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Novel antibody delivery system for biological response modifiers

a biological response modifier and antibody technology, applied in the field of monoclonal antibodies and biological response modifier conjugates, can solve the problems of inability to use specific delivery systems for targeting tissues or cells, inability to conjugate antibodies to biological response modifiers such as tumor necrosis factor, and insufficient prior art to treat a wide variety of human cancers, etc., to achieve the effect of reducing damage or injury, and reducing the risk of infection

Inactive Publication Date: 2005-05-12
ROSENBLUM MICHAEL G
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0022] It is a further object to provide a method of suppressing secondary cataract formation which comprises administration of the immunoconjugate of the present invention to an individual after the surgical replacement of the optic lens.

Problems solved by technology

Most of the commonly utilized alternative therapeutic modalities such as irradiation or chemotherapy do not confine their effects solely to the tumor cells and, although they have a proportionally greater destructive effect on malignant cells, often affect normal cells to some extent.
Although antibodies have been used as delivery systems for toxic moieties of plant toxins and other cytotoxic drugs, conjugation of antibodies to biological response modifiers such as tumor necrosis factor and the use of such conjugates as specific delivery system to target tissues or cells has not heretofore been possible.
The prior art remains deficient in the lack of effective means to treat a wide variety of human cancers.

Method used

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  • Novel antibody delivery system for biological response modifiers
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  • Novel antibody delivery system for biological response modifiers

Examples

Experimental program
Comparison scheme
Effect test

example 1

Purification of TNF

[0055] TNF may be purified by techniques known to these in the art. For instance, Aggarwal et al. describes the purification of TNF from various sources including several cell lines of monocytic origin. Aggarwal (1986) Methods of Enzymology 116:448, incorporated herein by reference. This method may also be utilized to purify TNF from other sources.

[0056] TNF is preferably obtained by recombinant technology known to those of skill in the art. Such a preparation is, for example, described in detail in U.S. Pat. No. 4,677,063 and European Publication EP 186,214. The TNF preparation used to obtain the following data depicted in the following Examples was obtained from Genentech Corp., South San Francisco, Calif. The human recombinant DNA derived material was purified to homogeneity from extracts of E. coli. TNF migrated as a single band with an approximate molecular weight of 18,000 daltons.

example 2

Assay of TNF Cytotoxic Activity

[0057] The TNF cytotoxic activity was monitored utilizing the following assay on L-929 cells. Forty thousand murine L-929 fibroblasts in MEM media containing 10% FCS were added to each well of a 96 well plate and incubated 24 hours at 37° C. (5% CO2). Cells were then treated with various amounts of either TNF or TNF-antibody conjugate in medium containing 0.5 g / ml Actinomycin-D for 24 hrs at 37° C. (5% CO2). The cells were washed with phosphate buffered saline (PBS), pH 7.2 and viable cells were stained with crystal violet. The plates were read at 590 nm to determine viable cell number. TNF with a specific activity no lower that 1×107 U / mg was used for conjugation with the antibodies. A unit of TNF activity is the amount of TNF protein which causes 50% inhibition of L-929 cell growth.

example 3

Modification of TNF with Iminothiolane

[0058] TNF in phosphate buffered saline was concentrated to approximately 2 milligrams / ml in a Centricon 10 microconcentrator. Triethanolamine hydrochloride (TEA / HCl), pH 8.0 and EDTA were added to a final concentration of 60 mM TEA / HCl and 1 mM EDTA pH 8.0. 2-Iminothiolane stock solution (20 mM) was added to a final concentration of 1 mM and the sample was incubated for 90 minutes at 4° C. under a stream of nitrogen gas.

[0059] Excess iminothiolane (IT) was removed by gel filtration on a column of Sephadex G-25 (1×24 cm) pre-equilibrated with 5 mM bis-tris / acetate buffer, pH 5.8 containing 50 mM NaCl and 1 mM EDTA. Fractions were analyzed for protein content in microtiter plates using the Bradford dye binding assay. Briefly, forty microliters of sample, 100 ul of phosphate buffered saline (PBS) and 40 ul of dye concentrate were added to each well. Absorbance at 600 nm was read on a Dynatech Microelisa Autoreader. TNF elutes at the void volume...

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Abstract

The present invention provides a novel conjugate comprising an antibody directed toward a cell surface associated antigen, wherein said antigen is selected from the group consisting of 15A8 antigen and ZME-018 antigen; and a biological response modifier moiety, wherein said moiety is selected from the group consisting of TNF-alpha, TNF-beta and Interleukin-1. In addition, the present invention also provides a method of treating proliferative cell diseases comprising administration of a cytocidally effective dose of the composition of claim 1 to an individual in need of said treatment.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation in part of U.S. Ser. No. 08 / 119,505, filed Sep. 10, 1993, which is a continuation of U.S. Ser. No. 07 / 951,357, filed Sep. 25, 1992, now abandoned, which is a continuation of U.S. Ser. No. 07 / 348,237, filed May 05, 1989, now abandoned.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates generally to the field of immunoconjugates. More specifically, the invention relates to conjugates of monoclonal antibodies and biological response modifiers. [0004] 2. Description of the Related Art [0005] Biological response modifiers exhibit a variety of effects upon a number of cell types. Mammalian cells produce lymphokines and cytokines to maintain homeostasis at the cellular level. At physiological or in pharmacological concentrations, biological response modifiers such as the interferons, the interleukins and tumor necrosis factor (TNF) all have cytotoxic effects. TNF ...

Claims

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Application Information

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IPC IPC(8): A61K47/48
CPCA61K47/48569A61K47/6851
Inventor ROSENBLUM, MICHAEL G.
Owner ROSENBLUM MICHAEL G
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