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EP-4735883-A1 - CELLULAR ASSAYS TO QUANTIFY TARGET-INDEPENDENT CLEARANCE OF THERAPEUTIC MOLECULES

EP4735883A1EP 4735883 A1EP4735883 A1EP 4735883A1EP-4735883-A1

Abstract

The present disclosure is directed to in vitro cell-based methods useful for predicting in vivo pharmacokinetic properties of candidate therapeutic proteins. In particular, the cell-based assays described herein are useful to predict in vivo non-target dependent clearance and subcutaneous bioavailability of candidate therapeutic proteins.

Inventors

  • TUHIN, Md Tariqul Haque
  • BRYNIARSKI, Mark A.
  • CONNER, Kip P.
  • SHOMIN, Carolyn D.
  • COOK, KEVIN D.

Assignees

  • Amgen Inc.

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. A method of predicting in vivo non-target mediated clearance of a biological molecule, said method comprising: providing a preparation of cells, wherein cells of the preparation do not express a target of the biological molecule; incubating the preparation of cells with culture media containing the biological molecule under conditions mimicking in vivo physiological conditions; determining an amount of the biological molecule taken up by the cells of the preparation after said incubating; and predicting in vivo non-target mediated clearance of the biological molecule based on said determining.
  2. 2. The method of claim 1, wherein the culture media during said incubating has a pH of about 7.0 to about 8.0.
  3. 3. The method of claim 1, wherein the culture media during said incubating has a pH of about 5.6 to about 7.0.
  4. 4. The method of claim 1 , wherein said incubating is carried out at 37°C for about 60 minutes.
  5. 5. The method of claim 1. wherein said determining comprises: permeabilizing cells of the preparation after said incubating; detecting the amount of biological molecule taken up by the cells of the preparation; and quantifying the amount of biological molecule taken up by the cells of the preparation based on said detecting, wherein predicting the in vivo non-target mediated clearance of the biological molecule is based on said quantifying.
  6. 6. The method of claim 5 further comprising: incubating, after said permeabilizing, the permeabilized cells with a detectable moiety that binds to said biological molecule, wherein the amount of detectable moiety bound to said biological molecule is detected.
  7. 7. The method of claim 5. wherein said quantifying comprises: comparing the detected amount of biological molecule taken up by the cells to one or more quantitative reference values.
  8. 8. The method of claim 7, wherein the one or more quantitative reference values comprise a calibration curve of biological molecule binding capacity.
  9. 9. The method of claim 6, wherein the detectable moiety is an anti-Fc antibody coupled to a label.
  10. 10. The method of claim 5, wherein said detecting is carried out using flow cytometry, immunoassay, or microscopy.
  11. 11. The method of claim 1, wherein the biological molecule is a human antibody, epitope-binding fragment of a human antibody, or a human antibody derivative.
  12. 12. The method of claim 1, wherein the biological molecule is a multispecific antibody.
  13. 13. The method of claim 1, wherein the biological molecule is a recombinant protein or fusion protein.
  14. 14. The method of claim 1, wherein cells of the preparation do not express an Fc receptor.
  15. 15. The method of claim 1, wherein cells of the preparation do not express human neonatal Fc receptor (hFcRn) and human 02m (h02m) complex.
  16. 16. The method of claim 1, wherein the preparation of cells is a preparation of cells selected from the group consisting of Chinese Hamster Ovary (CHO) Cells, Madin-Darby canine kidney (MDCK) cells, Vero cells, HUVECs. HEK293 cells, and primary endothelial cells.
  17. 17. A method of predicting in vivo non-target mediated clearance of an FcRn interacting molecule, said method comprising: providing a first cell preparation, wherein cells of the first preparation do not express hFcRn; providing a second cell preparation, wherein cells of the second preparation express a heterodimer of human neonatal Fc receptor (hFcRn) and human 02m (h02m), and wherein cells of the first and second preparations do not express a binding target of the FcRn interacting molecule; subjecting the first and second cell preparations to first and second incubation periods, wherein said first incubation period comprises incubating the cell preparations with media containing the FcRn interacting molecule under acidic and/or non-acidic conditions, and wherein said second incubation period comprises incubating the cell preparations, after said first incubation, with media lacking the FcRn interacting molecule under non-acidic conditions; determining an amount of the FcRn interacting molecule taken up by cells of the first and second preparations after said subjecting to the first incubation period and/or the second incubation period; measuring an amount of the FcRn interacting molecule in media from the second incubation period collected at the end of the second incubation period; quantifying non-specific endocytosis and FcRn recycling of the FcRn interacting molecule based on said determining and said measuring, respectively; and predicting the in vivo non-target mediated clearance of the FcRn interacting molecule based on said quantifying.
  18. 18. The method of claim 17, wherein said first incubation comprises incubating the first and second cell preparations with media containing the FcRn interaction molecule under acidic condition and non-acidic conditions.
  19. 19. The method of claim 17, wherein said first incubation comprises incubating the first and second cell preparations with media containing the FcRn interaction molecule under acidic or non-acidic conditions.
  20. 20. The method of any one of claims 17-19, wherein said non-acidic conditions comprise a media pH of 7.0 to 8.0.

