Novel humanized anti-CD22 antibody

An antibody, human-derived technology, applied in the field of bioengineering, can solve the problems of increasing antibody affinity and efficacy, and achieve the effect of reducing immunogenicity and avoiding HAMA reaction

Active Publication Date: 2013-07-24
BEIJING DONGFANG BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Researchers hoped to make mutations in the complement determining

Method used

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  • Novel humanized anti-CD22 antibody

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Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1, humanization of RFB4 antibody

[0049] The light and heavy chain genes of RFB4 are from NCBI, and the nucleic acid codes are: CS110728 and CS110730, respectively. After translating the DNA sequence into an amino acid sequence, the framework region (FR) and complementarity determining region (CDR) of the RFB4 variable region gene were analyzed according to the Kabat rules and with reference to relevant literature and patents.

[0050] Select the human antibody sequences Consensus KI and Consensus subIII with the highest homology to the framework region of the RFB4 mouse sequence in the Kabat database as the framework regions of the light chain and heavy chain of the humanized antibody, and connect the human framework region and the mouse CDR region Finally, as the initial humanized antibody hRFB4 sequence, the specific alignment results are as follows figure 1 shown.

[0051] Using the BLAST method and using PDB as the search database, the homology analys...

Embodiment 2

[0053] Embodiment 2, construction of YQ22-1 light and heavy chain expression vector

[0054] Entrust Shanghai Jierui Bioengineering Co., Ltd. to synthesize the variable region DNA sequences of YQ22-1 light chain and heavy chain. PCR primers were designed to amplify the antibody light chain and heavy chain genes respectively, and then they were subcloned into the expression vector pCDNA3.1 (Invitrogen Company) with EcoRI, PmlI and SalI, PmlI double digestion respectively,

[0055] Large-scale plasmids are prepared to transfect cells to prepare humanized antibodies. Its plasmid map is as image 3 shown.

Embodiment 3

[0056] Example 3, Expression and Purification of YQ22-1 Antibody

[0057] 1. Co-transfection of YQ22-1 light and heavy chain genes

[0058] The day before transfection, count and observe the cell viability with 6~7×10 5 293E cells were inoculated into 28ml FreeStyle 293 Expression Medium at a cell density of / ml. On the second day, the cell aggregates were shaken to break up, and samples were taken and added to trypan blue to count and observe the cell activity. The number of living cells accounted for more than 90%, and the transfection was carried out. Mix 30μg plasmid with 1ml Opti-MEMI, meanwhile, add 60μl Invitrogen 293 Fectin TM Mix reagent with 1ml Opti-MEMI. Stand at room temperature for 5 minutes. Then add the Opti-MEMI mixed with the plasmid to the Opti-MEMI mixed with the transfection reagent, and mix well to obtain a total of 2ml of the mixed solution. Let stand at room temperature for 20 minutes. At the same time, the 3×10 7 Place 2 cells in 28ml FreeStyle ...

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Abstract

The invention relates to the field of bioengineering, and specifically relates to a novel humanized anti-CD22 antibody. The novel humanized anti-CD22 antibody takes a murine antibody RFB4 as a parental antibody and uses a CDR implantation technique to carry out humanization; and the humanized new antibody hRFB4 has affinity similar with the parental antibody, and keeps the bonding capability of the parental antibody with the CD22 antigen and the cell internalizing activity of antigen-antibody complex caused by the antibody. Compared with the parental antibody, the humanized protein sequence proportion in the humanized hRFB4 antibody reaches over 90%, so that the immunogenicity is greatly reduced and the occurrence of HAMA reaction is avoided.

Description

Technical field: [0001] The invention relates to a novel humanized anti-CD22 antibody, which belongs to the technical field of bioengineering. Background technique: [0002] Non-Hodgkin lymphoma (NHL) is a group of malignant tumors originating from lymph nodes and other lymphoid tissues. It is a major type of lymphoma (except Hodgkin lymphoma). The main clinical manifestations can be summarized as follows: 1. Superficial lymph node enlargement or nodular mass formation; 2. Deep lymph node mass in the body; 3. Hyperplasia and mass of extranodal lymphoid tissue; 4. Systemic symptoms, including fever, night sweats, fatigue and weight loss. It is the second fastest growing cancer in the United States, with approximately 55,000 new cases per year. In recent years, the incidence of malignant lymphoma in my country has increased rapidly, and it has entered the ranks of the top ten high-incidence tumors. The incidence of NHL in my country has risen rapidly from 2 / 100,000 in the la...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/85C12N5/10A61K39/395A61P35/02A61P37/02
Inventor 尹琪王康郭晶晶高震孟艳敏刘志刚
Owner BEIJING DONGFANG BIOTECH
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