Method for making monoclonal antibodies and cross-reactive antibodies obtainable by the method

a monoclonal antibody and antibody technology, applied in the field of making monoclonal antibodies, can solve the problems of limited human use of therapeutic agents, and achieve the effect of reducing the number of animals

Inactive Publication Date: 2005-12-22
GENENTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] This method is thought to be useful for reducing the number of animals that need to be immunized ...

Problems solved by technology

Since murine monoclonal antibodies are derived from mice, their use as therapeutic agents in humans is limi...

Method used

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  • Method for making monoclonal antibodies and cross-reactive antibodies obtainable by the method
  • Method for making monoclonal antibodies and cross-reactive antibodies obtainable by the method
  • Method for making monoclonal antibodies and cross-reactive antibodies obtainable by the method

Examples

Experimental program
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example 1

Preparation of Immunogens

[0172] The receptor antigens in Examples 2 and 3 below were all receptors for Apo-2 ligand [Pitti et al., J. Biol. Chem., 271:12687-12690 (1996); and WO97 / 25428]. The Apo-2L receptors were: DR4 [Pan et al., Science, 276:111-113 (1997)]; Apo-2 [called “DR5” in Sheridan et al., Science 277:818-821 (1997)]; DcR1 [Sheridan et al., Science 277:818-821 (1997)]; and DcR2 [Marsters et al., Curr. Biol., 7:1003-1006 (1997)].

[0173] Receptor immunoadhesins (designated “DR4-IgG”, “Apo-2-IgG”, “DcR1-IgG” and “DcR2-IgG”) were prepared by fusing the extracellular domain sequence of each receptor to the hinge and Ec region of human immunoglobulin G1 heavy chain in pRK5 as described previously [Ashkenazi et al., Proc. Natl. Acad. Sci., 88:10535-10539 (1991)]. The immunoadhesin proteins were expressed by transient transfection into human 293 cells and purified from cell supernatants by protein A affinity chromatography, as described by Ashkenazi et al., supra. Purified immun...

example 2

Mixed Antigen Immunization

[0174] Animals in this example were immunized with the four receptor immunoadhesins of the preceding example. The mixed antigen immunization scheme used is shown in FIG. 1.

[0175] Balb / c mice (obtained from Charles River Laboratories) were immunized into each hind foot pad 14 times at 3-4 day intervals, with a mixture of DR4-IgG, Apo-2-IgG, DcR1-IgG and DcR2-IgG (1 μg each) suspended in monophosphoryl lipid A plus trehalose dicorynomycolate adjuvant (MPL-TDM; Ribi Immunochem. Research Inc., Hamilton, Mont.) at a 1:1 ratio of immunoadhesin:adjuvant (DcR2-IgG was only included in the mixture used for the final six immunizations).

[0176] Three days after the final boost, popliteal lymph node cells nodes were removed from the mice and a single cell suspension was prepared in DMEM media (obtained from Biowhitakker Corp.) supplemented with 1% penicillin-streptomycin. The lymph node cells were fused with murine myeloma cells P3X63AgU.1 (ATCC CRL 1597) using 35% p...

example 3

Single Antigen Immunization

[0180] The single antigen immunization scheme is shown in FIG. 2. The general method was almost the same as the mixed antigen immunization protocol in Example 2 above, except that only a single antigen was used as the immunogen and during the screening of hybridomas supernatant (180 μl) was collected only once to screen for the presence of positive monoclonal antibodies to the particular antigen and control CD4-IgG.

[0181] Balb / c mice (obtained from Charles River Laboratories) were immunized by injecting 0.5 μg / 50 μl of immunoadhesin protein (diluted in MPL-TDM adjuvant purchased from Ribi Immunochemical Research Inc., Hamilton, Mont.) 10 times into each hind foot pad at 3-4 day intervals. Three days after the final boost, popliteal lymph nodes were removed from the mice and a single cell suspension was prepared in DMEM media (obtained from Biowhitakker Corp.) supplemented with 1% penicillin-streptomycin. The lymph node cells were then fused with murine m...

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Abstract

A method of making monoclonal antibodies according to a mixed antigen immunization protocol is described. In addition, antibodies obtainable by the method are disclosed which specifically cross-react with two or more different receptors to which Apo-2 ligand (Apo-2L) can bind.

Description

RELATED APPLICATION [0001] This is a divisional of non-provisional application Ser. No. 09 / 329,633 filed Jun. 10, 1999 which claims priority under 35 USC 119(e) to provisional application No. 60 / 089,253 filed 12 Jun. 1998, the contents of which are incorporated herein by reference.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] This invention relates generally to a method for making monoclonal antibodies. The invention further pertains to antibodies obtainable by the method which specifically cross-react with two or more different receptors to which Apo-2 ligand (Apo-2L) can bind. [0004] 2. Description of Related Art [0005] Native antibodies are synthesized primarily by specialized lymphocytes called “plasma cells”. Production of a strong antibody response in a host animal is controlled by inducing and regulating the differentiation of B cells into these plasma cells. This differentiation involves virgin B cells (which have a modified antibody as a cell-surface a...

Claims

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Application Information

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IPC IPC(8): C07H21/04C07K14/705C07K16/28C12N5/06C12P21/08C12Q1/68G01N33/53
CPCA61K2039/505C07K14/70575C07K16/2875C07K2319/30C07K2316/95C07K2316/96C07K2319/00C07K16/2878C07K2317/73C07K2317/76
Inventor ASHKENAZI, AVICHUNTHARAPAI, ANANKIM, K.
Owner GENENTECH INC
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