Associating receptor occupancy with CXCR4 antagonist efficacy

Abstract

The present disclosure is based on a novel assay based on the determination of SDF-1 [alpha]-induced migration of CXCR4 primary human cells in the presence of an anti-CXCR4 polypeptide, correlating the degree of CXCR4 receptor occupancy (RO) of the anti-CXCR4 polypeptide with the efficacy of the anti-CXCR4 polypeptide. Migration inhibition as determined by an in vitro assay provides an alternative indicator of in vivo efficacy of the anti-CXCR4 polypeptide.

Description

Correlation between receptor occupancy and the efficacy of CXCR4 antagonists

[0001] This application claims priority to Australian application AU 2023902188 filed on 7 July 2023 and Australian application AU 2023903085 filed on 25 September 2023.

[0002] For all purposes, the full contents of the electronic submission of the sequence list are incorporated herein by reference in full.

[0003] All references cited or referenced in this document, as well as all references cited or referenced in the cited references, together with any manufacturer's instructions, descriptions, product specifications and product forms of any product mentioned in this document or any document incorporated herein by reference, are incorporated herein by reference in their entirety. Technical Field

[0004] This disclosure is based on a novel assay that measures SDF-1α-induced migration of primary human CXCR4 cells in the presence of an anti-CXCR4 peptide, correlating the degree of CXCR4 receptor occupancy (RO) of the anti-CXCR4 peptide with the efficacy of the anti-CXCR4 peptide. The migration inhibition measured by the in vitro assay provides a surrogate indicator of the in vivo efficacy of the anti-CXCR4 peptide. Background Technology

[0005] Chemokines (chemically induced cytokines) are a family of structurally and functionally related small proteins that, in addition to controlling other biological processes (such as angiogenesis, morphogenesis, autoimmunity, tumor growth, and metastasis), guide the migration of cells (such as leukocytes and / or lymphocytes and / or stem cells and / or neurons). Based on the presence and relative position of N-terminal cysteine ​​residues, chemokines are classified into different families (e.g., CC, CXC, CX3C, and C chemokines). Typically, the biological activity of chemokines is mediated by cell surface receptors, particularly by 7-transmembrane domain G protein-coupled receptors (GPCRs). Chemokine receptors are grouped and named according to the families of chemokines they bind to.

[0006] One member of the CXCR family is CXCR4, which is primarily expressed on lymphocytes and activates chemotaxis. CXCR4, also known as fusin, is an α-chemokine receptor specific for matrix-derived factor-1 (SDF-1, also known as CXCL12), a molecule with potent chemotactic activity towards leukocytes, including T cells.

[0007] CXCR4 plays a role in embryogenesis, homeostasis, fibrosis, and inflammation. Summary of the Invention

[0008] This disclosure is based on a novel assay that measures the migration of SDF-1α-induced CXCR4-expressing cells in the presence of an anti-CXCR4 peptide, correlating the degree of CXCR4 receptor occupancy (RO) of the anti-CXCR4 peptide with functional activity. Advantageously, the assay is performed in vitro, enabling simple and rapid determination of RO levels associated with functional activity in human cells. Using a PK / PD model, computer simulations can be performed, thereby estimating the dose and dosing regimen required to achieve the target RO level derived from the assay.

[0009] RO can be used as an alternative pharmacodynamic (PD) indicator in clinical trials because it assumes that drug efficacy is driven by inhibition of receptors upon drug binding (Liang M et al., Cytometry B: Clinical Cytomy, (2016) 90(2): 117-27). However, to confidently utilize RO as an alternative PD indicator, a relationship between RO and physiological response should be established to demonstrate that RO indeed inhibits functional effects and the level of RO required to elicit the desired biological effect.

[0010] In developing the assay, the inventors sought to determine the levels of CXCR4 RO necessary to establish the efficacy of their i-body support product AD-214 in the treatment of fibrosis. AD-214 comprises a CXCR4-binding polypeptide (i-body) fused to the Fc region. The CXCR4-binding polypeptide comprises an NCAM-based domain 1 and two binding ring regions (referred to as CDR1 and CDR3) as a scaffold. The generation and characterization of the CXCR4-binding polypeptide (referred to as AM3-114) are described in PCT / AU2016 / 050005, disclosed as WO 2016 / 109872, the entire contents of which are incorporated herein by reference.

[0011] CXCR4 / SDF-1α signaling is known to drive fibrosis; therefore, one mechanism of action of AD-214 is to inhibit the migration of immune cells to SDF-1α. Ideally, the relationship between RO and immune cell migration would be measured in vivo; however, this is not feasible in human studies. Furthermore, accurately estimating CXCR4 RO in mice is challenging. Additionally, it is unknown whether the RO levels of anti-CXCR4 peptides in mice are sufficient to reflect the required dose levels in humans.

[0012] Measurement of RO is important because for therapeutic drugs, the percentage of RO required to achieve therapeutic effect may be as high as 90% or as low as 10-20%. Typically, to achieve efficacy, the RO needs to be in the range of 60-90% at the drug's trough serum concentration.

[0013] In the work that conducted this study, a Phase I trial of AD-214 in healthy human volunteers revealed a sustained CXCR4 receptor occupancy (RO) following AD-214 infusion. However, it remains unknown what level of RO is necessary to achieve efficacy and what dose and dosing interval can maintain that level. These studies, at best, speculate on what doses might be safe in human volunteers and have not determined what doses are effective in humans.

[0014] Furthermore, this disclosure provides a method for correlating the CXCR4 receptor occupancy of AD-214 with efficacy. To the best of the inventors' knowledge, this is not taught or suggested in the prior art. The disclosed in vitro cell migration assay utilizes the inhibition of migration of primary human T cells. These cells, along with other CXCR4-expressing leukocytes, participate in inflammation and fibrosis by migrating to sites of injury. Therefore, the migration inhibition measured by the in vitro assay provides a surrogate indicator of in vivo efficacy. Surprisingly, the inventors have found that a low CXCR4 receptor occupancy of AD-214 (i.e., as low as 30%) is sufficient to substantially or meaningfully inhibit cell migration, thus demonstrating the efficacy of AD-214.

[0015] In a first aspect, a method is provided for stratifying subjects treated with an anti-CXCR4 peptide, the method comprising: (i) exposing CXCR4-expressing cells derived from the subject to the anti-CXCR4 peptide; (ii) measuring the CXCR4 receptor occupancy of the peptide on the cells derived from the subject; wherein if the CXCR4 receptor occupancy is greater than 30%, the subject is selected to be treated with the anti-CXCR4 peptide.

[0016] In one example, cells expressing CXCR4 are exposed to the anti-CXCR4 peptide in vivo or in vitro.

[0017] In one example, the cells expressing CXCR4 are human T cells. In another example, T cells are enriched from the erythrocyte sedimentation rate (ESR) amber layer of whole blood obtained from the subject. In another example, the T cells are CD3+ T cells. In yet another example, the cells expressing CXCR4 are fibroblasts, fibroblasts, or macrophages.

[0018] In one example, the subject had fibrotic disease. In another example, the subject had fibrosis of the lungs, kidneys, and / or eyes. In yet another example, the subject had idiopathic pulmonary fibrosis (IPF). In yet another example, the subject had cancer expressing CXCR4.

[0019] In some 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 another example, the CXCR4 receptor occupancy is between 60% and 85%. In one example, the CXCR4 receptor occupancy is approximately 85%.

[0020] In one example, the receptor occupancy was maintained for at least 7 days.

[0021] In a preferred example, 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 peptide; and (iii) determining the CXCR4 receptor occupancy by detecting the amount of bound anti-CXCR4 peptide relative to free CXCR4.

[0022] In one example, the T cells and the anti-CXCR4 peptide were combined for at least 15 minutes.

[0023] In one example, the receptor occupancy is determined by detecting and measuring the amount of anti-CXCR4 peptide bound to T cells relative to free CXCR4. In one example, the bound anti-CXCR4 peptide and free CXCR4 are detected by immunofluorescence staining using an antibody. In one example, the anti-CXCR4 peptide is detected using a labeled anti-Fc (e.g., Fc-FITC), and free CXCR4 is detected, for example, using an antibody 12G5 linked to a detectable label (e.g., 12G5-BV421). In another example, the anti-CXCR4 peptide is detected, for example, using anti-human Ig-AF647.

[0024] In one example, cells were stained with antibodies for at least 30 minutes. In another example, cells fixed with paraformaldehyde were subjected to flow cytometry. In yet another example, cell-fixed cells were analyzed by flow cytometry within 24 hours.

[0025] In a specific example, the anti-CXCR4 peptide is AD-214, which includes the sequence of SEQ ID NO:5. In another example, the anti-CXCR4 peptide is AM3-114, which includes the sequence of SEQ ID NO:2.

[0026] In one example, T cell migration inhibition was maximally suppressed. In another example, maximal inhibition was at least 80% suppression of T cell migration.

[0027] In one example, the receptor occupancy was maintained for at least 7 days.

[0028] In certain examples, the receptor occupancy is calculated as follows: RO% = 100 × (occupied / occupied + free). In some examples, the data are normalized to background fluorescence using a fluorescence minus one (FMO) control (i.e., subtracting 12G5-BV421 or AD-214 labeled cells from all antibodies).

[0029] In a second aspect, a method is provided for treating subjects with fibrotic diseases or cancers expressing CXCR4 by administering an anti-CXCR4 peptide at a dose that maintains a CXCR4 receptor occupancy (RO) greater than 30%.

