
               
               
                 2 pooled 
                 pG1D200158 and pKN100158 
                 3700 
               
               
                   
               
               
                 Legend: Antibody concentration was measured by ELISA in 3-day cultures of transfected COS 7 cells. COS cells were co-transformed with 10 μg each of the heavy and light chain chimeric expression vectors pG1D200158 and pKN100158. 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
           
               
                 TABLE 20 
               
               
                   
               
               
                 Amino acid sequence of 158 VH and 158 VK 
               
               
                 (SEQ ID NOS: 115-116) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 (SEQ ID NO: 115) 
               
               
                 VH 
                 DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGL 
               
               
                   
                 EWVAYISSGSSTIYYGDTVKGRFTISRDNPKNTLFLQMTSLRSED 
               
               
                   
                 TAMYYCAREGGYYYGRSYYTMDYWGQGTSVTVSS 
               
               
                   
               
               
                   
                 (SEQ ID NO: 116) 
               
               
                 VK 
                 DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKP 
               
               
                   
                 GQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGI 
               
               
                   
                 YYCFQGSHVPPTFGGGTKLEIK  
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 21 
               
             
             
               
                   
               
               
                 Best human VH framework VCI scores compared with 158 VH 
               
             
          
           
               
                 Kabat Number 6   
                 2 
                 24 
                 26 
                 27 
                 28 
                 29 
                 30 
                 37 
                 39 
                 45 
                 47 
                 48 
                 49 
                 67 
                 69 
                 71 
                 73 
                 78 
                 91 
                 93 
                 94 
                 103 
                   
               
               
                 Canonical Residue 8   
                 - 
                 1 
                 1 
                 1 
                 - 
                 1 
                 - 
                 - 
                 - 
                 - 
                 - 
                 - 
                 - 
                 - 
                 - 
                 2 
                 - 
                 - 
                 - 
                 - 
                 1 
                 - 
                   
               
               
                 Vernier Residue 7   
                 * 
                 - 
                 - 
                 * 
                 * 
                 * 
                 * 
                 - 
                 - 
                 - 
                 * 
                 * 
                 * 
                 * 
                 * 
                 * 
                 * 
                 * 
                 - 
                 * 
                 * 
                 - 
                   
               
               
                 Interface Residue 9   
                 - 
                 - 
                 - 
                 - 
                 - 
                 - 
                 - 
                 I 
                 I 
                 I 
                 I 
                 - 
                 - 
                 - 
                 - 
                 - 
                 - 
                 - 
                 I 
                 I 
                 - 
                 I 
                   
               
             
          
           
               
                 Sequence 
                 FW 
                 VCI 
                   
                   
               
               
                 name 
                 score 
                 score 
                 VCI Residues 
               
               
                   
               
             
          
           
               
                 158 VH 
                 87 
                 22 
                 V 
                 A 
                 G 
                 F 
                 T 
                 F 
                 S 
                 V 
                 Q 
                 L 
                 W 
                 V 
                 A 
                 F 
                 I 
                 R 
                 N 
                 L 
                 Y 
                 A 
                 R 
                 W 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 117) 
               
               
                   
               
               
                 38687 
                 79 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AB021520 
                 77 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AJ556669 
                 77 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 38672 
                 77 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 38673 
                 77 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 DQ322738 
                 77 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AB067108 
                 76 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AB021531 
                 76 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AB021532 
                 76 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AB063892 
                 76 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AB067237 
                 76 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AB021507 
                 76 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AF471177 
                 76 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AF471184 
                 76 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AF062243 
                 76 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AF174030 
                 76 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AF466141 
                 76 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AF466142 
                 76 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AJ245279 
                 76 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 AJ579216 
                 76 
                 22 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 118) 
               
               
                   
               
               
                 Legend: Canonical residues are numbered in this table according to which CDR they are associated with. 
               
               
                 FW score and VCI score are the number of residues in the FW or VCI definition respectively, which are identical to their counterpart in 158. 
               
