Linking receptor occupancy to efficacy of cxcr4 antagonists

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.

Description

[0001] "Linking Receptor Occupancy to Efficacy of CXCR4 antagonists"

[0002] This application claims priority from Australian application AU 2023902188 filed 7 July 2023 and Australian application AU 2023903085 filed 25 September 2023.

[0003] The entire content of the electronic submission of the sequence listing is incorporated by reference in its entirety for all purposes.

[0004] 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.

[0005] FIELD

[0006] 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.

[0007] BACKGROUND

[0008] 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.

[0009] 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.

[0010] CXCR4 plays a role in embryogenesis, homeostasis, fibrosis and inflammation. SUMMARY

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] In work leading up to the present studies, a phase I trial of AD-214 in healthy human volunteers revealed that a sustained CXCR4 receptor occupancy (RO) was obtained following infusion of AD-214. However, it was not known what level of RO was necessary for efficacy and what doses and dosing intervals could sustain this level. These studies however were at best a guess of what doses might be safe in human volunteers and did not determine what does are efficacious in humans.

[0017] Furthermore, the present disclosure provides methods to link CXCR4 receptor occupancy by AD-214 with efficacy. To the inventors’ knowledge, this has not been taught or suggested in the prior art. The in vitro cell migration assay disclosed uses inhibition of migration of primary human T cells. These cells, along with other CXCR4 expressing leukocytes are implicated in inflammation and fibrosis by migrating to sites of injury. Inhibition of migration as measured in the in vitro assay is therefore a surrogate for in vivo efficacy. Surprisingly, the inventors found that low CXCR4 receptor occupancy (i.e. as low as 30%) by AD-214 was sufficient to materially or meaningfully inhibit cell migration and therefore demonstrate efficacy of AD-214.

[0018] In a first aspect, there is provided a method of stratifying subjects for treatment with an anti-CXCR4 polypeptide, the method comprising:

[0019] (i) exposing CXCR4 expressing cells derived from the subject to the anti-CXCR4 polypeptide;

[0020] (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.

[0021] In one example, the CXCR4 expressing cells are exposed to the anti-CXCR4 polypeptide in vivo or in vitro.

[0022] In one example, the CXCR4 expressing cells are human T cells. In another example, the T cells are enriched from the buffy coat of whole blood obtained from the subject. In another example, the T cells are CD3+ T cells. In another example, the CXCR4 expressing cell is a fibrocyte, fibroblast or macrophage.

[0023] In one example, the subject has a fibrotic disease. In a further example, the subject has fibrosis of the lung, kidney and / or eye. In a further example, the subject has idiopathic pulmonary fibrosis (IPF). In another example, the subject as a CXCR4 expressing cancer.

[0024] In certain examples, 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%. In a further example, the CXCR4 receptor occupancy is between 60- 85%. In one example, the CXCR4 receptor occupancy is about 85%.

[0025] In one example, the receptor occupancy is maintained for at least 7 days.

[0026] In a preferred example, the receptor occupancy is measured by a method comprising:

[0027] (i) obtaining T cells from the subject; (ii) exposing the T cells to an anti-CXCR4 polypeptide;

[0028] (ill) determining the CXCR4 receptor occupancy by detecting the amount of bound anti- CXCR4 polypeptide versus free CXCR4.

[0029] In one example, the T cells and anti-CXCR4 polypeptide are combined for at least 15 mins.

[0030] In one example, the receptor occupancy is determined by detecting and measuring the amount of bound anti-CXCR4 polypeptide versus free CXCR4 on the T cells. In one example, the bound anti-CXCR4 polypeptide and free CXCR4 is detected by immunofluorescent staining using antibodies. In one example, the anti-CXCR4 polypeptide is detected using a labelled anti- Fc (e.g. Fc-FITC) and the free CXCR4 detected, for example, using antibody 12G5 attached to a detectable label (e.g. 12G5-BV421 ). In another example, the anti-CXCR4 polypeptide is detected, for example, using anti-human lg-AF647.

[0031] In one example, the cells are stained with the antibodies for at least 30 mins. In another example, the flow cytometry is performed on cells fixed with paraformaldehyde. In another example the cells fixed cells are analysed by flow cytometry within 24 hours.

[0032] In a particular example, the anti-CXCR4 polypeptide is AD-214 comprising the sequence of SEQ ID NO:5. In another example, the anti-CXCR4 polypeptide is AM3-114 comprising the sequence of SEQ ID NO:2.

[0033] In one example, the migration inhibition of the T cells is maximally inhibited. In a further example, maximum inhibition is at least 80% inhibition of migration of the T cells.

[0034] In one example, the receptor occupancy is maintained for at least 7 days.

[0035] In a particular example, the receptor occupancy is calculated as follows: %RO = 100 x (occupied / occupied + free)

[0036] In some examples, the data is normalised for background fluorescence using a fluorescence minus one (FMO) control (i.e. cells labelled with all antibodies minus 12G5-BV421 or minus AD-214).

[0037] In a second aspect, there is provided 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%.

[0038] In certain examples, 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%. In a further example, the CXCR4 receptor occupancy is between 60- 85%. In one example, the CXCR4 receptor occupancy is about 85%.

[0039] In one example, the anti-CXCR4 polypeptide is AD-214 comprising the sequence of SEQ ID NO:5. In another example, the anti-CXCR4 polypeptide is AM3-114 comprising the sequence of SEQ ID NO:2. In one example, wherein the dose maintains a serum concentration of between 0.7 - 1 nM for at least a week.

[0040] In one example, the RO is greater than or equal to 85% and the iv dose is between 5-10 mg / kg iv weekly.

[0041] In another example, the RO is greater than or equal to 85% and the iv dose is >5mg / kg weekly.

[0042] In another example, the RO is greater than or equal to 85% and the iv dose is 10-20mg / kg weekly.

[0043] In one example, the RO greater than or equal to 60% and the iv dose is between 3-10 mg / kg weekly.

[0044] In another example, the RO is greater than or equal to 60% and the iv dose is >3mg / kg weekly.

[0045] In one example, the RO is greater than or equal to 30% and the iv dose is between 1 -3 mg / kg weekly.

[0046] In another example, the RO is greater than or equal to 30% and the iv dose is >1 mg / kg weekly.

[0047] In one example, the RO is greater than or equal to 60% and the iv dose is >10mg / kg every two weeks.

[0048] In one example, the RO is greater than or equal to 30% and the iv dose is >3 mg / kg every two weeks.

[0049] In one example, the RO is greater than or equal to 85% and the sc dose is between 2-3 mg / kg weekly.

[0050] In another example the RO is greater or equal to 85% and the sc dose is >2mg / kg weekly.

[0051] In another example, the RO is greater than or equal to 85% and the sc dose is_>1 mg / kg every 3 days.

[0052] In one example, the RO is greater than or equal to 60% and the sc dose is >1 mg / kg weekly.

[0053] In one example, the RO is greater than or equal to 60% and the sc dose is between 1 -5 mg / kg weekly.

[0054] In one example, the RO is greater than or equal to 60% and the sc dose is between 0.03- 0.1 mg / kg daily.

[0055] In one example, the RO is greater than or equal to 85% and the sc dose is > 0.05 mg / kg daily.

[0056] In one example, the RO is greater than or equal to 60% and the sc dose is > 0.02 mg / kg daily. In one example, the RO is greater than or equal to 30% and the sc dose is > 0.01 mg / kg daily.

[0057] In one example, the RO is greater than or equal to 60% and the iv dose is between 210- 700 mg weekly.

[0058] In one example, the RO is greater than or equal to 60% and the iv dose is between 210- 700 mg every two weeks.

[0059] In one example, the RO is greater than or equal to 60% and the sc dose is between 70- 350 mg weekly.

[0060] In one example, the RO is greater than or equal to 60% and sc dose is between 2-7 mg daily.

[0061] In a particular example, the RO is greater than or equal to 85% and the sc dose is between 2-3 mg / kg weekly.

[0062] In one example, the fibrotic disease is idiopathic pulmonary fibrosis (IPF) or interstitial lung disease (ILD).

[0063] In a third aspect, there is provided an assay for determining receptor occupancy of a CXCR4 binding molecule and ability of the CXCR4 molecule to inhibit cellular migration, the method comprising:

[0064] (i) isolating human cells expressing CXCR4;

[0065] (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;

[0066] (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;

[0067] (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;

[0068] (v) comparing the relationship between % inhibition of migration of the cells versus % receptor occupancy by the CXCR4 binding molecule.

[0069] In one example, the assay is performed in vitro. In another example, the assay in performed in a cell culture plate.

[0070] In one example, the human cells are T cells from a non-diseased subject. In a further example, the T cells are isolated from the buffy coat of whole blood. In another example, the T cells are CD3+ T cells. In a further example, the T cells are obtained from a subject who has not received a CXCR4 antagonist.

