Unit dosage forms and methods comprising bl8040 for intravenous administration

Abstract

A unit dosage form comprising a peptide as set forth in SEQ ID NO: 1 (BL-8040) and a carrier is provided, wherein an amount of the peptide is 5-50 mg, at a concentration of 0.1-3 mg / ml formulated for intravenous administration. Also provided is a method of treating a CXCR4-related condition in a subject in need thereof, the method comprising intravenously administering to the subject a therapeutically effective amount of SEQ ID NO: 1 (BL-8040) wherein the therapeutically effective amount of BL-8040 is 0.05 mg / kg-0.75 mg / kg at a concentration of 0.1-3 mg / ml, thereby treating the CXCR4-related condition.

Description

[0001] UNIT DOSAGE FORMS AND METHODS COMPRISING BL8040

[0002] FOR INTRAVENOUS ADMINISTRATION

[0003] RELATED APPLICATION / S

[0004] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 531,033 filed on August 7, 2023, the contents of which are incorporated herein by reference in their entirety.

[0005] SEQUENCE LISTING STATEMENT

[0006] The XML file, entitled 100166 Sequence Listing. xml, created on July 29, 2024, comprising 10,712 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0007] FIELD AND BACKGROUND OF THE INVENTION

[0008] The present invention, in some embodiments thereof, relates to unit dosage forms and methods comprising BL8040 for intravenous administration.

[0009] BL-8040 is a peptide also known as 4F-benzoyl-TN14003 (4-fluoro-benzoyl-Arg-Arg-Nal- Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH2, SEQ ID NO: 1), Motixafortide or BKT140 is a cyclic peptide upon formation of a disulfide bond between the two Cys residues thereof.

[0010] U.S. Patent No. 7,423,007 describes peptides having CXC4 antagonism; including 4F- benzoyl-TN 14003. According to the teachings of U.S. Patent No. 7,423,007, 4F-benzoyl-TN14003 is manufactured by solid phase synthesis using DIPCDLHOBt as a coupling agent, in DMF (with amino acids added at 2.5 equivalents), followed by deprotection and cleavage using 1 M TMSBr- thioanisole / TFA with m-cresol and ethanedithiol, and cyclization by air oxidation. The peptide is indicated for the treatment of various medical conditions associated with CXCR4 such as cancer and specifically metastatic pancreatic adenocarcinoma.

[0011] Additional background art includes U.S. Patent Nos. 7,138,488 and 8,435,939, and International Patent Application Publications WO 2008 / 075369, WO 2008 / 075370, WO 2010 / 146578, WO 2012 / 095849, WO 2013 / 160895; W02017 / 009842, W02020 / 079692 and WO2022 / 144885.

[0012] SUMMARY OF THE INVENTION

[0013] According to an aspect of some embodiments of the present invention there is provided a unit dosage form comprising a peptide as set forth in SEQ ID NO: 1 (BL-8040) and a carrier, wherein an amount of the peptide is 5-50 mg, at a concentration of 0.1-3 mg / ml formulated for intravenous administration.

[0014] According to an aspect of some embodiments of the present invention there is provided a method of treating a CXCR4-related condition in a subject in need thereof, the method comprising intravenously administering to the subject a therapeutically effective amount of SEQ ID NO: 1 (BL-8040) wherein the therapeutically effective amount of BL-8040 is 0.05 mg / kg-0.75 mg / kg at a concentration of 0.1-3 mg / ml, thereby treating the CXCR4-related condition.

[0015] According to an aspect of some embodiments of the present invention there is provided a unit dosage form as described herein for use in treating a CXCR4-related condition.

[0016] According to some embodiments of the invention, the amount comprises 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg.

[0017] According to some embodiments of the invention, the amount comprises 5-50 mg (e.g., 15- 35 mg).

[0018] According to some embodiments of the invention, the intravenous administration is by way of infusion.

[0019] According to some embodiments of the invention, the infusion is over a time period of 0.5- 2 hours.

[0020] According to some embodiments of the invention, the therapeutically effective amount comprises 0.05 mg / kg, O.lmg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.30 mg / kg, 0.35 mg / kg, 0.40 mg / kg, 0.45 mg / kg, 0.50 mg / kg, 0.55 mg / kg, 0.60 mg / kg, 0.65 mg / kg or 0.70 mg / kg (e.g., 0.25-0.5mg / kg).

