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Complement component C5 antibodies

An antibody and complement technology, which is applied in the field of cell surface protein CD59 assembled by blocking MAC and can solve the problems such as loss of C5a functional activity and so on.

Active Publication Date: 2017-09-26
ALLERGAN INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In doing so, however, they also block the formation of C5a, resulting in loss of C5a functional activity that has been associated with tissue homeostasis (removal of opsonizing particles), neural survival, and anti-angiogenic responses

Method used

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  • Complement component C5 antibodies
  • Complement component C5 antibodies
  • Complement component C5 antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0300] Example 1 - Immunization and Hybridoma Formation

[0301] For the production of hybridomas and monoclonal antibodies, immunization and screening are performed basically as follows: Antibodies, Laboratory Manual, Cold Spring Harbor Laboratory. The procedure specific to the production of anti-C5 monoclonal antibodies as described in this application is briefly described as follows: 75 μg of human C5 in complete Freund's adjuvant was used by footpad injection ( Cat. No. A403), followed by a second boost on day 28 by intraperitoneal (I.P.) administration of 75 μg of C5 protein with incomplete Freund's adjuvant to immunize against complement C5 (Jackson Bar Harbor maine) deficient B10.D2-Hc O H2 d H2-T18c / 02SnJ mice. ELISA screening for serum titers for reactivity to C5 protein was performed 9-10 days after the second boost. For the initial group of fusions, mice showing favorable titers were immunized with a fusion booster (75 μg C5 in pBS, I.P.) on days 82, 83, a...

Embodiment 2

[0302] Example 2 - Hybridoma Culture

[0303] Hybridomas were maintained in DMEM containing 15% Fetal Clone II, OPI, HAT, non-essential amino acids, and recombinant mouse IL-6. Hybridoma supernatants were screened by enzyme-linked immunoassay (ELISA) to detect anti-human C5 antibodies. Positive cultures of C5 were expanded in DMEM containing 15% Fetal Clone II, OPI and non-essential amino acids and subcloned twice by effective dilution. Hybridomas were subcloned to isotype with the SBA Clonotyping System / HRP (SouthernBiotech) according to the manufacturer's protocol.

Embodiment 3

[0304] Example 3 - Cloning and sequencing of monoclonal variable heavy and light chain domains

[0305] After re-amplification by RT-PCR, the variable light (VL) and heavy (VH) domains were cloned. Briefly, using the Total RNA Isolation Kit Total RNA was isolated from selected subcloned hybridoma cell lines. Using First Strand cDNA Synthesis Kit Perform cDNA synthesis. The forward primers were specific for the N-terminal amino acid sequences of the VL and VH regions, and the LC and HC reverse primers were designed to anneal to regions in the constant light chain domain (CL) and constant heavy chain domain 1 (CH1). Primers used for recloning are listed below. Amplified VL or VH fragments are isolated and subcloned into Carrier ( life ) and sequenced using standard methods.

[0306]

[0307]

[0308] Perform PCR as follows:

[0309] cDNA5μL

[0310] 10x PCR buffer 5 μL

[0311] dNTP 1μL

[0312] Primer Mix 2.5 μL

[0313] Polymerase 1 μL

[0314] dH2O ...

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Abstract

The present disclosure relates to antibodies and polynucleotides encoding the same, that may be used to prevent, control, or reduce the activity of the complement pathway. In addition, the disclosure is directed to compositions and methods for diagnosing and treating diseases mediated by or involving complement C5. Specifically, the disclosure is related to C5 antibodies.

Description

[0001] cross reference [0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 61 / 768,374, filed February 20, 2014, and U.S. Provisional Application Serial No. 61 / 944,943, filed February 26, 2014, so Both provisional applications are incorporated herein by reference. [0003] invention technical field [0004] The present disclosure relates to antibodies and compositions thereof, polynucleotides encoding the antibodies, expression vectors and host cells for producing the antibodies, and compositions and methods for diagnosing and treating diseases mediated by complement. Background of the invention [0005] The complement system consists of nearly 50 individual proteins that function as part of the innate immune system, providing the initial stages of host defense, opsonization of foreign bodies, and tissue homeostasis. (Ricklin D., 2010, Complement: a Key system for immune surveillance and homeostasis. Nature: Immunology,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18A61K39/395A61P27/02
CPCA61K2039/505C07K16/18C07K2317/24C07K2317/565C07K2317/76A61K39/00A61P27/02A61P43/00A61K39/0005C07K2317/56A61K39/3955
Inventor P·C·巴修Y·梁J·古M·贝尔内特U·穆赫哈尔J·戴斯扎拉斯
Owner ALLERGAN INC
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