[0071] In one example, the receptor occupancy is determined for between 50,000 and 100,000 cells / well. In a further example, the cells are seeded in a 96 well plate in a suitable medium. In one example, the medium is FACS buffer (2% FCS / 2nM EDTA / 1 x PBS). In another example, the medium is 1%FCS / RPMI starvation media.

[0072] In a further example according to step (ii), the concentration gradient of CXCR4 binding molecule ranges from 10OnM to 0.001 nM.

[0073] In one example, for step (ii) the cells and CXCR4 binding agent are combined for at least 15 min.

[0074] In a further example, detecting the amount of CXCR4 binding molecule bound versus free CXCR4 is performed by flow cytometry. In another example, detection of the CXCR4 binding molecule is performed using a fluorescently labelled antibody. In another example, the CXCR4 antibody is AD-214 comprising the sequence of SEQ ID NO:5 and detection is performed using AF647 conjugated anti H + L secondary antibody. In another example, free CXCR4 is detected using a competing anti-CXCR4 antibody (e.g. 12G5) conjugated to a fluorescent label (e.g. BrilliantViolet421 ).

[0075] In a further example, the %RO is determined by the formula:

[0076] 100 x (occupied CXCR4 / occupied CXCR4 + free CXCR4).

[0077] In one example, between 50,000 and 200,000 cells are used in step (iv).

[0078] In a further example, step (iv) is performed in a transwell comprising a semi-permeable membrane.

[0079] In a further example according to step (iv), the cells are combined with the CXCR4 binding molecule and SDF-1 for a period of time to permit migration of the cells. In a further example, the cells are combined with the CXCR4 binding molecule and SDF-1 for at least 2.5 hours. In another example it is at least 5 hours, or at least 18 hours.

[0080] In a further example according to step (iv), the concentration gradient of CXCR4 binding molecule is 0 to 100,000nM.

[0081] In one example, the number of migrated cells is quantified by flow cytometry.

[0082] In one example, the % inhibition of migration is calculated as follows:

[0083] %inhibition = 100 x [1 - (X-MIN)ZMAX-MIN)], wherein MIN is number of cells that have migrated in the absence of SDF-1 , MAX is number of cells that have migrated in the presence of SDF-1 , and X is the number of migrated cells at a given anti CXCR4 polypeptide concentration or percent RO.

[0084] In one example, the CXCR4 binding molecule is an antagonist of CXCR4. In a particular example, the CXCR4 binding molecule is AD-214 comprising the sequence of SEQ ID NO:5. In one example, %RO at maximum inhibition is determinative of the efficacy of a given dose of the CXCR4 binding molecule. DESCRIPTION OF THE FIGURES

[0085] Figure 1 shows the percent CXCR4 receptor occupancy of human CD3+ T cells obtained from healthy donors administered AD-214. The lower limit of quantitation (LLOQ) is shown as a dotted line.

[0086] Figure 2 shows AD-214 binding and RO in U266 cells. CHO-hCXCR4 (induced with tetracycline) and U266 cells were seeded in 96 well plates and stained, blocked with anti-human Fc-block and then a concentration gradient of AD-214 diluted in 1%FCS / RPMI starvation media. Left panel = specific binding of AD-214; Middle panel = specific binding of anti-CXCR4 (12G5); Right panel = CXCR4 occupancy. Data is representative the mean of n=2 technical replicates in n=1 experiment.

[0087] Figure 3 shows SDF-1a induced migration of CD3+ T cells isolated from non-diseased control human buffy coats. (A and B) Isolated CD3+ T cells were seeded into transwell plates and SDF- 1 a loaded in the bottom chamber. Migration was quantified 2.5 h later. (A) and (B) represent independent experiments performed in triplicate. Error bars denote SEM.

[0088] Figure 4 RO and migration in CD3+ T cells from donor NDC02. CD3+ T cells isolated from buffy coats of healthy volunteer NDC02 were treated with a concentration gradient of AD-214 for 15 mins at 37°C. Cells were loaded onto a transwell plate with AD-214 present or washed and analysed for CXCR4 occupancy. For CXCR4 occupancy assays in (A), AD-214 was detected using AF647-conjugated anti-H+L secondary antibody and free CXCR4 using a competing anti- CXCR4 (clone 12G5)-BrilliantViolet421 antibody. For migration assays in (B), SDF-1 a (10 nM) induced migration across the transwell was quantified 2.5 h later. Data represent a single experiment with n=3 technical replicates (A-C) or pooled from this experiment and the previous migration assay in NDC02 (D).

[0089] Figure 5 RO and migration in CD3+ T cells from donor NDC06. CD3+ T cells isolated from buffy coats of healthy volunteer NDC06 were treated with a concentration gradient of AD-214 for 15 mins at 37°C. Cells were loaded onto a transwell plate with AD-214 present or washed and analysed for CXCR4 occupancy. For CXCR4 occupancy assays in (A), AD-214 was detected using AF647-conjugated anti-H+L secondary antibody and free CXCR4 using a competing anti- CXCR4 (clone 12G5)-BrilliantViolet421 antibody. For migration assays in (B), SDF-1 a (10 nM) induced migration across the transwell was quantified 2.5 h later. Data represent a single experiment with n=3 technical replicates (A-B). In (C), data are plotted as means and results were expressed as % inhibition by AD-214 relative to minimum (zero SDF-1a) and maximum (+SDF-1a) in donor NDC06.

[0090] Figure 6 RO and migration in CD3+ T cells from donor NDC09 and vendor PBMCs. CD3+ T cells isolated from buffy coats of healthy volunteer NDC09 and vendor human PBMCs were treated with a concentration gradient of AD-214 for 15 mins at 37°C. Cells were loaded onto a transwell plate with AD-214 present or washed and analysed for CXCR4 occupancy. For CXCR4 occupancy assays and expression in (A, D, G), AD-214 was detected using AF647-conjugated anti-H+L secondary antibody and free CXCR4 using a competing anti-CXCR4 (clone 12G5)- BrilliantViolet421 antibody. For migration assays in (B, E), SDF-1 a (10 nM) induced migration across the transwell was quantified 2.5 h later. Data represent a single experiment with n=3 technical replicates (A-B). In (C, F), data are plotted as means and results were expressed as % inhibition by AD-214 relative to minimum (zero SDF-1a) and maximum (+SDF-1a) in donor NDC09.

[0091] Figure 7 Structural pharmacokinetic / pharmacodynamic (PK / PD) model. Circles indicate compartments, solid lines denote drug flow with associated PK and PD parameters that were fixed according to previous results as described in the Methods.

[0092] Figure 8 AD-214 serum concentration vs time after IV or SC bolus dose in mice.

[0093] Figure 9 Fitting of mouse PK data.

[0094] Figure 10 Comparison of PK and RO fits between new model including total CXCR4 data (pink) and previous model fitted only on PK and RO data (blue, run082.1 model A).

[0095] Figure 11 Comparison of total CXCR4 fit between new model using total CXCR4 data (pink, run083, model B) and previous model fitted only on PK and RO data (blue, run82.1 , model A). Rtot,denvedconc% increase from baseline.

[0096] Figure 12 Predicted steady state CXCR4 RO at trough in a typical 70kg individual for different repeated SC and IV dose regimes. Horizontal dashed lines are the target RO of 30%, 60% and 85%. Left: model A (run 082.1 ) fitted to PK + RO data. Right: model B (run 083) fitted to PK + RO + total CXCR4 data. Figure 13 Predicted AD-214 serum concentration and CXCR4 RO after Qlweek IV and SC doses in a typical 70-kg individual. Predictions for a typical 70-kg individual. Horizontal dashed lines are the target RO of 30%, 60% and 85%. Model B (run 083) fitted to PK + RO + total CXCR4 data.

[0097] Figure 14 Predicted AD-214 serum concentration and CXCR4 RO after Q2week IV and SC doses in a typical 70-kg individual. Predictions for a typical 70-kg individual. Horizontal dashed lines are the target RO of 30%, 60% and 85%. Model B (run 083) fitted to PK + RO + total CXCR4 data.

[0098] Figure 15 Left: predicted AD-214 serum concentration and CXCR4 RO after Q1day SC (0.1 mg / kg) and Qlweek SC (1 -20 mg / kg) doses in a typical 70-kg individual. Right: AD-214 serum concentration and CXCR4 RO after Q1day SC doses of 0.001 to 0.1 mg / kg in a typical 70-kg individual. Horizontal dashed lines are the target RO of 30%, 60% and 85%. Model B (run 083) fitted to PK + RO + total CXCR4 data.

[0099] KEY TO SEQUENCE LISTING

[0100] SEQ ID NO:1 : amino acid sequence of domain 1 of NCAM

[0101] SEQ ID NO:2: amino acid sequence of AM3-114

[0102] SEQ ID NO:3: amino acid sequence of binding loop region (CDR1 )

[0103] SEQ ID NO:4: amino acid sequence of binding loop region (CDR3)

[0104] SEQ ID NO:5: amino acid sequence of AD-214

[0105] SEQ ID NO:6: nucleotide sequence of AD-214

[0106] DETAILED DESCRIPTION

[0107] General

[0108] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0109] A list or features including the phrase “and / or” between the second last and last feature means that any one or more of the listed features may be present in any combination.

