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Constitutively active uPAR variants and their use for the generation and isolation of inhibitory antibodies

A variant, antibody technology, applied in the direction of antibodies, peptide/protein components, antibody mimetics/scaffolds, etc., can solve problems such as no functional blocking activity, no defined binding site, etc.

Inactive Publication Date: 2014-07-16
IFOM FOND INST FIRC DI ONCOLOGIA MOLECOLARE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The binding site of the peptide in uPAR is not identified in the literature and there are no data on the function blocking activity of the peptide

Method used

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  • Constitutively active uPAR variants and their use for the generation and isolation of inhibitory antibodies
  • Constitutively active uPAR variants and their use for the generation and isolation of inhibitory antibodies
  • Constitutively active uPAR variants and their use for the generation and isolation of inhibitory antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Materials and Methods

[0112] Construction of expression vector

[0113] The expression vector for recombinant proteins with a human IgG constant region (hFc) tag was based on the pFRT / TO-Fc plasmid (Madsen et al., 2007), however, many modifications were introduced to facilitate shuffling of different coding regions and facilitate protein production. First, the XhoI restriction site located downstream of the vector sequence in the hFc coding region was destroyed by site-directed mutagenesis using oligonucleotides dXu / dXd. Second, a linker encoding the cleavage sequence of the PreScission protease prepared by annealing the oligonucleotides PreF / PreR was inserted into the XhoI site at the signal peptide / Fc junction. To remove the intron present in the Fc region of the construct (which was found to increase the yield of recombinant protein (our unpublished observation)), the vector was transfected into CHO cells, RNA was extracted, reverse transcribed , then amplified...

Embodiment 2

[0177] Materials and Methods

[0178] Antigen preparation

[0179] According to the detailed description of Example 1, the GFD uPAR-hFc and GFD uPAR-mFc was expressed and purified.

[0180] Immunization of mice

[0181] Contains 67 μg by intraperitoneal (i.p.) injection GFD 200 μl of a 1:1 emulsion of uPAR-hFc (100 μl of immunogen in PBS and 100 μl of complete Freund's adjuvant (CFA)) to immunize three 2-month-old male C57Bl / 6uPAR - / - Mice (Ms # 21574、Ms. # 1416 and Ms. # 1417). The immunized animals contained 67 μg by IP injection at 3-week intervals GFD 200 μl of a 1:1 emulsion of uPAR-hFc (100 μl of immunogen in PBS and 100 μl of incomplete Freund's adjuvant (IFA)) boosted 3 times. After a 7-week break, use 200 μg 4 days before fusion GFD uPAR-hFc in 200 μl PBS i.p. to Ms # 21574 applies a final pre-fusion immune boost. Contains 34 μg at 3-week intervals GFD 200 μl of 1:1 PBS / IFA emulsion of uPAR-hFc to Ms # 1416 and Ms. # 1417 applied three additional IP ...

Embodiment 3

[0227] Materials and Methods

[0228] mGFD Cloning of muPAR-Fc

[0229] coding mGFD The expression vector for muPAR-Fc was generated by amplifying mouse uPA cDNA with oligonucleotides muPAkf / mGFDr and mouse uPAR cDNA with oligonucleotides muL8f / MUPPFCR. The two PCR products were mixed, co-amplified with oligonucleotides muPAkf / MUPPFCR, and cloned with KpnI / XhoI into the vector pFRT / TO-Fc. The protein encoded by this vector ( mGFD muPAR-Fc, sequence 9 (SEQ ID NO: 17)) consists of 49 N-terminal residues of mouse uPA including a growth factor domain (GFD, sequence 9A (SEQ ID NO: 4)), a short linker (amino acid GGAGAAGG, sequence 9B (SEQ ID NO:8)), residues 1-273 of mouse uPAR (sequence 9C, corresponding to amino acids 1-273 of SEQ ID NO:2), the second short linker (amino acid VELEVLFQGPIE, sequence 9D (SEQ ID NO: 11)) and a human Fc tag (Sequence 1C (SEQ ID NO: 5)).

[0230] oligonucleotide sequence

[0231] muPAkf:5'-GGGGTACCATGAAAGTCTGGCTGGCGAG-3' (SEQ ID NO:54)

[02...

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Abstract

The invention relates to variants of the urokinase plasminogen activator receptor (uPAR) that display remarkably increased vitronectin (VN) binding activity, possibly caused by a more efficient exposure of the VN binding site. The present invention also refers to antibodies raised against said uPAR variants, able to bind to the VN binding site of uPAR and then acting as inhibitors of uPAR functions, acting as functional antagonists of VN activated-uPAR functions. In the present invention such antibodies are monoclonal, polyclonal, synthetic or recombinant derivatives thereof, as synthetic antibodies (scFv) from phage-display libraries. Antibodies of the invention act as competitive antagonists.

Description

Background technique [0001] The urokinase-type plasminogen activator receptor (uPAR, also known as CD87) is a membrane glycoprotein anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. The urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play important roles in physiological processes (such as wound healing, inflammation and stem cell mobilization) and severe pathological conditions (such as HIV-1 infection, tumor invasion and metastasis). ) plays an important role. The urokinase-type plasminogen activator receptor (uPAR) is a plasma membrane receptor overexpressed during inflammation and in almost all human cancers. The essential role of uPAR in tumor cell adhesion, migration, invasion and proliferation makes this receptor an attractive drug target in cancer therapy. In terms of inhibiting tumor growth, several therapeutic options have been or are being developed to inhibit the uPA system. In addition to uPAR's well-defined role...

Claims

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Application Information

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IPC IPC(8): A61K38/49
CPCA61K38/00A61K39/395C07K14/705A61K2039/505C07K2317/34C07K2317/565C07K2317/622C07K2317/73C07K2317/76C07K2319/30C07K16/2896A61P13/08A61P35/00C07K14/70596
Inventor N·西顿纽斯S·甘地
Owner IFOM FOND INST FIRC DI ONCOLOGIA MOLECOLARE
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