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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
  • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'

Definitions

• the pairings may be detected by quantifying a detectable moiety.
• a detectable moiety may be, for example, a protein binding site, a ligand binding site, or a tag comprising a further detectable moiety. Detection of the detectable moiety may comprise measurement of fluorescence, quenching, radioactivity or chemiluminescence.
• an environment comprising the HC:LC1 :LC2 is a complex molecular mixture, a cell supernatant, cytoplasm of a host cell, or a combination thereof.
• a mass of antibody with a particular light chain tag may be normalized by the amount of an isolated heavy chain fraction. An equivalent mass ratio may be evaluated for control antibodies.
• the second heavy chain polypeptide is labeled with a 6xHis tag and a second label.
• a detectable label may be a protein binding site, a ligand binding site, or a tag comprising a further detectable moiety. Detection of the detectable label may comprise measurement of fluorescence, quenching, radioactivity or chemiluminescence.
• the method can be performed as a high-throughput assay and is sensitive in that it measures the effects of small variations in the amino acid sequence of the IgG heavy and light chains.
• the method is generic in that it is capable of quantifying most heavy chain-light chain pairings without significant modification.
• residues within the CDRs of a converted antibody may be additionally substituted with other amino acids using conventionally known methods.
• no more than four amino acid residues in a CDR are changed, and most typically no more than two residues in the CDR will be changed, except for heavy chain CDR2, where as many as ten (10) residues may be changed.
• Changes in affinity can be measured by conventional methods such as those described herein (e.g., Biacore).
• Activity of modified antibodies may be determined using conventional assays based upon the specific target antigen.
• the term "specific” refers to a situation in which an antibody will not show any significant binding to molecules other than the antigen containing the epitope recognized by the antibody.
• the term is also applicable where for example, an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the antibody or antigen-binding fragment thereof carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.
• selectivity of a light chain polypeptide for one heavy chain polypeptide over the other is demonstrated when the resulting amount of light chain polypeptide paired with that heavy chain polypeptide is greater than the resulting amount of light chain polypeptide paired with the other heavy chain polypeptide when the light chain polypeptide and the two heavy chain polypeptides are co-expressed.
• the method allows quantitative analysis of the pairing of heavy chains with specific light chains within the mixture of co-expressed proteins.
• the method can be used to determine if one particular IgG heavy chain selectively associates with either one of the two IgG light chains when the heavy chain and light chains are co-expressed.
• the method can be used to determine if each of two different heavy chains selectively pairs with one of two light chains when the heavy and light chains are co-expressed.
• mice mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al, 1987, Genes and Devel. 1 :268-276), alp ha- fetoprotein gene control region which is active in liver (Krumlauf et al, 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al, 1987, Science 235:53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al, 1987, Genes and Devel.
• the set of constructs encoding the heavy and light chain polypeptides to be tested are co-expressed in host cells or in a cell-free protein expression system and are recovered from the culture or reaction medium. Co-expresion the set of constructs results in a set of polypeptide products.
• the set of polypeptide products secreted from the cell may include heavy chains paired with light chains, as well as light chain monomers and dimers.
• a method or assay described herein is useful for screening at least 50 different selective pairs per week per device. In some cases, a method or assay described herein is useful for screening at least 100 different selective pairs per week per device. In some cases, a method or assay is useful for screening at least 150 different selective pairs per week per device. In some cases, a method or assay is useful for screening at least 200 different selective pairs per week per device. Some methods described herein are useful for screening at least 300 different selective pairs per week per device.
• Figure 4 illustrates a plot of a doping experiment whereby known ratios of 100%
• heavy chain vector inserts consisting of 5'-EcoRlcutsite - HLA-A signal peptide - heavy chain clone - ABD2-His6tag - TGA stop - BamHl cutsite-3', were ligated into a pTT5 vector (ABD; albumin binding domain).
• the resulting vector + insert were also sequenced to confirm correct reading frame and sequence of the coding DNA.

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• 2013
  • 2013-10-03 EP EP13843363.6A patent/EP2904093B1/en active Active
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