[0110] Reference to the singular forms “a”, “an” and “the” is also understood to imply the inclusion of plural forms unless the context dictates otherwise.

[0111] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0112] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally- equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0113] Any example herein shall be taken to apply mutatis mutandis to any other example unless specifically stated otherwise.

[0114] Selected Definitions

[0115] The term “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0116] As used herein, the term “affinity” refers to the strength of binding of a single molecule to its ligands and is typically expressed as the equilibrium dissociation constant (KD) for the reversible binding of two agents. It is determined by the ratio of KOff / KOn, between the i-body or CXCR4 binding polypeptide of the present disclosure and CXCR4. KD and affinity are inversely related. The KD value relates to the concentration of i-body or CXCR4 binding polypeptide and so the lower the KD value (lower concentration), the higher the affinity of the antibody.

[0117] As used herein, the term “binds” in reference to the interaction of a CXCR4 binding molecule or polypeptide with a target means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the target. For example, a CXCR4 binding molecule or polypeptide recognizes and binds to a specific protein structure rather than to proteins generally.

[0118] As used herein, the terms “treating”, “treat” or “treatment” include administering a therapeutically effective amount of the polypeptide, nucleic acid molecule, conjugate or multimer of the present disclosure sufficient to reduce or eliminate at least one symptom of a specified disorder. In one example, the treatment involves administering a therapeutically effective amount of the i-body polypeptide to treat a CXCR4-related disease or disorder. In one example, treatment also refers to prophylactic treatment. The term “scaffold” or “i-body scaffold” as used herein is intended to refer to the sequence represented by the scaffold regions of the i-SET human NCAM1 Immunoglobulin (Ig) domain 1 (SEQ ID NO:1 ) defined by amino acids 1 to 26, 33 to 79 and 88 to 97.

[0119] The term “fibrosis” as used herein refers to a thickening or scarring of tissue from an organ such as the lungs, kidney or eye. Pulmonary fibrosis is a lung disease that occurs when lung tissue becomes damaged and scarred.

[0120] I-body scaffold and CXCR4 binding molecules

[0121] The present disclosure provides a binding polypeptide (or “i-body”), which comprises a scaffold with modified CDR1 and CDR3 regions. In one example, the scaffold region comprises Domain 1 of human NCAM1 as shown in SEQ ID NO:1 as follows:

[0122] LQVDIVPSQGEISVGESKFFLCQVAGPAKDKDISWFSPNGEKLTPNQQRISVVWNDDSSSTLT IYNANIDDAGIYKCVVTGEDGSESEATVNVKIFQ (SEQ ID NO:1 ).

[0123] NOAM (or Neural Cell Adhesion Molecule) is a glycoprotein of the l-SET domains or intermediate-set domains from the Immunoglobulin (Ig) superfamily. The extracellular domain of NCAM consists of five immunoglobulin-like (Ig) domains followed by two fibronectin type III (FNIII) domains.

[0124] Domain 1 of human NCAM has been produced as a recombinant polypeptide in a bacterial expression system (Frei et al. (1992) J. Cell Biol. 118:177-194).

[0125] Functional activity

[0126] The binding of a ligand, such as an agonist or SDF-1 to CXCR4 can result in signalling by this G protein coupled receptor, and the activity of G proteins as well as stimulating other intracellular signalling molecules. The CXCR4 / SDF-1 pathway has been implicated in organ vascularisation, as well as in the immune and hematopoietic systems (Tachibana K et al. (1998) Nature 393:591 -594). The inhibitory or stimulatory activity of a CXCR4 binding molecule or polypeptide of the present disclosure can be determined with or without a ligand in a suitable assay, as well as the assessment of the ability of the CXCR4 binding molecule or polypeptide to inhibit or stimulate the activity in the presence or absence of the ligand.

[0127] AD-214 has previously been demonstrated to inhibit migration of CXCR4 expressing cells induced by SDF-1 a. Migration assays are also described in this document. The present inventors have utilised the migration assay as a surrogate for determining and measuring the receptor occupancy of CXCR4 expressing cells bound by AD-214. Accordingly, what is described is a novel in vitro method for a determining the receptor occupancy. Because the receptor occupancy is linked to a functional activity (e.g. migration), the level of receptor occupancy required to elicit functional activity and therefore efficacy can be determined. This is further elaborated in the following experimental examples.

[0128] Examples Methods

[0129] CXCR4 blocking i-bodv

[0130] The generation of i-body AM3-114 which binds to CXCR4 has been previously described in PCT / AU2016 / 050005 published as WO2016 / 109872. Briefly, the modified Domain 1 of NCAM is used as the scaffold for the i-body. The scaffold sequence is represented by SEQ ID NO:1 below with the native binding loop sequences shown in the boxes.

[0131] LQVDIVPSQGEISVGESKFFLCQVAGIDAKDKDIISWFSPNGEKLTPNQQRISVVWNDDSSSTLT

[0132] I YN AN I DDAG I YKCVVffGEDGSESlEATVN VKI FQ (SEQ ID NO:1 ).

[0133] The i-body scaffold region corresponds to amino acid residues 1 to 26, 33 to 79 and 88 to 97 of SEQ ID NO:1.

[0134] The sequence of i-body AM3-114 is set forth in SEQ ID NO:2 below:

[0135] LQVDIVPSQGEISVGESKFFLCQVAGSLSG1RISWFSPNGEKLTPNQQRISVVWNDDSSSTLTI YNANIDDAGIYKCVVWRTGGYRHRYLVLGEATVNVKIFQ (SEQ ID NO:2).

[0136] The i-body comprises two binding loop regions consisting of the sequence SLSGIR (SEQ ID NO:3) and WRTGGYRHRYLVLG (SEQ ID NO:4). These binding loops are also referred to as CDR1 and CDR3 respectively. This i-body was generated by affinity maturation of i-body ADCX- 99 which is also described in PCT / AU2016 / 050005.

[0137] AD-214 comprises the i-body sequence of AM3-114 and an Fc region C-terminal to the scaffold sequence. The amino acid sequence of AD-214 is set forth in SEQ ID NO:5.

[0138] LQVDI VPSQG EISVG ESKFF LCQVA GSLSG IRISW FSPNG EKLTP NQQRI SVVWN DDSSS TLTIY NANID DAGIY KCVVW RTGGY RHRYL VLGEA TVNVK IFQDK THTCP PCPAP ELLGG PSVFL FPPKP KDTLM ISRTP EVTCV WAVS HEDPE VKFNW YVDGV EVHNA KTKPR EEQYN STYRV VSVLT VLHQD WLNGK EYKCK VSNKA LAAPI EKTIS KAKGQ PREPQ VYTLP PSRDE LTKNQ VSLTC LVKGF YPSDI AVEWE SNGQP ENNYK TTPPV LDSDG SFFLY SKLTV DKSRW QQGNV FSCSV MHEAL HNHYT QKSLS LSPGK

[0139] The corresponding nucleic acid sequence is set forth in SEQ ID NO:6.

[0140] AAGCTTACCGCCACCATGGAGTTCGGTCTGTCCTGGGTGTTCCTGGTCGCCATC ATCAAGGGCGTGCAGTGCCTCCAGGTCGACATCGTGCCGTCCCAGGGGGAGAT

[0141] CTCGGTGGGCGAATCGAAGTTCTTCCTCTGCCAGGTCGCGGGCAGTCTCTCCG

[0142] GGATCCGCATCTCCTGGTTCTCCCCTAACGGCGAGAAGCTCACCCCCAACCAG

[0143] CAACGAATCTCCGTCGTCTGGAACGACGATAGTTCCTCGACCCTGACCATCTAC

[0144] AACGCCAACATCGACGACGCGGGCATCTACAAGTGCGTGGTCTGGCGCACGGG

[0145] TGGGTACCGCCACCGCTACCTCGTCCTCGGCGAGGCCACCGTCAACGTCAAGA

[0146] TCTTCCAGGACAAGACCCACACCTGCCCGCCGTGCCCGGCCCCCGAGCTCCTC

[0147] GGCGGGCCCTCCGTGTTCCTGTTCCCCCCGAAGCCAAAGGACACCCTCATGAT

[0148] CTCGCGCACGCCGGAGGTCACGTGCGTCGTGGTCGCCGTCTCCCATGAGGAC

[0149] CCCGAGGTGAAGTTCAACTGGTACGTCGACGGGGTCGAGGTCCACAACGCCAA

[0150] GACCAAGCCCCGCGAGGAGCAGTACAACTCCACCTACCGGGTGGTGTCCGTGC

[0151] TGACGGTGCTCCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG

[0152] TCCAACAAGGCCCTCGCGGCACCCATCGAGAAGACGATCAGCAAGGCGAAGGG

[0153] GCAGCCCCGCGAGCCCCAGGTCTACACCCTGCCGCCCTCCCGCGACGAGCTG

[0154] ACGAAGAACCAGGTGTCGCTGACCTGCCTGGTCAAGGGCTTCTACCCGTCGGA

[0155] CATCGCCGTGGAGTGGGAGAGTAACGGGCAGCCCGAGAACAACTACAAGACGA

[0156] CGCCCCCGGTCCTGGACTCCGACGGCTCCTTCTTCCTCTACTCGAAGCTGACC

[0157] GTGGACAAGTCGCGCTGGCAACAGGGCAACGTCTTCTCCTGTTCCGTCATGCA

[0158] CGAGGCCTTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCCCCGGGGA

[0159] AGTGATAATCTAGAGT

[0160] The underlined portion in SEQ ID N0:5 and SEQ ID N0:6 in the protein / DNA sequence is the anti-CXCR4 i-body. The remaining protein / DNA sequence is the constant domains 2 and 3 of the Fc region of a mutated human lgG1 (DAPA).