[0030] In some 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 another example, the CXCR4 receptor occupancy is between 60% and 85%. In one example, the CXCR4 receptor occupancy is approximately 85%.

[0031] In one example, the anti-CXCR4 peptide is AD-214, which includes the sequence of SEQ ID NO:5. In another example, the anti-CXCR4 peptide is AM3-114, which includes the sequence of SEQ ID NO:2.

[0032] In one example, the dose is maintained at a serum concentration between 0.7 and 1 nM for at least one week.

[0033] In one example, the RO is greater than or equal to 85% and the IV dose is between 5 and 10 mg / kg IV per week.

[0034] In another example, the RO is greater than or equal to 85% and the IV dose is ≥ 5 mg / kg per week.

[0035] In another example, the RO is greater than or equal to 85% and the IV dose is 10 to 20 mg / kg per week.

[0036] In one example, the RO is greater than or equal to 60% and the IV dose is between 3 and 10 mg / kg per week.

[0037] In another example, the RO is greater than or equal to 60% and the IV dose is > 3 mg / kg per week.

[0038] In one example, the RO is greater than or equal to 30% and the IV dose is between 1 and 3 mg / kg per week.

[0039] In another example, the RO is greater than or equal to 30% and the IV dose is > 1 mg / kg per week.

[0040] In one example, the RO is greater than or equal to 60% and the IV dose is > 10 mg / kg every two weeks.

[0041] In one example, the RO is greater than or equal to 30% and the IV dose is > 3 mg / kg every two weeks.

[0042] In one example, the RO is greater than or equal to 85% and the sc dose is between 2 and 3 mg / kg per week.

[0043] In another example, the RO is greater than or equal to 85% and the sc dose is > 2 mg / kg per week.

[0044] In another example, the RO is greater than or equal to 85% and the sc dose is > 1 mg / kg every 3 days.

[0045] In one example, the RO is greater than or equal to 60% and the sc dose is > 1 mg / kg per week.

[0046] In one example, the RO is greater than or equal to 60% and the sc dose is between 1 and 5 mg / kg per week.

[0047] In one example, the RO is greater than or equal to 60% and the sc dose is between 0.03 and 0.1 mg / kg per day.

[0048] In one example, the RO is greater than or equal to 85% and the sc dose is > 0.05 mg / kg per day.

[0049] In one example, the RO is greater than or equal to 60% and the sc dose is > 0.02 mg / kg per day.

[0050] In one example, the RO is greater than or equal to 30% and the sc dose is > 0.01 mg / kg per day.

[0051] In one example, the RO is greater than or equal to 60% and the IV dose is between 210 and 700 mg / kg per week.

[0052] In one example, the RO is greater than or equal to 60% and the IV dose is between 210 and 700 mg every two weeks.

[0053] In one example, the RO is greater than or equal to 60% and the sc dose is between 70 and 350 mg per week.

[0054] In one example, the RO is greater than or equal to 60% and the sc dose is between 2 and 7 mg per day.

[0055] In a specific example, the RO is greater than or equal to 85% and the sc dose is between 2 and 3 mg / kg per week.

[0056] In one example, the fibrotic disease is idiopathic pulmonary fibrosis (IPF) or interstitial lung disease (ILD).

[0057] In a third aspect, a method is provided for determining the receptor occupancy of CXCR4-binding molecules and the ability of CXCR4 molecules to inhibit cell migration, the method comprising: (i) isolating human cells expressing CXCR4; (ii) combining the cells with a concentration gradient of the CXCR4-binding molecules for a time sufficient to allow the CXCR4-binding molecules to bind to CXCR4 on the cells; (iii) determining the receptor occupancy of the CXCR4-binding molecules by detecting the amount of bound CXCR4-binding molecules relative to free CXCR4 to obtain a receptor occupancy percentage value; (iv) determining the level of SDF-1α-induced migration and counting the number of migrating cells by combining the cells with a concentration gradient of the CXCR4-binding molecules in the presence or absence of SDF-1α, respectively; and (v) comparing the relationship between the inhibition percentage of cell migration and the receptor occupancy percentage of the CXCR4-binding molecules.

[0058] In one example, the assay is performed in vitro. In another example, the assay is performed in a cell culture plate.

[0059] In one example, the human cells are T cells derived from a non-disease-prone subject. In another example, the T cells are isolated from the erythrocyte sedimentation rate (ESR) layer of whole blood. In yet another example, the T cells are CD3+ T cells. In yet another example, the T cells are obtained from a subject who has not received a CXCR4 antagonist.

[0060] In one example, receptor occupancy was determined for between 50,000 and 100,000 cells / well. In another example, cells were seeded in a suitable medium in a 96-well plate. In one example, the medium was FACS buffer (2% FCS / 2 nM EDTA / 1x PBS). In another example, the medium was 1% FCS / RPMI starvation medium.

[0061] In another example, according to step (ii), the concentration gradient of CXCR4-binding molecules ranges from 100 nM to 0.001 nM.

[0062] In one example, for step (ii), the cells and the CXCR4 binder are combined for at least 15 minutes.

[0063] In another example, the amount of bound CXCR4 molecules relative to free CXCR4 is detected by flow cytometry. In another example, a fluorescently labeled antibody is used for the detection of CXCR4 bound molecules. In another example, the CXCR4 antibody is AD-214, comprising the sequence of SEQ ID NO:5, and is detected using an AF647-conjugated anti-H+L secondary antibody. In another example, free CXCR4 is detected using a competitive anti-CXCR4 antibody (e.g., 12G5) conjugated to a fluorescent label (e.g., BrilliantViolet421).

[0064] In another example, RO% is determined by the following formula: 100 × (occupied CXCR4 / occupied CXCR4 + free CXCR4).

[0065] In one example, between 50,000 and 200,000 cells are used in step (iv).

[0066] In another example, step (iv) is performed in a transwell that includes a semipermeable membrane.

[0067] In another example, according to step (iv), cells are combined with the CXCR4-binding molecule and SDF-1 for a period of time to allow cell migration. In another example, cells are combined with the CXCR4-binding molecule and SDF-1 for at least 2.5 hours. In yet another example, the time is at least 5 hours, or at least 18 hours.

[0068] In another example, according to step (iv), the concentration gradient of CXCR4-binding molecules ranges from 0 to 100,000 nM.

[0069] In one example, the number of migrating cells was quantified using flow cytometry.

[0070] In one example, the migration inhibition % is calculated as follows: inhibition % = 100 × [1-(X-MIN) / MAX-MIN)], where MIN is the number of cells that migrate in the absence of SDF-1, MAX is the number of cells that migrate in the presence of SDF-1, and X is the number of cells that migrate at a given concentration of anti-CXCR4 peptide or RO percentage.

[0071] In one example, the CXCR4 binding molecule is an antagonist of CXCR4. In a specific example, the CXCR4 binding molecule is AD-214, which includes the sequence of SEQ ID NO:5.

[0072] In one example, the RO% at maximum inhibition determines the efficacy of a given dose of CXCR4-binding molecules. Attached Figure Description

[0073] Figure 1 shows the percentage of CXCR4 receptor occupancy in human CD3+ T cells obtained from healthy donors administered AD-214. The lower limit of quantitation (LLOQ) is indicated by a dashed line.

[0074] Figure 2 illustrates AD-214 binding and RO in U266 cells. CHO-hCXCR4 (induced by tetracycline) and U266 cells were seeded in 96-well plates and stained, blocked with anti-human Fc blocking agent, and then blocked with a gradient of AD-214 diluted in 1% FCS / RPMI starvation medium. Left panel = specific binding of AD-214; Middle panel = specific binding of anti-CXCR4 (12G5); Right panel = CXCR4 occupancy. Data represent the mean of n = 2 technical replicates in n = 1 experiment.

[0075] Figure 3 shows SDF-1α-induced migration of CD3+ T cells isolated from the erythrocyte sedimentation rate (ESR) layer of non-disease-affected controls. (A and B) Isolated CD3+ T cells were seeded into transwell plates, and SDF-1α was loaded into the lower chamber. Migration was quantified after 2.5 hours. (A) and (B) represent independent experiments performed in triplicate. Error bars represent SEM images.

[0076] Figure 4. RO and migration in CD3+ T cells from donor NDC02. CD3+ T cells isolated from the erythrocyte sedimentation rate (ESR) amber layer of healthy volunteer NDC02 were treated with a concentration gradient of AD-214 at 37°C for 15 min. Cells were loaded onto transwell plates containing AD-214 or washed and analyzed for CXCR4 occupancy. For the CXCR4 occupancy assay in (A), AD-214 was detected using an AF647-conjugated anti-H+L secondary antibody, and free CXCR4 was detected using a competitive anti-CXCR4 (clone 12G5)-BrilliantViolet421 antibody. For the migration assay in (B), SDF-1α (10 nM)-induced migration across the transwell was quantified after 2.5 hours. Data represent single experiments with n = 3 technical replicates (AC) or pooled from this experiment and previous migration assays in NDC02 (D).

[0077] Figure 5. RO and migration in CD3+ T cells from donor NDC06. CD3+ T cells isolated from the erythrocyte sedimentation rate (ESR) amber layer of healthy volunteer NDC06 were treated with a concentration gradient of AD-214 at 37°C for 15 min. Cells were loaded onto transwell plates containing AD-214 or washed and analyzed for CXCR4 occupancy. For the CXCR4 occupancy assay in (A), AD-214 was detected using an AF647-conjugated anti-H+L secondary antibody, and free CXCR4 was detected using a competitive anti-CXCR4 (clone 12G5)-BrilliantViolet421 antibody. For the migration assay in (B), SDF-1α (10 nM)-induced migration across transwells was quantified after 2.5 hours. Data represent a single experiment with n = 3 technical replicates (AB). In (C), the data are plotted as mean, and the results are expressed as the suppression of AD-214 relative to the minimum (zero SDF-1α) and maximum (+SDF-1α) values ​​in the donor NDC06.

