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Crystal structure of neuropilin fragment in complex with neuropilin antibody

A complex and fragment technology, applied in the field of crystal structure of neuropilin fragment and neuropilin antibody complex, can solve problems such as expression inhibition

Inactive Publication Date: 2015-09-09
GENENTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Expression in bacterial cells is inhibited if the antibody or antigen-binding domain is not properly folded

Method used

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  • Crystal structure of neuropilin fragment in complex with neuropilin antibody
  • Crystal structure of neuropilin fragment in complex with neuropilin antibody
  • Crystal structure of neuropilin fragment in complex with neuropilin antibody

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Experimental program
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Embodiment approach

[0177] Based on the domain architecture and functional characteristics, the extracellular domain of Nrp is usually divided into three functional units: a1a2, b1b2, and c, which represent their main brain signal protein binding, VEGF binding, and dimerization regions ( Ellis, LM, Mol Cencer Ther 5, 1099-107 (2006)). Although structure-function studies describe the overall domain requirements of neuropilin function, the molecular details of these receptor / ligand complexes are still poorly understood. So far, structural information about Nrp is limited to the b1b2 domain of Nrp1 (Vander Kooi et al., supra; Lee, C. et al., J Biol Chem 281, 5702-10 (2006)). The crystal structure of Nrp1 b1b2 complexed with Tuftsin (a tetrapeptide homologous to the C-terminus of VEGF165) provides first-hand structural information about the interaction of Nrp with its binding partner VEGF (VanderKooi, et al., see above ; Von Wronski, MA, et al., J. Biol Chem 281, 5702-10 (2006)).

[0178] The present ...

Embodiment 1

[0309] Example 1: Anti-Nrp1 B And anti-pan Nrp A Construction and function of antibodies

[0310] Liang et al., J Mol Biol 366, 815-29 (2007) and Pan et al., Cancer Cell 11, 53-67 (2007) reported a strategy for developing phage-derived antibodies that selectively block brain signaling protein or VEGF binding to Nrp1. These monoclonal antibodies are designed as tools for distinguishing Nrp1-mediated various responses and for evaluating their potential as therapeutic agents in murine tumor models.

[0311] In short, a human synthetic antibody phage library was designed, which was built with VH / VL diversity on a single consensus scaffold. The details of the design and selection of this antibody library are described in Liang et al., see above, and can also be found in WO 03 / 102157 published on December 11, 2003, the complete disclosure of which is incorporated herein by the above. Since then, the library has generated functional blocking antibodies against human and murine NRP1. The...

Embodiment 2

[0314] Example 2: Structure study of neuropilin / antibody complex

[0315] Materials and Method

[0316] Functional assays and antibody binding affinity

[0317] The collapse assay and the determination of anti-Nrp antibody binding affinity were performed as previously described (He, Z and Tessier-Lavigne, M., Cell90, 739-51 (1997); Liang et al., supra and Pan et al., supra Text).

[0318] Expression and purification of proteins for crystallographic research

[0319] Nrp1-b1, Nrp1-b1b2, and Nrp2-b1b2 (see Table S1 for domain boundaries) were cloned into pET15b (Novagen) and expressed in E. coli at 37°C (Nrp1-b1 and -b1b2) or 16°C (Nrp2-b1b2) Incubate. All neuropilin b-type fragments are expressed as soluble proteins and do not require a refolding scheme. After the cells were lysed, the protein was purified using tweezers-nitrilotriacetic acid (Ni-NTA) resin in 50 mM Tris (pH 8.0), 300-500 mM NaCl, and 20 mM imidazole, and eluted in the same buffer plus 250 mM imidazole. Use thrombin...

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Abstract

The invention relates to a crystal structure of neuropil protein fragment and neuropil protein antibody compound, and provides a separate crystal structure or a crystal structure of neuropil protein 1 (Nrp1) and neuropil protein 2 (Nrp2) fragments which are combined with anti-neuropil protein antibodies and an application method thereof. The invention further provides an anti-Nrp antibody and a treatment application method thereof.

Description

[0001] This application is a divisional application of the invention application with the filing date of May 17, 2007, the Chinese application number being 200780053793.1, and the invention title being "Crystal Structure of Neuropilin Fragment and Neuropilin Antibody Complex". Invention field [0002] The present invention provides the crystal structure of neuropilin 1 (Nrp1) and neuropilin 2 (Nrp2) fragments alone or in combination with an anti-neuropilin antibody and their use. The present invention further provides antibodies that bind to Nrp1 and / or Nrp2 and methods of use thereof. Background of the invention [0003] The development of the vascular system is the basic requirement of many physiological and pathological processes. Actively growing tissues such as embryos and tumors require adequate blood supply. They meet this need by producing pro-angiogenic factors that promote the formation and maintenance of new blood vessels through a process commonly referred to as angio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K14/71G01N33/68
Inventor 布伦特.A.阿普尔顿克里斯琴.威斯曼吴雁
Owner GENENTECH INC
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