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EP-4740013-A1 - LINKING RECEPTOR OCCUPANCY TO EFFICACY OF CXCR4 ANTAGONISTS

EP4740013A1EP 4740013 A1EP4740013 A1EP 4740013A1EP-4740013-A1

Abstract

The disclosure is based on a novel assay which links the degree of CXCR4 receptor occupancy (RO) by an anti- CXCR4 polypeptide to the efficacy of the anti- CXCR4 polypeptide based on determining the SDF-1α induced migration of CXCR4 primary human cells in the presence of the anti-CXCR4 polypeptide. Inhibition of migration as measured in the in vitro assay provides a surrogate for in vivo efficacy of the anti-CXCR4 polypeptide.

Inventors

  • LYNCH, JASON
  • ORGAN, Louise
  • OLDHAM, Timothy
  • GAO, JIE

Assignees

  • AdAlta Limited

Dates

Publication Date
20260513
Application Date
20240705

Claims (20)

  1. 1. A method of stratifying subjects for treatment with an anti-CXCR4 polypeptide, the method comprising: (i) exposing CXCR4 expressing cells derived from the subject to the anti-CXCR4 polypeptide; (ii) measuring the CXCR4 receptor occupancy of the polypeptide on the cells obtained from the subject; wherein if the CXCR4 receptor occupancy is greater than 30%, the subject is selected for treatment with the anti-CXCR4 polypeptide.
  2. 2. The method of claim 1 wherein the cells are exposed in vivo or in vitro.
  3. 3. The method of claim 1 or 2, wherein the CXCR4 expressing cells are human T cells.
  4. 4. The method according to any one of claims 1 to 3, wherein the subject has a fibrotic disease or a CXCR4 expressing cancer.
  5. 5. The method according to any one of claims 1 to 4, wherein the CXCR4 receptor occupancy is greater than 40 %, greater than 50%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80% or greater than 85%.
  6. 6. The method of any one of claims 1 to 5, wherein the CXCR4 receptor occupancy is between 60-85%.
  7. 7. The method of any one of claims 1 to 6, wherein the receptor occupancy is measured by a method comprising: (i) obtaining T cells from the subject; (ii) exposing the T cells to an anti-CXCR4 polypeptide; (iii) determining the CXCR4 receptor occupancy by detecting the amount of bound anti- CXCR4 polypeptide versus free CXCR4.
  8. 8. The method of any one of claims 1 to 7, wherein the receptor occupancy is determined by detecting and measuring the amount of bound anti-CXCR4 polypeptide versus free CXCR4 on the T cells.
  9. 9. The method of claim 8, wherein the detecting and measuring is by flow cytometry.
  10. 10. The method of any one of claims 1 to 9, wherein the anti-CXCR4 polypeptide is AD-214 comprising the sequence of SEQ ID NO:5.
  11. 11. The method of any one of claims 1 to 10, wherein migration inhibition of the T cells is maximally inhibited.
  12. 12. A method of treating a subject with a fibrotic disease or CXCR4 expressing cancer by administering an anti-CXCR4 polypeptide at a dose that maintains a CXCR4 receptor occupancy (RO) of greater than 30%
  13. 13. The method according to claim 12, wherein the CXCR4 receptor occupancy is greater than 40 %, greater than 50%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, or greater than 85%.
  14. 14. The method of claim 12 or 13, wherein the anti-CXCR4 polypeptide is AD-214 comprising the sequence of SEQ ID NO:5.
  15. 15. The method of any one of claims 12 to 14, wherein: (i) the RO is greater than or equal to 85% and the iv dose is between 5-10 mg/kg weekly; or (II) the RO is greater than or equal to 85% and the iv dose is >5mg/kg weekly; or (ill) the RO is greater than or equal to 85% and the iv dose is 10-20 mg/kg weekly; or (iv) the RO is greater than or equal to 60% and the iv dose is between 3-10 mg/kg weekly; or (v) the RO is greater than or equal to 60% and the iv dose is >3mg/kg weekly; or (vi) the RO is greater than or equal to 30% and the iv dose is between 1 -3 mg/kg weekly; or (vii) the RO is greater than or equal to 30% and the iv dose is >1 mg/kg weekly; or (viii) the RO is greater than or equal to 60% and the iv dose is >10mg/kg every two weeks; or (ix) the RO is greater than or equal to 30% and the iv dose is >3 mg/kg every two weeks; or (x) the RO is greater than or equal to 85% and the sc dose is between 2-3 mg/kg weekly; or (xi) the RO is greater than or equal to 85% and the sc dose is >2mg/kg weekly; or (xii) the RO is greater than or equal to 60% and the sc dose is >1 mg/kg weekly; or (xiii) the RO is greater than or equal to 60% and the sc dose is >1 mg/kg weekly; or (xiv) the RO is greater than or equal to 60% and the sc dose is between 1 -5 mg/kg weekly; or (xv) the RO is greater than or equal to 60% and the sc dose is between 0.03-0.1 mg/kg daily; or (xvi) the RO is greater than or equal to 85% and the sc dose is > 0.05 mg/kg daily; or (xvii) the RO is greater than or equal to 60% and the sc dose is > 0.02 mg/kg daily; or (xviii) the RO is greater than or equal to 30% and the sc dose is > 0.01 mg/kg daily; or (xix) the RO is greater than or equal to 60% and the iv dose is between 210-700 mg weekly; or (xx) the RO is greater than or equal to 60% and the iv dose is between 210-700 mg every two weeks; or (xxi) the RO is greater than or equal to 60% and the sc dose is between 70-350 mg weekly; or (xxii) the RO is greater than or equal to 60% and sc dose is between 2-7 mg daily.
  16. 16. The method according to any one of claims 12 to 14, wherein the RO is greater than or equal to 85% and the sc dose is between 2-3 mg/kg weekly.
  17. 17. The method of any one of claims 12 to 16, wherein the fibrotic disease is idiopathic pulmonary fibrosis (IPF) or interstitial lung disease (ILD).
  18. 18. An assay for determining receptor occupancy of a CXCR4 binding molecule and ability of the CXCR4 molecule to inhibit cellular migration, the method comprising: (i) isolating human cells expressing CXCR4; (ii) combining the cells with a concentration gradient of the CXCR4 binding molecule for a time sufficient to permit binding of the CXCR4 binding molecule to bind to CXCR4 on the cells; (iii) measuring the receptor occupancy of the CXCR4 binding molecule by detecting the amount of CXCR4 binding molecule bound versus free CXCR4 to derive a percentage value of receptor occupancy; (iv) separately determining the level of SDF-1a induced migration by combining the cells with a concentration gradient of the CXCR4 binding molecule in the presence or absence of SDF- 1 a and counting the number of migrated cells; (v) comparing the relationship between % inhibition of migration of the cells versus % receptor occupancy by the CXCR4 binding molecule.
  19. 19. The method of claim 18 wherein the assay is performed in vitro.
  20. 20. The method of claim 18 or 19, wherein human cells are T cells from a non-diseased 5 subject.

