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Heteromultimers with reduced or silenced effector function

An effector function and multimer technology, applied in the field of polypeptides containing heterodimeric Fc regions, can solve problems such as adverse side effects

Inactive Publication Date: 2016-08-10
ZHENHUO BIOPHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, for certain disease indications, Fc region-mediated effector functions of antibodies may cause undesirable side effects, and therefore, efforts have been made to design antibodies with reduced or silenced effector functions

Method used

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  • Heteromultimers with reduced or silenced effector function
  • Heteromultimers with reduced or silenced effector function
  • Heteromultimers with reduced or silenced effector function

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0263] Example 1: Preparation and expression of antibody constructs (heteromultimers)

[0264] The following antibody constructs were prepared. All antibody constructs were based on the sequence of the wild-type anti-Her2 antibody trastuzumab (see Figure 5 , SEQ ID NO:2 is the amino acid sequence of the wild-type trastuzumab heavy chain, and SEQ ID NO:3 is the amino acid sequence of the wild-type trastuzumab light chain), introducing the following modifications added in the heavy chain CH3 domain , to promote the formation of a heterodimeric Fc domain with increased stability compared to a CH3 domain that does not contain amino acid mutations.

[0265] Chain A: T350V / L351Y / S400E / F405A / Y407V, and

[0266] Chain B: T350V / T366L / N390R / K392M / T394W

[0267] This construct with the above modifications was designated v791. All sequences described herein are numbered using the EU numbering system.

[0268] Additional variants were constructed based on v791 with amino acid modific...

Embodiment 2

[0280] Example 2: Trastuzumab-based asymmetric antibodies do not bind FcγRs

[0281] The ability of asymmetric antibody constructs to bind to FcyRIIaH, FcyRIIaR, FcyRIIb FcyRIIIaF, FcyRIIIaV and FcyRIa was assessed by surface plasmon resonance (SPR).

[0282] Affinity of FcyRs to antibody Fc was measured by SPR using a ProteOn XPR36 using 10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.05% Tween 20 at 25°C. Recombinant HER-2 was captured on an activated GLM sensor chip as follows: 4.0 μg / mL recombinant HER-2 in 10 mM NaOAc (pH 4.5) was injected at 25 μL / min until approximately 3000 resonance units were immobilized. , RU), to quench the remaining active groups. After injection with buffer to establish a stable baseline, 40 μg / mL of purified HER-2 / neu-based antibody was captured indirectly with an injection at 25 μL / min for 240 s (resulting in approximately 500 RU). FcyRs were injected at 60 μL / min for 120 seconds with a 180 second dissociation phase to obtain a set of bi...

Embodiment 3

[0294] Example 3: Trastuzumab-based asymmetric antibodies do not bind C1q

[0295] Asymmetric antibody constructs were tested for their ability to bind CIq as follows. Human C1q was purchased from GenWay Biotech (San Diego, CA). SPR chip immobilization of antibodies was performed as described in Example 2. Also as described in Example 2, 30 nM C1q was injected over the mAb variants captured on the surface of the HER2 SPR using standard protocols. The results are shown in Table C below.

[0296] Table C: Results of the C1q binding assay

[0297] Variants

C1q 1

WT

yes

Control / 1051

NB

AAC1

part

AAC2

NB

AAC3

NB

AAC4

NB

AAC5

NB

AAC6

NB

AAC7

NB

AAC8

NB

[0298] 1. C1q is a hexamer of heterotrimers with a possible stoichiometric ratio of mAb:C1q of 6:1. Binding kinetics are very complex and the appropriate Kd may not be determined. Receptors were tes...

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Abstract

Provided herein are heteromultimer constructs with reduced or silenced effector function. In an embodiment is provided a heteromultimer construct comprising an IgG Fc construct having a first and a second Fc polypeptide, each Fc polypeptide comprising a modified lower hinge region wherein: the modified lower hinge region of said first Fc polypeptide comprises at least one amino acid modification, the modified lower hinge region of said second Fc polypeptide comprises at least one amino acid modification which is different from at least one amino acid modification of said first Fc polypeptide, and the IgG Fc construct displays reduced binding to all Fc Gamma receptors and to C1q protein as compared to a corresponding parent IgG Fc construct. Also provided are methods of producing such heteromultimer constructs, and methods of reducing ADCC for an antibody construct by reducing effector function.

Description

[0001] cross reference [0002] This application claims the benefit of US Provisional Application No. 61 / 829,973, filed May 31, 2013, which is hereby incorporated by reference in its entirety for all purposes. [0003] introduce 2.1 Field of Invention [0004] The present invention relates to the field of therapeutic antibody design, and in particular to polypeptides comprising a heterodimeric Fc region that has been modified to silence effector functions mediated by the Fc region. 2.2 Background of the invention [0005] Therapeutic antibodies have been developed to treat a variety of disease indications. In some of these cases, the efficacy of a therapeutic antibody is due at least in part to the ability of the antibody Fc region to mediate one or more effector functions. These effector functions are due to the interaction of antibodies and antibody-antigen complexes with cells of the immune system to stimulate a variety of responses, including antibody-dependent cell-me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46A61K39/395C07K16/00C07K16/28C12N15/13
CPCC07K16/2887C07K16/32C07K2317/526C07K2317/71C07K2317/92C07K2317/94C07K16/00C07K2317/53A61P17/00A61P17/02A61P17/06A61P19/02A61P25/00A61P25/04A61P25/28A61P29/00A61P3/00A61P37/00A61P7/04A61P9/00A61P9/04A61P9/10C07K16/468C07K2317/41
Inventor E·埃斯科巴-卡布瑞拉
Owner ZHENHUO BIOPHARMACEUTICAL CO LTD
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