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WO2008135259A2 - Composition de molécules d'anticorps - Google Patents

Composition de molécules d'anticorps Download PDF

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Publication number
WO2008135259A2
WO2008135259A2 PCT/EP2008/003586 EP2008003586W WO2008135259A2 WO 2008135259 A2 WO2008135259 A2 WO 2008135259A2 EP 2008003586 W EP2008003586 W EP 2008003586W WO 2008135259 A2 WO2008135259 A2 WO 2008135259A2
Authority
WO
WIPO (PCT)
Prior art keywords
antibody molecule
antibody
cells
heavy chain
variable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2008/003586
Other languages
English (en)
Other versions
WO2008135259A3 (fr
Inventor
Steffen Goletz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Glycotope GmbH
Original Assignee
Glycotope GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Glycotope GmbH filed Critical Glycotope GmbH
Publication of WO2008135259A2 publication Critical patent/WO2008135259A2/fr
Publication of WO2008135259A3 publication Critical patent/WO2008135259A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

  • Antibody molecule composition
  • the cDNA sequence was extended by Ncol/Nhel in the case for the VL and Ncol/Sall in the case of the VH and the cDNA was generated.
  • the NcoI/Sall-cut variable heavy chain cDNA fragment VH was cloned into the Ncol/Sall- cut BS-Leader vector as described in WO2004/065423.
  • the BS-Leader vector includes a cloning cassette to introduce the T cell receptor signal peptide sequence at the 5' end and a splice donor sequence at the 3' end of the variable domains.
  • variable light chain VL of the corresponding antibody was designed with an additional splice donor site at the 3 'end of the coding cDNA and was cloned via Ncol/Nhel into the likewise digested BS-Leader vector. Thereafter, each Hindlll/BamHI fragment from the BS- Leader vector was cloned into the corresponding eukaryotic expression vector.
  • vectors (pEFpuroC ⁇ lV H , pEFdhfrCicVu pEFdhfr mu tC ⁇ V L ) comprise EF-l ⁇ -promoter and HCMV enhancer, SV40 origin, polyadenylation signal, puromycin resistance gene in the vector for the heavy chain and the murine dihydrofolase gene (dhfr) for CHO cell expression or SEQ ID No 1
  • EEKGIKYKFEVYEKKD SEQ ID No 1) for NM-F9, K562, NM-H9, NM-D4, or NM- H9D8 expression for selection and gene amplification in the vector for the light chain, as well as the genomic sequences of the human constant ⁇ l region for the heavy chain or the human constant K region for the light chain.
  • Variable Heavy chain SEQ ID No 1) for NM-F9, K562, NM-H9, NM-D4, or NM- H9D8 expression for selection and gene amplification in the vector for the light chain, as well as the genomic sequences of the human constant ⁇ l region for the heavy chain or the human constant K region for the light chain.
  • CHOdhfr- ATCC No. CRL-9096 cells were co-transfected with a mixture of above described vectors for the heavy and light chains (1 :1 to 1 :3) by lipofectamin or electroporation for the adherent cell line.
  • growth medium was changed to selection medium (DMEM + 10% dialysed FCS + 2 mM L- glutamine + 5 ⁇ g/ml puromycin + 50 mM methotrexate) for 1 week.
  • selection medium DMEM + 10% dialysed FCS + 2 mM L- glutamine + 5 ⁇ g/ml puromycin + 50 mM methotrexate
  • NM-H9D8 (DSM ACC2806) and NM-F9 (DSM ACC2606), adapted to serum-free medium X-Vivo 20 was performed under serum-free conditions using electroporation. Two days post-transfection, growth medium was changed to selection medium (X- Vivo 20 + 0.75 ⁇ g/ml puromycin + 50 nM methotrexate) for 1 week.
  • the stably transfected cells secreting the Cetuximab were cultivated in serum free medium until a cell density of about 1 to 2 x 10 6 cells/ml was reached.
  • the chimeric antibody was purified using a protein A column (HiTrap r-protein A FF, Amersham Pharmacia Biotech).
  • the purified antibody fraction eluted by sudden pH change was re-buffered in PBS and concentrated using Centriprep centrifuge tubes (cut off 50 kDa, Millipore).
  • PBMC peripheral blood mononuclear cells
  • ADCC antibody-dependent cellular cytotoxicity
  • Target cells (LS174T, 3 x 10 6 ) were incubated for 6 minutes on ice in 100 ⁇ l of europium buffer (50 mM HEPES, pH 7.4, 93 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 10 mM diethylenetriaminepentaacetic acid, 2 mM europium(III)acetate), electroporated in a Nucleofector II (Amaxa) with program A-OI l , and subsequently incubated on ice for another 6 min.
  • europium buffer 50 mM HEPES, pH 7.4, 93 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 10 mM diethylenetriaminepentaacetic acid, 2 mM europium(III)acetate
  • the cells were washed 5 times in RPMI 1640/5% FCS and seeded in a 96- well round-bottom plate (Nunc; 5 x 10 3 cells in 100 ⁇ l per well).
  • a 96- well round-bottom plate (Nunc; 5 x 10 3 cells in 100 ⁇ l per well).
  • human peripheral blood cells 80 ⁇ l per well were added as effector cells, using an effector/target cell ratio of 80:1.
  • 80 ⁇ l RPMI/FCS with no effector cells was added to determine spontaneous release. Maximum release was determined after complete lysis of the target cells with 1% Triton X-100.
  • the ADCC activity of the Cetuximab isolated from NM-H9D8 is significantly higher than the ADCC activity of the Cetuximab isolated from the CHOdhfr- cells.
  • Glycans of at least lOO ⁇ g purified antibody were cleaved by acid hydrolysis.
  • Sialic acids were labelled specifically by conjugation with the fluorescence dye DMB.
  • Analysis was performed by HPLC e.g. on an Asahipak-NH2 column to separate the saccharides. Identification and calculation of sialic acids was performed by standard substances of appropriate sialic acids.
  • Lectins which bind preferentially to alpha2-6 or alpha2-3 linked sialic acids were used to characterize the antibody sialylation by ELISA or Western blot analysis.
  • ELISA experiments were performed by coating the purified antibodies expressed in the different cell lines in wells of microtiter plates (2 ⁇ g/ml, 50 ⁇ l per well) and detecting by appropriate biotinylated lectins.
  • Dependence of lectin binding by sialylation was checked by neuraminidase treatment (0.1U/ml and incubation at room temperature for lhour). Results are illustrated in figure 1 and/or 2.
  • Cetuximab and Erbitux are used as synonyms and mean both the antibody variable sequences of light and heavy chain accessible in public data bases independent of the cell or cell line producing the antibody.
  • Figure legends are used as synonyms and mean both the antibody variable sequences of light and heavy chain accessible in public data bases independent of the cell or cell line producing the antibody.
  • FIG. 1 ELISA analysis was performed to identify the differently sialylated antibody molecule compositions expressed in CHOdhfr-, NM-F9, or NM-H9D8. Sialylation was analysed (A) by SNA which detects alpha2-6 sialylation with or without neuraminidase treatment and (B) by MAL I which detects alpha2-3 sialylation.
  • FIG. 2 Western blot analysis was performed to identify the differently sialylated heavy chain of antibody molecule compositions expressed in CHOdhfr- or NM-H9D8. Proteins were transfered to nitrocellulose and visualized by SNA which detects 2-6 sialylation.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un anticorps comprenant au moins une chaîne lourde variable et une chaîne légère variable. La chaîne lourde variable est: QVQLKQSG PGLVQPSQSLS ITCTVSGFSLTNYGVHWVRQS PGKGLEWLGVIWS GGNTDYN TPFTSRLSiNKDNSKSQVFFKMNSLQSH Γ;TAI Y YCARALT YYDYEFAYWGQGTLVTVSTASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS 7YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQ QGNVFSCSVMHEGLHNHYTQKSLSLSPGK; et/ou la chaîne légère variable est: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTKGSPRLLIKYASESISGIPS RFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFI FPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
PCT/EP2008/003586 2007-05-04 2008-05-05 Composition de molécules d'anticorps Ceased WO2008135259A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP07090094.9 2007-05-04
EP07090094 2007-05-04