Description

"Linking Receptor Occupancy to Efficacy of CXCR4 antagonists" This application claims priority from Australian application AU 2023902188 filed 7 July 2023 and Australian application AU 2023903085 filed 25 September 2023. The entire content of the electronic submission of the sequence listing is incorporated by reference in its entirety for all purposes. All documents cited or referenced herein, and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference in their entirety. FIELD The disclosure is based on a novel assay which links the degree of CXCR4 receptor occupancy (RO) by an anti- CXCR4 polypeptide to the efficacy of the anti- CXCR4 polypeptide based on determining the SDF-1a induced migration of CXCR4 primary human cells in the presence of the anti-CXCR4 polypeptide. Inhibition of migration as measured in the in vitro assay provides a surrogate for in vivo efficacy of the anti-CXCR4 polypeptide. BACKGROUND Chemokines (chemoattractant cytokines) are a family of structurally and functionally related small proteins that direct migration of cells (e.g., leukocytes and/or lymphocytes and/or stem cells and/or neurons) in addition to controlling other biological processes, such as angiogenesis, morphogenesis, autoimmunity, tumor growth and metastasis. Chemokines are grouped into families based on the presence and relative position of amino terminal cysteine residues (e.g. CC, CXC, CX3C and C chemokines). Generally, the biological activity of a chemokine is mediated by a cell surface receptor, in particular a 7-transmembrane-domain G protein-coupled receptor (GPCR). The chemokine receptors are grouped and named according to the family of chemokine(s) to which they bind. One member of the CXCR family is CXCR4 that is predominantly expressed on lymphocytes and that activates chemotaxis. CXCR4, also called fusin, is an alpha-chemokine receptor specific for stromal-derived-factor-1 (SDF-1 , also called CXCL12), a molecule endowed with potent chemotactic activity for leukocytes including T cells. CXCR4 plays a role in embryogenesis, homeostasis, fibrosis and inflammation. SUMMARY The present disclosure is based on a novel assay which links the degree of CXCR4 receptor occupancy (RO) by an anti-CXCR4 polypeptide to a functional activity based on determining the SDF-1a induced migration of CXCR4 expressing cells in the presence of the anti-CXCR4 polypeptide. Advantageously, the assay is performed in vitro enabling a simple and rapid determination of the level of RO that is associated with functional activity in human cells. Using PK/PD models, in silica simulations can be performed whereby doses and dosing regimens required to achieve target RO levels derived from this assay can be estimated. RO can be used as a surrogate pharmacodynamics (PD) readout in clinical trials, as it assumes that the efficacy of the drug is driven by inhibition of the receptor, whilst the drug is bound (Liang M et al., Cytometry B Clin Cytom (2016) 90(2):117-27). However to confidently utilise RO as a surrogate PD readout, the relationship between RO and a physiological response should be established to show the RO does indeed inhibit functional effects and what degree of RO is required to elicit a desired biological effect. In developing the assay, the inventors sought to determine the level of CXCR4 RO that would be necessary to establish efficacy of their i-body enabled product, AD-214, in the treatment of fibrosis. AD-214 comprises a CXCR4 binding polypeptide (i-body) fused to an Fc region. The CXCR4 binding polypeptide comprises a scaffold based on Domain 1 of NCAM and two binding loop regions (referred to as CDR1 and CDR3). The generation and characterisation of the CXCR4 binding polypeptide (referred to as AM3-114) is described in PCT/AU2016/050005 published as WO 2016/109872, the entire contents of which are incorporated by reference herein. CXCR4/SDF-1a signalling is known to drive fibrosis and therefore one mechanism of action of AD-214 is the inhibition of immune cell migration towards SDF-1a. Ideally, the relationship between RO and immune cell migration would be measured in vivo, however human studies are not feasible. Additionally, there are challenges with accurately estimating CXCR4 RO in mice. Furthermore, it was not known whether the level of RO by the anti-CXCR4 polypeptide in the mouse would adequately reflect the dosage levels required in humans. Determination of RO is important because for therapeutic drugs, the percentage RO needed to achieve a therapeutic effect may need to be as high as 90% or could be as low as 10- 20%. Typically, RO is required to be in the range of 60-90% at trough serum concentration of the drug for efficacy. In work leading up to the present studies, a phas