               
                 Residues identical to those in 158 VH are indicated by a dot. 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
             
           
               
                 TABLE 22 
               
               
                   
               
               
                 Sequences of best VCI-scoring human VH, compared with 158 VH (SEQ ID NOS: 119-135) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Kabat 
                           1         2         3           4         5            6         7         8 
               
               
                 Number 6   
                 -12345678901234567890123456789012345AB67890123456789012ABC345678901234567890123456789012ABC34 
               
               
                 Canonical 
                                         1 11 1    1                    2   22               2 
               
               
                 Vernier 
                  *                         ****                  ***                    * * * *    * 
               
               
                 Interface 
                                                    I   I I     I I 
               
               
                 Kabat CDR 
                                                *******              ******************* 
               
               
                   
               
               
                 158 VH 
                 -DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMH--WVRQAPEKGLEWVAYISS--GSSTIYYGDTVKGRFTISRDNPKNTLFLQMTSLRS 
               
               
                   
               
               
                 AB021520 
                 -EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYWMS--WVRQAPGKGLEWVANIKQ--DGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRA 
               
               
                   
               
               
                 AJ556669 
                 -EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMS--WVRQAPGKGLEWVANIKE--DGGEKFYVDSVKGRFTISRDNPKNSLFLQMNSLRA 
               
               
                   
               
               
                 DQ322738 
                 PLVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMS--WVRQAPGKGLEWVAVIWY--DGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA 
               
               
                   
               
               
                 AB067108 
                 -EVQLVESGGGVVQPGGSLRLSCAASGFTFSNYAMH--WVRQAPGKGLEWVAVISY--DGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA 
               
               
                   
               
               
                 AB021531 
                 -QVQLVESGGGVVQPGRSLKLSCAASGFTFSSYAMH--WVRQAPGKGLEWVAVISY--DGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA 
               
               
                   
               
               
                 AB021532 
                 -QVQLVESGGGVVQPGRSLKLSCAASGFTFSSYAMH--WVRQAPGKGLEWVAVISY--DGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA 
               
               
                   
               
               
                 AB063892 
                 -EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMS--WVRQAPGKGLEWVANIKQ--DGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRA 
               
               
                   
               
               
                 AB067237 
                 -EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMS--WVRQAPGKGLEWVANIKQ--DGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRA 
               
               
                   
               
               
                 AB021507 
                 -QVQLVESGGGVVQPGRSLKLSCAASGFTFSSYAMH--WVRQAPGKGLEWVAVISY--DGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA 
               
               
                   
               
               
                 AF471177 
                 -EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMS--WVRQAPGKGLEWVANIKQ--DGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRA 
               
               
                   
               
               
                 AF471184 
                 -EVQLVESGGGLVQPGSSLRLSCAASGFTFSSYWMS--WVRQAPGKGLEWVANIKQ--DGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRA 
               
               
                   
               
               
                 AF062243 
                 CEVQLVESGGGLVQPGGSLRLSCSASGFTFSTYWMT--WVRQAPGKGLEWVANIKP--HGSEQYYVDSVKGRFTISRDNAKNSLFLQMSSLRA 
               
               
                   
               
               
                 AF174030 
                 CEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMS--WVRQAPGKGLEWVANIKQ--DGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRA 
               
               
                   
               
               
                 AF466141 
                 -QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMH--WVRQAPGKGLEWVAVIWY--DGSNKYYADSAKGRFTISRDNSKNTLFLQMNSLRA 
               
               
                   
               
               
                 AF466142 
                 -QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMH--WVRQAPGKGLEWVAVIWY--DGSNKYYADSAKGRFTISRDNSKNTLFLQMNSLRA 
               
               
                   
               
               
                 AJ245279 
                 -QVQLVESGGGVVQPGGSLRLSCAASGFTFSSYGMH--WVRQAPGKGLEWVAVIWY--DGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA 
               
               
                   
               
             
          
           
               
                   
                 Kabat 
                      9        10                   11 
                   
               
               
                   
                 Number 6   
                 5678901234567890ABCDEFGHIJKlmnopqrstuv1234567890123 
                   
               
               
                   
                 Canonical 
                          1 
                   
               
               
                   
                 Vernier 
                         **                              * 
                   
               
               
                   
                 Interface 
                       I I I     I                       I 
                   
               
               
                   
                 Kabat CDR 
                           ****************************** 
                   
               
               
                   
               
               
                   
                 158 VH 
                 EDTAMYYCAREGGYYYGRSYYT---------------MDYWGQGTSVTVSS 
                 (SEQ ID NO: 119) 
               
               
                   
               
               
                   
                 AB021520 
                 EDTAVYYCARPDDSSGYYSAEY---------------FQHWGQGTLVTVSS 
                 (SEQ ID NO: 120) 
               
               
                   
               
               
                   
                 AJ556669 
                 EDTAVYYCARERGHDFWSIYYTH--------------FDYWGOGALVTVSS 
                 (SEQ ID NO: 121) 
               
               
                   
               
               
                   
                 DQ322738 
                 EDTAVYYCARDGGSI----------------------FDYWGQGTLVTVSS 
                 (SEQ ID NO: 122) 
               