[0021] According to some embodiments of the invention, the CXCR4-related condition is selected from the group consisting of cancer, thrombocytopenia, risk of bone marrow suppression, and HIV infection.

[0022] According to some embodiments of the invention, the cancer is a solid tumor.

[0023] According to some embodiments of the invention, the cancer is acute myeloid leukemia (AML).

[0024] According to some embodiments of the invention, the condition is cancer and further comprising at least one of chemotherapy and an immune modulator.

[0025] According to some embodiments of the invention, the immune modulator is a checkpoint inhibitor.

[0026] According to some embodiments of the invention, the checkpoint is PD-1 or PD-L1.

[0027] According to some embodiments of the invention, the cancer is metastatic cancer. According to some embodiments of the invention, the cancer is pancreatic cancer optionally pancreatic adenocarcinoma.

[0028] According to some embodiments of the invention, the treating or treatment is first-line treatment.

[0029] According to some embodiments of the invention, the cancer is a chemotherapy-resistant cancer.

[0030] According to some embodiments of the invention, the peptide is in a composition-of-matter comprising the BL-8040 (SEQ ID NO: 1) and at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88, wherein the relative retention time is determined using a first mobile phase which is an aqueous solution of 0.017 M perchlorate at a pH in a range of from about 3.0 to about 3.3, a second mobile phase which is acetonitrile, a gradient whereby a concentration of the second mobile phase increases by about 10 % in about 50 minutes from an initial concentration of about 20 %, a C18 reverse phase column, an injection volume in a range of from about 5 to about 20 pl, and a flow rate of about 1 ml per minute, at a temperature of about 40 °C, and wherein a relative retention time of the BL-8040 (SEQ ID NO: 1) is defined as 1.

[0031] According to some embodiments of the invention, the at least one compound represents at least 20 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter as determined according to absorption at a wavelength at about 226 nm.

[0032] According to some embodiments of the invention, the at least one compound represents at least 50 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter as determined according to absorption at a wavelength at about 226 nm.

[0033] According to some embodiments of the invention, the at least one compound comprises a compound having the same molecular weight as BL-8040 (SEQ ID NO: 1) and / or a compound having a molecular weight which is higher than the molecular weight of BL-8040 (SEQ ID NO: 1) by about 80 Da.

[0034] According to some embodiments of the invention, a total concentration ratio of the at least one compound to the BL-8040 (SEQ ID NO: 1) is at least about 0.01 %.

[0035] According to some embodiments of the invention, a total concentration ratio of the at least one compound to the BL-8040 (SEQ ID NO: 1) is no more than about 1 %.

[0036] According to some embodiments of the invention, a total concentration ratio of the at least one compound to the BL-8040 (SEQ ID NO: 1) is in a range of from about 0.075 % to about 0.225 %. According to some embodiments of the invention, the composition-of-matter further comprises at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73.

[0037] According to some embodiments of the invention, the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 represents at least 10 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter as determined according to absorption at a wavelength at about 226 nm.

[0038] According to some embodiments of the invention, a total concentration ratio of the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 to the BL- 8040 (SEQ ID NO: 1) is at least about 0.01 %.

[0039] According to some embodiments of the invention, the total concentration ratio of the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 to the BL- 8040 (SEQ ID NO: 1) is in a range of from about 0.03 % to about 0.15 %.

[0040] According to some embodiments of the invention, a total concentration of the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 and the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 represents at least 50 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter as determined according to absorption at a wavelength at about 226 nm.

[0041] According to some embodiments of the invention, a concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) in the composition-of-matter is no more than 50 % of a total concentration of the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88.

[0042] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0043] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0044] The present invention, in some embodiments thereof, relates to unit dosage forms and methods comprising BL8040 for intravenous administration. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0045] In order to improve the compliance with treatment and reduce adverse effects caused by subcutaneous administration of BL8040, the present inventors formulated novel dosage forms for intravenous administration which have specific advantage in chronic treatment. As used herein “chronic treatment” means repeated doses (more than 2) for several administrations. Advantage is that the AE's might be "bearable" when given once or twice but if given on a weekly bases for several weeks this becomes a real issue and people might not want to take the drug anymore. So it is also a compliance advantage

[0046] According to an aspect of the invention there is provided a unit dosage form comprising a peptide as set forth in SEQ ID NO: 1 (BL-8040) and a carrier, wherein an amount of the peptide is 5-50 mg, at a concentration of 0.1-3 mg / ml formulated for intravenous administration.

