Integrin antagonists with enhanced antibody dependent cell-mediated cytoxicity activity

a technology of cytoxicity activity and integrin antagonist, which is applied in the direction of peptides, drug compositions, and infusion cells, can solve the problems of pathological conditions, increased destruction, and ineffective current treatment options, such as surgery, chemotherapy and radiation treatment, and achieves enhanced and reduced adcc and/or cdc activity.

Inactive Publication Date: 2006-02-23
MEDIMMUNE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0034] It is a further object of the present invention to provide Fc variants that have enhanced ADCC and/or CDC activity. In one embodiment, Fc variants of the invention have ADCC and/or CDC activity that is at least 2 fold greater then that of a comparable molecule (e.g., an antibody prior to Fc modification). In another embodiment, the Fc variants of the invention have ADCC and/or CDC activity that is between about 2 fold and about 100 fold greater then that of a comparable molecule. In yet another embodiment, the Fc variants of the invention have ADCC and/or CDC activity that is between 2 fold and 100 fold greater then that of a comparable mol

Problems solved by technology

Cancerous cells destroy the part of the body in which they originate and then spread to other part(s) of the body where they start new growth and cause more destruction.
Current treatment options, such as surgery, chemotherapy and radiation treatment, are oftentimes either ineffective or present serious side effects.
Different integrins play different roles in each of these biological processes and the inappropriate regulation of their function or activity can lead to various pathological conditions.
All of these approaches pose significant drawbacks for the patient.
Surgery, for example, may be contraindicated due to the health of the patient or may be unacceptable to the patient.
Additionally, surgery may not completely remove the neoplastic tissue.
Radiation therapy is only effective when the neoplastic tissue exhibits a higher sensitivity to radiation than normal tissue, and radiatio

Method used

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  • Integrin antagonists with enhanced antibody dependent cell-mediated cytoxicity activity
  • Integrin antagonists with enhanced antibody dependent cell-mediated cytoxicity activity
  • Integrin antagonists with enhanced antibody dependent cell-mediated cytoxicity activity

Examples

Experimental program
Comparison scheme
Effect test

example 1

7.1 Example 1

Construction and Expression of Novel Fc Variants of Antibodies

[0278] Based on the structural information available for the Fc-FcγRIIIB complex, each of the putative FcγR contact residues of the IgG1 Fc portion was randomly mutated by using degenerated oligonucleotides incorporating all possible single mutations. The contact residues were divided into four regions (RI: Leu234, Leu235, Gly236, Gly237, Pro238, Ser239; RII: Asp265, Ser267, Glu269; RIII: Ser298; and RIV: Ala327, Leu328, Pro329, Ala330, and Ile332). Primers used for the amplification and library construction are listed in table 4. The IgG1 of antibody Vitaxin™, converted into scFv-Fc format, was used as the model for this study. The DNA and corresponding amino acid sequences of the variable regions of the Vitaxin® heavy and light chains used to generate the scFv-Fc are shown in FIG. 1 (panels A and B, respectively). The scFv-Fc was then harnessed as the template to build three Fc mutant libraries containing...

example 2

7.2 Example 2

Construction and Expression of the Extracellular Domains of FcγRIIIA and FcγRIIB

[0284] To facilitate the binding studies of the Fc variants to FcγRs the extracellular domains of FcγRIIIA and FcγRIIB were subcloned for expression as strepavidin fusion proteins in E. coli and for expression in mammalian cells. The FcγRIIIA prepared for analysis is the low affinity (F158) allotype. Two forms of FcγRIIIA and FcγRIIB were prepared, a “tetramer” form, generated as as Strepavidin fusion, and a “monomer” form generated as a Flag-tagged.

7.2.1 Materials and Methods

[0285] Construction and Bacterial Expression of the Extracellular Domains of FcγRIIIA- and FcγRIIB-Strepavidin Fusion Proteins (Tetramer): Primer pairs SA1 / SA2, A1 / A2, and B1 / B2 (see primer list, Table 4) were used to PCR amplify streptavidin and the extracellular domains of FcγR IIIA and FcγR IIB, respectively. The cDNA library of human bone marrow (Clontech) was used as a template for FcγR IIIA and FcγR IIB ampli...

example 3

7.3 Example 3

Characterization of the Fc Variants

[0287] After mutagenesis of the Fc domain (see example 1 supra) Fc variants, in the scFV-Fc fusion format, were screened for enhanced binding to FcγRIIIA tetramer by ELISA as detailed below. The results for several clones are shown in FIG. 5. In addition, the ADCC activity of these clones was determined against M21 cells. The results for several clones are shown in FIG. 6. Based on these studies three substitutions were chosen for further study, S239D, A330L and I332E. These substitutions were introduced into the Fc region of the intact Vitaxin® IgG1 heavy chain and coexpressed with Vitaxin® light chain to produce full length Vitaxin® Fc variant IgG1 molecules. The Vitaxin® Fc variant having the I332E substitution was designated Vitaxin®-1M, the Vitaxin® Fc variant having the S239D, A330L, I332L triple substitution was designated Vitaxin®-3M.

[0288] A panel of Vitaxin® Fc variants, in IgG format, was generated in which each of the st...

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Abstract

The present invention relates to novel Fc variants of antibodies that immunospecifically binds to Integrin αvβ3. The Fc variants comprise a variable region that immunospecifically binds to Integrin αvβ3 and a Fc region that further comprises at least one novel amino acid residue which may provide for enhanced effector function. More specifically, this invention provides Fc variants that have modified binding affinity to one or more FcγR and/or C1q. Additionally, the Fc variants have altered antibody dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) activity. The invention further provides methods and protocols for the application of said Fc variants of an antibody that immunospecifically binds to Integrin αvβ3, particularly for therapeutic purposes.

Description

1. CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit under 35 U.S.C. §119(e) of the following U.S. Provisional Application Nos. 60 / 601,634, filed, Aug. 16, 2004 and 60 / 608,852, filed, Sep. 13, 2004. The priority applications are hereby incorporated by reference herein in their entirety for all purposes.2. FIELD OF THE INVENTION [0002] The present invention provides novel antibodies comprising immunologically active fragments of immunoglobulin molecules and an Fc region that further comprises at least one novel amino acid residue of the invention. The present invention also relates to novel antibodies comprising a variable region, or fragment thereof, that immunospecifically binds to Integrin αvβ3 and a Fc region that further comprises at least one high effector function amino acid residue (e.g., 239D, 330L, 332E). The present invention further relates to novel variants of antibodies that immunospecifically bind to Integrin αvβ3 (e.g., VITAXIN® (Wu et...

Claims

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Application Information

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IPC IPC(8): C07K16/28G01N33/53C12P21/06C12N5/06
CPCC07K16/00C07K16/2848C07K16/2866C07K2317/622C07K2317/72C07K2317/52C07K2317/92C07K2319/30G01N33/566G01N33/574G01N2500/00C07K2317/732A61P1/04A61P11/00A61P11/06A61P17/06A61P29/00A61P35/00A61P35/02A61P35/04A61P43/00A61P9/00
Inventor WU, HERRENGAO, CHANGSHOU
Owner MEDIMMUNE LLC
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