               
                   
               
               
                   
                 AB067108 
                 EDTAVYYCARARDYYYYP-------------------MDVWGQGTTVTVSS 
                 (SEQ ID NO: 123) 
               
               
                   
               
               
                   
                 AB021531 
                 EDTAVYYCARDQSWSRIAAAGTPPSL-----------FDPWGQGTLVTVSs 
                 (SEQ ID NO: 124) 
               
               
                   
               
               
                   
                 AB021532 
                 DEDTAVYYCARARNYYDSSGYS---------------FDYWGOGTLVTVSS 
                 (SEQ ID NO: 125) 
               
               
                   
               
               
                   
                 AB063892 
                 EDTAVYYCARVRRGS----------------------GDSWGQGTLVTVSS 
                 (SEQ ID NO: 126) 
               
               
                   
               
               
                   
                 AB067237 
                 EDTAVYYCAREQQLGPHNW------------------FDPWGQGTLVTVSS 
                 (SEQ ID NO: 127) 
               
               
                   
               
               
                   
                 AB021507 
                 EDTAVYYCARDQETGTTFDYYYYG-------------MDVWGOGTTVTVSS 
                 (SEQ ID NO: 128) 
               
               
                   
               
               
                   
                 AF471177 
                 EDTAVYYCARDPMTTVVKPSLAT--------------NDYWGQGTLVTVSS 
                 (SEQ ID NO: 129) 
               
               
                   
               
               
                   
                 AF471184 
                 EDTAVYYCARDCVGALGA-------------------FDIWGQGTMVTVSS 
                 (SEQ ID NO: 130) 
               
               
                   
               
               
                   
                 AF062243 
                 EDTAVYYCARANS------------------------LDVWGQGTTVTVSS 
                 (SEQ ID NO: 131) 
               
               
                   
               
               
                   
                 AF174030 
                 EDTAVYYCARDGDIGDWW-------------------FDPWGQGTLVTVSS 
                 (SEQ ID NO: 132) 
               
               
                   
               
               
                   
                 AF466141 
                 EDTAVYYCARDKGYYDYVWGSYRSNPKNDA-------FDIWGQGTMVTVSS 
                 (SEQ ID NO: 133) 
               
               
                   
               
               
                   
                 AF466142 
                 EDTAVYYCARDKGYYDYVWGSYRSNPKNDA-------FDIWGQGTMVTVSS 
                 (SEQ ID NO: 134) 
               
               
                   
               
               
                   
                 AJ245279 
                 EDTAVYYCARDRFF-----------------------FDNWGQGTLVTVSS 
                 (SEQ ID NO: 135) 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
             
           
               
                 TABLE 23 
               
               
                   
               
               
                 Alignment of 158 VH with the best VCI-scoring human VH (SEQ ID NO: 136-147) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Legend: Residues identical to 158 VH are represented by a dot. CDRs are grey-shaded 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 24 
               
               
                   
               
               
                 VH signal peptide selection-5.5.1 V-gene alignment of 158 VH, 
               
               
                 human AF062243 and human germline M99649 (VH3-07)(SEQ ID NOS: 148-150) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 DVQLVESGGGLVQPGGSRLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYIS 
                 158 VH 
                 (SEQ ID NO: 148) 
               
               
                   
               
               
                 E................LR...S......TYW.T......G.......N.K 
                 AF062243 
                 (SEQ ID NO: 149) 
               
               
                   
               
               
                 E................LR...........YW.S......G.......N.K 
                 M99649 
                 (SEQ ID NO: 150) 
               
               
                   
               
               
                 SGSSTIYYGDTVKGRFTISPDNPKNTLFLQMTSLRSEDTAMYYCAR 
                 158 VH 
                 (SEQ ID NO: 148) 
               
               
                   
               
               
                 PHG.EA..V.S...........A..S.....S...A....V..... 
                 AF062243 
                 (SEQ ID NO: 149) 
               
               
                   
               
               
                 QDG.EK..V.S...........A..S.Y...N...A....V..... 
                 M99649 
                 (SEQ ID NO: 150) 
               
               
                   
               
               
                 Legend: V-gene residues identical to 158 VH are represented by a dot. 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
           
               
                 TABLE 25 
               
               
                   
               
               
                 Signal peptide of M99649 human germline VH gene 
               
               
                 (SEQ ID NOS: 151-152) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 DNA 
                 ATGGAATTGGGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTA 
               
               
                   
                 GAAGGTGTCCAGTGT (SEQ ID NO: 151) 
               
               
                   
               
               
                 protein 
                 MELGLSWVFLVAILEGVQC (SEQ ID NO: 152) 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 26 
               
             
             
               
                   
               
               
                 M99649 signal peptide cutting prediction (SEQ ID NO: 153) 
               
             
          
           
               
                 # Measure  
                 Position 
                 Value 
                 Cutoff  
                 signal peptide? 
               