[0161] Antibodies and dyes i-body AD-214 (AM3-114-Fc) 924 mg / ml, MW 73.8 kDa (Batch PPP.20.140) and 21 H5- Fc 22.69 mg / ml, MW 72.04 kDa (GenScript: U813ZHD260-3 / P9HF001 ) were used in this study. The following anti-human antibodies were used for flow cytometry: CD3-PE (552127, BD), CD4- V450 (560346, BD), CD8-FITC (561947, BD), CD19-PE-Cy7 (25019941 , BD), CD14- BrilliantViolet650 (301836, Biolegend), anti-Fc-FITC (309-096-008, AffiniPure F(ab')2Fragment Rabbit Anti-Human IgG, Fey fragment specific, Jackson ImmunoResearch), anti-human H+L- AF647 (A-21445, Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647, Invitrogen). A viability dye live / dead aqua (AF405 Invitrogen) was used to exclude dead cells.

[0162] Cell lines

[0163] The T-REx™-CHO Cell Line stably transfected with human CXCR4, hereafter referred to as CHO- hCXCR4, or mouse CXCR4, hereafter referred to as CHO-mCXCR4, were generated previously by the Foley lab. THP-1 and U266 cells were obtained from the Foley lab. CHO lines were grown in DMEM / F12 1 :1 (Gibco) with 10% FCS (SFBS, Bovogen), 1% penicillin / streptomycin (Gibco) and hygromycin B (Gibco, 500 pg / mL final concentration). Tetracycline (1 pg / mL final concentration) was added to the media for a minimum of 18 hours to induce hCXCR4 expression. THP-1 and U266 cells were grown in RPM1 with 10% FCS and 1% pen ici 11 i n / streptomyci n .

[0164] Primary human T cell isolation

[0165] Human buffy coat samples from healthy volunteers were prepared by the RedCross and stored in liquid nitrogen. In one experiment, pre-isolated human peripheral blood mononuclear cells (PBMCs) obtained from Stemcell Technologies (70025.3) were used. Briefly, cryovials of buffy coat sample or PBMCs from individual donors were thawed quickly in a 37°C water bath and transferred to the media used in downstream migration assays, hereafter referred to as starvation media (RPMI 1640 (11875119, Gibco), 1% FCS, 0.1% penicillin / streptomycin), followed by pipetting to resuspend the cells. For the buffy coats, debris, likely clotted blood, not in suspension was removed. Unless otherwise stated, all centrifugation steps were performed at 1 ,500 rpm for 5 min at 4°C. To lyse red blood cells in the buffy coats, the pellet was resuspended in a 10x volume of red blood cell lysis buffer (555899, BD Pharm Lyse™ Lysing Buffer) and incubated for 15 min at room temperature. White blood cells from the human buffy coats and the vendor sourced pre-isolated PBMCs were washed by pelleting and resuspending in FACS buffer (2% FCS, 1x PBS, 2 mM EDTA) two times and counted by trypan blue exclusion using a haemocytometer. CD3+ T cells were immunomagnetically sorted using the EasySep™ Human T Cell Isolation Kit (17951 , Stemcell Technologies) following the manufacturer’s instructions.

[0166] Receptor occupancy (RO) and assay development

[0167] For RO analysis, the protocol generated was based upon the methodology described in Junker F et al., (2021 ) Cytometry Part A 99(8):832-43). Cell lines and primary human CD3+ T cells were seeded in 96 well plates. Cell seeding densities including 50,000, 100,000 and 200,000 cells per well were tested. A concentration gradient of AD-214 (200nM to low pM) was added to the cells and the following incubation times and temperatures tested: 15 min, 37°C; 30 min, 37°C; 1 h, 4°C. After incubation, cells were washed twice then incubated for 30 min at 4SC with a cocktail of antibodies including (i) anti-human lgG-AF647 (A-21445, Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647, Invitrogen) or Fc-FITC (309-096- 008, AffiniPure F(ab')2Fragment Rabbit Anti-Human IgG, Fey fragment specific, Jacskon ImmunoResearch), (II) a viability dye (live / dead aqua, L34963, ThermoFisher Scientific), (iii) antihuman CXCR4 12G5-APC (306509) or -BrilliantViolet(BV)421 (306518) (both Biolegend). For human buffy coat T cells, CD3-PE (552127, BD) was incorporated into the staining cocktail. Cells were washed a further two times before being fixed with 4% paraformaldehyde (PFA), washed and a minimum of 5,000 live cells acquired per sample on a flow cytometer within 24 h. Receptor occupancy was calculated as follows: %RO=100x(normalizedOCCUPIED / normalizedOCCUPIED + normalizedFREE) i.e. %RO = 100 x (normalizedAD MFI / normalizedAD-214 MFI + normalized12G5 MFI), where MFI = median fluorescence intensity. Fluorescence minus one controls (FMO) were used to both determine positive staining for AD- 214 / H+L-AF647 and 12G5-BV421 , and normalise for background fluorescence (e.g. caused by non-specific binding of the secondary antibody). This was done by staining cells with all antibodies / dyes minus the antibody / fluorophore in question. The median fluorescence intensity (MFI) of the FMO control was then subtracted from the respective test conditions to give the true fluorescence of AD-214 / H+L-AF657 or Fc-FITC and 12G5-APC or -BV421.

[0168] SDF-1a induced migration assay

[0169] For migration assays, cells were loaded into transwell plates in RPMI / 1% FCS at 100,000 or 200,000 cells / well. For CD3+ T cells, CLS3387 Corning® HTS Transwell-96 Permeable Support with 5.0 pm pore polycarbonate membrane plates were used. For U266 cells, CLS3384 HTS Transwell®-96 Permeable Support with 8.0 pm Pore Polyester Membrane plates were used. SDF-1 a (Recombinant Human / Rhesus Macaque / Feline, 350-NS), was added to the bottom chamber in RPMI / 1% FCS, and migration across the transwell was quantified 2.5 h later for CD3+ T cells and 6 h for U266 cells. Concentration gradients as indicated in each Figure were tested to determine maximum level of migration achieved for U266 cells and CD3+ T cells. In each assay, non-SDF-1a / background migration was determined by including a 0 nM SDF-1a control. After 2.5h of migration, cells plus media in the bottom well of the transwell were transferred to a V bottom plate, centrifuged, and resuspended in 100 uL of 2% PFA. 90 uL of the cell suspension was acquired on flow cytometer within 2-3 days. Debris were gated out during acquisition.

[0170] RO and subsequent migration assay in primary human T cells

[0171] To link RO and migration, 250,000 CD3+ T cells isolated from human buffy coats or PBMCs as described above were incubated with a concentration gradient of AD-214 for 15 min at 37 °C. 200,000 cells with or without (i.e., by washing twice) AD-214 were then seeded into transwell plates and SDF-1a (10 nM) induced migration monitored as described above. The remaining 50,000 cells were washed twice, and RO was assayed as described above. For the human CD3+ T cells, because of variability in response between donors, data are expressed a % inhibition of migration to account for differences in migrated cell number in the controls i.e., no SDF-1a / no AD-214 (minimum) and +SDF-1a / no AD-214 (maximum).

[0172] Data analysis and software Flow cytometry data were analysed using BD FACSDiva™ and FlowJo v10 software. Graphs were generated using GraphPad Prism v9.

[0173] Example 1 Phase I safety study of AD-214 in human health volunteers

[0174] AD-214 was administered as a single dose to heathy volunteers (men and women) with a body weight range between 49.5 and 92.5 kg. The following dosages were administered as a single iv dose: 0.01 mg / kg, 0.02 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg and 20 mg / kg to 33 subjects. 6 subjects were given an iv dose of 5 mg / ml Q2W x 3. This was a safety study only and efficacy was not assessed.