[0078] Figure 6. RO and migration in CD3+ T cells from donor NDC09 and supplier PBMC. CD3+ T cells isolated from the erythrocyte sedimentation rate (ESR) tannin layer of healthy volunteer NDC09 and supplier human PBMC were treated with a concentration gradient of AD-214 at 37°C for 15 min. Cells were loaded onto transwell plates containing AD-214 or washed and analyzed for CXCR4 occupancy. For CXCR4 occupancy assays in (A, D, G), AD-214 was detected using an AF647-conjugated anti-H+L secondary antibody, and free CXCR4 was detected using a competitive anti-CXCR4 (clone 12G5)-BrilliantViolet421 antibody. For migration assays in (B, E), SDF-1α (10 nM)-induced transwell migration was quantified after 2.5 hours. Data represent a single experiment with n = 3 technical replicates (AB). In (C, F), the data are plotted as mean, and the results are expressed as the suppression of AD-214 relative to the minimum (zero SDF-1α) and maximum (+ SDF-1α) of the donor NDC09.

[0079] Figure 7. Structural pharmacokinetic / pharmacodynamic (PK / PD) model. Circles indicate compartments, and solid lines represent drug flow rates, where the relevant PK and PD parameters are fixed based on previous results described in the Methods section.

[0080] Figure 8. Relationship between serum AD-214 concentration and time after IV or SC administration in mice.

[0081] Figure 9. Fitting of mouse PK data.

[0082] Figure 10. Comparison of PK and RO fit between the new model (pink) containing total CXCR4 data and the previous model (blue, running 082.1 model A) that only fits the PK and RO data.

[0083] Figure 11 Comparison of total CXCR4 fit between the new model (pink, run 083, model B) using total CXCR4 data and the previous model (blue, run 82.1, model A) that only fitted PK and RO data. % increase relative to baseline.

[0084] Figure 12. Steady-state CXCR4RO predicted at trough for a typical 70 kg individual under different repeated SC and IV dosing regimens. The horizontal dashed lines represent the target RO at 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.

[0085] Figure 13. Predicted serum AD-214 concentrations and CXCR4 RO in a typical 70 kg individual after weekly IV and SC doses. Predictions were made for a typical 70 kg individual. The horizontal dashed lines represent the target RO at 30%, 60%, and 85%. Model B (run 083) was fitted to the PK + RO + total CXCR4 data.

[0086] Figure 14. Predicted serum AD-214 concentrations and CXCR4 RO in a typical 70 kg individual following every two weeks of IV and SC doses. Predictions were made for a typical 70 kg individual. The horizontal dashed lines represent the target RO at 30%, 60%, and 85%. Model B (run 083) was fitted to the PK + RO + total CXCR4 data.

[0087] Figure 15. Left: Predicted serum AD-214 concentrations and CXCR4 RO following once-daily SC (0.1 mg / kg) and once-weekly SC (1 to 20 mg / kg) doses in a typical 70 kg individual. Right: Serum AD-214 concentrations and CXCR4 RO following once-daily SC doses ranging from 0.001 to 0.1 mg / kg in a typical 70 kg individual. Dashed horizontal lines represent the target RO at 30%, 60%, and 85%. Model B (run 083) was fitted to the PK + RO + total CXCR4 data.

[0088] Sequence List Symbol Explanation

[0089] SEQ ID NO:1: Amino acid sequence of domain 1 of NCAM

[0090] Amino acid sequence of SEQ ID NO:2: AM3-114

[0091] SEQ ID NO:3: Amino acid sequence of the binding loop region (CDR1)

[0092] SEQ ID NO:4: Amino acid sequence of the binding loop region (CDR3)

[0093] SEQ ID NO:5: Amino acid sequence of AD-214

[0094] SEQ ID NO:6: Nucleotide sequence of AD-214 Detailed Implementation

[0095] Overview

[0096] The term “and / or”, for example, “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be regarded as providing explicit support for both or either of these meanings.

[0097] A list or feature containing the phrase "and / or" between the penultimate and last features means that any one or more of the listed features can exist in any combination.

[0098] Unless the context otherwise specifies, references to the singular forms “a,” “one,” and “the” are also understood to imply the inclusion of the plural forms.

[0099] Throughout this specification, unless otherwise expressly stated or required by the context, references to a single step, composition of matter, group of steps, or group of composition of matter shall be deemed to cover one or more (i.e., one or more) of those steps, compositions of matter, groups of steps, or groups of composition of matter.

[0100] Those skilled in the art will understand that variations and modifications can be made to the invention described herein, in addition to those specifically described. It should be understood that the invention encompasses all such variations and modifications. The invention also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, as well as any and all combinations of any two or more of said steps or features.

[0101] The scope of this disclosure is not limited to the specific embodiments described herein, which are intended for illustrative purposes only. As described herein, functionally equivalent products, compositions, and methods are clearly defined within the scope of this disclosure.

[0102] Unless otherwise expressly stated, any example in this document should be considered applicable to any other example after necessary modifications.

[0103] Selected definition

[0104] The word “comprise” or variations such as “comprises” or “comprising” should be understood to imply inclusion of the stated element, whole or step or group of elements, whole or steps, but does not exclude any other element, whole or step or any other group of elements, whole or steps.

[0105] As used in this article, the term "affinity" refers to the strength of the binding between a single molecule and its ligand, and is usually expressed as the equilibrium dissociation constant (K0) for the reversible binding of two reagents. D The constant is determined by the K-linked peptide between the i-body or CXCR4-binding peptide and CXCR4 as disclosed herein. off / K on The ratio determines K. D It is inversely proportional to affinity. K D The value relates to the concentration of the i-body or CXCR4-binding peptide, therefore K D The lower the value (the lower the concentration), the higher the affinity of the antibody.

[0106] As used herein, the term "binding" in relation to the interaction between CXCR4-binding molecules or peptides and their targets means that the interaction depends on the presence of a specific structure on the target (e.g., an antigenic determinant or epitope). For example, CXCR4-binding molecules or peptides recognize and bind to specific protein structures rather than binding to proteins in general.

[0107] As used herein, the terms “treating,” “treat,” or “treatment” include the administration of a therapeutically effective amount of the disclosed polypeptide, nucleic acid molecule, conjugate, or polymer sufficient to reduce or eliminate at least one symptom of a particular condition. In one example, treatment involves administering a therapeutically effective amount of the i-body polypeptide to treat a CXCR4-related disease or condition. In one example, treatment also refers to preventative treatment.

[0108] As used herein, the term “scaffold” or “i-body scaffold” is intended to refer to the sequence represented by the scaffold region 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.

[0109] As used in this article, "fibrosis" refers to the thickening or scarring of tissues originating from organs such as the lungs, kidneys, or eyes. Pulmonary fibrosis is a lung disease that occurs when lung tissue is damaged and scarred.

[0110] i-body scaffold and CXCR4 binding molecules

[0111] This disclosure provides a binding polypeptide (or "i-body") comprising a scaffold having modified CDR1 and CDR3 regions. In one example, the scaffold region includes domain 1 of human NCAM1 as shown in SEQ ID NO:1, as follows: .

[0112] NCAM (or neural cell adhesion molecule) is a glycoprotein derived from the I-SET domain or intermediate domain of the immunoglobulin (Ig) superfamily. The extracellular domain of NCAM consists of five immunoglobulin-like (Ig) domains followed by two type III fibronectin (FNIII) domains.

[0113] Domain 1 of human NCAM has been produced as a recombinant peptide in bacterial expression systems (Frei et al. (1992), J. Cell Biol. 118: 177-194).

[0114] Functional activity

[0115] Binding of a ligand (e.g., an agonist or SDF-1) to CXCR4 can trigger signal transduction of the G protein-coupled receptor, as well as the activity of the G protein and stimulation of other intracellular signaling molecules. The CXCR4 / SDF-1 pathway is involved in organ angiogenesis and the immune and hematopoietic systems (Tachibana K et al. (1998), Nature 393:591-594). The inhibitory or stimulatory activity of the CXCR4-binding molecules or peptides disclosed herein can be determined by appropriate assays in the presence or absence of a ligand, and the ability of CXCR4-binding molecules or peptides to inhibit or stimulate activity in the presence or absence of a ligand can be evaluated.

[0116] It has been previously demonstrated that AD-214 inhibits the migration of SDF-1α-induced CXCR4-expressing cells. A migration assay is also described herein. The inventors have used this migration assay as an alternative to determine and measure the receptor occupancy of CXCR4-expressing cells bound by AD-214.

[0117] Therefore, a novel in vitro method for determining receptor occupancy is described. Because receptor occupancy is related to functional activity (e.g., migration), the level of receptor occupancy required to induce functional activity can be measured, and thus efficacy can be determined. This is further illustrated in the following experimental examples.

[0118] Example

[0119] method

[0120] CXCR4 blocks i-body

[0121] The generation of the i-body AM3-114, which incorporates CXCR4, has been previously described in PCT / AU2016 / 050005, disclosed as WO2016 / 109872. In simple terms, the NCAM-modified domain 1 serves as the scaffold of the i-body. The scaffold sequence is represented by the following SEQ ID NO:1, where the natural binding loop sequence is shown in the box.