               
                   
               
             
          
           
               
                 max. 
                 C 
                 20 
                 0.909 
                 0.32 
                 YES 
               
               
                 max. 
                 Y 
                 20 
                 0.836 
                 0.33 
                 YES 
               
               
                 max. 
                 S 
                 13 
                 0.953 
                 0.87 
                 YES 
               
               
                 mean 
                 S 
                 1-19 
                 0.859 
                 0.48 
                 YES 
               
               
                   
                 D 
                 1-19 
                 0.848 
                 0.43 
                 YES 
               
               
                   
               
               
                 # Highest probability for cleavage is between amino acid residue 19-20: VQC-DV. 
               
             
          
         
       
     
     
       
         
               
             
               
             
           
               
                 TABLE 27 
               
               
                   
               
               
                 Generation of 158RHA protein sequence (SEQ ID NOS: 154-163) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
           
               
                 TABLE 28 
               
               
                   
               
             
             
               
                 Generation of 158RHA DNA sequence - Generation of 158RHAss DNA sequence 
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Legend: Human VH3-07 leader and AF062243VH FWs intercalated with 158 VH CDRs (grey-shaded) to generate 158RHAss. (relate to Tables 27 and 28) 
               
             
          
         
       
     
     
       
         
               
             
               
             
           
               
                 TABLE 29 
               
               
                   
               
               
                 DNA and protein sequence of 158RHAss (SEQ ID NOS: 173-174) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Legend: Splice donor sites predicted by Lasergene 6.0 GeneQuest analysis, together with their score, using the human_ds_2 matrix with a threshold of 4.2. 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 30 
               
               
                   
               
               
                 Mutations in 158RHA removing splice sites in 158RHAss 
               
               
                 (SEQ ID NOS: 175-176) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 (SEQ ID NO: 175) 
                   
               
               
                 1 
                 ATGGAATTGGGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAGAGGGAGT 
                 158RHA 
               
               
                   
                 (SEQ ID NO: 176) 
                   
               
               
                 1 
                 ............................................A..T.. 
                 158RHAss 
               
               
                   
               
               
                 51 
                 CCAGTGCGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTG 
                 158RHA 
               
               
                 51 
                 ......T..G........................................ 
                 158RHAss 
               
               
                   
               
               
                 101 
                 GGGGGTCCCTGAGACTCTCCTGTTCAGCCTCTGGATTCACCTTTAGTAGC 
                 158RHA 
               
               
                 101 
                 .................................................. 
                 158RHAss 
               
               
                   
               
               
                 151 
                 TTTGGAATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGT 
                 158RHA 
               
               
                 151 
                 ............................................G..... 
                 158RHAss 
               
               
                   
               
               
                 201 
                 GGCCTACATTAGTAGTGGCAGTAGTACCATCTACTATGGAGACACCGTGA 
                 158RHA 
               
               
                 201 
                 .............................................A.... 
                 158RHAss 
               
               
                   
               
               
                 251 
                 AGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTTTCTG 
                 158RHA 
               
               
                 251 
                 .................................................. 
                 158RHAss 
               
               
                   
               
               
                 301 
                 CAAATGAGCAGCCTGAGAGCCGAGGACACGGCCGTGTATTATTGTGCGAG 
                 158RHA 
               
               
                 301 
                 .................................................. 
                 158RHAss 
               
               
                   
               
               
                 351 
                 AGAGGGGGGATATTACTACGGAAGGAGTTACTATACTATGGACTACTGGG 
                 158RHA 
               
               
                 351 
                 .....................T............................ 
                 158RHAss 
               
               
                   
               
               
                 401 
                 GCCAAGGGACCACGGTCACCGTCTCC 
                 158RHA 
               
               
                 401 
                 .......................... 
                 158RHAss 
               
               
                   
               
               
                 Legend 158RHA DNA sequence compared to 158RHAss (Table 5.7.2) which contains predicted splice sites. Positions identical to 158RHA are identified as a dot. 
               
