Anti-IgM monoclonal antibodies and methods of their use

Details Anti-IgM monoclonal antibodies and methods of their use Anti-IgM monoclonal antibodies and methods of their use

1994-02-07

2001-10-23

Caputa, Anthony C (Department: 1642)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Using tissue cell culture to make a protein or polypeptide

C424S178100, C424S183100, C530S391100, C530S391300, C530S391700, C435S449000

Reexamination Certificate

active

06306626

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of immunology and monoclonal antibody production. More particularly, the present invention concerns anti-IgM monoclonal antibodies and methods for their use.
2. Description of the Related Art
Since the initial description of monoclonal antibodies, the development of technology to produce immortalized lymphocytes capable of producing antibodies of predetermined specificity has had a major impact on both clinical and basic scientific research as well as the therapeutic modalities available for the diagnosis and treatment of a wide variety of pathological conditions.
Antibodies are endogenous proteins produced by the immune system in response to antigenic stimuli. These proteins specifically bind to antigen molecules at defined sites (epitopes). Polyclonal antibodies are derived from immunization of animals with antigens and bind to these antigens at multiple epitopes. Monoclonal antibodies, on the other hand, are a specific, defined set of antibodies which are derived from a single clone (monoclone) of cells producing a specific antibody. In contrast to polyclonal antibodies monoclonal antibodies bind to only one specific epitope on the antigen molecule.
Although the technology for the generation of monoclonal antibodies has existed for some time, the current methodology is time-consuming, laborious and often results in the production of antibodies which, although specific for the target antigen, are of relatively low affinity for the antigen, and thus are of limited usefulness in a wide variety of applications.
Among the difficulties encountered in the production of useful and clinically relevant monoclonal antibodies is the abundance of antibodies of the IgM sub-type obtained from hybridomas produced by standard in vivo or in vitro immunization procedures. The IgM sub-type antibodies are generally of low affinity, are difficult to purify and often comprise the bulk of antibodies produced by hybridomas. In addition, in mixed cultures of IgM and IgG secreting hybridoma cells, IgM secreting cells often overgrow the IgG secreting hybrid cells.
Part of the laborious procedure for the production of hybridomas is the elimination of the IgM producing hybridoma cells produced after a cell fusion. This is generally done by cloning the cells by limiting dilution, growing up the individual cells into colonies, and testing each colony individually to determine which colonies produce IgG sub-type antibodies. Generally, the IgG producing hybridoma cells are then further analyzed to determine the antigen specificity of the antibodies produced.
Although antibodies have been reported which are directed against epitopes on the IgM antibody, all tested to date were also reactive with IgG sub-type antibodies.
Linking cytotoxic agents to antibodies to make “immunotoxins” has been disclosed by the applicants and others. Recent interest has centered on immunotoxins of monoclonal antibodies conjugated to the enzymatically active portions (A chains) of toxins of bacterial or plant origin via hetero-bifunctional agents. Nevelle, D. M. and Youle, R. J.,
Immunol Rev
(1982) 62: 75-91; Ross, W. C. J., et al.,
European J Biochem
(1980) 104; Vitteta, E. S., et al.,
Immunol Rev
(1982) 62: 158-183; Raso, V., et al.,
Cancer Res
(1982) 42: 457-464; Trowbridge, I. W. and Domingo, D. L.,
Nature
(
Lond
) (1981) 294: 171-173.
SUMMARY OF THE INVENTION
A principal aspect of the invention concerns rat monoclonal antibodies that: (a) bind selectively to IgM sub-type antibodies; (b) are IgGs; and (c) do not bind to IgG
1
or IgG
2
sub-type. The preferred embodiment of these antibodies is one designated 2G10, and functional equivalents thereof.
The rat x rat hybridomas that produce the above described antibodies and progeny of those hybridomas are other aspects of the invention.
The invention also includes a method of preparing a hybridoma as defined above comprising fusing rat tumor cells with rat splenocytes obtained from a rat immunized with murine IgM sub-type immunogen and selecting for hybridomas producing antibody as defined above.
A further aspect of the invention is a method of producing antibody as defined above comprising culturing a hybridoma having the ability to produce such antibody, or optionally a hybridoma which has been prepared by effecting a method as described above.
Another aspect of the invention relates to immunotoxins and their preparation by conjugating (a) the above described monoclonal antibodies, and (b) a cytotoxic moiety or magnetic beads.
Another aspect of the invention concerns labeled derivatives of the above described monoclonal antibodies that are labeled with a detectable label that permits the derivatives to be used in targeting, specific selection or sorting.
Another aspect of the invention concerns a method of killing IgM producing hybridoma or B cells by contacting the cells with a cytocidally effective amount of one or more of the above described immunotoxins.
Other aspects of the invention are direct and indirect immunoassays for determining whether a cell is producing IgM antibodies or to determine whether an antibody is of the IgM isotype. These assays involve incubating the cells with the monoclonal antibodies or labeled derivatives thereof. When the labeled derivatives are used, the presence of labeled binary immune complexes on the cells is read directly. When an unlabeled antibody is used, the cells are further incubated with a labeled antibody against the monoclonal antibody and the presence of labeled ternary immune complexes on the cells is read.


REFERENCES:
Julius, Eur. J. Immunol, 14:753-7 1984.*
Kung et al., J. Immunol. 127:873-6 1981.*
Lambert et al. J. Biol. Chem. 260:12035-41, 1985.*
Taylor et al., Nature New Biology 233:225-9, 1971.*
Uletta et al. Science 238: 1098-1104, 1987.*
DeClercq et al. Mtds Inenzymology 121: 234-238, 1986.*
Brady et al., J Radation Oncol., Biol., Phys 13: 1535-44, 1987.

Affiliated with

Also associated with

Rating

Anti-IgM monoclonal antibodies and methods of their use does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Anti-IgM monoclonal antibodies and methods of their use, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Anti-IgM monoclonal antibodies and methods of their use will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2617474