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Humanized anti-CD20 monoclonal antibody with high affinity

A monoclonal antibody, affinity technology, applied in the field of genetic engineering, can solve problems such as low immunogenicity, and achieve the effect of low immunogenicity

Inactive Publication Date: 2017-08-11
LIAOCHENG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] It can be seen from the above that when the predecessors modified antibodies, they only modified one of them, and they have not found that both of them are designed and modified at the same time to obtain antibody drugs with both low immunogenicity and high affinity.

Method used

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  • Humanized anti-CD20 monoclonal antibody with high affinity
  • Humanized anti-CD20 monoclonal antibody with high affinity
  • Humanized anti-CD20 monoclonal antibody with high affinity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] A high-affinity humanized anti-CD20 monoclonal antibody, said antibody comprises optimized CD20H, CD20L nucleotide sequence or optimized CD20H, amino acid sequence encoded by CD20L, wherein, CD20H is CD20 heavy chain variable region and constant The CD20L is obtained by joining the Fc sequence of the CD20 light chain variable region and the constant region; the nucleotide sequences of CD20H and CD20L are respectively optimized by CHO cell preferred codons, and the eukaryotic expression original Kozak sequence is added at the N-terminus , signal peptide sequence and start codon ATG, and add a Hind III restriction site at the 5' end, add an EcoRI site at the 3' end to obtain optimized CD20H and CD20L nucleotide sequences, and optimize CD20H nucleotides The sequence is SEQ ID NO: 1, the optimized CD20L nucleotide sequence is SEQ ID NO: 2, the optimized CD20H encoded amino acid sequence is SEQ ID NO: 3, and the optimized CD20L encoded amino acid sequence is SEQ ID NO: 4.

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Embodiment 2

[0140] Testing methods and results of final products

[0141] Detection of CD20mRNA Expression by Reverse Transcription PCR

[0142] G418 and Zeocin were used for pressurized screening of co-transferred CHO-k1 cells, and total RNA was extracted from the stable expression cell lines obtained by pressurized screening (see Figure 6A ). The total RNA was then reverse-transcribed to obtain cDNA, which was then identified by PCR. The identification result shows that the specific amplification size is 1423bp, the target gene of 717bp (see Figure 6B ). This indicated that the constructed recombinant expression vector was successfully transfected into CHO-k1 cells and expressed successfully.

[0143] Determination of antibody content in supernatant of 17 monoclonal cell lines by ELISA

[0144] Use Her 2 antibody as a standard curve (see Figure 7 ), the antibody content in the supernatant was measured to be between 0.45-4.72 μg / mL (see Table 2-3). It can be seen from the table...

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Abstract

The invention relates to the technical field of genetic engineering and discloses a humanized anti-CD20 monoclonal antibody with high affinity. The antibody comprises optimized CD20H and CD20L nucleotide sequences, wherein CD20H is obtained by connecting a CD20 heavy chain variable region with a constant region Fc sequence, and CD20L is obtained by connecting a CD20 light chain variable region with a constant region Fc sequence; the CD20H and CD20L nucleotide sequences are optimized by virtue of a CHO cell preference codon, an eukaryotic expression original Kozak sequence, a signal peptide sequence and an initiation codon ATG are added at the tail end of N, a Hind III restriction enzyme cutting site is added at a 5' terminal, and an EcoRI site is added at a 3' terminal, so that the optimized CD20H and CD20L nucleotide sequences are obtained. According to the invention, by virtue of a large number of experiments, the humanized anti-CD20 monoclonal antibody with high affinity is finally determined, and the antibody has low immunogenicity and high affinity at the same time.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a high-affinity humanized anti-CD20 monoclonal antibody. Background technique [0002] Non-Hodgkin lymphoma (NHL) is a common malignant tumor of the lymphoid hematopoietic system in clinical practice, and non-Hodgkin lymphoma is a common B-cell lymphoma. With the increasing incidence of NHL year by year, chemotherapy drugs are the most important way to treat NHL. Although, chemoradiotherapy drugs are of great significance to effectively control the patient's condition and prolong the remission period. However, when patients undergo radiotherapy and chemotherapy, it can not only kill tumor cells, but also have a certain killing effect on normal cells in the human body, and then affect the patient's digestive function and bone marrow function to varying degrees, and at the same time produce platelets and white blood cells A series of adverse reactions such as hair los...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/85A61K39/395A61P35/00A61P35/02
CPCA61K2039/505C07K16/2887C07K2317/64C07K2317/732C07K2317/734C07K2317/92C12N15/85C12N2800/22
Inventor 郭尚敬张倩倩陈坤
Owner LIAOCHENG UNIV
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