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EP-3720959-B1 - SELECTING FOR DEVELOPABILITY OF POLYPEPTIDE DRUGS IN EUKARYOTIC CELL DISPLAY SYSTEMS

EP3720959B1EP 3720959 B1EP3720959 B1EP 3720959B1EP-3720959-B1

Inventors

  • MCCAFFERTY, JOHN
  • PERERA, Rajika
  • DYSON, MICHAEL RICHARD
  • PARTHIBAN, Kothai
  • SYRJANEN, Johanna Liinamaria

Dates

Publication Date
20260506
Application Date
20181205

Claims (14)

  1. A method of distinguishing or ranking binders according to their solubility and/or resistance to self-association in solution, and/or enriching for binders exhibiting greater solubility and/or greater resistance to self-association in solution, comprising (i) providing a library of higher eukaryotic cell clones each containing DNA encoding a binder, (ii) culturing the clones in vitro under conditions for expression of the binders, wherein the binders are presented on the cell surface, (iii) determining surface presentation levels, measured in terms of number of displayed binders per cell, of the binders on the clones, by labelling the binders with an agent incorporating a detectable label, (iv) selecting one or more clones that exhibit higher surface presentation of binders compared with other clones, and (v) identifying binders encoded by the one or more selected clones as having good solubility and/or resistance to self-association in solution, and optionally providing the selected clones for use in one or more further screening steps, wherein the binders are transmembrane domain-containing polypeptides.
  2. A method according to claim 1, wherein the detectable label is a fluorescent label.
  3. A method according to claim 1 or 2, wherein the agent binds to a constant region of the binders, optionally wherein the binders comprise an Fc region and the agent binds to the Fc region.
  4. A method according to any of claims 1 to 3, comprising sorting cells into a collected fraction and a discarded fraction according to the level of surface presentation of binders on the cells, whereby cells displaying surface presentation above a pre-determined threshold are sorted into the collected fraction and cells displaying surface presentation below the pre-determined threshold are sorted into the discarded fraction.
  5. A method according to claim 4, wherein discarded fraction comprises cells expressing comparator polypeptides that have a critical concentration of at least 10 mg/ml and wherein the collected fraction comprises cells expressing binders that have a critical concentration at least 1.5-fold higher than the comparator polypeptides in the discarded fraction.
  6. A method according to claim 4 or claim 5, wherein sorting is performed by a fluorescence activated cell sorter (FACS).
  7. A method according to any of claims 1 to 6, wherein step (ii) comprises culturing the clones of the library as a mixture in one vessel.
  8. A method according to any of claims 1 to 6, wherein step (ii) comprises culturing each clone of the library in a separate vessel.
  9. A method according to any one of the preceding claims, wherein the binders are sequence variants of a parent binder.
  10. A method according to claim 9, wherein the parent binder has been identified as requiring improvement in solubility or resistance to self-association in solution.
  11. A method according to claim 9 or claim 10, wherein the method comprises generating sequence variants of the parent binder and integrating DNA encoding the sequence variants into cellular DNA of higher eukaryotic cells to provide the library of cell clones containing DNA encoding the binders, optionally wherein the method comprises analysing the polypeptide sequence of the parent, identifying one or more amino acid residues that are predicted to promote self-association and/or reduce solubility, and generating mutation at the one or more amino acid residues.
  12. A method according to any of claims 9 to 11, wherein the parent binder has a critical concentration of less than 50 mg/ml in phosphate buffered saline solution (PBS) and/or has a solubility limit of less than 50 mg/ml in phosphate buffered saline solution (PBS), and/or wherein the method comprises identifying binders encoded by the one or more selected clones as having a critical concentration and/or a solubility limit at least 1.5 fold higher than that of the parent binder.
  13. A method according to any one of the preceding claims, comprising (i) simultaneously determining surface presentation levels of the binders and levels of target binding by the binders, and co-selecting clones displaying cognate binders exhibiting higher surface presentation compared with other clones; or (ii) simultaneously determining surface presentation levels of the binders and levels of non-specific binding to non-target molecules, and co-selecting clones displaying binders exhibiting higher surface presentation and lower non-specific binding compared with other clones; or (iii) simultaneously determining levels of target binding and levels of non-specific binding to non-target molecules by the binders, and co-selecting clones displaying cognate binders exhibiting lower non-specific binding compared with other clones.
  14. A method according to any one of the preceding claims, comprising determining the sequence of the DNA encoding the binder from the one or more selected clones, and providing isolated nucleic acid encoding the binder.

