Antibodies against human CCN1 and uses thereof

An antibody and application technology, applied in the field of antibodies against human CCN1 and its application, can solve the problems of mucin, loss, etc.

Inactive Publication Date: 2012-04-11
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because CCN1 possesses 10% cysteine ​​residues and thus presents a very

Method used

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  • Antibodies against human CCN1 and uses thereof
  • Antibodies against human CCN1 and uses thereof
  • Antibodies against human CCN1 and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] immunity

[0069] a) Immunization of mice with human mutant CCN1

[0070] 50 μg of recombinant human CCN1 containing a single amino acid substitution E173D or F185L was injected intraperitoneally with complete Freund's adjuvant on day 0, day 28 and day 56 (both with incomplete Freund's adjuvant) and Balb / c and NMRI mice were immunized with 50 μg of recombinant protein on day 84 together with incomplete Freund's adjuvant. Blood was collected on days 91 and 108 and serum was prepared for titer determination by ELISA (see below). Animals with the highest titers were selected for boosting on day 112 by intravenous injection of 50 μg of recombinant human CCN1.

[0071] b) Immunization of NZW rabbits with human mutant CCN1

[0072] 100 μg of recombinant human CCN1 containing a single amino acid substitution E173D or F185L was used on day 0 with complete Freund's adjuvant and with 100 μg protein on day 21, day 43, day 65 and day 85 with incomplete Freund's adjuvant immuniz...

Embodiment 2

[0082] Recombinant expression of membrane-bound CCN1 and CCN1 domains

[0083] Adherently grown cells were transfected with a recombinant vector encoding CCN1 or each CCN1 domain fused at the C-terminus to the transmembrane domain of the PDGF receptor (human platelet-derived growth factor receptor beta-type, UniProt accession number P09619, amino acids 513 to 561). Mouse NIH 3T3 cells (CRL-1658 TM ) or suspension-adapted human HEK293 (CRL-1573 TM )cell.

[0084] Cell binding assays, measured by FACS, were performed using suspension-adapted HEK293 cells. Adherently grown NIH 3T3 cells were used for cell binding assays analyzed in fluorescence microscopy.

Embodiment 3

[0086] Antibody binding to human CCN1 and fragments determined by ELISA

[0087] Human and mouse CCN1 ELISA

[0088] Antibody binding to human and mouse CCN1 was determined by ELISA. Recombinant human CCN1 or mouse recombinant CCN1 containing single amino acid substitutions E173D or F185L were immobilized on 384-well Nunc Maxisorp plates at 2.5 μg / ml in PBS, 25 μl / well by incubation overnight at 2-8°C. Plates were blocked with PBS / 1% BSA for 1 hour at room temperature, followed by two washing steps (0.1% Tween-20 in PBS), and incubated at room temperature with different concentrations of anti-CCN1 antibody or hybridomas in blocking buffer. The supernatants were incubated together for 1 hour. After washing four more times, anti-mouse-HRP (Amersham #NA9310), anti-rabbit-HRP (Jackson Immunoresearch #711-036-152) or anti-human IgG-HRP (Jackson Immunoresearch #109-036-097) was used to detect antibodies for 1 hour at room temperature. Signals were visualized by adding 25 μl TMB ...

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Abstract

Method for the recombinant expression of mammalian cell membrane-bound human CCN1 or a CCN1 domain thereof in mammalian cells, characterized in transforming a mammalian host cell with a vector encoding CCN1 or a CCN1 domain thereof C-terminally fused to a mammalian cell transmembrane domain (CCN1 fusion protein), expressing said CCN1 or CCN1 domain fusion protein in said host cell and recovering said membrane bound CCN1 or said CCN1 domain thereof. And use of such membrane bound CCN1 and domain for the generation of antibodies. An antibody against human CCN1 is useful for the treatment of diseases.

Description

Background of the invention [0001] The present invention relates to an antibody against human CNN1 (CCN1 antibody), its production method, a pharmaceutical composition containing the antibody and its use. [0002] CCN1 (CYR61, GIG-1, IGFBP-10, SwissProt O00622) is a growth factor-inducible immediate early gene, a member of the CCN protein family, and is involved in cell adhesion, angiogenesis, apoptosis, and growth arrest or growth stimulation (Reviewed in Chen Y. and Xiao-Yan D., J. Cell. Biochem. 100 (2007) 1337-1345 and Kubota, K. and Tagikawa, M., Angiogenesis 10 (2007) 1-11). CCN1 consists of a modular structure and contains 38 conserved cysteine ​​residues. CCN1 shares a common modular structure with other CCN proteins (except CCN5, which lacks a C-terminal module). CCN1 contains an insulin-like growth factor binding protein (IGFBP)-like motif (domain I, IGF-BP, amino acids 26-97), a von Willebrand C-type domain (domain II, VWC, amino acids 98- 164), the thrombospondi...

Claims

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Application Information

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IPC IPC(8): C07K14/475C07K16/18A61P35/00
CPCC07K16/18C07K2317/565C07K2317/92C07K14/475A61K2039/505C07K2319/03C07K2317/55C07K2317/73C07K2317/56C07K2316/96C07K2317/76A61P35/00C12N15/63A61K39/395
Inventor H.诺特金H-W.克雷尔S.米勒J.尼沃纳D.庞赛尔E.托克索兹
Owner F HOFFMANN LA ROCHE & CO AG
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