Monoclonal antibodies directed to cd20

a monoclonal antibody and cd20 technology, applied in the field of monoclonal antibodies, can solve the problems of limited veterinary medicine arsenal, affecting the effect of veterinary medicine, and generally only addressing symptoms

Inactive Publication Date: 2017-01-05
ARATANA THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The arsenal of veterinary medicine is thus limited when it comes to addressing immune conditions and cancer.
Additional drawback of these treatments is that they generally only address symptoms and they are associated with serious side effects as large doses have to be administered repeatedly for a long period of time with cumulative effects that often tend to be worse than the disease itself.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning of Canine and Feline CD20

I. Cloning of Canine CD20.

[0154]The canine CD20 gene can be cloned into a mammalian expression vector and the corresponding plasmid DNA transfected into mammalian cells. Cells expressing CD20 can be used for immunization and cell-screening based assays.

[0155]CD20 are isolated from canine peripheral blood mononuclear cells (PBMC). Total RNA is extracted from 1 million canine PBMC using the MasterPure™ RNA Purification Kit (Epicentre Biotechnology). The first-strand cDNA is synthesized from 2 μg of total RNA using the First-Strand Synthesis System for RT-PCR kit (Invitrogen) according to the manufacturer's instructions. The coding region is amplified with primers of SEQ ID NO:3 and SEQ ID NO:4 and a fragment thereof encompassing the large extracellular domain (loop) are amplified with primers of SEQ ID NO:5 and SEQ ID NO:6 by PCR. The samples are denatured at 94° C. for 5 min followed by amplifications for 35 cycles (94° C. for 30 s, 62° C. for 20 s, 7...

example 2

Immunization with CD20 and Generation of Murine Monoclonal Antibodies to Canine CD20

[0159]To generate monoclonal antibodies to canine CD20, CHO-DG44 (Chinese hamster ovary cells, dihydrofolate reductase deficient ATCC CRL-9096) and NIH:3T3 (ATCC CRL-1658) are transfected with an expression vector encoding the full-length canine CD20 protein. Magnetic Proteoliposome Particles (MPLs) containing CD20, such that the native conformation of the transmenbrane receptor is maintained are prepared for immunizations and panning. In brief, recombinant canine CD20 that contains an epitope tag are solubilized from a transfected CD20-expressing cell line using the detergent CHAPSO and the protein is captured on magnetic beads via the epitope tag. A lipid membrane is reconstituted during removal of the detergent, such that the native membrane conformation of CD20 is maintained, to create the CD20-MPLs.

[0160]Anti-CD20 monoclonal antibodies are generated by immunization of mice to raise immunoglobuli...

example 3

Heterochimeric Antibodies

[0166]The following EXAMPLE provides general representations of heterochimeric antibodies, which are constructed according to standard techniques using the sequences and general patterns illustrated below. In the examples listed below, the CDRs are defined using the Kabat nomenclature.

I. Antibody Variable Domains.

[0167]Illustrated in Table 1, are diagrammatic representations of the heterochimerization for the light chain (AVD1 to AVD10) and heavy chain (AVD11 to AVD13) antibodies, showing contiguous sequences of discrete immunoglobulin domains. Additional antibody variants are constructed by flanking the variable regions from the donor species with any of the constant domains from the target species.

TABLE 1AVD 1:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4T-Lambda-CT-LambdaAVD 2:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4T-Kappa-CT-LambdaAVD 3:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4T-Lambda-CT-KappaAVD 4:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4T-kappa-CT-KappaAVD 5:FR1T-Lambda-CDR1-FR2-CDR2-FR3-CDR3-FR4...

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Abstract

The invention provides antibody to canine or feline or equine antigens. Specifically, antibodies directed to canine CD20 which have been caninized or felinized are provided. Also provided are methods for preparing high affinity antibodies to canine and feline CD20 as well as methods for treating B cell disorders in companion animals.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of priority under 35 USC §119 of U.S. Provisional Application Ser. No. 61 / 310,440 filed Mar. 4, 2010, the contents of which are incorporated herein by reference.FIELD OF THE INVENTION[0002]The present invention generally relates to monoclonal antibodies, including portions or variants thereof, directed to CD20 for the treatment of diseases, e.g., in mammals and particularly in companion animals, such as dogs, cats and horses. More particularly, the invention provides antibody constructs, and antibodies encoded by the constructs, which react with CD20 and are useful for detection of targets, diagnosis of disease and treatment of companion animals. Further disclosed herein are methods for the treatment of B cell disorders in companion animals. These methods are based upon the administration of an anti-CD20 antibody or antibodies targeting the CD20 of a target animal for the modulation of B-lymphocytes.BAC...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/28A61K39/395
CPCC07K16/2887C07K2317/94C07K2317/732C07K2317/734C07K2317/62C07K2317/24C07K2317/52C07K2317/76A61K2039/505A61K2039/552A61K2039/572C07K2317/565C07K2317/567C07K2317/92A61K39/3955C07H21/00C07K16/28A61K39/395C07K16/2896
Inventor HANSEN, GENEVIEVE
Owner ARATANA THERAPEUTICS
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