EP1107990A1 - Anticorps a forte affinite - Google Patents

Anticorps a forte affinite

Info

Publication number
EP1107990A1
EP1107990A1 EP99940355A EP99940355A EP1107990A1 EP 1107990 A1 EP1107990 A1 EP 1107990A1 EP 99940355 A EP99940355 A EP 99940355A EP 99940355 A EP99940355 A EP 99940355A EP 1107990 A1 EP1107990 A1 EP 1107990A1
Authority
EP
European Patent Office
Prior art keywords
antibody
samples
sample
hour
antigen
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.)
Withdrawn
Application number
EP99940355A
Other languages
German (de)
English (en)
Inventor
Peter John Harrison
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.)
KS Biomedix Ltd
Original Assignee
KS Biomedix Ltd
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 KS Biomedix Ltd filed Critical KS Biomedix Ltd
Publication of EP1107990A1 publication Critical patent/EP1107990A1/fr
Withdrawn legal-status Critical Current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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6815—Enzymes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • This invention relates to antibodies and their therapeutic use. Background to the Invention
  • Antibodies have long been regarded as potentially powerful tools in the treatment of cancer and other diseases. However, although there have been some notable exceptions, this potential has not generally yet been realised.
  • a high-affinity antibody has affinity characterised by:
  • the maximum pH in step (iii) is 2.5, more preferably 2.0.
  • Antibodies or antibody fragments with the "acid- resistant" properties are expected to favour association rather than dissociation and they therefore have longer localisation times at target sites, which results in a higher concentration of antibodies localised at the target sites.
  • this invention relates to the production of a high affinity single-chain Fv antibody fragment.
  • This ScFv has particular advantages in that it allows better targeting to a site in vivo . Description of the Drawing
  • Figure 1 illustrates the results achieved for acid- resistance of sheep and mouse monoclonal antibodies and single-chain Fvs with affinity to carcinoembryonic antigen at various pH values. Description of the Invention
  • the acid-resistant monoclonal antibodies according to the present invention may be obtained using various techniques. For example, classical hybridoma technology can be applied, comprising the fusion of B-lymphocytes from immunised animals secreting high-affinity antibodies with an appropriate fusion partner.
  • An alternative method is to purify the mRNA from selected lymphocytes and use the technique of PCR to amplify the antibody genes required.
  • Phage display technology and other techniques for the display of antibody fragments may also be used to obtain the antibody genes from naive or immunised libraries after appropriate selection procedures.
  • the antibody gene can be co-expressed with or otherwise chemically linked to toxins, radioisotopes or enzymes or any other desirable molecules to provide a fusion protein with strong binding characteristics.
  • the antibodies may be produced by transgenic animals as described in US-A-5770429.
  • the antibody may be a whole antibody, comprising heavy and light chains, and constant and variable regions.
  • the antibody is an antibody fragment, e.g. F(ab') 2.
  • Fab, Fv or single-chain Fv fragments provided that at least part of the variable region is present which confers the property of "acid resistance”.
  • the antibody may also be an animal, chimeric or humanised antibody. A suitable method for producing humanised antibodies is disclosed in O-A-92/15699.
  • the antibody is a single-chain Fv fragment.
  • the single-chain Fv fragment comprises both heavy chain and light chain variable regions linked by a suitable peptide.
  • the antibodies of the present invention may be defined by their acid-resistant properties, which can be characterised by an acid-washed enzyme-linked im unosorbent assay (EIA) , as described above.
  • EIA enzyme-linked im unosorbent assay
  • the A 405 value obtained by EIA will represent antibody binding of >50% for a sample at pH 3 or below, compared to the value for the sample at pH 7.2.
  • the A 40s value of a sample at pH 2 will represent antibody binding of >60% more preferably 70% of that obtained at pH 7.2.
  • the animal that is subjected to immunisation is not a rodent, but is chosen to give higher affinity antibodies. Any large mammal may be used and suitable animals include rabbits, goats, cows and sheep.
  • An antibody of the invention may be used in therapy and may be formulated into any suitable composition with a physiologically-acceptable excipient, diluent or carrier. The following Examples illustrate the invention. Example 1.
  • CEA carcinoembryonic antigen
  • lymph node cells were then washed and fused with sheep heteromyeloma fusion partner SFP3.2. Fused cells were plated out at a total density of approximately 10 b per ml in medium containing HAT (Life Technologies) . These samples were then screened for hybridomas secreting high- affinity antibodies to the specified antigen using both a normal EIA and an acid-washed EIA.
  • Standard EIA screening assays were carried out as follows: Maxisorb assay plates (NUNC) were coated with CEA (0.4 ⁇ g/ml in phosphate-buffered saline at pH 7.2), lOO ⁇ l per well and left overnight at 4°C. The plates were then washed three times using phosphate buffered saline at pH 7.2 with 0.01% Tween 20 detergent. Any remaining reactive sites on the plates were blocked by the addition of 200 ⁇ l per well of 0.2% fat-free milk protein in PBS at pH 7.2 at 37°C for hour. The plates were then washed in PBS as described above and 45 ⁇ l of the antibody samples were added to the wells of the plates.