[0122] .

[0123] 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.

[0124] The sequence of i-body AM3-114 is listed in SEQ ID NO:2 below: .

[0125] 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 is generated through affinity maturation of i-body ADCX-99, as described in PCT / AU2016 / 050005.

[0126] AD-214 includes the i-body sequence of AM3-114 and the Fc region at the C-terminus of the scaffold sequence. The amino acid sequence of AD-214 is shown in SEQ ID NO:5.

[0127]

[0128] The corresponding nucleic acid sequence is shown in SEQ ID NO:6.

[0129]

[0130] The underlined portions in SEQ ID NO:5 and SEQ ID NO:6 of the protein / DNA sequence are the anti-CXCR4 i-body. The remaining protein / DNA sequence consists of constant domains 2 and 3 of the Fc region of the mutant human IgG1 (DAPA).

[0131] Antibodies and dyes

[0132] In this study, i-body AD-214 (AM3-114-Fc) 924 mg / ml, MW 73.8 kDa (batch PPP.20.140) and 21H5-Fc 22.69 mg / ml, MW 72.04 kDa (GenScript: U813ZHD260-3 / P9HF001) were used. 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')2 fragment rabbit anti-human IgG, Fcγ fragment specific, Jackson ImmunoResearch), and anti-human H+L-AF647 (A-21445, goat anti-human IgG (H+L) cross-adsorption secondary antibody, Alexa Fluor™ 647, Invitrogen). Dead cells were excluded using a viability dye live / dead cell staining reagent (AF405, Invitrogen).

[0133] cell lines

[0134] The T-REx™-CHO cell line, stably transfected with human CXCR4 (hereinafter referred to as CHO-hCXCR4) or mouse CXCR4 (hereinafter referred to as CHO-mCXCR4), was previously generated by Foley's laboratory. THP-1 and U266 cells were obtained from Foley's laboratory. The CHO line was grown in DMEM / F12 1:1 (Gibco) containing 10% FCS (SFBS, Bovogen), 1% penicillin / streptomycin (Gibco), and hygromycin B (Gibco, 500 μg / mL final concentration). Tetracycline (1 μg / mL final concentration) was added to the medium for at least 18 hours to induce hCXCR4 expression. THP-1 and U266 cells were grown in RPM1 containing 10% FCS and 1% penicillin / streptomycin.

[0135] Primary human T cell isolation

[0136] Human erythrocyte sedimentation rate (ESR) amber samples from healthy volunteers were prepared by RedCross and stored in liquid nitrogen. In one experiment, pre-isolated human peripheral blood mononuclear cells (PBMCs) (70025.3) obtained from Stemcell Technologies were used. In short, frozen vials of ESR amber samples or PBMCs from individual donors were rapidly thawed in a 37°C water bath and transferred to the medium used in downstream migration assays, hereinafter referred to as starvation medium (RPMI 1640 (11875119, Gibco), 1% FCS, 0.1% penicillin / streptomycin), and then pipetted to resuspend the cells. For the ESR amber layer, debris not in the suspension, possibly clotted blood, was removed. Unless otherwise specified, all centrifugation steps were performed at 1,500 rpm for 5 minutes at 4°C. To lyse the erythrocyte sedimentation rate (ESR) amber layer, the precipitate was resuspended in 10 volumes of erythrocyte lysis buffer (555899, BD Pharm Lyse™ lysis buffer) and incubated at room temperature for 15 minutes. White blood cells from the human ESR amber layer and pre-isolated PBMCs from the supplier were washed twice with FACS buffer (2% FCS, 1x PBS, 2mM EDTA) and counted using a hemocytometer via trypan blue exclusion. CD3+ T cells were immunomagnetically sorted using the EasySep™ Human T Cell Isolation Kit (17951, Stemcell Technologies) according to the manufacturer's instructions.

[0137] Receptor occupancy (RO) and assay development

[0138] For RO analysis, the resulting protocol was based on the method 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 of 50,000, 100,000, and 200,000 cells / well were tested. A concentration gradient of AD-214 (200 nM to low pM) was added to the cells, and the following incubation times and temperatures were tested: 15 min, 37°C; 30 min, 37°C; 1 h, 4°C. After incubation, the cells were washed twice and then incubated at 4°C for 30 minutes with an antibody mixture comprising (i) anti-human IgG-AF647 (A-21445, goat anti-human IgG (H+L) cross-adsorption secondary antibody, Alexa Fluor™ 647, Invitrogen) or Fc-FITC (309-096-008, AffiniPure F(ab')2 fragment rabbit anti-human IgG, Fcγ fragment specific, Jackon ImmunoResearch), (ii) viability dye (live / dead cell staining reagent, L34963, ThermoFisherScientific), and (iii) anti-human CXCR4 12G5-APC (306509) or anti-human CXCR4 12G5-BrilliantViolet(BV)421 (306518) (both from Biolegend). For human erythrocyte sedimentation rate (ESR) brown-yellow T cells, CD3-PE (552127, BD) was incorporated into the staining mixture. Cells were washed twice, fixed with 4% paraformaldehyde (PFA), washed, and flow cytometry was performed on each sample to obtain at least 5,000 viable cells within 24 hours. Receptor occupancy was calculated as follows: RO% = 100 × (normalized occupied / normalized occupied + normalized free), i.e., RO% = 100 × (normalized AD MFI / normalized AD-214 MFI + normalized 12G5 MFI), where MFI = median fluorescence intensity. A fluorescence minus one control (FMO) was used to identify positive staining for AD-214 / H+L-AF647 and 12G5-BV421 and to normalize (e.g., due to non-specific binding of secondary antibodies) background fluorescence. This was done by subtracting the antibody / fluorophore-stained cells from all antibodies / dyes. Then, the median fluorescence intensity (MFI) of the FMO control was subtracted from each of the respective test conditions to obtain the true fluorescence of AD-214 / H+L-AF657 or Fc-FITC and 12G5-APC or 12G5-BV421.

[0139] SDF-1α-induced migration assay

[0140] 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 supports with 5.0 μm pores of polycarbonate membrane were used. For U266 cells, CLS3384 HTS Transwell®-96 permeable supports with 8.0 μm pores of polyester membrane were used. SDF-1α (recombinant human / rhesus monkey / cat, 350-NS) was added to the lower chamber of RPMI / 1% FCS, and migration across transwells of CD3+ T cells was quantified after 2.5 hours and U266 cells after 6 hours. The concentration gradients shown in each figure were tested to determine the maximum migration levels achieved by U266 cells and CD3+ T cells. Non-SDF-1α / background migration was measured in each assay by including a 0 nM SDF-1α control. After 2.5 hours of migration, cells from the bottom wells of the transwell were transferred to a V-shaped plate with culture medium, centrifuged, and resuspended in 100 μL of 2% PFA. A 90 μL cell suspension was obtained by flow cytometry over 2–3 days. Debris was removed during collection.

[0141] RO in primary human T cells and subsequent migration assay

[0142] To correlate RO and migration, 250,000 CD3+ T cells isolated from human erythrocyte sedimentation rate (ESR) amber layer or PBMCs as described above were incubated with a concentration gradient of AD-214 at 37°C for 15 minutes. Then, 200,000 cells, with or without AD-214 (i.e., after two washes), were seeded into Transwell plates, and SDF-1α (10 nM)-induced migration was monitored as described above. The remaining 50,000 cells were washed twice, and RO was measured as described above. For human CD3+ T cells, due to the variability in response between donors, data are expressed as migration inhibition percentage (%) to account for differences in the number of migrating cells in the control group, i.e., no SDF-1α / no AD-214 (minimum) and +SDF-1α / no AD-214 (maximum).

[0143] Data analysis and software

[0144] Using BD FACSDiva TM Flow cytometry data were analyzed using FlowJo v10 software. Graphs were generated using GraphPadPrism v9.

[0145] Example 1: Phase I safety study of AD-214 in healthy human volunteers

[0146] AD-214 was administered as a single dose to healthy volunteers (men and women) weighing between 49.5 and 92.5 kg. The following doses were administered as single intravenous doses to 33 subjects: 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. Six subjects received an intravenous dose of 5 mg / ml Q2W × 3. This was a safety study only and efficacy was not evaluated.

[0147] Receptor occupancy on CD3+ T cells and plasma AD-214 concentration were measured. The results of receptor occupancy are shown in Figure 1. The results indicated that at a dose of 5 mg / kg, a receptor occupancy rate higher than 70% was maintained for 1 week. At a dose of 10 mg / kg, a receptor occupancy rate higher than 80% was maintained for 1 week. At a dose of 20 mg / kg, a receptor occupancy rate higher than 60% was maintained for 3 weeks.

[0148] This study also determined that 1 nM of AD-214 could be detected in the serum of healthy human subjects 72 hours after intravenous administration of 10 mg / kg AD-214.

[0149] While this data demonstrates that AD-214 provides a surprisingly high duration of CXCR4 receptor occupancy, the minimum receptor occupancy level required to achieve therapeutic efficacy (i.e., inhibition of the fibrotic process) remains unknown, especially since CXCR4 receptor occupancy cannot be obtained from in vivo mouse efficacy studies due to various technical difficulties in performing these measurements. Therefore, it is unclear whether CXCR4 receptor occupancy in mice is sufficiently correlated with CXCR4 receptor occupancy in humans. Consequently, the dosage and administration regimen of AD-214 necessary to achieve effective CXCR4 receptor occupancy levels remain unknown.