             
          
         
       
     
     
       
         
               
             
               
             
           
               
                 TABLE 31 
               
               
                   
               
               
                 DNA and protein sequence of 158RHA (SEQ ID NOS: 177-178) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 32 
               
               
                   
               
               
                 Best VCI scores of human VK compared with 158 VK 
               
               
                 (SEQ ID NOS: 179-182) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Kabat Number 6   
                 2 
                 4 
                 35 
                 36 
                 38 
                 44 
                 46 
                 47 
                 48 
                 49 
                 64 
                 66 
                 68 
                 69 
                 71 
                 87 
                 98 
                   
               
               
                 Canonical Residue 8   
                 1 
                   
                   
                   
                   
                 2 
                 2 
                   
                 1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Vernier Residue 7   
                 * 
                 * 
                 * 
                 * 
                   
                 * 
                 * 
                 * 
                 * 
                 * 
                 * 
                 * 
                 * 
                 * 
                   
                 * 
                   
                   
               
               
                 Interface Residue 9   
                   
                   
                 I 
                 I 
                 I 
                 I 
                   
                   
                   
                 I 
                 I 
               
               
                   
               
             
          
           
               
                   
                 Fw 
                 VCI 
                   
                   
               
               
                 Sequence 
                 score 
                 score 
                 VCI residues 
               
               
                   
               
             
          
           
               
                 158 VK 
                 80 
                 17 
                 V 
                 M 
                 W 
                 Y 
                 Q 
                 P 
                 L 
                 L 
                 I 
                 Y 
                 G 
                 G 
                 G 
                 T 
                 F 
                 Y 
                 F 
                 (SEQ ID NO: 179) 
               
               
                   
               
               
                 AB064054 
                 71 
                 17 
                 V 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 180) 
               
               
                   
               
               
                 AB063934 
                 70 
                 17 
                 V 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 180) 
               
               
                   
               
               
                 AB064105 
                 70 
                 17 
                 V 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 180) 
               
               
                   
               
               
                 AY941999 
                 70 
                 17 
                 V 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 180) 
               
               
                   
               
               
                 AX805665 
                 69 
                 17 
                 V 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 180) 
               
               
                   
               
               
                 AB064104 
                 69 
                 17 
                 V 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 180) 
               
               
                   
               
               
                 AY942057 
                 69 
                 17 
                 V 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 180) 
               
               
                   
               
               
                 AB064055 
                 68 
                 17 
                 V 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 180) 
               
               
                   
               
               
                 AX742B74 
                 68 
                 17 
                 V 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 180) 
               
               
                   
               
               
                 AY685343 
                 67 
                 17 
                 V 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 180) 
               
               
                   
               
               
                 AY685353 
                 67 
                 17 
                 V 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 180) 
               
               
                   
               
               
                 DQ187506 
                 70 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 DQ187679 
                 70 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 AY043107 
                 69 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 AJ388639 
                 69 
                 16 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 V 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 AJ388646 
                 69 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 AJ388642 
                 69 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 M74470 
                 69 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 X72466 
                 69 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 U95244 
                 69 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 AAA51016 
                 69 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 X89054 
                 69 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 DQ187505 
                 69 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 DQ187683 
                 69 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 DQ187691 
                 69 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 AX805669 
                 68 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 AF455562 
                 68 
                 16 
                 I 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 . 
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 Legend: Canonical residues are numbered in this table according to which CDR they are associated. 
               
               
                 FW score and VCI score are the number of residues in the FW or VCI definition respectively, which are identical to their counterpart in 158. 
               
               
                 Residues identical to 158 VK are indicated by a dot. 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 33 
               
               
                   
               
               
                 Sequences of best VCI-scoring human VK, cormpared with 158 VK (SEQ ID NO: 183-209) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Kabat 
                          1         2               3         4         5         6         7         8 
               
               
                 number 6   
                 123456789012345678901234567ABCDEF89012345678901234567890123456789012345678901234567890 
               
               
                 Canonical 8   
                  1                      1   1     1   1              2               2      1 
               
               
                 Vernier 7   
                  * *                                    **         ****              * * ** * 
               
               
                 Interface 5   
                                                        F F F     F F 
               
               
                 VCI 
                  1 *                    1   2     1   1F*F F     F ****              * * ** * 
               
               
                 Kabat CDR 
                                        *****************               ******* 
               
               
                   
               
               
                 158 VK 
                 DVLMTQTPLSLPVSLGDQASISCRSSQSIVHS-NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AB064054 
                 DVVMTQSPLSLPVTPGAPASISCRSSQSLLHT-NGVNFLDWYLQKPGQSPKLLIYLASHRASGVPDRFSGSGSGTDFTLRISRVEA 
               
               
                   
               
               
