Antibodies directed against amyloid-beta peptide and methods using same
An amyloid, antibody-based technology for the treatment and/or prevention of disease that can address issues such as delays in human clinical trials
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Embodiment 1
[0283] Example 1 Determination of Binding Affinity of Antibody 9TL and Its Variants
[0284] A. General method
[0285] The following general method was used in this example.
[0286] Expression vectors for clonal analysis
[0287] Expression of antibody Fab fragments was carried out under the control of the IPTG-inducible lacZ promoter, similar to Barbas (2001) Phage display: a laboratory manual, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press pg2.10.Vector pComb3X However, modifications include the addition and expression of the following additional domains: human kappa light chain constant domain and CHI constant domain of IgG2a human immunoglobulin, Ig gamma-2 chain C region, protein number P01859; immunoglobulin kappa light chain (human), protein number CAA09181.
[0288] Small scale Fab preparation
[0289] Small-scale Fab expression in 96-well plates was performed as described below. Starting from E.coli transformed with the Fab library, colonies ...
Embodiment 2
[0312] Example 2 Analysis of antigenic determinants of Aβ1-40TL
[0313] To determine the epitope on the Aβ polypeptide recognized by antibody 9TL, surface plasmon resonance (SPR, Biacore 3000) binding analysis was used. Conjugate biotin (Glocal PeptideServices, CO) Aβ 1-40 The peptides were immobilized on a streptavidin-coated chip (SA chip). In the presence or absence of different soluble fragments of Aβ peptide (10 μM, from American PeptideCompany Inc., CA), Aβ antibody Fab fragment (50nM) and immobilized Aβ 1-40 combination. Aβ 1-40 , Aβ 1-42 and Aβ 1-43 The amino acid sequence of is shown in Table 4. Display antibody 9TL Fab fragment and Aβ 1-40 The bound Aβ peptides are Aβ 28-40 , Aβ 1-40 , Aβ 33-40 and Aβ 17-40 (figure 2). Therefore, antibody 9TL and Aβ 1-40 C-terminal peptide (33-40) binding. As shown in Figure 2, Aβ 1-28 , Aβ 28-42 , Aβ 28-35 , Aβ 1-16 , Aβ 1-43 and Aβ 1-38 Peptides did not inhibit the binding of antibody 9TL Fab fragments, sugge...
Embodiment 3
[0317] Example 3 Production of monoclonal antibody 2H6 and deglycosylated 2H6
[0318] A. Production and Analysis of Monoclonal Antibody 2H6
[0319] 25-100 μg peptide (Aβ 1-40 Amino acids 28-40 of the amino acid 28-40), with the interval of about 16 consecutive weeks to immunize mice, such as Geerligs HJ et al., 1989, J.Immunol.Methods 124: 95-102, Kenney JSet al., 1989, J.Immunol . Methods 121: 157-166 and Wicher K et al., 1989, Int. Arch. Alergy Appl. Immunol. 89: 128-135. Mice were first deimmunized with 50 μg of peptide in CFA (complete Freud's excipient). 21 days later, mice were immunized a second time with 25 μg of peptide in IFA (Incomplete Freud's excipient). Twenty-three days after the second immunization, a third immunization was performed with 25 μg of peptide in IFA. 34 days after the third immunization, a fourth immunization was performed with 25 μg of peptide in IFA. Thirty-two days after the fourth immunization, a final boost was performed with 100...
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