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Engineered fc

A technology of antigen-binding molecules and amino acids, which is applied in the field of molecular biology and can solve problems such as contamination of antibody preparations

Pending Publication Date: 2021-06-04
HUMMINGBIRD BIOSCI HLDG PTE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such antibody preparations are often contaminated with fucosylated antibodies, limiting the increase in Fcγ receptor binding activity on fucosylated antibody preparations to ~3-fold (see e.g. Chung et al., MAbs (2012) 4 (3): 326-340)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0307] Example 1: Preparation of Antigen Binding Molecules Comprising Engineered Fc Regions

[0308] The present inventors prepared antigen-binding molecules comprising heavy chains including amino acid substitutions at positions in the CH2 and / or CH3 regions to study the consequences of the substitutions on Fc effector functions.

[0309] An antigen-binding molecule is prepared comprising: (i) a light chain comprising a light chain variable region (VL) and a constant region light chain (CK) of an antibody specific to HER3, and (ii) a heavy chain comprising an antibody specific to HER3 Chain variable region (VH) and human immunoglobulin G1 (G1m3 allotype) heavy chain constant region 1 (CH1), hinge region, heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3).

[0310] The CH2 and CH3 regions are either unsubstituted or have the following substitution combinations:

[0311]

[0312] Antigen-binding molecules were expressed using 1) Expi293 Transient ...

Embodiment 2

[0325] Example 2: Thermal Stability Analysis of Antigen Binding Molecules Comprising Engineered Fc Regions by Differential Scanning Fluorescence analysis

[0326] The thermal stability of the antigen-binding molecules prepared as described in Example 1 was evaluated by differential scanning fluorescence.

[0327] Briefly, reaction mixtures of 0.2 mg / ml of antibody and Sypro orange dye (Thermo Fisher) were prepared in 25 μl PBS in triplicate and transferred to MicroAmp Optical 96-well reaction plates (Thermo Fisher). wells and sealed with MicroAmp Optical Adhesive Film (Thermo Fisher). Melting curves were run on a 7500 Fast Real-Time PCR System (Applied Biosystems) with TAMRA selected as the reporter gene and ROX as the reference fluorescence. The thermal profile included an initial step of 2 minutes at 25°C and a final step of 2 minutes at 99°C with a ramp rate of 1.2%. Plot the first derivative of the raw data as a function of temperature to obtain a derivative melting ...

Embodiment 3

[0337] Example 3: Antigen-binding molecules containing an engineered Fc region directed against the human Fc receptor FcγRIIIA-158V Affinity analysis

[0338] Antigen binding molecules prepared as described in Example 1 were evaluated for binding to the human Fc receptor FcyRIIa by biolayer interferometry (BLI) using the Pall ForteBio Octet Red384 system.

[0339] Anti-penta-his (His1k) biosensors were purchased from Forte Bio (18-5120) and incubated in PBS buffer (pH 7.2) for 60 s to obtain the first baseline, followed by grouping in PBS pH 7.2 Amino acid-tagged human FcγRIIIa-158V was loaded for 120 seconds. After loading, the biosensor was incubated in PBS buffer (pH 7.2) for 60 seconds to obtain a second baseline, and then the antigen-binding molecules were tested in a diluted series (at concentrations ranging from 15.6 nM to 500 nM) in PBS, pH 7.2. ) for 60 seconds to obtain an association curve. Finally, the biosensor was incubated in PBS pH 7.2 for 120 s to obtain...

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Abstract

Antigen-binding molecules comprising an Fc region comprising a polypeptide having: (i) C at the position corresponding to position 242, and C at the position corresponding to position 334, and (ii) one or more of: A at the position corresponding to position 236, D at the position corresponding to position 239, E at the position corresponding to position 332, L at the position corresponding to position 330, K at the position corresponding to position 345, and G at the position corresponding to position 430 are disclosed. Also discloses are constituent polypeptides of such Fc regions, nucleic acids encoding such antigen- binding molecules and polypeptides, compositions comprising such antigen-binding molecules, polypeptides and nucleic acids, and methods using the same.

Description

[0001] This application claims priority from GB 1817354.2, filed 25 October 2018, the contents and elements of which are hereby incorporated by reference for all purposes. [0002] field of invention [0003] The present invention relates to the field of molecular biology, more specifically antibody technology. [0004] Background of the invention [0005] Two main (non-mutually exclusive) strategies to modulate (enhance or attenuate) antibody FC effector functions (ADCC, ADCP, CDC) are by altering Fc:Fc receptor and Fc:complement component 1q (C1) interactions. [0006] The most common approach involves providing amino acid substitutions to the polypeptide chain of the Fc region to generate a symmetrical (homodimeric) IgG molecule. [0007] Alternatively, antibodies can be glycoengineered; the most common strategies include modification of N-linked oligosaccharides by manipulating the glycan biosynthetic pathway in host cells, and glycan remodeling in vitro. Modifications in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K16/32A61K39/395A61P35/00
CPCC07K16/32C07K16/00A61K2039/505C07K2317/72C07K2317/732A61P35/00C07K2317/92C07K2317/524C07K2317/526C07K16/283
Inventor J·D·博伊德-柯克普P·英格拉姆V·桑塞农
Owner HUMMINGBIRD BIOSCI HLDG PTE LTD
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