                 AB063934 
                 DVVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPKLLIYLGSHRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AB064105 scFv 
                 DVVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AY941939 scFv 
                 DVVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AX805665 patent 
                 DVVMTQSPLSLPVTPGEPASISCRSSQSIVHS-NGNTYLQWYLQKPGQSPQLLIYKVSNRLYGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AB064104 
                 DVVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDGFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AY942057 scFv 
                 DVVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AB064055 
                 DVVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYSDWYLQKPGQSPQLLIYLGSSRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AX742874 
                 DVVMTQTPLSLSVTPGQPASISCRSSQSLLHS-DGMTYFSWYLQKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AY685343 
                 DVVMTQSPLSLAVTPGEPASISCTSSQSVVFT-NGKNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AY685353 
                 DVVMTQSPLSLAVTPGEPASISCTSSQSVVFT-NGKNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 DQ187506 
                 DIVMTQTPLSLPVTPGEPASISCRSSQSLLES-HGYNYLDWYLQKPGQSPQLLIYLASNRPSGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 DQ187679 
                 DIVMTQTPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AY043107 
                 DIVMTQTPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AJ388639 
                 DVVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYFAWYLQRPGQSPQLLVYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AJ388646 
                 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AJ388642 
                 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 M74470 
                 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGFNYLHWYLQKPGQSPRLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 X72466 
                 DIVMTQSPLSLPVTPGEPASISCTSSQSLLHN-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDRTLKISRVEA 
               
               
                   
               
               
                 U95244 
                 DIVMTQSPLSLVPTPGEPASISCRSSQSLLYS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AAA51016 
                 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGFNYLHWYLQKPGQSPRLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 X89054 
                 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYFDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVFA 
               
               
                   
               
               
                 DQ187505 
                 DIVMTQSPLSLPVTPGEPASISCRSSQSLLES-HGYNYLDWYLQKPGQSPQLLIYLASNRPSGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 DQ187683 
                 DIVMTQTPLSLPVTPGEPASISCRSSQSLLHG-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 DQ187691 
                 DIVMTQTPLSLPVTPGEPASISCRSSQSLLHG-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
               
                 AX805669 
                 DIVMTQSPLSLPVTPGEPASISCRSSQSIVHS-NGNTYLQWYLQKPGQSPQLLIYDVSNRLYGVPDRFSGSGSGTDFTLKISRVEA 
               
               
                   
               
             
          
           
               
                   
                 Kabat 
                          9              10 
               
               
                   
                 number 6   
                 123456789012345ABCDEF678901234567 
               
               
                   
                 Canonical 8   
                          3    3       3 
               
               
                   
                 Vernier 7   
                                        * 
               
               
                   
                 Interface 5   
                       F F F          F F 
               
               
                   
                 VCI 
                       F F3F  33  *   F3F 
               
               
                   
                 Kabat CDR 
                         *************** 
               
               
                   
               
               
                   
                 158 VK 
                 EDLGIYYCFQGSHVP------PTFGGGTKLEIK (SEQ ID NO: 183) 
               
               
                   
               
               
                   
                 AB064054 
                 EDVGIYYCMQGLQTP------FTFGPGTKLEIK (SEQ ID NO: 184) 
               
               
                   
               
               
                   
                 AB063934 
                 EDVGVYYCMQQLQTP------LTFGGGTKVEIK (SEQ ID NO: 185) 
               
               
                   
               
               
                   
                 AB064105 scFv 
                 EDVGVYYCMQALQTP------YTFGQGTKLEIK (SEQ ID NO: 186) 
               
               
                   
               
               
                   
                 AY941939 scFv 
                 EDVGVYYCMQALQTP------YTFGQGTKLEIK (SEQ ID NO: 187) 
               
               
                   
               
               
                   
                 AX805665 patent 
                 EDVGVYYCFQGSHVP------WTFGQGTKVEIK (SEQ ID NO: 188) 
               
               
                   
               
               
                   
                 AB064104 
                 EDVGVYYCMQALQTP------HTFGQGTKLEIK (SEQ ID NO: 189) 
               
               
                   
               
               
                   
                 AY942057 scFv 
                 EDVGVYYCMQALQSP------PTFGRGTKVEIK (SEQ ID NO: 190) 
               
               
                   
               
               
                   
                 AB064055 
                 EDVGVYYCMQALQTP------FTFGPGTKVDIK (SEQ ID NO: 191) 
               
               
                   
               
               
                   
                 AX742874 
                 EDVGVYYCMQNIQLP------WTFGQGTKVEIK (SEQ ID NO: 192) 
               
               
                   