[0175] The receptor occupancy of CD3+ T cells was measured as well as plasma AD-214 concentration. The results of receptor occupancy are presented in Figure 1. The results show that a % receptor occupancy above 70% was maintained for 1 week for a dosages of 5 mg / kg. The % receptor occupancy above 80% was maintained for 1 week for a dosage of 10 mg / kg. The % receptor occupancy above 60% was maintained for 3 weeks at a dosage of 20 mg / kg.

[0176] This study also determined that 1 nM of AD-214 was detectable in the serum of healthy human subjects 72 hours following administration of 10 mg / kg of AD-214 intravenously.

[0177] Whilst this data demonstrated AD-214’s ability to deliver a surprising duration of high CXCR4 receptor occupancy, it was still not known what minimum level of receptor occupancy was required for therapeutic efficacy (i.e. inhibition of fibrotic processes), the more so because CXCR4 receptor occupancy was not available from in vivo mouse efficacy studies due to the technical difficulties associated with making these measurements. Hence, it was not clear whether CXCR4 receptor occupancy in the mouse would be adequately correlated with CXCR4 receptor occupancy in human in vivo. Therefore, the doses and dosing regimens of AD-214 necessary to achieve efficacious levels of CXCR4 receptor occupancy remained unknown.

[0178] Example 2 Receptor occupancy (RO) development and validation in CHO-hCXCR4 cells

[0179] To link receptor occupancy (RO) to a functional behaviour in a cell, the inventors needed to determine whether the RO assay is suitable for in vitro based assays in which cells are directly treated with a dose titration of AD-214. This would then enable the same cells to be assessed in a migration assay to correlate receptor occupancy to the degree of migration inhibition. This was first optimised with hCXCR4-CHO lines as a stable high expressing CXCR4 cell before moving into human cell lines / primary human PBMCs, which have lower CXCR4 levels. To enable RO and migration to be simultaneously measured, the inventors also needed to validate that the RO assay was unaffected by conditions required for the cell migration assay, such as temperature and use of media vs FACS buffer, and serum starvation. The inventors previously developed an assay to analyse binding of AD-214 to hCXCR4 in CHO-hCXCR4 cells that used anti- human Fc-FITC or H+L-AF647 secondary antibodies to detect AD-214 bound to the cell surface. Flow cytometry is then employed to measure the median fluorescence intensity (MFI) of the secondary antibody signal as a readout of AD-214 binding. Using this method, the inventors determined a KD for AD-214 in the range of 70-300 pM. Herein, the inventors sought to adapt the assay to measure both CXCR4 occupied by AD- 214 and unoccupied or free CXCR4. As the goal here was to link RO with subsequent migration, to ensure this RO assay also works for conditions required for the migration assay, the following conditions were tested in the RO assay: (i) effect of using 37°C culture media vs. 4SC in FACS buffer (current method); (ii) shorter incubation times with AD-214 to minimise risk of internalisation (comparing 15 min and 30 min to 60 min currently used) (ill) cell seeding densities required for migration assay (50,000 vs 100,000 vs 200,000) and (iv) effect of serum starvation media prior to performing assay, as this is required for the migration assay.

[0180] In the first assay, (i-iii) were added simultaneously. Changing the temperature, incubation time and cell seeding density did not have an appreciable effect on AD-214 binding. Assay conditions selected for further assay development were 200,000 cells and 37°C, 15 min.

[0181] In the second assay, (iv) the effect of incubating cells with AD-214 diluted in serum starvation media was tested. The inventors compared AD-214 diluted in the current buffer (i.e. FACS buffer (2% FCS / 2 mM EDTA / 1 x PBS) and 1% FCS / RPMI starvation media. No change in AD-214 binding or KD was observed. Therefore, 1% FCS / RPMI starvation media was used in further assay development steps.

[0182] Example 3 Characterisation of cryopreserved non-disease control human buffy coat

[0183] To provide a stronger translational link to the clinic for the RO / migration data, the inventors sought to establish migration and RO assays in primary human T-cell using stocks of cryopreserved buffy coats obtained from non-diseased donors (NDC) from The Alfred hospital.

[0184] First, the inventors sought to determine recovery of cells post-thaw, expression of CXCR4 and T-cell yields per vial. White blood cells were prepared from NDC01 by washing; removal of debris, likely clotted blood and red blood cells, through lysis; and further washing as described in the methods. After counting, cells were seeded in plates and stained to characterize 12G5 (CXCR4) expression on CD3+ T cells and CD3- cells (i.e., other lymphocytes / immune cells). Donor NDC01 contains the following: 45% of cells are live lymphocytes; -75% of live lymphocytes = CD3 / 12G5++, -91% of CD3+ cells are 12G5+. Notably, expression of CXCR4 is likely low on T cells vs other cells based on their MFI: CD3+ 12G5 MFI = -520; CD3- 12G5 MFI = -2000. In a second experiment, buffy coats from donor NDC01 were further characterized pre- and post-immunomagnetic enrichment of CD3+ lymphocytes. Staining for CD4 and CD8, two T cell markers confirmed that the CD3+ population is indeed T cells and magnetic sorting of CD3+ cells led to enrichment to >86% of the total population in the sample. 40% of CD3+ cells were lost during the process of magnetic sorting, and it was determined that a single vial of NDC01 buffy coat could yield 200-300,000 CD3+ T cells.

[0185] Example 4 Testing AD-214 RO in THP-1 and U266 cell lines

[0186] Whilst the CHO-hCXCR4 cells provide a useful tool for assessing AD-214 binding and assay optimisation, they are an artificially induced cell with very high levels of CXCR4 expression. Therefore, the inventors also wanted to validate their assays in additional human cell lines that endogenously express CXCR4 and have previously been used in the migration assay. The THP- 1 (monocytic cell line, leukaemia patient) and U266 (peripheral blood B cell, myeloma patient) CXCR4 positive multiple myeloma cells lines have previously been used in the Foley lab for both AD-114 and AD-214 studies. Using the conditions established from the CHO-hCXCR4 cells described above the inventors performed the same AD-214 RO assay in THP-1 and U266 cell lines to determine if this assay is suitable for cells with lower CXCR4 expression.

[0187] Non-specific binding of Fc-FITC antibody to THP1 cells was observed. AD-214 competed for 12G5 binding in U266 cells indicating specific binding to CXCR4. The KD was 40- 50 pM for AD-214 in U266 cells. Expression of CXCR4 was lower on U266 cells compared to CHO-hCXCR4 cells as expected. These data (Figure 2A and B) demonstrate proof of principle for this method to measure RO in cell lines.

[0188] Example s Validation of RO assay using H+L-AF647 (AD-214 secondary) and antihuman 12G5-BrilliantViolet421 in RO assay.

[0189] In experiments subsequent to those shown in the prior example, the Fc-FITC antibody did not pass QC, therefore the investors elected to use a different secondary anti-Fc antibody. Hence, a H+L-AF647 was obtained and titrated. As the 12G5 antibody being used in the RO assay was conjugated to APC, a version with a different fluorophore, BrilliantViolet421 , was obtained and titrated. This revised RO assay was then validated in two buffy coat donors which showed the KD and IC50 values for AD-214 in the expected picomolar range. An observation was made that the presence of the H+L antibody lowers detection of CD3-PE; however, T cells can still be gated in the analysis using the combination of a lymphocyte gate and CD3+.

[0190] Example s SDF-1a induced migration of CD3+ T cells isolated from non-diseased control human buffy coats

[0191] The inventors sought to validate an SDF-1a induced migration assay using CD3+ T cells isolated from non-diseased control human buffy coats. In the first experiment, shown in Figure 3A, CD3+ T cells were enriched from buffy coats of donors NDC01 and NDC02 to 60-80% of cells. A 2.5 h migration assay was performed using 200,000 or 100,000 CD3+ T cells from each donor seeded in the top well of a 96 well 5 urn transwell plate with a concentration gradient of SDF1 (100-0 nM) placed in the bottom well. For both donors, migration was highest with 5 or 10 nM SDF1 and seeding 200,000 cells led to greater cell migration but did not appreciably increase background, i.e. migration in the media (non-SDF1 ) wells. In a second experiment, shown in Figure 3B, the inventors validated the finding that CD3+ T cells from donor NDC02 seeded 200,000 cells / well led to greater migration than 5 nM and showed that 24 well 5 urn transwell plates gave a similar level of migration to 96 well 5 urn plates. The chemotaxis index, fold change over media (i.e. no SDF-1 ) is a useful method to normalise for differences in background migration and was plotted in these experiments revealing a 6-fold increase in migration with 10 nM SDF-1. Therefore, seeding density of 200,000 cells and 10 nM SDF-1 were the selected conditions for future SDF-1 migration assays using human buffy coat derived CD3+ T cells.

