CD19 antibodies and their uses
An antibody and application technology, applied in the field of CD19 antibody and its application, can solve the problem of human anti-mouse and other problems
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[0353] Preparation of human monoclonal antibody-producing hybridomas
[0354] To prepare hybridomas that produce the human monoclonal antibodies of the present invention, spleen cells and / or lymph node cells are isolated from the immunized mice and fused with a suitable immortalized cell line (e.g., a mouse myeloma cell line). The hybridomas obtained are screened based on the production of antigen-specific antibodies. For example, 50% PEG can be used to fuse a single cell suspension of splenic lymphocytes from immunized mice with one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580). Alternatively, a CytoPulse large chamber cell fusion electroporator (CytoPulse Sciences, Inc., Glen Burnie Maryland) can be used to fuse a single cell suspension of splenic lymphocytes from immunized mice using an electric field-based electrofusion method. Divide the cells to approximately 2×10 5 The density was inoculated in a flat-bottomed microtiter plate, and th...
Embodiment 1
[0440] Example 1: Preparation of anti-CD19 human monoclonal antibody
[0441] antigen
[0442] B-cell tumor cell lines Raji (ATCC deposit number CCL-86) and Daudi (ATCC deposit number CCL-213) were used as antigens for immunization.
[0443] Transgenic transchromosome KM-mouse
[0444] A fully human monoclonal antibody against CD19 was prepared using transgenic transchromosomal KM mice expressing human antibody genes. In this mouse line, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al. (1993) EMBO J.12: 811-820, and has been implemented as described in PCT Publication WO 01 / 09187 Example 1 homozygously destroys the endogenous mouse heavy chain gene for HuMab mice. The mouse carries the human kappa light chain transgene KCo5, as described in Fishwild et al. (1996) Nature Biotechnology 14:845-851. This mouse also carries the human heavy chain transchromosome SC20, as described in PCT Publication WO 02 / 43478.
[0445] KM-mouse Im...
Embodiment 2
[0453] Example 2: Structural characterization of human monoclonal antibodies 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8
[0454] The cDNA sequences of the heavy and light chain variable regions of monoclonal antibodies encoding 21D4 and 21D4a were obtained from 21D4 hybridomas using standard PCR technology, and sequenced using standard DNA sequencing technology. Note that the 21D4 hybridoma produces antibodies in which the heavy chain is paired with one of the two light chains (SEQ ID NOs: 8 and 9). Two antibodies (ie V with SEQ ID NOs: 1 and 8 respectively H And V L 21D4 of the sequence, and V with SEQ ID NOs: 1 and 9, respectively H And V L The 21D4a) of the sequence binds to CD19. The cDNA sequences encoding the heavy and light chain variable regions of the 47G4, 27F3, 3C10, 5G7, 13F1, and 46E8 monoclonal antibodies were obtained from 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1, and 46E8 hybridomas using standard PCR techniques, respectively. Obtained and sequenced using ...
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