[0047] According to an aspect of the invention there is provided a method of treating a CXCR4- related condition in a subject in need thereof, the method comprising intravenously administering to the subject a therapeutically effective amount of SEQ ID NO: 1 (BL-8040) wherein the therapeutically effective amount of BL-8040 is 0.05 mg / kg-0.75 mg / kg at a concentration of 0.1- 3 mg / ml, thereby treating the CXCR4-related condition.

[0048] As used herein “a peptide set forth in SEQ ID NO: 1” which is referred to as “BL-8040” or “BL8040” (previously named “B KT 140”) or drug name: Motixafortide, is a highly selective CXCR4 antagonist, a novel therapy for the treatment of cancer. The peptide is a 14-residue, cyclic, synthetic peptide capped with an aromatic ring that binds to CXCR4 with high affinity (IC500.54- 4.5 nM) and inhibits its function [Tamamura H, Hiramatsu K, Kusano S, Terakubo S, Yamamoto N, Trent JO, et al. Synthesis of potent CXCR4 inhibitors possessing low cytotoxicity and improved biostability based on T140 derivatives. Org Biomol Chem 2003;1:3656-3662].

[0049] For sequence listing purposes, SEQ ID NO:1-

[0050] <210> 1

[0051] <211> 14

[0052] <212> PRT

[0053] <213> Artificial

[0054] <220>

[0055] <223> synthetic peptide

[0056] <220>

[0057] <221> MOD_RES <222> (1)..(1)

[0058] <223> 4-fluorobenzoyl-arginine

[0059] <220>

[0060] <221> MOD_RES

[0061] <222> (3)..(3)

[0062] <223> 3-((2-naphthyl) alanine

[0063] <220>

[0064] <221> MOD_RES

[0065] <222> (6)..(6)

[0066] <223> citrulline

[0067] <220>

[0068] <221> MOD_RES

[0069] <222> (8)..(8)

[0070] <223> D-lysine

[0071] <220>

[0072] <221> MOD_RES

[0073] <222> (12)..(12)

[0074] <223> citrulline

[0075] <220>

[0076] <221> MOD_RES

[0077] <222> (14)..(14)

[0078] <223> C AMIDATED

[0079] <400> 1

[0080] Xaa Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg

[0081] 1 5 10

[0082] According to a specific embodiment, the BL-8040 is manufactured as a white to off-white powder synthetic polypeptide, freely soluble in water and in 0.45 % Sodium Chloride (half normal saline). It is manufactured in accordance with current Good Manufacturing Practice (cGMP) by BioConnection B.V. (previously MSD), Kloosterstraat 9, 5349 AB Oss, Netherlands.

[0083] WO2022 / 144885 describes a composition of BL8040 according to some embodiments of the invention.

[0084] According to some embodiments the peptide is in a composition-of-matter comprising BL- 8040 (SEQ ID NO: 1) and at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 and / or at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73, as determined according to any of the embodiments described herein relating to relative retention time.

[0085] Herein throughout, the term “BL-8040” refers to a cyclic peptide having SEQ ID NO: 1. Herein, a “cyclic” peptide refers to a peptide comprising a pair of cysteine residues linked by a disulfide bond. For example, “cyclic peptide having SEQ ID NO: 1” refers to SEQ ID NO: 1 in which the two cysteine residues thereof are linked by a disulfide bond.

[0086] According to some of any of the embodiments described herein, the composition-of-matter comprises at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88, according to any of the respective embodiments described herein (e.g., in an amount according to any of the respective embodiments described herein). In some such embodiments, the relative retention time of the at least one compound is in a range of from 0.865 to 0.88. In some embodiments, the relative retention time is in a range of from 0.868 to 0.878. In some embodiments, the relative retention time is in a range of from 0.870 to 0.876.

[0087] According to some of any of the embodiments described herein, the composition-of-matter comprises at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73, according to any of the respective embodiments described herein (e.g., in an amount according to any of the respective embodiments described herein). In some such embodiments, the relative retention time of the at least one compound is in a range of from 0.715 to 0.73. In some embodiments, the relative retention time is in a range of from 0.718 to 0.728. In some embodiments, the relative retention time is in a range of from 0.719 to 0.726.