[0192] Example 7 RO and subsequent migration in human CD3+ T cells

[0193] The inventors sought to link inhibition of cell migration and RO as a function of AD-214 concentration in CD3+ T cells isolated from donor NDC02. Thousands of cells migrated in response to SDF-1 a as expected. Dose dependent inhibition of migration was observed for AD- 214 treated cells. However, a lower-than-expected Bmax of AD-214 / Fc-FITC which suggests either AD-214 or the Fc-FITC antibody may be a problem and require further QC.

[0194] Example 8 Titration of H+L-AF647 (AD-214 secondary)

[0195] To overcome the challenges with the Fc-FITC, a different secondary antibody against the Fc was obtained on a different fluorophore. Anti-human H+L-AF647, as an alternative AD-214 secondary antibody, was titrated in cells occupied with AD-214 at increasing concentrations from 1 :100 to 1 :25. In hCXCR4 cells, 1 :25 was required to detect maximal AD-214 binding. The apparent KD -460 pM was somewhat higher than previously determined for hCXCR4-CHO cells and therefore titration in the target cell type (i.e. T cells) should also be conducted. In this assay, binding to CHO-mCXCR4 was also tested. A titration was also performed against mouse CXCR4. In CHO-mCXCR4 cells, 1 :25, the highest concentration of H+L-AF647, was required to detect AD-214. The data suggest that higher concentrations of AD-214 are required to achieve saturation in mCXCR4-CHOs and the KD is likely in the 1 pM range.

[0196] Example 9 Validation of H+L-AF647 (AD-214 secondary) and anti-human 12G5- BrilliantViolet421 in RO assay In the previous iteration of the assay the secondary for AD-214 was Fc-FITC and 12G5 conjugated to APC was used, however, the Fc-FITC was not performing optimally. To overcome this, a new secondary against the Fc was obtained, as described above, which also required a new 12G5 antibody using a suitable complementary fluorophore to be used for the RO assay. A 12G5 antibody conjugated to BrilliantViolet421 was obtained for this purpose and was titrated at 1 :50 and 1 :100 dilution together with H+L AF647 in the AD-214 RO assay using CHO-hCXCR4 cells and buffy coat CD3+ cells isolated from donor NDC08 to assess performance in the RO assay. In CHO-hCXCR4 cells, at 1 :50 dilution of BV421 -12G5 there was a loss of H+L-AD214 signal that did not occur in PBMC donor NDC08, whereas 1 :100 dilution showed comparable results to the previous RO assays, therefore was selected as the optimal dilution for future use. In donor NDC08, either 1 :100 or 1 :50 are adequate for measurement of CXCR4 expression. It was observed that CD3-PE expression is lower in the staining cocktail compared to the single colour control for CD3-PE, it is unclear whether this is due to presence of AD-214 or other antibodies in the cocktail. This revised RO assay was then validated in a different donor, NDC07 which showed the Kd for AD-214 of 45 pM and IC50 of 38 pM , which is in the expected range based on historical data. This also showed an RO with an EC50 of 80 pM as expected. The presence of the H+L antibody appears to lower detection of CD3-PE; however, CD3+ T cells can still be gated in the analysis. These data which are a repeat of the previous assay in donor NDC08 suggest that this RO assay is robust and fit for purpose using the new antibodies for 12G5 and human Fc.

[0197] Example 10 Linking RO and migration in CD3+ T cells from donor NDC02 using the validated H+L-AF647 and 12G5-BV421 RO assay.

[0198] Having validated an RO assay, the inventors performed a RO and subsequent migration assay in CD3+ T cells isolated from donor NDC02. As shown in Figure 4A, the RO assay performed as expected with a KD in the pM range. Compared to the prior AD-214 migration assay performed in this buffy coat donor NDC02, fewer cells migrated in the control wells, particularly 0 nM AD-214 + 10 nM SDF-1a (Figure 4B). A concentration dependent relationship between AD-214 and migration was observed at most concentrations of AD-214 but at low concentrations this was not the case (Figure 4B-C). Migration data has been generated in n=2 experiments. In Figure 4D, data were pooled between the two experiments performed for donor NDC02 to enable more accurate estimation of RO vs migration inhibition. The graphs of %RO vs % inhibition of migration was plotted. The data are an average of technical replicates for RO and migration, respectively. Example 11 Linking RO and migration in CD3+ T cells from donor NDC06

[0199] To ensure a more accurate measurement of RO and migration in primary T-cells, the assay needs to be performed in multiple donors, therefore it was repeated in NDC06. Originally, this assay was to be performed in triplicate with 8 concentrations of AD-214; however, fewer cells were recovered from this donor, therefore the protocol was adjusted for duplicate wells, 6 AD- 214 concentrations tested, and 21 H5-Fc was not tested. The KD for AD-214 was in the expected range, 500 pM. Migration was low with approximately 2x the number of migrated cells in the +SDF-1a control as compared to the no SDF-1a control. AD-214 inhibited migration in a concentration dependent manner. Interpolation of the RO x migration curve showed that to achieve maximal migration inhibition, the % RO and concentration of AD-214 was 57% and 0.8 nM, respectively. Results are shown in Figure 5.

[0200] Example 12 Linking RO and migration in CD3+ T cells from donor NDC09 and purified PBMCs.

[0201] Based on the number of available frozen buffy coats, the assay was repeated on donor NDC09 and a vial of human PBMCs from a vendor, Stemcell technologies. Modest cell number was recovered from NDC09 and therefore triplicate wells were run with 6 AD-214 concentrations tested.

[0202] 21 H5-Fc was not tested. For NDC09, 85% RO = 1 nM AD-214 was required to achieve maximum migration inhibition. For Stemcell Technologies donor, 60-70% RO = 1 nM AD-214 was required to achieve maximum migration inhibition. Low CXCR4 expression was observed on CD3+ T cells from the Stemcell Technologies derived CD3+ T cells relative to the other donors.

[0203] Results are shown in Figure 6.

[0204] A summary of % RO and AD-214 concentrations required to achieve maximal migration inhibition and IC50 values is shown in Table 1 for each donor tested in previous examples. Data are interpolated from RO and % migration inhibition curves from each donor.

[0205] Table 1 %R0 and AD-214 concentrations to achieve maximal migration inhibition

[0206] Over n-3 experiments / donors, to achieve 100% migration inhibition: o RO- - 57-85% (AVE AGE = 66%)

[0207] = 0.7-1 nM (AVERAGE 0.83 nM) [AD-214]

[0208] It was concluded that 100% migration inhibition required a RO of between 57-85%, the average being 66%. The concentration range of AD-214 was between 0.7 and 1 nM, the average being 0.83nM. Earlier studies had determined that 1 nM of AD-214 was detectable in the serum of healthy human subjects 72 hours after administration of 10 mg / kg of AD-214 intravenously.

[0209] In summary, these ex vivo studies showed that:

[0210] • CXCR4 receptor occupancy measured in the model system at different AD-214 concentrations was consistent with the receptor occupancy observed in phase I clinical trials at the same circulating blood concentrations of AD-214.

[0211] • Maximal migration inhibition was achieved at CXCR4 receptor occupancy of 60-85% (average 66%) while a meaningful 50% migration inhibition was achieved at CXCR4 receptor occupancy of 11 -37% (average 30%).

[0212] • Maximal migration inhibition (and corresponding required receptor occupancy) was achieved at AD-214 concentrations of 0.05-0.07 pg / ml with 50% migration inhibition achieved at AD-214 concentrations five to ten fold lower.

[0213] • Blood concentrations of AD-214 exceed 0.07 pg / ml for approximately 72 hours following a single 10 mg / kg intravenous administration of AD-214, well after primary distribution from the blood.

[0214] Taken together, these results help identify target levels of CXCR4 receptor occupancy, and hence circulating concentrations of AD-214, that may be needed for efficacy in fibrotic indications. Maximal T cell migration inhibition, and hence potential efficacy against fibrosis, can be achieved by maintaining CXCR4 receptor occupancy above 60-85% and meaningful inhibition can be achieved as low as 11 -37%. Due to the very tight binding of AD-214 to CXCR4, these levels of receptor occupancy can be achieved at very low circulating concentrations of AD-214. The Phase I clinical study shows that concentrations for maximal inhibition of T cell migration are maintained for several days after intravenous administration, and for meaningful inhibition for much longer.

[0215] Example 13 Simulating intravenous and subcutaneous administration of AD-214

[0216] The aims of this study was to predict what is the likely minimum i.v. dose weekly, every two weeks and every three weeks to maintain 30, 60 or 85% receptor occupancy (RO) at trough and predict what s.c. dose and dosing frequency of AD-214 would be required to achieve the same outcome.

[0217] Methods

[0218] Study design

[0219] The population PK / PD model was developed based on data from a phase I study in healthy volunteers, men and women mixed. Body weight ranged from 49.5 to 95.2kg. The study had 2 parts, with different subjects in both parts:

[0220] • Single Ascending Dose (SAD) part (N = 33): 7 cohorts (A1 to A7), with single IV doses of 0.01 , 0.02, 0.1 , 1 , 5, 10 and 20 mg / kg.