  • Example 2 A single-chain Fv fragment was produced from the hybridoma 6H9 above, as follows: mRNA was purified from the cultured hybridoma cells using oligo-dT cellulose. Single-stranded DNA complementary to the mRNA (cDNA) was synthesized by reverse transcription. Universal primers, designed from the constant regions of sheep heavy and light chain antibody genes, were used in separate reverse transcription reactions to synthesise the cDNA for the antibody variable regions.
  • the cDNA was then amplified by the polymerase chain reaction to make double-stranded DNA using primers designed from the heavy and light chain variable framework sequences. Separate polymerase chain reactions were used to amplify the heavy and light chain regions. The products were then analysed by agarose gel electrophoresis and the DNA bands equivalent to light and heavy chain genes were cut from the gel and purified. Equi olar amounts of variable heavy and light chain DNA were mixed together with an oligonucleotide linker DNA. The linker DNA coded for the amino acid sequence (Gly 4 Ser) 3 with additional nucleotides complementary to the 3 ' end of the heavy chain variable region and the 5' end of the light chain variable region. The three DNA molecules were denatured, annealed and extended in the first stage (without primers) of a two-stage PCR reaction so that the fragments were joined, thereby assembling the single-chain Fv.
  • the single-chain Fv DNA was amplified in the second stage of the PCR using a pair of primers derived from the heavy and light chain variable region termini with the addition of the restriction enzyme recognition sites for A1W44 ⁇ and Notl .
  • the single-chain Fv gene product was analysed by agarose gel electrophoresis and purified.
  • the single-chain Fv was then digested with the restriction enzymes AlW44 i and Notl and cloned into an expression vector.
  • the vector was then used to transform E . coli HB 2151, and protein expression was allowed to occur.
  • the vector was designed so as to include a hexa-histidine tag at the COOH terminus of the SFv.
  • the single-chain Fv was purified using nickel-chelate affinity chromatography and analysed by SDS-PAGE.
  • the amino acid sequence for the heavy chain variable region and the light chain variable region is disclosed in SEQ ID Nos. 2 and 4, respectively.
  • An acid-wash EIA was also carried out to determine the acid-resistant properties of the single-chain Fv.
  • Acid-wash EIA was carried out as follows: Carcinoembryonic antigen (CEA) -coated microtitre plates were prepared as described previously.
  • Single-chain Fv samples (6H9) were diluted to a range of concentrations between lng/ml and lOOng/ml in PBS at pH 7.2 containing 1% bovine serum albumin (BSA) .
  • BSA bovine serum albumin
  • lOO ⁇ l samples were added to the microtitre plate wells and incubated for 1 hour at 37°C.
  • the plates were then washed, 200 ⁇ l per well of citrate added, and the plates incubated for 1 hour at 37°C.
  • the acid preparations were made using a stock solution of lOOmM citrate diluted to pH values of 4.0, 3.5, 3.0, 2.5 and 2.0 in the reaction mixture. PBS at pH 7.2 was used as a reference control.
  • the plates were then washed and lOO ⁇ l per well of mouse anti-tetra-histidine antibody (Qiagen) (lOOng/ml diluted in PBS at pH 7.2 with 1% BSA) added and incubated for 1 hour at 37°C.
  • Qiagen mouse anti-tetra-histidine antibody
  • sFv samples were incubated with PBS at pH 7.2 to generate an EIA response curve for the SFv samples.
  • a concentration of 10-20ng/ml of the SFv sample gave an absorbance (A 405 ) of 1.0-1.5 and was therefore used to determine the amount of antibody bound in the acid washed samples as a percentage of the amount bound in the reference sample.
  • the acid-resistant properties of the 6H9 whole antibody and the 6H9 single-chain Fv were compared with that for the mouse-derived anti-carcinoembryonic antigen whole antibody, A5B7 and the single-chain Fv MFE.
  • the results are shown in Figure 1, with the antigen-binding of the mouse-derived antibodies being substantially reduced at pH 3.5 and less than 5% at pH 2.5.
  • the 6H9 antibodies retain >70% antigen at pH 3.5, >60% at pH 2.5 and >50% at pH 2.0.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Cette invention se rapporte à des anticorps monoclonaux à forte affinité, cette affinité se caractérisant par les opérations suivantes: (i) on laisse incuber un premier et un second échantillon de l'anticorps dans les alvéoles d'une plaque de microtitration recouverte d'antigène, à une concentration choisie de façon à se situer dans la partie linéaire d'une courbe standard à un pH de 7,2 pendant 1 heure à 37 °C; (ii) on retire des deux échantillons tout anticorps non lié; (iii) on laisse incuber le premier échantillon avec un soluté tampon de phosphate à un pH de 7,2 pendant une heure à 37 °C, et on abaisse le pH du second échantillon jusqu'à atteindre un pH égal ou inférieur à 3, et on laisse incuber ce premier échantillon pendant 1 heure à 37 °C; (iv) on retire des deux échantillons tout anticorps non lié; (v) on laisse incuber les deux échantillons avec un conjugué de phosphatase alcaline anti-anticorps pendant 1 heure à 37 °C; Cvi) on retire des deux échantillons tout conjugué non lié; et (vii) on ajoute aux échantillon sun substrat PNPP, on mesure l'absorbance des échantillons à 405nm et on détermine la quantité d'anticorps liée à l'antigène, la quantité liée du second échantillon constituant plus de 50 % de celle du premier échantillon.
EP99940355A 1998-08-28 1999-08-20 Anticorps a forte affinite Withdrawn EP1107990A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GBGB9818915.2A GB9818915D0 (en) 1998-08-28 1998-08-28 Antibodies
GB9818915 1998-08-28
PCT/GB1999/002729 WO2000012556A1 (fr) 1998-08-28 1999-08-20 Anticorps a forte affinite