[0150] Example 2: Development and validation of receptor occupancy (RO) in CHO-hCXCR4 cells

[0151] To correlate receptor occupancy (RO) with functional behavior in cells, the inventors needed to determine whether the RO assay was suitable for in vitro-based assays where cells were directly titrated with AD-214. This would allow for the evaluation of the same cell in migration assays to correlate receptor occupancy with the degree of migration inhibition. This was first optimized in the hCXCR4-CHO cell line (which is a stable, high-expressing CXCR4 cell line) and then extended to human cell lines / primary human PBMCs with lower CXCR4 levels. To simultaneously measure RO and migration, the inventors also needed to verify that the RO assay was unaffected by the conditions required for cell migration assays, such as temperature, the use of culture medium and FACS buffer, and serum starvation.

[0152] The inventors previously developed an assay for analyzing the binding of AD-214 to hCXCR4 in CHO-hCXCR4 cells, which uses anti-human Fc-FITC or H+L-AF647 secondary antibodies to detect AD-214 bound to the cell surface. The median fluorescence intensity (MFI) of the secondary antibody signal was then measured using flow cytometry as an indicator of AD-214 binding. Using this method, the inventors determined the KD of AD-214 to be in the range of 70 to 300 pM. In this paper, the inventors attempt to adapt the assay to measure both AD-214-occupied and unoccupied or free hCXCR4. Since the objective of this study was to correlate RO with subsequent migration, the following conditions were tested in the RO assay to ensure that it was also applicable to the conditions required for migration assays: (i) the effect of using 37°C medium versus 4°C medium in FACS buffer (current method); (ii) shortening the incubation time with AD-214 to minimize the risk of internalization (comparing 15 min and 30 min with the currently used 60 min); (iii) the cell seeding density required for migration assays (50,000 vs 100,000 vs 200,000); and (iv) the effect of using serum-starved medium prior to the assay, as this condition is required for migration assays.

[0153] In the first assay, (i through iii) were added simultaneously. Variations in temperature, incubation time, and cell seeding density had no significant effect on AD-214 binding. The assay conditions selected for further assay development were 200,000 cells at 37°C for 15 minutes.

[0154] In the second assay, (iv) the effect of incubating cells with AD-214 diluted in serum-starved medium was tested. The inventors compared AD-214 diluted in the current buffer (i.e., FACS buffer (2% FCS / 2 mM EDTA / 1x PBS)) and in 1% FCS / RPMI starved medium. No changes in AD-214 binding or KD were observed. Therefore, 1% FCS / RPMI starved medium was used in the further assay development steps.

[0155] Example 3: Characterization of the brown-yellow layer of erythrocyte sedimentation rate in cryopreserved non-disease control individuals

[0156] In order to provide a stronger translational link between RO / migration data and clinical applications, the inventors attempted to establish migration and RO assays in primary human T cells using cryopreserved ESR (erythrocyte sedimentation rate) stock solution obtained from non-disease donors (NDCs) at Alfred Hospital.

[0157] First, the inventors sought to determine the cell recovery rate, CXCR4 expression, and T cell yield per vial after thawing. Leukocytes were prepared from NDC01 cells by washing; debris (potentially clotted blood and red blood cells) was removed by lysis; and further washing was performed as described in the method. 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). The donor NDC01 cells contained the following: 45% were viable lymphocytes; approximately 75% viable lymphocytes = CD3 / 12G5++, and approximately 91% of CD3+ cells were 12G5+. Notably, based on their MFI, CXCR4 expression on T cells may be lower relative to other cells: CD3+ 12G5 MFI = approximately 520; CD3- 12G5 MFI = approximately 2000. In the second experiment, the erythrocyte sedimentation rate (ESR) amber layer from donor NDC01 was further characterized before and after immunomagnetic enrichment of CD3+ lymphocytes. Staining with two T cell markers, CD4 and CD8, confirmed that the CD3+ population was indeed T cells, and magnetic sorting of CD3+ cells resulted in an enrichment rate of >86% of the total population in the sample. 40% of CD3+ cells were lost during the magnetic sorting process, and it was determined that a single vial of NDC01 ESR amber layer could generate 200 to 300,000 CD3+ T cells.

[0158] Example 4: Testing AD-214 RO in THP-1 and U266 cell lines

[0159] While CHO-hCXCR4 cells provide a useful tool for assessing AD-214 binding and assay optimization, these cells are artificially induced cells with very high levels of CXCR4 expression. Therefore, the inventors also wished to validate their assay in other human cell lines that endogenously express CXCR4 and have previously been used for migration assays. The THP-1 (monocyte cell line, leukemia patient) and U266 (peripheral blood B cells, myeloma patient) CXCR4-positive multiple myeloma cell lines have previously been used in the Fry laboratory for studies of both AD-114 and AD-214. Using the conditions established by the aforementioned CHO-hCXCR4 cells, the inventors performed the same AD-214 RO assay in the THP-1 and U266 cell lines to determine whether the assay was suitable for cells with lower CXCR4 expression.

[0160] Non-specific binding of the Fc-FITC antibody to THP1 cells was observed. AD-214 competed for 12G5 binding in U266 cells, indicating specific binding to CXCR4. The KD of AD-214 in U266 cells was 40 to 50 pM. As expected, CXCR4 expression was lower in U266 cells compared to CHO-hCXCR4 cells. These data (Figures 2A and B) provide evidence of the principle for measuring RO in cell lines using the described method.

[0161] Example 5: Use of H+L-AF647 (AD-214 secondary antibody) and anti-human 12G5- in RO assay BrilliantViolet421 validates RO assay.

[0162] In experiments following those shown in the previous examples, the Fc-FITC antibody failed QC, so the investor opted to use a different secondary anti-Fc antibody. Therefore, H+L-AF647 was obtained and titrated. Since the 12G5 antibody used in the RO assay is conjugated to APC, a version with a different fluorophore, BrilliantViolet421, was obtained and titrated. The revised RO assay was then validated in two ESR (erythrocyte sedimentation rate) donors, showing that the KD and IC50 values ​​for AD-214 were within the expected picomolar range. The presence of the H+L antibody was observed to reduce the detection of CD3-PE; however, T-cell gating using a combination of lymphocyte-gated and CD3+ gating was still possible in the analysis.

[0163] Example 6: SDF-1α-induced migration of CD3+ T cells isolated from the erythrocyte sedimentation rate (ESR) layer of non-disease-affected controls.

[0164] The inventors sought to validate an SDF-1α-induced migration assay using CD3+ T cells isolated from the erythrocyte sedimentation rate (ESR) amber layer of non-disease-positive controls. In the first experiment, as shown in Figure 3A, CD3+ T cells were enriched to 60% to 80% cellularity from the ESR amber layer of donors NDC01 and NDC02. A 2.5-hour migration assay was performed using 200,000 or 100,000 CD3+ T cells from each donor seeded in the top wells of a 96-well 5 μm Transwell plate, with a concentration gradient of SDF1 (100 to 0 nM) placed in the bottom wells. For both donors, migration was highest at 5 or 10 nM SDF1, and seeding 200,000 cells induced even greater cell migration without a significant increase in background, i.e., migration in the culture medium (non-SDF1) wells. In the second experiment, as shown in Figure 3B, the inventors verified the following finding: seeding CD3+ T cells from donor NDC02 at 200,000 cells / well induced migration greater than 5 nM, and a 24-well 5 μm Transwell plate showed similar migration levels to a 96-well 5 μm plate. The chemotactic index (fold change relative to the culture medium (i.e., without SDF-1)) is a useful method for normalizing background migration differences and was plotted in these experiments, showing that 10 nM SDF-1 increased migration by 6-fold. Therefore, a seeding density of 200,000 cells and 10 nM SDF-1 are the selected conditions for future SDF-1 migration assays using CD3+ T cells derived from human erythrocyte sedimentation rate (ESR) amber layer.

[0165] Example 7: RO in human CD3+ T cells and subsequent migration

[0166] The inventors sought to correlate cell migration inhibition with RO (reactivity) as varying with the concentration of AD-214 in CD3+ T cells isolated from the donor NDC02. As expected, thousands of cells migrated in response to SDF-1α. For AD-214-treated cells, dose-dependent inhibition of migration was observed. However, the Bmax of AD-214 / Fc-FITC was lower than expected, suggesting that the AD-214 or Fc-FITC antibody may be a problem requiring further QC.

[0167] Example 8: Titration of H+L-AF647 (AD-214 secondary antibody)

[0168] To overcome the challenges of Fc-FITC, different secondary antibodies targeting Fc were obtained on different fluorophores. Anti-human H+L-AF647 was titrated in increments of 1:100 to 1:25 as an alternative to AD-214 in cells occupied by AD-214. In hCXCR4 cells, a dilution of 1:25 was required to detect maximum AD-214 binding. The apparent KD of approximately 460 pM was slightly higher than previously measured in hCXCR4-CHO cells, therefore titrations for the target cell type (i.e., T cells) should also be performed. Binding to CHO-mCXCR4 was also tested in the assay. Titrations were also performed for mouse CXCR4. In CHO-mCXCR4 cells, the highest concentration of H+L-AF647, at 1:25, was required to detect AD-214. Data suggest that saturation in mCXCR4-CHO requires higher concentrations of AD-214, and the KD is likely in the 1 μM range.

[0169] Example 9: Validation of H+L-AF647 (AD-214 secondary antibody) and anti-human 12G5-BrilliantViolet421 in RO assay.

