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Novel anti-human NGF antibody

An antibody and fragment technology, applied in the field of anti-human NGF antibodies, can solve the problem of high incidence of arterial thrombosis, and achieve the effects of expanding the dosing interval, reducing the risk of side effects and reducing the dosage of the drug

Active Publication Date: 2014-04-23
ASTELLAS PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has also been reported that patients with metastatic cancer who received chemotherapy and Abastin had a higher incidence of arterial thrombosis compared with the placebo group who received chemotherapy alone (Non-Patent Document 20)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] (Example 1: Immunization to VelocImmune mice)

[0110]Antibodies to human NGF were obtained by immunizing VelocImmune mice. In order to increase the diversity of the obtained antibodies, the present inventors conducted research on various immunization methods, administration routes, adjuvants, immunization time, and the like. Using human βNGF (R&D System Co., Ltd.) as an immunogen, the method of dissolving human βNGF and mixing it with an adjuvant for immunization, and heat denaturation of human βNGF (under 0.5% SDS solution, 80°C, 10 minutes) and The method of immunization with adjuvant mixture was studied. As the route of administration, plantar administration and intraperitoneal administration were investigated. As adjuvants, TiterMax Gold (CytRx), complete Freund's adjuvant (Sigma), incomplete Freund's adjuvant (Sigma), and RIBI adjuvant (Corixa) were investigated. In addition, as further added immune activators, CpG oligonucleotides and aluminum phosphate gel (B...

Embodiment 2

[0112] (Example 2: Production of hybridomas producing anti-human NGF antibodies)

[0113] Final immunization (intravenous or intraperitoneal administration of the antigen) was performed on the mice selected after confirming the increase in the antibody titer. Lymphocytes were collected by removing the spleen, lymph nodes, etc. of the immunized mice according to a conventional method, and they were fused with mouse myeloma cells SP2 / 0 to prepare hybridomas. The hybridomas were limitedly diluted and monoclonalized, and on this basis, antibodies were purified from the supernatant using protein A or protein G columns (GE HEALTHCARE).

Embodiment 3

[0114] (Example 3: Evaluation of NGF-trkA binding inhibition)

[0115] Human βNGF (R&D System Company) and EZ-LINK5-(biotinamide) pentylamine (PIERCE Company) were reacted at room temperature and in the dark for 30 minutes to carry out biotin labeling, and excess biotin was removed with a desalting column to obtain biotin Tagged human βNGF. In addition, in the following Examples 6 and 7, it was confirmed that the biological activity of the produced biotin-labeled human βNGF was equivalent to that of native human βNGF.

[0116] Inhibitory activity was measured using the following method. 60 ng of human trkA (R&D Systems) was added to each well of a white Maxisorp384 plate (Nunc), and incubated overnight at 4°C to immobilize it. On the next day, after washing the plate once with TBST washing solution, a blocking agent (TBST containing 20% ​​Blocking One (Nacalai Tesque)) was added, and left to stand at room temperature for 1 hour. Next, the biotin-labeled human βNGF (0.2 μg / m...

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PUM

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Abstract

Provided are an anti-human NGF antibody which is reduced in the influence on fetuses and the risk of adverse side effects including thrombosis while keeping a high neutralizing activity, and which has excellent safety, or an antigen-binding fragment thereof; and a means, utilizing the antibody or an antigen-binding fragment thereof , for preventing or treating various diseases for which human NGF is involved in the development of a disease state. An Fab' fragment of an anti-human NGF antibody comprises a heavy-chain variable region comprising the amino acid sequence represented by SEQ ID NO: 6 and a light-chain variable region comprising the amino acid sequence represented by SEQ ID NO: 4.

Description

technical field [0001] The present invention relates to novel anti-human NGF antibodies, more specifically to Fab' fragments of anti-human NGF antibodies. Background technique [0002] Nerve growth factor (NGF) is one of the humoral factors collectively called neurotrophic factors, which play an important role in the occurrence, differentiation and function maintenance of neurons in organisms. As receptors for NGF, there are known high-affinity trkA receptors (receptor-type tyrosine kinases) and low-affinity p75NTR receptors. Among them, it has been reported that p75NTR binds to all neurotrophic factors and is involved in apoptosis in the process of neurogenesis, but its role has not been fully clarified. On the other hand, it is known that NGF and trkA receptor knockout mice show the same phenotype (Non-Patent Document 1), and it is considered that the physiological action of NGF is mainly expressed through the trkA receptor. [0003] In 1993, it was reported that pain co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K16/22C12N1/15C12N1/19C12N1/21C12N5/10C12P21/08
CPCC07K16/22C07K2317/33C07K2317/21C07K2317/76C07K2317/94A61K2039/505A61K47/48215C12N2510/02C07K2317/55C07K2317/24A61K47/60C07K2317/51C07K2317/515C07K2317/565
Inventor 蒲原正纯田中祐嗣神谷由香里高崎淳米泽敦夫吉见英治
Owner ASTELLAS PHARMA INC
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