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Novel method for producing antibodies

A technology for antibodies and antigens, applied in the field of preparation of fully human antibodies

Active Publication Date: 2020-05-01
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, people have carried out in-depth research and development on in vitro immunization technology, making this technology no longer require animal immunization, thus reducing its cost, making the operation more convenient and quicker, and obtaining fully human antibodies without any humanization steps. However, Few successful antibody preparations using the above methods have been reported

Method used

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  • Novel method for producing antibodies
  • Novel method for producing antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0162] Example 1: Materials and methods

[0163] Material

[0164] Lymphocyte Separation Medium: LSM, Lymphocyte Separation Medium (MP, cat.V0111A)

[0165] L-leucine methyl ester: LLME, L-leucyl-L-leucine methyl ester (BacheM, cat.G-2550.0001)

[0166] Ham's F-12 Nutrient Mixture: Ham's F-12 Nutrient Mixture (Gibco, cat.11765047)

[0167] Heparin anticoagulation tube: Heparin anticoagulation tube (BD, cat.367878)

[0168] Disposable blood collection needle: Disposable blood collecting needle (BD, cat.367237)

[0169] IL2 / Interleukin-2 / lymphokine / TCGF: IL2 / Interleukin-2 / lymphokine / TCGF(sinobiological, cat.11848-HNAY1-50)

[0170] BCGF-1 / BCGF1 / BSF-1 / BSF1 / IL-4 / Interleukin-4: BCGF-1 / BCGF1 /

[0171] BSF-1 / BSF1 / IL-4 / Interleukin-4 (sinobiological, cat. GMP-11846-HNAE-100)

[0172] CD154 / CD40 Ligand: CD154 / CD40 Ligand (sinobiological, cat.10239-H01H-50)

[0173] OX40L (sinobiological, cat.13127-H04H-100)

[0174] Human ICOS Ligand / B7-H2 / ICOSLG (histidine tag): Human ICOS Li...

Embodiment 2

[0226] Embodiment 2: IL2 stimulates PBMC proliferation

[0227] PBMCs include populations of antibody-producing B cells, T cells, and dendritic cells. Expansion of these cells in vitro can form germinal center-like structures. The result is as figure 1 shown. In the graph, "control group" represents cells without antigen or any stimulator; all other bars represent cells treated with antigen TrkA as well as different factors. It is to be noted that IL2 is the most potent stimulator of cell proliferation.

Embodiment 3

[0228] Example 3: ICOSL is a key stimulator for inducing antibody production

[0229] During PBMC expansion, ICOSL was added to the culture medium along with the antigen TrkA and other stimulators. We found that a stimulator cocktail including ICOSL, in combination with other key components CD40L, IL2, IL21, and CpG-ODN, enhanced human antibody (IgM and IgG) synthesis / production in B cells after 10-14 days in culture. ICOSL is also a key stimulator, which induced the highest antibody levels. The result is as figure 2 As shown in A-2B, it shows that ICOSL and CD40L synergistically promote the production of IgG, far exceeding the effect of ICOSL or CD40L alone.

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PUM

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Abstract

Disclosed is a method for producing an antibody or an antigen-binding fragment thereof comprising a step of cultivating PBMCs in a medium comprising CD40L, ICOSL, ICOS, and / or TLR agonist. Also provided herein is a method for inducing proliferation of PBMCs, B cell activation and differentiation, and / or B cell maturation, comprising a step of cultivating PBMCs in a medium comprising IL2. Also provided herein is a method for promoting class switch in an antibody-producing PBMC to produce IgG, comprising a step of cultivating the antibody-producing PBMC in a medium comprising IL21.

Description

[0001] field of invention [0002] The present invention mainly relates to a new method for preparing antibodies, especially an in vitro method suitable for preparing fully human antibodies. [0003] Background of the invention [0004] Antibody preparation methods are widely used in laboratories and clinics. The antibody preparation process involves hybridoma technology, transgenic animal models and in vitro immunization. Traditional hybridoma technology has been accepted by most people as a mainstream and mature technology, usually including animal immunization, lymphocyte separation, fusion of lymphocytes and immortalized cells such as myeloma, antibody humanization and affinity maturation and other steps. Antibodies can also be prepared by high-throughput screening methods, but they also face disadvantages such as high cost, long production cycle, low affinity, and unpredictable pairing of heavy chain and light chain variable regions. Transgenic animal models are a relati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078C07K16/00
CPCC07K16/00C07K2317/14C12N5/0634C07K16/28
Inventor 鲁白姚虹洋郭炜
Owner TSINGHUA UNIV
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