Antibody derivatives

a technology of antibodies and derivatives, applied in the field of antibodies derivatives, can solve the problems of marked reduction of production yield of these disulfide, hampered wide-spread development of bispecific antibodies, and difficulty in producing materials of sufficient quality and quantity

Inactive Publication Date: 2014-05-08
MEDIMMUNE LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0056]It is not excluded that antibodies bind to p40 and inhibit IL-23 yet do inhibit IL-12—such antibodies are included within the scope of “anti-IL-23 antibodies”.
[0057]In an embodiment the present invention provides anti_IL-23/IL-12 antibodies, or de

Problems solved by technology

The widespread development of bispecific antibodies has been hampered by the difficulty of producing materials of sufficient quality and in sufficient quantity for in vivo preclinical and clinical studies.
However, a marked reduction in production yield has been reported for these disulfide-stabilized bispecific diabodies in E-coli.
Providing such therapies in the form of a bispecific construct (e.g. one that is specific fo

Method used

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Examples

Experimental program
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Effect test

example 1

Construction of IL6 and IL23 Expression Clones and Purification of Fusion Proteins

[0404]Human IL-6 (DQ891463; ABM82389.1, SEQ ID NOs. 343 and 344) was expressed as a 3×FLAG-IL6-Avi fusion. The expression construct was generated by a two-step polymerase chain reaction (PCR) amplification. The product was inserted into the plasmid p3xFLAG-CMV-23 (Sigma), downstream of the CMV promoter and in frame with the pre-pro-trypsin leader sequence and triple FLAG tag. The expressed IL-6 (SEQ ID NO. 1) contained an amino terminal triple FLAG tag (amino acid sequence dhdgdykdhdidykdddd; SEQ ID NO. 345) and a carboxyl-terminal Avi tag (glndifeaqkiewhe; SEQ IS NO. 346) (ALLO66-MM4-80) The IL6 coding region was amplified by PCR and inserted into p3xFLAG-CMV-23 to express 3×FLAG-IL6 and 3×FLAG-IL6-myc fusions. (SEQ ID NO. 2)

[0405]The coding region of the macaca mulatta IL-6 (mmIL-6; NM—001042733.1; NP—001036198; SEQ ID NO. 346 and SEQ ID NO. 347) was generated by overlapping oligomers and PCR (DNA2.0...

example 2

Determination of Antibody Affinity

[0411]Equilibrium dissociation constants were determined by surface Plasmon resonance using a SensiQ Pioneer (ICx Nomadics, Stillwater, Okla.) and a carboxylated COOH1 sensor (Ibid) amenable for amine coupling. Protein G (6510-10, Biovision, Mountain View, Calif.) was coupled to the COOH1 sensor using amine coupling reagents (Sigma Aldrich (N-Hydroxysuccinimide (NHS, 56480), N-(3-Dimethylaminopropyl)-B′-ethylcarbodiimide hydrochloride (EDC, E7750), Ethanolamine (398136), St. Louis, Mo.) or with the Biacore Amine Coupling Kit (BR-1000-50, GE Healthcare, Waukesha, Wis.).

[0412]Briefly, the carboxylated surface is activated with 2 mM EDC and 0.5 mM NHS for a contact time ranging between 2-10 minutes. Protein G, in variable concentrations ranging between 20-400 ug / mL, was diluted into 10 mM acetate buffer, pH 4.3 (sodium acetate, BP334-1; glacial acetic acid, A490-212; Thermo Fisher Scientific, Waltham, Mass.), and injected over the activated sensor for ...

example 3

Generation of Rabbit Anti Human IL-6 Monoclonal Antibodies

3.1 Rabbit Immunization:

[0416]One New Zealand White rabbit was immunized with 100 μg of IL-6 protein (Recombinant E. Coli-derived human IL-6, Ref. Seq. accession NP000591.1, obtained from ProSpec-Tany TechnoGene Ltd., Rehovot, Israel (Cat. # CYT-213i)) in Sigma Adjuvant System (Sigma S6322), at days 0, 21, and 42. The rabbit was boosted not less than 10 days prior to bleeding. Rabbits were maintained at R & R Research Laboratories (Stanwood, Wash., USA) in accordance with NIH, USDA and IACUC guidelines.

3.2 B Cell Cloning:

[0417]30 ml of blood were harvested from each rabbit by venipuncture. Peripheral Blood Mononuclear cells (PBMC) were prepared by density centrifugation (Lympholyte-rabbit, Cat. # CL5050, Cedarlane Laboratories Ltd., Ontario, Canada).

[0418]The neutralizing activity of immune rabbit serum against human IL-6 was assayed after 3 immunizations.

[0419]Human IL-6 is titered from 1 ng / ml with and without a 1:3200 dilu...

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Abstract

The invention relates inter alia to a bivalent, bispecific construct comprising an anti-IL-6 antibody, or derivative thereof, and an anti-IL-23 antibody, or derivative thereof and its use in therapy. The invention also relates to useful anti-IL-6 antibodies and anti-IL-23 antibodies.

Description

RELATED APPLICATIONS[0001]The present application is a Continuation of co-pending PCT Application No. PCT / EP2011 / 065697 filed Sep. 8, 2011, which in turn, claims priority from U.S. Provisional Application Ser. No. 61 / 381,789, filed Sep. 10, 2010. Applicants claim the benefits of 35 U.S.C. §120 as to the PCT application and priority under 35 U.S.C. §119 as to the said U.S. Provisional application, and the entire disclosures of both applications are incorporated herein by reference in their entireties.[0002]This disclosure relates to novel antibody derivatives, methods for preparing them, compositions containing them, and their use in therapy.INTRODUCTIONBispecific Antibodies[0003]Monospecific antibodies, such as the naturally-occurring IgG, have two identical antigen binding paratopes, as they are made of two identical heavy chains and two identical light chains. Bispecific antibodies are engineered immunoglobulin derivatives that have two different binding paratopes, usually directe...

Claims

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Application Information

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IPC IPC(8): C07K16/46
CPCC07K16/468A61K2039/505A61P1/00A61P3/10A61P17/06A61P19/02A61P25/00A61P29/00A61P37/02A61P37/06A61P43/00C07K16/244C07K16/248C07K2317/24C07K2317/31C07K2317/33C07K2317/40C07K2317/565C07K2317/567C07K2317/622C07K2317/76C07K2317/92A61K2039/507C07K2317/56
Inventor GRABSTEIN, KENNETHBRADY, WILLIAMKING, GORDONNAIRN, NATALIE WINBLADESHANEBECK, KURT DAVIDSLAGLE, PAUL HEFFNERTHORNTON, KENNETH CHRISTOPHERVANBRUNT, MICHAEL PETERWANG, ANDREAXU, HENGYU
Owner MEDIMMUNE LTD
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