[0221] • Multiple Ascending Dose (MAD) part (N = 6): 1 cohort (B1 ), with IV doses of 5 mg / kg Q2w x3.

[0222] Bioassavs

[0223] The concentration of AD-214 was measured in serum using ELISA and was validated by acceptance criteria defined in acceptance protocols. The LLOQ in 100% serum was 19.53 ng / mL and any sample above 1250 ng / mL could be further diluted up to 100-fold. Free CXCR4 receptors were measured on Peripheral blood mononuclear cells (PBMCs) by staining cells for anti-CXCR4 on APC channel. AD-214-bound CXCR4 receptors were measured on PBMCs using fluorescence quantitation, which was performed using Simply Cellular standards (Bangs Laboratory), consisting of a single population of antibody-coated microspheres of known Antibody Binding Capacity (ABC). Background subtraction was performed for both occupied receptor and free receptor ABC values. Total CXCR4 counts were derived as free CXCR4 counts + occupied CXCR4 counts. RO was calculated as 100 * occupied CXCR4 counts / total CXCR4 counts. Total CXCR4 receptors were also measured with another assay, but these data were not used for the current analysis (only total derived CXCR4 data were used).

[0224] PK study in mice

[0225] AD-214 (10 mg / kg) was administered to BALB / c mice as a single IV or SC bolus injection. Two samples per animal and three animals per timepoint. Blood samples were processed to serum by centrifugation. The PK assay is a sandwich ELISA using an anti-NCAM antibody (MAB24081 clone) as the capture antibody and an anti-Human Fc-HRP (Sigma Aldrich; A0170- 1 ML) antibody as the detection antibody - LLOQ 86 ng / mL.

[0226] Data programming and QC

[0227] Data from all cohorts were merged into a single PK / PD analysis data set using a standard modeling data format as required for analysis with Monolix software. Nominal dosing times, infusion durations and observation times were used. Total CXCR4 concentrations were calculated as: where WCC = White cell counts, NA=6.022-10A23 = Avogadro Number and c = estimated conversion factor.

[0228] An automated quality control (QC) check of the analysis data sets was performed using the script Data_QC.1 .34. R to ensure formal correctness of the data set for population PK / PD modeling with Monolix. The QC output indicated no errors in the data set programming. The analysis data set included the PK / PD and dosing information for 39 subjects. The total number of PK observations were 642, of these 213 (33.18%) were BLQ (Below the Limit of Quantitation) (see Handling of missing and outlier data). The total number of RO observations were 272 and the total number of total CXCR4 observations were 305.

[0229] Handling of missing and outlier data

[0230] A small number of outlier data was excluded where there was obvious assay errors such as abnormally high baseline values or there was some physically explainable reason. No other outliers were identified via visual inspection of the data and no further steps were taken to identify and handle outliers. CXCR4 RO and total CXCR4 data from cohort B1 were not included in the model, because of differences with cohort A5 testing the same dose of 5 mg / kg. PK data of cohort B1 were consistent with cohort A5 data and were included for modeling. The analysis data set had no missing values. Data below the limit of quantification (BLQ) were included in the population PK analysis using the censored methodology available with Monolix (Samson A et al, (2006) Extension of the SAEM algorithm to left-censored data in nonlinear mixed-effects model: Application to HIV dynamics model. Computational Statistics & Data Analysis. 51 (3):1562-74). Parameter estimation

[0231] Population parameters were estimated using the SAEM algorithm implemented in Monolix. The minimum and maximum number of iteration steps for the exploratory and smoothing phase of the algorithm were set to 150- 2000 and 50-500, respectively. It was confirmed with the SAEM convergence plots that the number of iteration steps was sufficiently large to have convergence to stable parameter estimates in all cases. The initial estimates for the fixed effects parameters were determined by fitting the model manually to the data using the 'Check initial estimates' functionality in Monolix. The initial standard deviations of the random effects were set to 1. The standard errors of the parameter estimates were derived from the Fisher information matrix using stochastic approximation. For the individual parameters, the conditional mean and standard deviation were computed using default settings in Monolix. The -2 log-likelihood (-2LL) was computed using importance sampling with a Monte Carlo chain length of 10'000. It was confirmed via the standard error of the -2LL estimates that this Monte Carlo chain was sufficiently long.

[0232] Structural model

[0233] The model to describe AD-214 PK, CXCR4 RO and total CXCR4 in human was three- compartmental with parallel linear first-order elimination and non-linear Michaelis-Menten elimination from the central compartment. There was constant production and first-order internalization of CXCR4 in the central compartment. Binding of AD-214 to CXCR4 was modelled with forward and backward reactions in the central compartment. The AD-214:CXCR4 complex was internalized at a different rate than free CXCR4 (Figure 7). The non-linear Michaelis-Menten elimination term was thought to represent AD-214 elimination in the liver, independent of the general non-specific elimination of the i-body and independently of CXCR4-mediated elimination. Both models A (run 082.1 ) and B (run 083) had the same structure. The main difference was that model A was fitted to PK and CXCR4 RO data only, while model B was fitted to PK, CXCR4 RO and total CXCR4 data. Another difference was that the baseline CXCR4 concentration (R0) was estimated in model A, while model B used the individual observed baseline CXCR4 counts and an estimated conversion factor to concentration (representing the number of receptors per measured count).

[0234] Statistical model

[0235] Individual parameters were modeled as random variables, with log-normal distributions. The equation for an individual parameter was: where (ppopwas the population typical parameter and nJ was a normally distributed random variable with mean 0 and standard deviation co. Combined error models were used for modeling the AD-214 PK, CXCR4 RO and total CXCR4 observations: y obs Vpred T (fl + Vpred ' where yObs was the observation, ypred the model prediction, s was an independent random variable, normally distributed with mean 0 and variance 1. The parameter a described the standard deviation of the constant error and the parameter b the proportional coefficient. No covariates were included in the model.

[0236] Model development approach

[0237] For model development and selection, the diagnostic plots, the standard error of the parameter estimates and the -2LL were used as selection criteria. Diagnostic plots were constructed in Monolix. Additionally, simulations using population-typical parameter estimates were run with Simulx and overlaid with the observed data to evaluate how well model predictions agreed with observations.

[0238] Simulation methodology

[0239] PK simulations were done in R using the Simulx API. The simulations were done for a typical individual, i.e. , they did not include between-individual variability, nor observational error. The simulations included the covariate effects. A body weight of 70 kg was used to calculate the administered amount in mg from the mg / kg dose. Simulations were performed for 1 -h IV infusion and for SC administration.

[0240] Values for SC absorption rate ka and SC bioavailability F were assumed based on the literature:

[0241] • F = 0.8, based on values reported for similar compounds (Richter WF et al., (2012) Mechanistic determinants of biotherapeutics absorption following SC Administration. The AAPS Journal, 14(3), pp. 559-570. doi:10.1208 / s12248-012-9367-0, Richter WF et al., (2014) Subcutaneous absorption of biotherapeutics: Knowns and unknowns. Drug Metabolism and Disposition, 42(11 ), pp. 1881-1889. doi:10.1124 / dmd.114.059238, Temrikar ZH et al., (2020) Pharmacokinetics and clinical pharmacology of monoclonal antibodies in pediatric patients. Pediatric Drugs, 22(2), pp. 199-216. doi:10.1007 / s40272-020-00382-7.

[0242] • ka = 0.721 day-1 , based on a published relationship between molecular weight and absorption half-life in mouse (Richter 2014) and scaling the absorption rate based on body weight and a scaling coefficient of -0.25. These values from the literature were compared with estimates based on sponsor data in mouse (small study of one SC and IV dose level) and NHP (AD-114-PAS, which is the parent molecule without Fc fusion) that were shown to be consistent. For the plots of CXCR4 RO at trough vs dose, the RO at the end of a dosing interval at steady-state was extracted. It was visually checked that steady-state had been reached.

[0243] Software and computer system

[0244] All data programming, data exploration, model building, and simulations were done on a desktop computer (PowerCrunch-9) running Windows 10 Professional. For PK / PD parameter estimation and diagnostic plots, a validated version Monolix Suite 2023R1 was used (Monolix (2023) version 2023R1. Antony, France: Lixoft SAS). For the simulations, the Simulx API (Monolix Suite 2023R1 ) and a validated version of R 4.3.0 (R Development Core Team (2008) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051 -07-0, 2008, URL http: / / www.Rproject.org) were used.

[0245] Guidelines

[0246] The data analysis was in accordance with the FDA Guidance for Industry on Population Pharmacokinetics (FDA 2022), the EMA guideline on PopPK analyses (EMA (2007) Guideline on Reporting the Results of Population Pharmacokinetic Analyses. Doc. Ref. CHMP / EWP / 185990 / 06) and the LYO-X standard operating procedures (SOP) from the QMS Version 3.97.