Publications (1)

Publication Number Publication Date
EP1107990A1 true EP1107990A1 (fr) 2001-06-20

Family

ID=10838074

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99940355A Withdrawn EP1107990A1 (fr) 1998-08-28 1999-08-20 Anticorps a forte affinite

Country Status (21)

Country Link
EP (1) EP1107990A1 (fr)
JP (1) JP2002525082A (fr)
KR (1) KR20010089174A (fr)
CN (1) CN1315966A (fr)
AU (1) AU5435099A (fr)
BG (1) BG105294A (fr)
BR (1) BR9913303A (fr)
CA (1) CA2340865A1 (fr)
EA (1) EA200100287A1 (fr)
GB (1) GB9818915D0 (fr)
HR (1) HRP20010149A2 (fr)
HU (1) HUP0103233A3 (fr)
ID (1) ID28905A (fr)
IL (1) IL141523A0 (fr)
MX (1) MXPA01002248A (fr)
NO (1) NO20011006D0 (fr)
PL (1) PL346472A1 (fr)
TR (1) TR200100643T2 (fr)
WO (1) WO2000012556A1 (fr)
YU (1) YU15401A (fr)
ZA (1) ZA200101573B (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0031284D0 (en) * 2000-12-21 2001-01-31 Ks Biomedix Ltd High affinity antibodies
GB0112844D0 (en) * 2001-05-25 2001-07-18 Psimei Pharmaceuticals Plc Neutron capture therapy
WO2003105757A2 (fr) * 2002-06-12 2003-12-24 Genencor International, Inc. Methodes et compositions de liaison dependant du milieu d'un agent cible a une cible
US20030232401A1 (en) * 2002-06-12 2003-12-18 Pugia Michael J. Bacterial test method by glycated label binding
JP4982183B2 (ja) * 2003-12-12 2012-07-25 ジェネンコー・インターナショナル・インク Ｃａｂ分子
CN111848790B (zh) * 2020-08-07 2022-02-22 上海交通大学 一种牛源抗金黄色葡萄球菌的单链抗体及其制备和应用
CN112724255A (zh) * 2021-01-28 2021-04-30 成都金昆生物科技有限公司 靶向癌胚抗原的小分子抗体

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5081235A (en) * 1989-07-26 1992-01-14 City Of Hope Chimeric anti-cea antibody
GB9014932D0 (en) * 1990-07-05 1990-08-22 Celltech Ltd Recombinant dna product and method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0012556A1 *

Also Published As

Publication number Publication date
ID28905A (id) 2001-07-12
CN1315966A (zh) 2001-10-03
CA2340865A1 (fr) 2000-03-09
NO20011006L (no) 2001-02-27
IL141523A0 (en) 2002-03-10
HUP0103233A2 (hu) 2001-12-28
YU15401A (sh) 2003-07-07
BG105294A (bg) 2001-12-29
TR200100643T2 (tr) 2001-07-23
MXPA01002248A (es) 2002-05-08
HRP20010149A2 (en) 2002-02-28
EA200100287A1 (ru) 2001-08-27
BR9913303A (pt) 2001-10-09
PL346472A1 (en) 2002-02-11
GB9818915D0 (en) 1998-10-21
HUP0103233A3 (en) 2003-10-28
AU5435099A (en) 2000-03-21
KR20010089174A (ko) 2001-09-29
WO2000012556A1 (fr) 2000-03-09
JP2002525082A (ja) 2002-08-13
NO20011006D0 (no) 2001-02-27
ZA200101573B (en) 2002-02-26

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