               
               
                   
                 AY685343 
                 DDVGVYYCMHAVQAP------WTFGQGTKVEIK (SEQ ID NO: 193) 
               
               
                   
               
               
                   
                 AY685353 
                 DDVGVYYCMHAVQAP------WTFGQGTKVEIK (SEQ ID NO: 194) 
               
               
                   
               
               
                   
                 DQ187506 
                 EDVGIYYCMQNLQTP------YSFGQGTKLEIR (SEQ ID NO: 195) 
               
               
                   
               
               
                   
                 DQ187679 
                 EDVGVYYCMQALQTP------HSFGQGTKLEIK (SEQ ID NO: 196) 
               
               
                   
               
               
                   
                 AY043107 
                 EDVGVYYCMQALQTP------LTFGGGTKVEIK (SEQ ID NO: 197) 
               
               
                   
               
               
                   
                 AJ388639 
                 EDVGIYYCMQVLQTP------YTFGQGTKLEIS (SEQ ID NO: 198) 
               
               
                   
               
               
                   
                 AJ388646 
                 EDVGVYYCMQALQTP------LTFGGGTKVEIK (SEQ ID NO: 199) 
               
               
                   
               
               
                   
                 AJ388642 
                 EDVGVYYCMQALQTP------PTFGGGTKVEIK (SEQ ID NO: 200) 
               
               
                   
               
               
                   
                 M74470 
                 DDVGIYYCMQALASP------YTFGQGTKLEIK (SEQ ID NO: 201) 
               
               
                   
               
               
                   
                 X72466 
                 EDVGVYYCMQVLQIP------LTFGGGTKVEIK (SEQ ID NO: 202) 
               
               
                   
               
               
                   
                 U95244 
                 EDVGDYYCMQALQSP------LTFGGGTKVEIK (SEQ ID NO: 203) 
               
               
                   
               
               
                   
                 AAA51016 
                 DDVGIYYCMQALQSP------YTFGQGTKLEIK (SEQ ID NO: 204) 
               
               
                   
               
               
                   
                 X89054 
                 EDVGVYYCMQALQTP------LTFGGGTKVEIK (SEQ ID NO: 205) 
               
               
                   
               
               
                   
                 DQ187505 
                 EDVGIYYCMQNLQTP------YSFGQGTKLEIR (SEQ ID NO: 206) 
               
               
                   
               
               
                   
                 DQ187683 
                 EDVGVYYCMQALQTP------RTFGQGTKVEIK (SEQ ID NO: 207) 
               
               
                   
               
               
                   
                 DQ187691 
                 EDVGVYYCMQALQTP------RTFGQGTKVEIK (SEQ ID NO: 208) 
               
               
                   
               
               
                   
                 AX805669 
                 EDVGVYYCFQGSHVP------WTFGQGTKVEIK (SEQ ID NO: 209) 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
             
           
               
                 TABLE 34 
               
               
                   
               
               
                 Alignment of 158 VK with the best VCI-scoring human VK 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Legend: CDR 1, 2 and 3 are grey shaded. 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 35 
               
               
                   
               
               
                 VK signal peptide selection - Alignment of 158 VK 
               
               
                 with human AB064054 and human germline A19 (SEQ ID NOS: 234-236) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSN 
                 158 VK 
                 (SEQ ID NO: 234) 
               
               
                   
               
               
                 ..V...S......TP.AP.........LL.T..VNF.D................LA.H 
                 AB064054 
                 (SEQ ID NO: 235) 
               
               
                   
               
               
                 .IV...S......TP.EP.........LL....YN..D...........Q....LG.. 
                 A19 
                 (SEQ ID NO: 236) 
               
               
                   
               
               
                 RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIK 
                 158 VK 
                 (SEQ ID NO: 234) 
               
               
                   
               
               
                 .A..................R........V.....M..LQT.F...P......N 
                 AB064054 
                 (SEQ ID NO: 235) 
               
               
                   
               
               
                 .A...........................V.V...M.ALQT. 
                 A19 
                 (SEQ ID NO: 236) 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
           
               
                 TABLE 36 
               
             
             
               
                   
               
               
                 Signal peptide of human A19 
               
               
                 (VK2-28; X63397) germlie VK (SEQ ID NOS: 237-238) 
               
             
          
           
               
                   
                 VK A19 leader sequence 
               
               
                   
               
               
                 DNA 
                 ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGG 
               
               
                   
                 GTCTCTGGATCCAGTGGG (SEQ ID NO: 237) 
               
               
                   
               
               
                 protein 
                 MRLPAQLLGLLMLWVSGSSG (SEQ ID NO: 238) 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 37 
               
             
             
               
                   
               
               
                 A19 signal peptide cutting prediction (SEQ ID NO: 239) 
               
             
          
           
               
                 &gt;Sequence 
                 length = 50 
               
             
          
           
               
                 # Measure  
                 Position 
                 Value 
                 Cutoff  
                 signal peptide? 
               
