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Multimeric hybrid fc proteins for replacement of ivig

A protein and duplex technology, applied in hybrid immunoglobulin, anti-animal/human immunoglobulin, immunoglobulin, etc., can solve the problem of short serum half-life

Pending Publication Date: 2021-08-17
JN BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Fc-μTP-L309C potently inhibits inflammatory arthritis and ITP in mice, it has a short serum half-life in human FcRn transgenic mice (3.1 hours) and rats (2.5 to 3 hours)

Method used

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  • Multimeric hybrid fc proteins for replacement of ivig
  • Multimeric hybrid fc proteins for replacement of ivig
  • Multimeric hybrid fc proteins for replacement of ivig

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1: General Methods and Materials

[0090] Using standard laboratory techniques (e.g. Green and Sambrook (Molecular Cloning, A Laboratory Manual, Fourth Edition, 2012, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Greenfield (Antibodies, A Laboratory Manual, Second Edition, 2014, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Kostelny et al. (Int. J. Cancer 93:556-565, 2001), Cole et al. (J. Immunol. 159:3613-3621, 1997), and Tsurushita Manipulation of recombinant DNA and expression, purification and characterization of recombinant proteins were performed as described by et al. (Methods 36:69-83, 2005).

[0091] Mammalian expression vector pVF101 ( figure 1 ) was designed to generate a multimeric hybrid Fc protein comprising an artificial signal peptide (sp) from N-terminus to C-terminus, a hinge, the CH2 and CH3 regions of the human IgG1 isotype, followed by human Cμ3 and Cμ4 region, containing the following genetic components. ...

Embodiment 2

[0100] Example 2: Expression and purification of multimer hybrid Fc protein

[0101] The expression vectors pVF102 and pVF103 were introduced into the chromosome of Chinese hamster ovary cell line CHO-K1 to obtain cell lines that stably produce LS41K-Fc.S and LS41K-Fc.SL, respectively. CHO-K1 cells were grown in SFM4CHO medium (GE Healthcare, Chicago, IL) at 37°C in 7.5% CO 2 Grow in an incubator. It was stably transfected into CHO-K1 by electroporation. Before transfection, each expression vector was linearized using FspI. In a typical experiment, approximately 10 7 Each cell was transfected with 20 μg of linearized plasmid, suspended in SFM4CHO medium, and seeded into several 96-well plates after appropriate dilution of cells. After 48 hours, puromycin was added to isolate stable transfectants. Approximately twelve days after initiation of selection, culture supernatants from transfectants were assayed for antibody production.

[0102] Expression of LS41K-Fc.S and LS41...

Embodiment 3

[0104] Example 3: Analysis of Pharmacokinetics (PK) and Pharmacodynamics (PD) of LS41K-Fc.S in Mice

[0105] Fifty (50) μg of mouse monoclonal anti-human CD122 IgG1 antibody ABC2 was intracardially administered to each group in the presence and absence of 400 μg LS41K-Fc.S in 50 μl of PBS (groups A and B, respectively) in three Balb / c mice. On the day before dosing (Day -1), two hours after dosing (2HR), one day (Day 1), three days (Day 3), five days (Day 5) and eight days (Day 8) Serum samples were then collected from these mice.

[0106] ABC2 concentrations in serum samples were measured by ELISA as described above. ABC2 concentrations at each time point (Day 1, Day 3, Day 5, and Day 8) were normalized to the concentration in the 2HR sample of each mouse. The data is plotted on image 3 middle. Mean relative concentrations of ABC2 (ABC2 only) in Group A were 100% (2HR), 52.0% (Day 1), 36.7% (Day 3), 29.9% (Day 5) and 19.3% (Day 8) . In contrast, the mean percentage co...

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PUM

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Abstract

The hybrid Fc proteins of this invention include IgG and IgM Fc components. The IgG Fc component includes at least a portion of a hinge region and CH2 and CHS regions. The IgM component includes C[mu]3 and C[mu]4 regions of a C[mu] constant region. The hybrid Fc proteins can form duplexes by interchain disulfide bonding between cysteines in their hinge regions. The hybrid Fc proteins can be used for treating immune disorders mediated by endogenous IgG, such as those previously treated with intravenous immunoglobulin.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US 62 / 767,303, filed November 14, 2018, which is hereby incorporated by reference in its entirety for all purposes. [0003] sequence listing [0004] This application includes a 85KB sequence listing written in txt file 538890WO-ST25, created on October 24, 2019, incorporated by reference. Background technique [0005] Intravenous immunoglobulin (IVIG), a pooled IgG preparation from thousands of healthy human donors, has been used as a human therapy for immunodeficiency and immune-mediated diseases (Nimmerjahn et al., Annu. Rev. Immunol. 26:513-533,2008; Nagelkerke et al.,Front.Immunol.5:Article674,2015;Mitrevski et al.,Front.Immunol.6:Article 4,2015;Seite et al.,Arthritis Rheum.67:595 -603, 2015; Afonso et al., Biomolecules 6:15, 2016; Lazarus, Chapter 6 (Chapter 6) in Imbach (eds), Antibody Therapy, Springer, 2018). IVIG, administered at a dosage range of 200 to 500 mg / kg b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395C07K16/28C07K16/46
CPCC07K2317/52C07K2317/524C07K2317/71C07K16/00C07K2317/526A61K2039/505C07K2317/90C07K2317/94C07K2317/41C07K2317/14C07K2317/24A61K2039/507C07K16/2866C07K16/2863A61K38/00C07K14/47
Inventor 鹤下直哉J·云·曹
Owner JN BIOSCI
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