[0088] According to some of any of the embodiments described herein, the composition-of-matter comprises both at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88, according to any of the respective embodiments described herein (e.g., in an amount according to any of the respective embodiments described herein) and at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73, according to any of the respective embodiments described herein (e.g., in an amount according to any of the respective embodiments described herein).

[0089] Herein throughout, whenever a range of from 0.86 to 0.88 or from 0.71 to 0.73 is indicated, the range encompasses a range of from 0.860 to 0.880 or from 0.710 to 0.730, respectively, as well as any intermediate values (which may serve as an upper limit or lower limit) and subranges therebetween.

[0090] Herein, the term “relative retention time” refers to a retention time of a compound (e.g., a component of a composition-of-matter described herein) when performing high performance liquid chromatography (HPLC) relative to a retention time (under the same conditions) of a reference compound, which herein is BL-8040 (SEQ ID NO: 1). Thus, the relative retention time of BL-8040 (SEQ ID NO: 1) is by definition equal to 1. In addition, the term “relative retention time” is determined according to the time at which the greatest concentration of compound is retained (e.g., the mode). Thus, for example, a compound defined as having a retention time of 0.87 may include individual molecules exiting a chromatography column at a retention time ranging from 0.84 to 0.90 (e.g., in a bell curve distribution). However, the retention time of all molecules of such a compound will be considered as being 0.87, and therefore “in a range of from 0.86 to 0.88”.

[0091] The relative retention time is preferably determined using a C18 reverse phase column, optionally a silica-based C18 column (e.g., a Hypersil Gold™ C18 column). In some of any of the embodiments described herein, the reverse phase column is characterized by at least one of, at least two of, or all of the following features: a length of about 250 mm length, an inner diameter of about 4.6 mm, and a particle size of about 5 pm.

[0092] The relative retention time according to any of the respective embodiments described herein is preferably determined using an aqueous solution of about 0.017 M perchlorate at a pH in a range of from about 3.0 to about 3.3 as a first mobile phase which is, acetonitrile as a second mobile phase, and a gradient whereby a concentration of the second mobile phase increases by about 10 % (e.g., by 10.0 %) in about 50 minutes from an initial concentration of about 20 % (e.g., increasing from 19.3 % to 29.3 % or from 20 % to 30 %).

[0093] Relative retention time is preferably determined at a temperature of about 40 °C, an injection volume in a range of from about 5 to about 20 pl, and a flow rate of about 1 ml per minute. Detection of compounds may optionally be effected by determining absorption at a wavelength of about 226 nm. In exemplary embodiments, an Agilent™ 1200 or Agilent™ 1100 HPLC system is used.

[0094] A compound in a composition-of-matter characterized by an indicated relative retention time (according to any of the respective embodiments described herein) may optionally be present in a concentration which is at least about 0.01 % of the concentration of the BL-8040 (SEQ ID NO: 1), as determined according to absorption at a wavelength at about 226 nm.

[0095] Herein, the phrase “as determined according to absorption” means that a relative concentration of compound (e.g., in calculating a percentage described herein) is determined by quantification of absorption (e.g., optical density) at a given wavelength (e.g., 226 nm). Thus, for example, two fractions (e.g., representing two different compounds in a composition-of-matter) would be considered as having the same concentration of compound if they absorb to the same degree, regardless of whether they contain the same concentration of molecules of the compound(s); and a compound which does not absorb at all at the indicated wavelength would be considered as having a concentration of zero. In some of any of the embodiments described herein, the composition-of-matter is characterized by a relatively low amount of compounds other than BL-8040 (SEQ ID NO: 1) and the at least one compound characterized by a relative retention time of 0.71 to 0.73 and / or 0.86 to 0.88 (according to any of the respective embodiments described herein), e.g., such that the at least one compound characterized by a relative retention time of 0.71 to 0.73 and / or 0.86 to 0.88 represents a relatively large proportion (e.g., as determined according to absorption at a wavelength at about 226 nm, as defined herein) of all compounds other than BL-8040 (SEQ ID NO: 1).

