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A kind of anti-human rank1 humanized antibody and its pharmaceutical composition and application

A humanized antibody, selected technology, applied in drug combinations, antibody mimics/scaffolds, antibodies, etc., can solve problems such as decreased antibody affinity and conformational changes in CDRs regions

Active Publication Date: 2019-03-08
GENOR BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have proved that among all the amino acids in the FRs region, the humanized substitution of most amino acids has only a slight impact on the conformation of the CDRs region, and will not have a serious impact on the antibody affinity, but there are also a few key amino acids, Once these key amino acids are replaced by the corresponding amino acids of adults, it will cause a large change in the conformation of the CDRs region, which will cause a serious decrease in antibody affinity.

Method used

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  • A kind of anti-human rank1 humanized antibody and its pharmaceutical composition and application
  • A kind of anti-human rank1 humanized antibody and its pharmaceutical composition and application
  • A kind of anti-human rank1 humanized antibody and its pharmaceutical composition and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] Embodiment 1, the preparation of human source RANKL

[0152] By constructing a human RANKL expression vector and stably transfecting CHO cells, a cell line capable of stably and highly expressing human RANKL was screened. The cell line was cultivated on a large scale, the cell supernatant was collected, and purified by a nickel column to prepare human RANKL protein, which was used for mouse immunization, clone screening and functional identification in Examples 2, 3, 4, 5, 6 and 7.

[0153] CHO cells were purchased from Invitrogen Company, RAW264.7 cells were purchased from Shanghai Chinese Academy of Sciences Cell Bank, the expression vector was provided by our company, T4DNA ligase, protein molecular weight standard marker, restriction endonuclease, etc. were purchased from NEB Company; gel recovery kit was purchased from From Invitrogen; 302 medium, trypsin and FBS were purchased from Invitrogen; Phenylsepharose6FF-low sub, SP-Sepharose FF, Ni-NTA Sepharose FF gel we...

Embodiment 2

[0165] Embodiment 2, mouse immunization and titer determination

[0166] The antigen used for immunization (recombinant human RANKL) was from Example 1, and the BALB / c mice were purchased from Beijing Tonglihua Experimental Animal Technology Co., Ltd. Monoclonal antibody against RANKL was obtained by immunizing BALB / c mice several times. The way of immunization was plantar injection, and the immunization dose was 10 μg / 50 μl / mouse, 25 μl per foot. A total of 10 mice were immunized.

[0167] For the first immunization, mix 10 μg human RANKL with an equal volume of TiterMax (Sigma, Oakville, ON) for 10 subsequent immunizations. Mix 10 μg human RANKL with an equal volume of 100 μg alum (Sigma, Oakville, ON) and 10 μl pyrogen-free CpG-containing D-PBS without adjuvant . Immunization of BALB / c mice occurred on days 0, 5, 10, 15, 20, 25, 30, 35, 40 and 44, respectively, and four mice with the highest serum titers were taken for fusion on day 44.

[0168] After the fifth (20th ...

Embodiment 3

[0172] Example 3, Generation of anti-human RANKL mouse monoclonal

[0173] Immunize mice with CO 2 After euthanasia, the cervical vertebrae were dislocated, the lymph nodes were isolated, and the lymph nodes from different mice were combined. Put it into DMEM medium for grinding, collect the supernatant, centrifuge to obtain lymphocytes, and count them with a hemocytometer.

[0174] Wash the B cells obtained above, and mix them with non-secretory myeloma cells P3X63Ag8.653 (ATCC, Cat#CRL1580) at a ratio of 1:1, centrifuge the cell mixture at 800g, remove the supernatant gently, and add 2-4ml Pronase solution (CalBiochem, cat.#53702; 0.5mg / ml in PBS), the action does not exceed 2 minutes, add 3-5ml fetal bovine serum to terminate the enzyme reaction, add cell electrofusion solution ECFS (0.3M Sucrose, Sigma, Cat#S7903, 0.1mM MagnesiumAcetate, Sigma, Cat#M2545, 0.1mM Calcium Acetate, Sigma, Cat#C4705) Adjust the volume to 40ml, centrifuge, remove the supernatant, resuspend the...

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Abstract

The present invention provides an anti-human RANKL humanized antibody and its pharmaceutical composition and use. The heavy chain variable region of the humanized antibody is selected from the amino acid sequence shown in SEQ ID NO: 6, 23, 25, 27 or 29 , the light chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 14, 31, 33, 35, 37 or 39, preferably, the heavy chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 29, The light chain variable region is selected from the amino acid sequence shown in SEQ ID NO:31. The antibody can be used to prepare medicine for treating bone loss diseases.

Description

[0001] This application claims priority from PCT / CN2014 / 095236 filed on December 28, 2014. Background technique [0002] Human bone is a dynamic and ever-changing tissue. After osteoclasts absorb bone, osteoblasts form bone, completing a normal bone remodeling cycle. The rate of bone remodeling in adults is 2-10% per year. Under normal circumstances, bone resorption and bone formation (bone metabolism) maintain a dynamic balance. After the balance between osteoclasts and osteoblasts is broken, it will cause too much bone (osteosclerosis) or too little bone (osteoporosis) . [0003] Osteoclast differentiation signals are transmitted by osteoblast / mesenchymal stem cells. Various factors that stimulate bone resorption transmit the signal to induce osteoclast formation to osteoblast / mesenchymal stem cells, and induce the expression of osteoclast differentiation factor-1 (Osteoclast differentiation factor, ODF-1) on the membrane, also known as the nucleus Factor-κB receptor acti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28A61K39/395A61P19/10A61P19/02A61P19/08
CPCA61P19/02A61P19/08A61P19/10A61P29/00A61P37/02C07K16/2875C07K2317/24C07K2317/33C07K2317/622C07K2317/76C07K2317/92C07K2319/30C07K2317/51C07K2317/515C07K2317/52C07K2317/56C07K2317/565
Inventor 周清李玲云
Owner GENOR BIOPHARMA
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