[0247] Results

[0248] Data from the mouse PK study are shown in Tables 2 and 3 and displayed graphically in Figure 8.

[0249] Table 2 AD-214 PK data after IV administration -10mg / kg AD-214 in mice

[0250] Table 3 AD-214 PK data after SC administration -10mg / kg AD-214 in mice

[0251] Non-compartmental PK parameters are shown for each route of administration in Table 4 below. Table 4 AD-214 PK parameters after IV and SC administration -10mg / kg AD-214

[0252] In mice, after IV administration, the PK profile showed an initial very rapid distribution followed by a slower elimination phase. 2 / 3 samples were below LLOQ (negative values reported) at 144h post-dose and this time point was excluded from the PK analysis. The terminal half-life was estimated to be 24h although the fit to the data was quite poor (R2<0.9) (Figure 8). SC administered AD-214 was absorbed very rapidly with Tmax at 4h post-dose. Thereafter, the PK profile was consistent with that observed after IV administration. 2 / 3 samples were below LLOQ (negative values reported) at 144h post-dose and this time point was excluded from the PK analysis. In addition, the terminal elimination phase could not be accurately quantified (value 28h, R20.69). Overall, AD-214 is absorbed rapidly after SC administration in mice with Cmax 404 ng / mL and absolute bioavailability is estimated to be about 79% based on AUC(O-inf). Given the uncertainty in estimating a terminal half-life, an absolute bioavailability of 73% was also estimated using AUC(0-72h).

[0253] There was a good fit of mouse PK data by a 2-compartmental model with linear elimination, but V1 estimate (84 mL) was much larger than expected in mouse (plasma volume = 1 mL). F and ka estimates were consistent with the values assumed in human (Figure 9).

[0254] A PK / PD model (Version A) was built based on PK and RO data obtained from volunteers that received single i.v. doses of AD-214 of 0.02, 0.1 , 1 , 5, 10 and 20 mg / kg and volunteers that received an iv dose of 5 mg / ml Q2W x 3. A 3-compartment model with parallel linear and nonlinear elimination (Michaelis-Menten approximation) as well as explicit receptor binding + complex internalization (with KD and konfixed to SPR measured values), was required to fit the AD-214 concentration and CXCR4 receptor occupancy (RO) data (see Figure 7). A good fit of PK and RO observations was observed for all tested dose levels, except some slight underprediction of CXCR4 RO at 7 and 14 days after 20 mg / kg. Standard errors of the estimates were reasonably small, but the model is complex with risk of model over-parametrization and overfitting to the data. The need for 3 compartments is not unusual and improved the fit of the PK observations, especially for the one observation >LLOQ at 7 days and 20 mg / kg. The need for an additional non-linear elimination process supports the hypothesis that another specific elimination route, for example in the liver, might be responsible for the observed AD-214 concentration-time profiles, in addition to CXCR4 on the cells where RO was measured. This is mostly dictated by the CXCR4 RO data, as a simpler model was able to fit the PK observations. A second PK / PD model (Version B) was then created to additionally predict the increase in total CXCR4 receptors observed in the Phase I trial. Total CXCR4 data was added to the model, using individual observed baseline CXCR4 and an estimated conversion factor c (number of receptors per count) without variability on c, Q and V2.

[0255] As noted, Version A of the PK / PD model was derived by fitting only available PK and RO data and does not accurately model total CXCR4 receptor numbers. Version B of the model potentially has lower precision / wider confidence interval because total receptor numbers is derived from a difference in two indirect assay measurements (unlike the RO assay which, as a ratio measurement, self-corrects some of its variability. Version A and Version B produce similar predictions are both therefore useful in predicting target doses.

[0256] Comparisons between the Version A and Version B models including total CXCR4 data were performed. As shown in Figure 10 for the comparison of PK and RO fits between new model including total CXCR4 data (pink, run083) and previous model fitted only on PK and RO data (blue, run082.1 ), very similar PK fits were observed. Similar RO fits were also observed, but slightly faster decrease for the new model was observed, especially for the low doses. Hence, the new model is correctly fitting the PK and RO data. As shown in Figure 11 for the comparison of total CXCR4 fit between new model including total CXCR4 data (pink, run083) and previous model fitted only on PK and RO data (blue, run082.1 ) (Rtot,dertvedconcincrease from baseline). As expected, model Version B fits the observed total receptor increase.

[0257] The inventors then performed simulations and predictions of the likely achievable receptor occupancy with different dosing regimens with an IV administration. The model was used to simulate the required IV dose to reach a trough RO of either 30, 60 or 85% for a dosing regimen of once every week, once every two weeks or once every three weeks. These trough RO levels were selected based on the potential to inhibit T cell migration (and therefore impact a therapeutically relevant mode of action) at 50% inhibition, maximum inhibition in at least some individuals and maximum inhibition in all individuals respectively. In addition, to then understand what dosing and frequency would be required to achieve these RO troughs (30, 60 and 85%) using a subcutaneous administration route, the model was applied with the inclusion of subcutaneous kinetics for biological molecules of appropriate molecular weight. Bioavailability factors and rate absorption constants were estimated using the results of a pilot study of AD-214 SC and IV administration in rodents, literature values for similar molecules and standard allometric scaling.

[0258] Simulations were performed to predict CXCR4 RO at trough after Q1day, Qlweek, Q2week and Q3week IV and SC doses of AD-214 as shown in Figure 12. Predictions were performed for a typical 70 kg individual. 1 -h IV infusion. F=0.8 and ka = 0.721 days-1 for SC. Horizontal dashed lines are the target RO of 30%, 60%, and 85% at trough. 10 mg / kg represents a 28 mL IV infusion for a 70 kg patient using current clinical formulation. 1 mg / kg represents a 0.9 mL SC injection for a 70 kg patient at maximum AD-214 concentration for which stability is available to date. Target dosing parameters for RO: minimum of 30% at trough (and greater than 60% for 75% of dosing window); IV: 2 week minimum interval between doses; SC: daily or weekly. Daily or weekly SC administration could offer a more patient convenient route of administration and lower total protein load.

[0259] Predications for AD-214 plasma concentrations and CXCR4 RO were then performed for Q2 week i.v. and s.c. doses as shown in Figure 13. Predications were performed for a typical 70kg individual following 1 h i.v. infusion or s.c. injection. Q2w doses required to reach targets: 30% RO at trough: 5-10mg / kg i.v; 20 mg / kg s.c.

[0260] 60% RO at trough: 20 mg / kg i.v.

[0261] >60% RO at trough for .75% of dosing window: 10 mg / kg i.v; 20 mg / kg s.c.

[0262] Predications for AD-214 plasma concentrations and CXCR4 RO were then performed for Q1 week i.v. and s.c. doses as shown in Figure 14. Predictions were performed for a typical 70kg individual following 1 hr i.v. infusion or s.c. injection. Q1w doses required to reach targets: 30% RO at trough: 1 -3 mg / kg i.v or s.c.

[0263] 60% RO at trough: 5 mg / kg i.v. or s.c.

[0264] >60% RO for >75% of dosing wondow: 3mg / kg i.v; 1 mg / kg s.c.

[0265] Predictions for AD-214 plasma concentrations and CXCR4 RO were then performed for Q1day and Q1w SC doses as shown in Figure 15. Predictions for a typical 70-kg individual - F = 0.8 and ka = [0.3; 0.721 ; 1.1]days-1; 1 -20 mg / kg Q1w; 0.001 mg / kg Q1 d. Required doses to reach targets:

[0266] 30% RO at trough: 0.01 mg / kg Q1d, 1 -3 mg / kg Q1w;

[0267] 60% RO at trough: 0.03 mg / kg Q1d; 3-5 mg / kg Q1w;

[0268] >60% RO for >75% of dosing window: 0.01 -0.03 Q1d; 1 -3 mg / kg Q1w; 80% RO at trough: 0.1 mg / kg Q1d; 10-20 mg / kg d1w.

Claims

CLAIMS:

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. The method of claim 1 wherein the cells are exposed in vivo or in vitro.

3. The method of claim 1 or 2, wherein the CXCR4 expressing cells are human T cells.

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. 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. The method of any one of claims 1 to 5, wherein the CXCR4 receptor occupancy is between 60-85%.

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. 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. The method of claim 8, wherein the detecting and measuring is by flow cytometry.

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. The method of any one of claims 1 to 10, wherein migration inhibition of the T cells is maximally inhibited.

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. 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. The method of claim 12 or 13, wherein the anti-CXCR4 polypeptide is AD-214 comprising the sequence of SEQ ID NO:5.

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. 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. 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. 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. The method of claim 18 wherein the assay is performed in vitro.

20. The method of claim 18 or 19, wherein human cells are T cells from a non-diseased 5 subject.

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