Description

FIELD OF THE INVENTION The present invention relates to identification of candidate polypeptide drugs having desirable developability characteristics such as solubility, capability to be formulated at high concentrations, low propensity for non-specific binding, and optimal half-life. The invention further relates to screening of binders in drug discovery, including antibody discovery. BACKGROUND Antibodies have proven to be a very successful class of drug with over 70 therapeutic antibodies approved to date and many more in the development pipeline. However, the production and formulation of polypeptide drugs such as antibodies is in many respects a more complex endeavour than for small molecule pharmaceuticals. A number of factors affect the practicality of developing polypeptide drugs such as antibodies, influencing whether a lead candidate antibody will be successfully developed into a drug that is not only efficacious but also manufacturable, stable and safe. Such factors include chemical stability (resistance to e.g., fragmentation, deamidation, oxidation and isomerisation), physical stability (e.g., conformational stability, propensity of the protein to unfold, aggregate and/or precipitate, colloidal stability), and solution properties (e.g., solubility, tendency to reversibly self-associate in solution, and viscosity at high concentrations). The ability of a polypeptide to be expressed at high yield in cell culture is also a relevant consideration, as the amount of polypeptide secreted from the host cells determines the yield of product that is recoverable from the culture medium. The immunogenicity of an antibody or polypeptide drug is also a consideration. All these factors can collectively be referred to as "developability" characteristics of the product. These are important considerations as they impact the product's cost and practicality of manufacture, safety profile, dosing schedule and mode of administration. Where developability problems are encountered, they can affect the utility or commercial success of antibodies, and may cause a lead molecule to fail during the development process. Aspects of antibody drug developability and methods to measure them have been reviewed1-3. Production of stable, soluble protein at high concentration is desired for administration to patients. For example antibody concentrations of 50 mg/ml and greater are sought for subcutaneous administration where a relatively large dose has to be administered in a low volume. An ideal polypeptide for therapeutic use is highly soluble in aqueous buffered solution and thus able to be concentrated to high levels. Success in producing soluble protein at high concentration depends at least in part on the polypeptides' resistance to self-association which can otherwise lead to increased viscosity and/or precipitation. When the solubility limit of a molecule is reached, further increase in dissolved concentration is not possible and undesirable effects occur such as precipitation of the molecule out of solution. As the solubility limit is approached, a solution may become viscous and/or the dissolved polypeptide may self-associate in solution (reversibly or otherwise) - such effects complicate handling and formulation of the solution. Even worse, the formation of aggregates by poorly soluble and/or unstable polypeptides can present a heightened risk of immunogenicity when the product is administered to patients4,5. An anti-drug immune response (e.g., anti-drug antibodies) may neutralise the therapeutic effect, and hypersensitivity can result in morbidity or mortality. The ability of a polypeptide to be expressed at high levels can affect the economics of production. During the early steps of antibody discovery, yields achieved from optimised transient expression in mammalian cell culture can reach 50 µg/ml or greater. Transient expression is a temporary expression system in which a plasmid encoding the product of interest is transfected into a cell and is expressed for a period of time, usually declining after 2 - 7 days as the plasmid is lost from the cell. To achieve stably expressing recombinant cells for drug manufacture, the encoding DNA must be stably integrated into the cell genome. Following creation of stable manufacturing cell lines, cells may be cultured for one or two weeks to obtain higher yields, which may be around 1 mg/ml or better. Low yields from transient expression may indicate potential developability problems with a candidate drug. For example an anti-angiopoetin antibody ("Ang2") that was prone to aggregation was reported to give yields of only 10 µg/ml, whereas an optimised variant of the antibody yielded 260 µg/ml6. However, even when good yields are obtained from transient expression (e.g., > 50 µg/ml) and/or from stable cell lines (e.g., > 1 mg/ml), biophysical problems may emerge when a polypeptide is concentrated above 1 mg/ml. Dobson et al (20167) found that the anti-NGF antibody MEDI1912 had sign