[0170] In the previous iteration of the assay, the secondary antibody for AD-214 was Fc-FITC, using a 12G5 conjugated to APC; however, Fc-FITC performed poorly. To overcome this, a new secondary antibody against Fc was obtained as described above, which also required a new 12G5 antibody with a suitable complementary fluorophore for the RO assay. For this purpose, a 12G5 antibody conjugated to BrilliantViolet421 was obtained and titrated with H+LAF647 at 1:50 and 1:100 dilutions in an AD-214 RO assay using CHO-hCXCR4 cells isolated from donor NDC08 and erythrocyte sedimentation rate (ESR) amber CD3+ cells to evaluate the performance of the RO assay. In CHO-hCXCR4 cells, a loss of H+L-AD214 signal was observed at a 1:50 dilution of BV421-12G5, which was not observed in PBMC donor NDC08, while a 1:100 dilution showed results comparable to previous RO assays and was therefore selected as the optimal dilution for future use. In donor NDC08, 1:100 or 1:50 was sufficient to measure CXCR4 expression. Lower CD3-PE expression was observed in the staining mixture compared to the monochromatic control of CD3-PE; it is unclear whether this is due to the presence of AD-214 or other antibodies in the mixture. This revised RO assay was then validated in a different donor, NDC07, which showed a Kd of 45 pM and an IC50 of 38 pM for AD-214, which were within the expected range based on historical data. This also showed an RO of 80 pM EC50 as expected. The presence of H+L antibodies appears to reduce the detection of CD3-PE; however, CD3+ T cells can still be gated in the analysis. These data, as a replication of previous assays in donor NDC08, demonstrate that this RO assay is robust and suitable for use with novel antibodies against 12G5 and human Fc.

[0171] Example 10: Using the validated H+L-AF647 and 12G5-BV421 RO assay to correlate CD3 from donor NDC02. RO and migration in + T cells.

[0172] After validating the RO assay, the inventors performed RO and subsequent migration assays on CD3+ T cells isolated from donor NDC02. As shown in Figure 4A, the RO assay proceeded as expected, with KD in the pM range. Fewer cells migrated in the control wells compared to the previously performed AD-214 migration assay on the erythrocyte sedimentation rate (ESR) tannin donor NDC02, specifically 0 nM AD-214 + 10 nM SDF-1α (Figure 4B). A concentration-dependent relationship between AD-214 and migration was observed at most AD-214 concentrations, but not at low concentrations (Figures 4B-C). Migration data were generated in n = 2 experiments. In Figure 4D, data were pooled between the two experiments performed on donor NDC02 to allow for a more accurate estimation of RO and migration inhibition. A graph of RO% versus migration inhibition% is plotted. The data are the averages of technical replicates of RO and migration, respectively.

[0173] Example 11: Association of RO and migration in CD3+ T cells from donor NDC06

[0174] To ensure more accurate measurement of RO and migration in primary T cells, the assay needed to be performed on multiple donors; therefore, the assay was repeated in NDC06. Initially, this assay was performed in triplicate with eight concentrations of AD-214; however, due to the limited number of cells recovered from the donors, the protocol was adjusted to test six AD-214 concentrations in duplicate wells, and 21H5-Fc was not tested. The KD of AD-214 was within the expected range at 500 pM. Migration was low, with approximately two-fold migration in the +SDF-1α control compared to the no-SDF-1α control. AD-214 inhibited migration in a concentration-dependent manner. Interpolation of the RO × migration curves showed that the optimal concentrations for RO% and AD-214 were 57% and 0.8 nM, respectively, to achieve maximum migration inhibition. The results are shown in Figure 5.

[0175] Example 12: Association of RO and migration in CD3+ T cells from donor NDC09 and purified PBMCs.

[0176] Based on the amount of available frozen erythrocyte sedimentation rate (ESR) brown-yellow layer, the assay was repeated on donor NDC09 and a vial of human PBMCs from supplier Stemcell Technologies. Since the number of cells recovered from NDC09 was not large, triplicate assays were performed, and six AD-214 concentrations were tested.

[0177] 21H5-Fc was not tested. For NDC09, 85% RO = 1 nM AD-214 was required to achieve maximum migration inhibition. For Stemcell Technologies donors, 60-70% RO = 1 nM AD-214 was required to achieve maximum migration inhibition. Low CXCR4 expression was observed in CD3+ T cells derived from Stemcell Technologies compared to other donors.

[0178] The results are shown in Figure 6.

[0179] For each donor tested in the previous examples, a summary of the RO% and AD-214 concentrations and IC50 values ​​required to achieve maximum migration inhibition is shown in Table 1. Data were interpolated from the RO and migration inhibition % curves for each donor.

[0180] Table 1. RO% and AD214 concentration for achieving maximum migration inhibition

[0181] The study concluded that 100% migration inhibition required a RO concentration between 57% and 85%, with an average of 66%. The concentration range of AD-214 was between 0.7 and 1 nM, with an average of 0.83 nM. Previous studies determined that 1 nM of AD-214 was detectable in the serum of healthy human subjects 72 hours after intravenous administration of 10 mg / kg.

[0182] In summary, these in vitro studies show that: ● The CXCR4 receptor occupancy measured in the model system at different AD-214 concentrations is consistent with the receptor occupancy observed in a phase I clinical trial at the same circulating blood concentration of AD-214.

[0183] ● Maximum migration inhibition was achieved when the CXCR4 receptor occupancy was 60% to 85% (mean 66%), while meaningful 50% migration inhibition was achieved when the CXCR4 receptor occupancy was 11% to 37% (mean 30%).

[0184] ● Maximum migration inhibition (and corresponding desired receptor occupancy) was achieved at AD-214 concentrations of 0.05 to 0.07 μg / ml, and 50% migration inhibition was achieved at AD-214 concentrations five to ten times lower.

[0185] ● Following a single intravenous administration of 10 mg / kg of AD-214, blood concentrations of AD-214 exceeded 0.07 μg / ml for approximately 72 hours, much later than the initial distribution from the blood.

[0186] In summary, these results help identify the target levels of CXCR4 receptor occupancy that may be required for efficacy in the fibrosis indication, and thus the circulating concentrations of AD-214. Maximum T-cell migration inhibition and thus potential efficacy against fibrosis can be achieved by maintaining CXCR4 receptor occupancy at levels above 60% to 85%, with meaningful inhibition reaching as low as 11% to 37%. These receptor occupancy levels can be achieved at very low circulating AD-214 concentrations due to the very tight binding of AD-214 to CXCR4. Phase I clinical studies have shown that, following intravenous administration, maximum inhibitory concentrations of T-cell migration are maintained for several days, and for meaningful inhibition, for a much longer duration.

[0187] Example 13: Simulated intravenous and subcutaneous administration of AD-214

[0188] The aim of this study was to predict the lowest possible intravenous dose for weekly, bi-weekly, and tri-weekly administrations to maintain receptor occupancy (RO) of 30%, 60%, or 85% at troughs, and to predict the AD-214 sc injection dose and dosing frequency required to achieve the same effect.

[0189] method

[0190] Research Design

[0191] A population PK / PD model was developed based on data from a Phase I study of healthy male and female volunteers. Body weight ranged from 49.5 to 95.2 kg. The study consisted of two parts with different participants: ● Single Incremental Dose (SAD) Part (N = 33): 7 concurrent cohorts (A1 to A7) with single IV doses of 0.01, 0.02, 0.1, 1, 5, 10, and 20 mg / kg.

[0192] ● Multiple escalation dose (MAD) portion (N = 6): 1 concurrent group (B1) with an IV dose of 5 mg / kg Q2w × 3.

[0193] Bioassay

[0194] Serum AD-214 concentrations were measured using ELISA and validated against acceptance criteria defined in the acceptance protocol. The LLOQ in 100% serum was 19.53 ng / mL, and any sample with a concentration higher than 1250 ng / mL could be further diluted up to 100-fold. Free CXCR4 receptors on peripheral blood mononuclear cells (PBMCs) were measured using anti-CXCR4-stained cells targeting the APC channel. AD-214-bound CXCR4 receptors on PBMCs were measured using quantitative PCR using simple cell standards (Bangs Laboratory) consisting of a single population of antibody-coated microspheres with known antibody-binding capacity (ABC). Background subtraction was performed for occupied and free receptor ABC values. The total CXCR4 count was derived as free CXCR4 count + occupied CXCR4 count. RO was calculated as 100. Possible CXCR4 count / Total CXCR4 count. Total CXCR4 receptors were also measured using an alternative assay, but these data were not used in the current analysis (only CXCR4 data derived from the population were used).

[0195] PK study in mice

[0196] AD-214 (10 mg / kg) was administered to BALB / c mice via a single IV or SC bolus. Two samples were collected from each animal, and three animals were tested at each time point. Blood samples were processed into serum by centrifugation. The p-pharmacokinetics assay was a sandwich ELISA using an anti-NCAM antibody (MAB24081 clone) as the capture antibody and an anti-human Fc-HRP antibody (Sigma-Aldrich; A0170-1ML) as the detection antibody – LLOQ 86 ng / mL.

[0197] Data programming and QC

[0198] Data from all cohorts were merged into a single PK / PD analysis dataset using the standard modeling data format required for Monolix software analysis. Nominal dosing time, infusion duration, and observation time were used. Total CXCR4 concentration was calculated as follows:

[0199] Where WCC = white blood cell count, N A = 6.022·10^23 = Avogadro's number, and c = the estimated conversion factor.

