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Method for selecting biological binding molecules

A technology that combines molecules and biology, applied in the fields of biomaterial analysis, biological testing, chemical instruments and methods, etc.

Active Publication Date: 2021-08-13
AVA LIFESCIENCE GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such antibodies are not known in the prior art and no methods exist for making or obtaining them by selection

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The amino acid sequence of the mutant receptor is known from genetic studies on CLL. The receptor consists of two amino acid chains, a light chain (LC) and a heavy chain (HC).

[0044] R110 HC: SEQ ID NO 1

[0045] EVQLVESGGGLVKPGGSLRLSCAASGFTFRSYSMNWVRQAPGKGLEWVSSIISSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARDQNAMDVWGQGTTVTVSSDSASAPTLFPLVSCENSPSDTSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRQHPSVLRGGKYNNPLHAATSQVLLPSKDPNGNKVKVGT

[0046] R110 LC: SEQ ID NO 02

[0047] IRSLEATMAWTVLLLGLLSHCTGSVTSYELTQPPSVSVAPGKTARITCAGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSGSDHPWVFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS*EFRPS

[0048] The corresponding phylotype BCR has the following sequence

[0049] WT HC: SEQ ID NO 03

[0050] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDQNAMDVWGQGTTVTVSSDSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSD...

Embodiment 2

[0089] The same hybridoma as in Example I was used. Only the selection method is different. Selection in this case was performed using TKO cells that had previously been incubated to expose to BCR antigen (5 pg / ml, 5 minutes). This forms the binding of the antigen to the BCR. Activation of the BCR (and actual internalization of the BCR) does not occur because SLP65 is not induced. Cells are then separated. A portion was measured in a BD Fortessa II FACS device for functional testing. This device allows the measurement to be interrupted and the recording to be repeated with new parameters. Most other FACS equipment cannot do this. For this, cells were incubated with indole-1 according to the manufacturer's instructions for 45 min prior to measurement. Indole-1 produces a fluorescent signal upon calcium influx into the cell, which can then be measured by the FACS device. Measure these cells for one minute to generate a baseline. 4-OH-Tamoxifen (2mM) was then added, there...

Embodiment III

[0091] Cells were then incubated with potential inhibitors (5 μg / ml, 5 min) prior to stimulation. These cells are then stimulated with an agonist (antigen, see Example 2) (or in the case of autonomously active cells without stimulation, since the stimulator is on the cell itself) and the subsequent Ca signaling will be inhibited with an inhibitor Compared to the Ca signal of cells without inhibitor. Suppression means that the CA signal is significantly smaller (at least 50% of the net contrast signal) in cells with the inhibitor.

[0092] To screen in this case the following were used: (induced means, the cells had been previously treated with hydroxytamoxifen)

[0093] Induced control group (transformation vector without BCR)

[0094] Induced cells: transformed with a vector carrying DNA encoding the CLL-R110G BCR

[0095] Induced cells: transformed with a vector carrying DNA encoding for the germline-type BCR to form R110G

[0096] In the case of BCRs that are not autono...

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PUM

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Abstract

The present invention relates to the field of producing, identifying, and selecting biological binding molecules, e.g. in particular antibodies or fragments thereof, which selectively bind to somatically hypermutated B-cell receptors or B-cell receptor complexes. The method is used in order to select a biological binding molecule which specifically binds to a B-cell receptor having hypermutated regions as the target receptor, but not to a B-cell receptor without hypermutated regions, and is carried out in a cell-based system using immature B cells which are in the pro / pre stage and cause a 'Triple Knockout' of the genes for RAG2 or RAG1, lambda5, and SLP65.

Description

technical field [0001] The present invention relates to the field of preparation, identification and selection of biological binding molecules, such as in particular antibodies or fragments thereof which selectively bind to somatically hypermutated B cell receptors or B cell receptor complexes. Background technique [0002] In biochemistry, a protein or protein complex is called a receptor (Latin for recipere, meaning " accepted" or "accepted"). Receptors can receive signals from the outside and be located at the surface of biofilms, or they can be detected in the cytosol. Receptors have specific binding sites for their physiological agonists or antagonists (binding partners, ligands). [0003] Membrane receptors are located at the surface of biological membranes and consist of proteins that are often modified (eg carbohydrate chains). They have specific passage shapes for small molecules (so-called ligands) or for parts of larger molecules that bind to the receptor struc...

Claims

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Application Information

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IPC IPC(8): G01N33/50
CPCG01N33/5052G01N33/56972C07K16/2803C07K2317/565G01N33/566G01N2500/10G01N33/57484G01N33/6854
Inventor H·克洛夫特M·杜伦-冯·明登
Owner AVA LIFESCIENCE GMBH
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