Publications (2)

Publication Number Publication Date
WO2008135259A2 true WO2008135259A2 (fr) 2008-11-13
WO2008135259A3 WO2008135259A3 (fr) 2009-04-09

Family

ID=39944059

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2008/003586 Ceased WO2008135259A2 (fr) 2007-05-04 2008-05-05 Composition de molécules d'anticorps

Country Status (1)

Country Link
WO (1) WO2008135259A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106470697A (zh) * 2014-09-16 2017-03-01 依姿魅力有限公司 抗egfr抗体以及其用途

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2450289A1 (fr) * 2003-03-20 2005-05-19 Imclone Systems Incorporated Methode de production d'un anticorps ciblant le recepteur du facteur de croissance epidermique
US20050142133A1 (en) * 2003-12-03 2005-06-30 Xencor, Inc. Optimized proteins that target the epidermal growth factor receptor
CA2557725C (fr) * 2004-02-13 2015-06-30 Glycotope Gmbh Conditions de traitement de glycoproteines extremement actives et methode efficace de production associee
EP1931709B1 (fr) * 2005-10-03 2016-12-07 Xencor, Inc. Variants de fc dotés de propriétés de liaison aux récepteurs fc optimisées
HRP20150307T1 (hr) * 2006-09-10 2015-04-24 Glycotope Gmbh Upotreba ljudskih stanica podrijetlom iz ljudske leukemije za eksprimiranje protutijela

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106470697A (zh) * 2014-09-16 2017-03-01 依姿魅力有限公司 抗egfr抗体以及其用途
EP3110447A4 (fr) * 2014-09-16 2017-08-23 Ease Charm Limited Anticorps anti-egfr et leurs utilisations
CN106470697B (zh) * 2014-09-16 2019-10-25 兴盟生物医药(苏州)有限公司 抗egfr抗体以及其用途
US10759860B2 (en) 2014-09-16 2020-09-01 Synermore Biologics Co., Ltd. Anti-EGFR antibody and uses of same

Also Published As

Publication number Publication date
WO2008135259A3 (fr) 2009-04-09

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