[0200] An automated quality control (QC) check was performed on the analysis dataset using the script Data_QC.1.34.R to ensure the correct format of the dataset used for population PK / PD modeling with Monolix. The QC output indicated no errors in the dataset programming. The analysis dataset contained PK / PD and dosing information for 39 subjects. The total number of PK observations was 642, of which 213 (33.18%) were BLQ (below the limit of quantitation) (see [link to relevant documentation]). Handling missing and outlier data The total number of RO observations was 272, and the total number of CXCR4 observations was 305.

[0201] Handling missing and outlier data

[0202] A small number of outliers were excluded, including those with significant measurement errors, such as unusually high baseline values, or those with physically explainable causes. No other outliers were identified through visual inspection, and no further steps were taken to identify and address them. CXCR4 RO and total CXCR4 data from concordance group B1 were not included in the model because they differed from those of concordance group A5, which tested the same dose at 5 mg / kg. PK data from concordance group B1 were consistent with those from concordance group A5 and were included for modeling. No missing values ​​were found in the analyzed dataset. Data below the limit of quantitation (BLQ) were included in population PK analyses using censoring methods available in Monolis (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).

[0203] Parameter estimation

[0204] Population parameters were estimated using the SAEM algorithm implemented in Monolis. The minimum and maximum number of iterations for the exploration and smoothing periods were set to 150–2000 and 50–500, respectively. The SAEM convergence plot confirmed that the number of iterations was large enough to converge to stable parameter estimates in all cases. Initial estimates of the fixed effects parameters were determined by manually fitting the model to the data using Monolis's 'Check Initial Estimates' function. The initial standard deviation for the random effects was set to 1. The standard errors of the parameter estimates were derived from the Fisher information matrix using a stochastic approximation. For each parameter, the conditional mean and standard deviation were calculated using the default settings in Monolis. The -2 log-likelihood (-2LL) was calculated using importance sampling with a Monte Carlo chain length of 10,000. The standard errors of the -2LL estimates confirmed that the Monte Carlo chain was sufficiently long.

[0205] Structural Model

[0206] A three-compartment model was used to describe AD-214 PK, CXCR4 RO, and total CXCR4 in the human body, with the central compartment employing parallel linear first-order elimination and nonlinear Michaelis-Menten elimination. Constant production and first-order internalization of CXCR4 occurred in the central compartment. AD-214 binding to CXCR4 was modeled using both forward and reverse reactions in the central compartment. The AD-214:CXCR4 complex was internalized at a different rate than free CXCR4 (Figure 7). The nonlinear Michaelis-Menten elimination term was considered to represent AD-214 elimination in the liver, independent of general nonspecific elimination in the i-body and independent of CXCR4-mediated elimination.

[0207] Both models, A (run 082.1) and B (run 083), have the same structure. The main difference is that model A fits only to PK and CXCR4 RO data, while model B fits to PK, CXCR4 RO, and total CXCR4 data. Another difference is that model A estimates baseline CXCR4 concentration (R0), while model B uses individually observed baseline CXCR4 counts and an estimated concentration conversion factor (representing the number of receptors for each measured count).

[0208] Statistical Model

[0209] Individual parameters are modeled as random variables with a log-normal distribution. The equation for the individual parameters is:

[0210] in pop η_i is a typical parameter of the population, and η_i is a normally distributed random variable with a mean of 0 and a standard deviation of ω.

[0211] The combined error model was used to model the AD-214 PK, CXCR4 RO, and total CXCR4 observations:

[0212] Where y obs It is the observed value, y pred This is a model prediction. ε is an independent random variable, normally distributed, with a mean of 0 and a variance of 1. Parameter 'a' describes the standard deviation of the constant error, and parameter 'b' describes the proportionality coefficient. The model does not include covariates.

[0213] Model Development Methodology

[0214] For model development and selection, diagnostic plots, the standard errors of parameter estimates, and -2LL were used as selection criteria. Diagnostic plots were constructed in Monolux. Additionally, simulations using population canonical parameter estimates were run in Simulx and overlaid with observed data to evaluate the consistency between model predictions and observations.

[0215] Simulation method

[0216] PK simulations were performed in R using the Simulx API. The simulations were performed on a typical individual basis, meaning they do not include inter-individual variability or observational errors. Covariate effects were included. A body weight of 70 kg was used to calculate the dosage in mg based on a mg / kg dose. Simulations were performed for 1-hour IV infusions and SC administration.

[0217] The values ​​of SC absorption rate ka and SC bioavailability F were based on the following assumptions from the literature: ● F = 0.8, based on the values ​​reported for similar compounds (Richter WF et al., (2012) "Mechanistic determinants of biotherapeutics absorption following SC Administration", 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.

[0218] ● ka = 0.721 / day, based on the publicly available relationship between molecular weight and absorption half-life in mice (Richter2014) and the absorption rate scaled based on body weight and a scaling factor of -0.25.

[0219] These values ​​from the literature were compared with estimates based on sponsor-based data from mice (a small study at SC and IV dose levels) and NHP (AD-114-PAS, which is the parental molecule without Fc fusion), and these estimates showed consistency. For the CXCR4 RO versus dose plot at the trough, the RO at the end of the dosing interval at steady state was extracted. Visual inspection confirmed that steady state had been reached.

[0220] Software and computer systems

[0221] All data programming, data exploration, model building, and simulation were performed on a desktop computer (PowerCrunch-9) running Windows 10 Professional. For PK / PD parameter estimation and diagnostic plots, a validated version of Monolix Suite 2023R1 was used (Monolix (2023) version 2023R1, Antoine: Lixoft SAS, France). For simulations, the Simulx API (Monolix Suite 2023R1) and a validated version of R 4.3.0 were used (R Core Development Group (2008), "R: A Language and Environment for Statistical Computing," Foundation for Statistical Computing, Vienna, Austria, ISBN 3-900051-07-0, 2008, URL http: / / www.Rproject.org).

[0222] Guidelines

[0223] Data analysis was conducted in accordance with the FDA Population Pharmacokinetics Industry Guidance (FDA 2022), the EMA PopPK Analysis Guidance (EMA (2007), Population Pharmacokinetic Analysis Results Reporting Guidance Doc.Ref.CHMP / EWP / 185990 / 06), and the LYO-X Standard Operating Procedure (SOP) from QMS version 3.97.

[0224] result

[0225] Data from the mouse PK study are shown in Tables 2 and 3 and graphically presented in Figure 8.

[0226] Table 2. PK data of AD-214 in mice after IV administration of 10 mg / kg AD-214.

[0227] Table 3. PK data of AD-214 in mice after administration of 10 mg / kg AD-214 by SC.

[0228] Table 4 below shows the non-compartmental PK parameters for each administration route.

[0229] Table 4. PK data of AD-214 after IV and SC administration of 10 mg / kg AD-214

[0230] In mice, PK curves showed a very rapid initial distribution following IV administration, followed by a slower elimination phase. Two-thirds of the samples were below the LLOQ (reported as negative) at 144 hours post-administration, and this time point was excluded from the PK analysis. Although the data fit was quite poor (R0.05...), the results were not satisfactory. 2 < 0.9), but the terminal half-life was still estimated as 24 hours (Figure 8). AD-214 administered via SC was absorbed very rapidly, with Tmax at 4 hours post-dose. Thereafter, the PK curves were consistent with those observed after IV administration. At 144 hours post-dose, two-thirds of the samples had a lower LLOQ (reported negative value), and this time point was excluded from the PK analysis. Furthermore, the terminal elimination period (value 28 hours, R) could not be accurately quantified. 2 0.69). Overall, AD-214 was rapidly absorbed in mice after SC administration, with a Cmax of 404 ng / mL, and the absolute bioavailability was estimated to be approximately 79% based on AUC (0-inf). Given the uncertainty in estimating the terminal half-life, the absolute bioavailability was also estimated to be 73% using AUC (0-72 hours).

[0231] The mouse PK data were well fitted by a 2-compartment model with linear elimination, but the V1 estimate (84 mL) was much larger than the expected value in mice (plasma volume ≈ 1 mL). The F and ka estimates were consistent with the assumed human values ​​(Figure 9).

[0232] Based on PK and RO data obtained from volunteers receiving single IV doses of AD-214 at doses of 0.02, 0.1, 1, 5, 10, and 20 mg / kg, and volunteers receiving an IV dose of 5 mg / ml Q2W × 3, a PK / PD model (version A) was established. Parallel linear and nonlinear elimination (Michaelis-Menten approximation) and explicit receptor binding + complex internalization (K...) are required. D and k onA three-compartment model (fixed to SPR measurements) was used to fit AD-214 concentration and CXCR4 receptor occupancy (RO) data (see Figure 7). A good fit was observed for both PK and RO observations across all tested dose levels, except for slight underestimation of CXCR4 RO at days 7 and 14 after 20 mg / kg. The standard errors of the estimates were fairly small, but the model is complex and carries the risk of overparameterization and overfitting of the data. The need for a three-compartment model is not uncommon and improved the fit for PK observations, particularly for the observations at days 7 and 20 mg / kg that were > LLOQ. The need for an additional nonlinear elimination process supports the hypothesis that, in addition to CXCR4 present on the cells where RO was measured, there may be another specific elimination pathway (e.g., in the liver) that may contribute to the observed AD-214 concentration-time curve. This is primarily determined by the CXCR4 RO data, as a simpler model was able to fit the PK observations.

[0233] 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. This was done using individual-observed baseline CXCR4 and estimated conversion factors. (Number of receptors per count) The total CXCR4 data is added to the model, and variability is not considered for c, Q, and V2.