               
                   
               
             
          
           
               
                 max. 
                 C 
                 21 
                 0.853 
                 0.32 
                 YES 
               
               
                 max. 
                 Y 
                 21 
                 0.831 
                 0.33 
                 YES 
               
               
                 max. 
                 S 
                 13 
                 0.990 
                 0.87 
                 YES 
               
               
                 mean 
                 S 
                 1-20 
                 0.932 
                 0.48 
                 YES 
               
               
                   
                 D 
                 1-20 
                 0.881 
                 0.43 
                 YES 
               
               
                   
               
               
                 # Most likely cleavage site between pos. 20 and 21: SSG-DV 
               
             
          
         
       
     
     
       
         
               
             
               
             
           
               
                 TABLE 38 
               
               
                   
               
               
                 Generation of 158RKA Protein Sequence (SEQ ID NOS: 240-249) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
             
               
               
             
               
             
           
               
                 TABLE 39 
               
               
                   
               
               
                 Generation of 158RKAss DNA Sequence - Intercalation of 158VK into AB064054 FW 
               
               
                 (SEQ ID NOS: 250-258) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
             
          
           
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
           
               
                 Legend: A19 leader and AB064054 FW with intercalated 158 VK                          
CDRs to generate 158RKAss 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
             
           
               
                 TABLE 40 
               
               
                   
               
               
                 158RKAss DNA sequence (SEQ ID NOS: 259-260) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Legend: Splice donor sites predicted by Lasergene 6.0 GeneQuest analysis, together with their score, using the human_ds_2 matrix with a threshold of 4.2. 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 41 
               
               
                   
               
               
                 Mutations in 158RKA removing splice sites in 158RKA (SEQ ID NOS: 261-262) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGGTCTCTGGAAGCAGTGGG 
                 158RKA 
                 (SEQ ID NO: 261) 
               
               
                 1 
                 ...................................................TC....... 
                 158RKAss 
                 (SEQ ID NO: 262) 
               
               
                   
               
               
                 61 
                 GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGCGCCGGCCTCC 
                 158RKA 
                   
               
               
                 61 
                 ............................................................ 
                 158RKAss 
                   
               
               
                   
               
               
                 121 
                 ATCTCCTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAGTGG 
                 158RKA 
                   
               
               
                 121 
                 ........................................................A... 
                 158RKAss 
                   
               
               
                   
               
               
                 181 
                 TATCTTCAAAAGCCAGGGCAGTCTCCAAAGCTCCTGATCTATAAAGTTTCCAACCGATTT 
                 158RKA 
                   
               
               
                 181 
                 .....G..G................................................... 
                 158RKAss 
                   
               
               
                   
               
               
                 241 
                 TCTGGAGTCCCTGACAGGTTCAGTGGAAGTGGATCAGGCACAGATTTTACACTGAGAATC 
                 158RKA 
                   
               
               
                 241 
                 ..........................C.....G........................... 
                 158RKAss 
                   
               
               
                   
               
               
                 301 
                 AGCAGAGTGGAGGCTGAGGATGTTGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCT 
                 158RKA 
                   
               
               
                 301 
                 ............................................................ 
                 158RKAss 
                   
               
               
                   
               
               
                 361 
                 CCGACGTTCGGCCCTGGGACCAAATTGGAAATCAAA 
                 158RKA 
                   
               
               
                 361 
                 .................................... 
                 158RKAss 
               
               
                   
               
               
                 Legend: 158RKA DNA sequence compared to 158RKAss (Table 5.13.2) which contains predicted splice sites. Residues identical to 158RKA are identified by a dot. 
               
             
          
         
       
     
     
       
         
               
             
               
             
           
               
                 TABLE 42 
               
               
                   
               
               
                 DNA and protein sequence of 158RKA (SEQ ID NOS: 263-264) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
             
           
               
                 TABLE 43 
               
               
                   
               
               
                 Further version of humanized 158VHA (158RHB,158RHC,158RHD) (SEQ ID NO: 265-268) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
             
           
               
                 TABLE 44 
               
               
                   
               
               
                 Further version of humanized 158VKA (158RKB,158RKC) (SEQ ID NO: 269-271)