Description

CELLULAR ASSAYS TO QUANTIFY TARGET-INDEPENDENT CLEARANCE OF THERAPEUTIC MOLECULES [0001] The benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos. 63/523,868 filed June 28, 2023, and 63/547,184 filed November 3, 2023, is hereby claimed, and the disclosures thereof are hereby incorporated by reference herein. FIELD [0002] The present disclosure relates to cell-based methods for predicting in vivo targetindependent clearance of a biological molecule. INCORPORA TION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY [0003] Incorporated by reference in its entirety herein is a nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 8 kilobyte XML document named “10587-W001-SEC_SequenceListing.xml,” created on June 27, 2024. BACKGROUND OF VARIOUS EMBODIMENTS [0004] The pharmacokinetics (PK) of monoclonal (mAbs) and multispecific antibodies is determined by both target-dependent and target-independent clearance (CL) pathways. Target independent CL (CLind) as pertains to mAbs is described mathematically by first-order, linear kinetics (i.e. (dA/dt)/[Drug] = CLind). Mechanistically, the rate of CLind results from the interplay of at least two competing processes, where non-specific endocytosis is balanced by intracellular salvage/recycling via the neonatal Fc receptor (FcRn) that returns drug to the systemic circulation (Ovacik, M., and Lin, K. (2018) Tutorial on Monoclonal Antibody Pharmacokinetics and Its Considerations in Early Development. Clin Transl Sci 11, 540-552; Ryman, J. T., and Meibohm, B. (2017) Pharmacokinetics of Monoclonal Antibodies. CPT Pharmacometrics Syst Pharmacol 6, 576-588; Huisinga, et al., Target-Driven Pharmacokinetics of Biotherapeutics, in Pharmaceutical Sciences Encyclopedia, pp 1-15, and Meno-Tetang, G. M. L. Target-Driven Pharmacokinetics of Biotherapeutics, in Pharmaceutical Sciences Encyclopedia, pp 1-12). Non-specific endocytosis of therapeutic proteins can be driven by fluid-phase uptake and non-specific adsorptive internalization. Fluid-phase uptake is a mechanism whereby solutes are internalized along with the extracellular fluid whose extent is directly proportional to the solute concentration (Steinman, et al. (1983) Endocytosis and the recycling of plasma membrane. J Cell Biol 96, 1 -27, and Besterman, J. M., and Low, R. B. (1983) Endocytosis: a review of mechanisms and plasma membrane dynamics. Biochem J 210, 1-13). Non-specific adsorptive endocytosis occurs when a protein non-specifically interacts with the cell membrane, such as charge-based attraction, that results in its internalization. Fluid-phase is constitutive and likely cell-type specific where nonspecific adsorption is protein-dependent and can be influenced by factors that includes local charge (Lloyd and Williams (1984) Non-specific adsorptive pinocytosis. Biochem. Soc. Trans. 12(3): 527-28). FcRn is the alpha chain of anon-covalent, heterodimeric complex with p2-microglobulin (P2m) that is chiefly localized within endosomal membranes under basal condition (Praetor, A., and Hunziker, W. (2002) beta(2)-Microglobulin is important for cell surface expression and pH- dependent IgG binding of human FcRn. J Cell Sci 115, 2389-2397; Simister, N. E., and Mostov, K. E. (1989) An Fc receptor structurally related to MHC class I antigens. Nature 337, 184-187; Antohe, et al. (2001) Expression of functionally active FcRn and the differentiated bidirectional transport of IgG in human placental endothelial cells. Hum Immunol 62. 93-105; Roberts, et al. (1990) Isolation and characterization of the Fc receptor from the fetal yolk sac of the rat. J Cell Biol 111, 1867-1876; Dickinson, et al. (1999) Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line. J Clin Invest 104, 903-911; D'Hooghe. et al. (2017) Cell surface dynamics and cellular distribution of endogenous FcRn. PLoS One 12, eO 182695). FcRn exhibits increased affinity to its endogenous ligands albumin and immunoglobulin G (IgG) at acidic pH (Chaudhury, et al. (2003) The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan. J Exp Med 197, 315-322; Raghavan, et al. (1995) Analysis of the pH dependence of the neonatal Fc receptor/immunoglobulin G interaction using antibody and receptor variants. Biochemistry 34, 14649-14657; Andersen, et al. (2012) Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor. Nat Commun 3, 610; Ober, et al. (2001) Differences in promiscuity7 for antibody-FcRn interactions across species: implications for therapeutic antibodies. Int Immunol 13, 1551-1559; Martin, et al. (2001) Crystal structure at 2.8 A of an FcRn/heterodimeric Fc complex: mechanism of pH- dependent binding. Mol Cell 7, 867-877), and therefore selectively retains these proteins by way of intracellular trafficking away from lysosomal degradation and towards the plasma membrane to facilitate a