[0234] As noted, the PK / PD model in version A was derived by fitting only existing PK and RO data and cannot accurately model the total CXCR4 receptor number. The model in version B potentially has lower precision / wider confidence intervals because the total receptor number is derived from the difference between two indirect assays (unlike the RO assay, which, as a ratio measure, self-corrects for some of its variability). Versions A and B produce similar predictions, therefore both can be used to predict target dose.

[0235] A comparison was made between version A and version B models that included total CXCR4 data. As shown in Figure 10, the PK and RO fits of the new model (pink, run 083) including total CXCR4 data were compared with those of the previous model (blue, run 082.1) that only fitted PK and RO data. Very similar PK fits were observed. Similar RO fits were also observed, but the new model showed slightly faster decay, especially at low doses. Therefore, the new model fits the PK and RO data correctly. As shown in Figure 11, the total CXCR4 fits of the new model (pink, run 083) including total CXCR4 data and those of the previous model (blue, run 082.1) that only fitted PK and RO data were compared. (% increase relative to baseline). As expected, model version B fitted the observed increase in total receptors.

[0236] The inventors then simulated and predicted the receptor occupancy that could be achieved under different IV administration regimens. Models were used to simulate the IV doses required to achieve 30%, 60%, or 85% trough RO at once-weekly, once-every-two-week, or once-every-three-week administration regimens. These trough RO levels were chosen based on the following: the potential to inhibit T cell migration at 50% inhibition (and thus affect the treatment-related mode of action), the achievement of maximal inhibition in at least some individuals, and the achievement of maximal inhibition in all individuals. Furthermore, to further understand the doses and frequencies required to achieve these RO troughs (30%, 60%, and 85%) using the subcutaneous administration route, models of subcutaneous kinetics incorporating biomolecules of appropriate molecular weights were applied. Bioavailability factors and rate absorption constants were estimated using preliminary results from AD-214 SC and IV administration in rodents, literature values ​​for similar molecules, and the standard allometric growth scale.

[0237] As shown in Figure 12, simulations were performed to predict trough CXCR4 RO after daily, weekly, bi-weekly, and tri-weekly IV and SC doses of AD-214. Predictions were made for a typical 70 kg individual with a 1-hour IV infusion. For SC, F = 0.8 and ka = 0.721 / day. The horizontal dashed lines represent target ROs of 30%, 60%, and 85% at trough. 10 mg / kg represents a 28 mL IV infusion in a 70 kg patient using the current clinical formulation. 1 mg / kg represents a 0.9 mL SC injection in a 70 kg patient at the highest AD-214 concentration with achievable stability to date. Target dosing parameters for RO: minimum 30% at trough (and greater than 60% for a 75% dosing window); IV: minimum interval of 2 weeks between doses; SC: daily or weekly. Daily or weekly SC administration provides patients with a more convenient route of administration and a lower total protein load.

[0238] AD-214 plasma concentrations and CXCR4 RO were then predicted for every two-week IV and SC doses, as shown in Figure 13. Predictions were made for a typical 70 kg individual one hour after IV infusion or SC injection. The required every-week dose to achieve the target was: 30% RO at trough: 5–10 mg / kg IV; 20 mg / kg SC.

[0239] 60% RO at trough: 20 mg / kg IV.

[0240] For a 0.75% dosing window at trough > 60% RO: 10 mg / kg IV; 20 mg / kg SC.

[0241] AD-214 plasma concentrations and CXCR4 RO were then predicted for weekly IV and SC doses, as shown in Figure 14. Predictions were made for a typical 70 kg individual one hour after IV infusion or SC injection. The weekly dose required to reach the target: 30% RO at trough: 1–3 mg / kg IV or SC.

[0242] 60% RO at trough: 5 mg / kg IV or SC.

[0243] For a dosing window of >75%, >60% RO: 3 mg / kg IV; 1 mg / kg SC.

[0244] Then, AD-214 plasma concentrations and CXCR4 RO were predicted for once-daily and once-weekly SC doses, as shown in Figure 15. Predictions were made for a typical 70 kg individual – F = 0.8 and ka = [0.3; 0.721; 1.1] / day; 1–20 mg / kg once weekly; 0.001 mg / kg once daily. Target doses: 30% RO at trough: 0.01 mg / kg once daily, 1–3 mg / kg once weekly; 60% RO at trough: 0.03 mg / kg once daily; 3–5 mg / kg once weekly; for a dosing window >75%, >60% RO: 0.01–0.03 mg / kg once daily; 1–3 mg / kg once weekly; 80% RO at trough: 0.1 mg / kg once daily; 10–20 mg / kg once weekly.

Claims

1. A method for stratifying subjects treated with an anti-CXCR4 peptide, the method comprising: (i) Exposing CXCR4-expressing cells derived from the subject to the anti-CXCR4 peptide; (ii) Measure the CXCR4 receptor occupancy of the polypeptide obtained from the subject's cells; wherein if the CXCR4 receptor occupancy is greater than 30%, the subject is selected to be treated with the anti-CXCR4 polypeptide.

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

3. The method according to claim 1 or 2, wherein the cell expressing CXCR4 is a human T cell.

4. The method according to any one of claims 1 to 3, wherein the subject suffers from fibrotic disease or cancer expressing CXCR4.

5. The method according to any one of claims 1 to 4, wherein the CXCR4 receptor occupancy rate 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 according to any one of claims 1 to 5, wherein the CXCR4 receptor occupancy is between 60% and 85%.

7. The method according to any one of claims 1 to 6, wherein the receptor occupancy is determined by means of: (i) obtaining T cells from the subject; (ii) exposing the T cells to an anti-CXCR4 peptide; and (iii) determining the CXCR4 receptor occupancy by detecting the amount of bound anti-CXCR4 peptide relative to free CXCR4.

8. The method according to any one of claims 1 to 7, wherein the receptor occupancy is determined by detecting and measuring the amount of anti-CXCR4 peptide bound to the T cell relative to free CXCR4.

9. The method of claim 8, wherein the detection and measurement are performed by flow cytometry.

10. The method according to 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 according to any one of claims 1 to 10, wherein the T cell migration inhibition is suppressed to the greatest extent.

12. A method for treating a subject with fibrotic disease or cancer expressing CXCR4, wherein the treatment is performed by administering an anti-CXCR4 peptide at a dose that maintains a CXCR4 receptor occupancy (RO) greater than 30%.

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

5.

15. The method according to 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 and 10 mg / kg per week; or (ii) The RO is greater than or equal to 85% and the IV dose is > 5 mg / kg per week; or (iii) The RO is greater than or equal to 85% and the IV dose is 10 to 20 mg / kg per week; or (iv) The RO is greater than or equal to 60% and the IV dose is between 3 and 10 mg / kg per week; or (v) The RO is greater than or equal to 60% and the IV dose is > 3 mg / kg per week; or (vi) The RO is greater than or equal to 30% and the IV dose is between 1 and 3 mg / kg per week; or (vii) The RO is greater than or equal to 30% and the IV dose is > 1 mg / kg per week; or (viii) The RO is greater than or equal to 60% and the IV dose is > 10 mg / 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. mg / kg; or (x) the RO is greater than or equal to 85% and the IV dose is between 2 and 3 mg / kg per week; or (xi) the RO is greater than or equal to 85% and the SC dose is > 2 mg / kg per week; or (xii) the RO is greater than or equal to 60% and the SC dose is > 1 mg / kg per week; or (xiii) the RO is greater than or equal to 60% and the SC dose is > 1 mg / kg per week; or (xiv) the RO is greater than or equal to 60% and the SC dose is between 1 and 5 mg / kg per week; or (xv) the RO is greater than or equal to 60% and the SC dose is between 0.03 and 0.1 mg / kg per day; or (xvi) the RO is greater than or equal to 85% and the SC dose is > 0.05 mg / kg per day; or (xvii) the RO is greater than or equal to 60% and the SC dose is > 0.02 mg / kg per day; or (xviii) the RO is greater than or equal to 30% and the SC dose is > 0.01 mg / kg per day. mg / kg; or (xix) the RO is greater than or equal to 60% and the IV dose is between 210 and 700 mg per week; or (xx) the RO is greater than or equal to 60% and the IV dose is between 210 and 700 mg every two weeks; or (xxi) the RO is greater than or equal to 60% and the SC dose is between 70 and 350 mg per week; or (xxii) the RO is greater than or equal to 60% and the SC dose is between 2 and 7 mg per day.

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 and 3 mg / kg per week.

17. The method according to any one of claims 12 to 16, wherein the fibrotic disease is idiopathic pulmonary fibrosis (IPF) or interstitial lung disease (ILD).

18. A method for determining the receptor occupancy of CXCR4-binding molecules and the ability of said CXCR4 molecules to inhibit cell migration, the method comprising: (i) Isolate human cells expressing CXCR4; (ii) Combining the cells with a concentration gradient of the CXCR4-binding molecules for a sufficient time to allow the CXCR4-binding molecules to bind to CXCR4 on the cells; (iii) Determining the receptor occupancy of the CXCR4-binding molecules by detecting the amount of bound CXCR4-binding molecules relative to free CXCR4 to obtain a receptor occupancy percentage value; (iv) Determining the level of SDF-1α-induced migration and counting the number of migrating cells by combining the cells with a concentration gradient of the CXCR4-binding molecules in the presence or absence of SDF-1α; (v) Comparing the relationship between the inhibition % of cell migration and the receptor occupancy % of the CXCR4-binding molecules.

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

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

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