Chimeric anti-VEGF-D antibodies and humanized anti-VEGF-D antibodies and methods of using same

a technology of vegf-d and chimeric antivegfd, which is applied in the field of humanized anti-vegf-d antibodies and chimeric antivegf-d antibodies, can solve the problems of embryonic lethality around midgestation, little is known about the mechanisms leading to metastasis via bloodstream or lymphatics, and the ligand for tie has not yet been identified, so as to reduce unwanted angiogenesis, improve the state of the animal, and reduce tumor cell survival

Inactive Publication Date: 2006-02-02
VEGENICS PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0061] The subject treated by the methods of the invention may be human, or any non-human animal model for human medical research, or an animal of importance as livestock or pets, (e.g., companion animals). In one variation, the subject has a disease or condition characterized by a need for modulation of angiogenesis or lymphangiogenesis, and administration of a composition comprising a humanized anti-VEGF-D antibody substance, antibody or polypeptide improves the animal's state, for example, by palliating disease symptoms, reducing unwanted angiogenesis or lymphangiogenesis, reducing tumor cell survival, or otherwise improving clinical symptoms. In a preferred embodiment, the subject to be treated is human.

Problems solved by technology

); however, the ligand for Tie has not yet been identified.
Disruption of the VEGFR genes results in aberrant development of the vasculature leading to embryonic lethality around midgestation.
However, in spite of its clinical relevance, little is known about the mechanisms leading to metastasis via the bloodstream or via the lymphatics.

Method used

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  • Chimeric anti-VEGF-D antibodies and humanized anti-VEGF-D antibodies and methods of using same
  • Chimeric anti-VEGF-D antibodies and humanized anti-VEGF-D antibodies and methods of using same
  • Chimeric anti-VEGF-D antibodies and humanized anti-VEGF-D antibodies and methods of using same

Examples

Experimental program
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example 1

Cloning of Murine / Human Heavy and Light V-Region Genes

[0288] The following procedures pertain to construction of a chimeric antibody wherein variable regions from a mouse anti-VEGF-D antibody are assembled into human constant region to create a chimeric, humanized antibody.

[0289] The monoclonal antibody used for generating a chimeric antibody was the mouse anti-VEGF-D antibody produced by the hybridoma VD1 / 4A5 [deposited in the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, on Apr. 16, 1999 (ATCC No. HB-12698]. The deposit was made under the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Production of the VD1 / 4A5 antibody is described in U.S. Pat. No. 6,383,484 (Achen et al.), incorporated herein by reference.

[0290] In order to begin constructing a chimeric VEGF-D antibody, it was first necessary to clone and sequence the light chain and heavy chai...

example 2

Construction of Human IgG1 VEGF-D Expression Vectors

[0296] Next, an expression vector comprising the mouse anti-VEGF-D antibody variable regions and human immunoglobulin constant region was assembled.

[0297] PCR was used to modify the ends of the anti-VEGF-D light chain (SEQ ID NO: 36 and 37) and heavy chain (SEQ ID NO: 38 and 39) sequences listed in FIGS. 1A and 1B. The PCR primers used to modify the 5′ and 3′ sequences flanking the cDNA sequences are set out in Table 5. The 5′ end of the cDNA flanking sequences were modified by adding a HindIII restriction site followed by a standard Kozak sequence (GCCGCCACC, SEQ ID NO: 40), (Kozak, Nucleic Acids Res 15: 8125-48, 1987). All cDNA sequences was modified at the 3′ end to include a splice donor site and an intron after the last amino acid of the variable domain. A BamHI restriction site was included in the intron for insertion into the expression vector. Thus, each heavy and light chain construct comprised a HindIII restriction site...

example 3

Chimeric Antibody Expression and Initial Characterization of Binding Activity

[0298] The chimeric anti-VEGF-D antibody construct was expressed by transient gene expression in HEK293 cells according to the method published by Meissner and colleagues (Meissner et al., Biotechnol Bioeng. 75:197-203, 2001).

[0299] Modified HEK 293-EBNA cells (Meissner et al., supra) were cultured in Ex-Cell V Pro media (Lexena, USA). Transfection of suspension adapted HEK293-EBNA cells was carried out in DMEM / F12 medium supplemented with 29 mM sodium bicarbonate, 10 mM HEPES, 2.5 mg / L human transferrin, 2.5 mg / L insulin, 0.1 mM diethanolamine, 0.1 mM L-proline, and 1% FCS (hereafter DMEM-based medium). Prior to transfection, cells were expanded in 0.5 L spinner flasks in 293G medium (Bio-Whittaker, Walkersville, Md., USA) supplemented with 1% FCS. For transfection, cells were centrifuged in 250 ml bottles for 5 minutes at 400 g and 200 ml spinner flasks were inoculated with 1×106 cells / ml freshly resusp...

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Abstract

The present invention relates to materials and methods for modulating angiogenesis and lymphangiogenesis. The compositions of the invention provide chimeric and/or humanized VEGF-D antibody substances, antibodies, polypeptides and fragments thereof useful for modulating angiogenesis and lymphangiogenesis in a subject.

Description

[0001] The present application claims the priority benefit of U.S. Provisional Application No. 60 / 550,441, filed Mar. 5, 2004, incorporated herein by reference in its entirety.FIELD OF THE INVENTION [0002] The present invention provides materials and methods relating to modulators of vascular endothelial growth factors with respect to vascularization and angiogenesis. The invention also provides therapeutic compositions for the modulation of lymphangiogenesis, and methods for ameliorating tumor growth and metastasis in patients with cancer. BACKGROUND OF THE INVENTION [0003] Angiogenesis is a fundamental process required for normal growth and development of tissues, and involves the proliferation of new capillaries from pre-existing blood vessels. Angiogenesis is not only involved in embryonic development and normal tissue growth, repair, and regeneration, but is also involved in the female reproductive cycle, establishment and maintenance of pregnancy, and in repair of wounds and f...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C07K16/44C07K16/22
CPCA61K2039/505C07K16/22C07K2316/96C07K2317/73C07K2317/56C07K2317/565C07K2317/24A61P29/00A61P35/00C07K2317/76
Inventor ACHEN, MARCSTACKER, STEVENRENNER, CHRISTOPH
Owner VEGENICS PTY LTD
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