[0096] Herein, the term “all compounds other than BL-8040” refers to a sum of compounds in a composition-of-matter described herein which are not BL-8040 (SEQ ID NO: 1), a counter-ion thereof, or a solvent (e.g., buffered aqueous solution). The compounds other than BL-8040 (SEQ ID NO: 1) may include, for example, compounds differing in chemical composition and / or molecular configuration (e.g., stereoisomers of BL-8040 (SEQ ID NO: 1)). It is to be appreciated that some compounds which are not BL-8040 (SEQ ID NO: 1) may fail to absorb at an indicated wavelength, and thus fail to contribute substantially to “all compounds other than BL-8040” as determined according to absorption (as defined herein).

[0097] In some of any of the respective embodiments described herein, the term “all compounds other than BL-8040” includes only compounds having a relative retention time of at least 0.1, optionally from 0.1 to 2, as determined under conditions described herein; that is, compounds having a relative retention time outside the indicated range are in such embodiments excluded (e.g., for convenience of measurement) from the definition of “compounds other than BL-8040” (according to any of the respective embodiments described herein), even if they are not a counterion or solvent. In some embodiments, the term “all compounds other than BL-8040” includes only compounds having a relative retention time of at least 0.5, optionally from 0.5 to 1.5.

[0098] In some of any of the respective embodiments described herein, the term “all compounds other than BL-8040” refers to the sum of peptide compounds other than BL-8040 (SEQ ID NO: 1); that is, compounds which are not peptides (i.e., compounds which do not include at least two amino acid residues linked by an amide bond) are in such embodiments excluded from the definition of “compounds other than BL-8040” (according to any of the respective embodiments described herein), even if they have the indicated relative retention time.

[0099] Without being bound by any particular theory, it is believed that peptides, by nature, which are somewhat similar to BL-8040 (SEQ ID NO: 1), may come in a wide variety of variants (e.g., one or more amino acid residue(s) in a peptide may independently be substituted, degraded and / or replaced by another residue, such as a stereoisomer thereof), some of which may provide an advantageous activity, whereas non-peptide compounds are unlikely to provide an advantageous activity and are therefore of less interest.

[0100] According to some of any of the embodiments, the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 (according to any of the respective embodiments described herein) comprises a compound having the same molecular weight as BL- 8040 (SEQ ID NO: 1) and / or a compound having a molecular weight which is higher than the molecular weight of BL-8040 (SEQ ID NO: 1) by no more than 100 Da, e.g., by about 80 Da. As exemplified herein, the molecular weight of a compound may be determined by mass spectrometry, optionally using liquid chromatography-mass spectrometry, according to techniques known in the art.

[0101] According to some of any of the embodiments, the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 (according to any of the respective embodiments described herein) consists of one or more compounds having the same molecular weight as BL-8040 (SEQ ID NO: 1) and / or one or more compounds having a molecular weight which is higher than the molecular weight of BL-8040 (SEQ ID NO: 1) by about 80 Da; that is, compounds which do not have the aforementioned molecular weight are in such embodiments excluded from the definition of “at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88” (according to any of the respective embodiments described herein), even if they have the indicated relative retention time. In some such embodiments, the at least one at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 consists of one or more compounds having the same molecular weight as BL-8040 (SEQ ID NO: 1).

[0102] In some of any of the embodiments described herein, a total concentration ratio of the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 (according to any of the embodiments described herein) to BL-8040 (SEQ ID NO: 1) is at least about 0.01 %. In some such embodiments, the total concentration ratio is at least about 0.02 %. In some embodiments, the total concentration ratio is at least about 0.03 %. In some embodiments, the total concentration ratio is at least about 0.05 %. In some embodiments, the total concentration ratio is at least about 0.075 %. In some embodiments, the total concentration ratio is at least about 0.1 %. In some embodiments, the total concentration ratio is at least about 0.2 %. In some embodiments, the total concentration ratio is at least about 0.3 %. In some embodiments, the total concentration ratio is at least about 0.4 %. In some embodiments, the total concentration ratio is at least about 0.5 %. Herein the term “total concentration ratio” refers to a ratio of a total concentration of (one or more) compounds (e.g., compounds having a relative retention rate within an indicated range according to any of the respective embodiments described herein) to another concentration (e.g., a concentration of BL-8040 (SEQ ID NO: 1)), which represents unity (or 100 % when the ratio is defined as a percentage); wherein the concentrations are determined by absorption as defined herein.

[0103] In some of any of the embodiments described herein, a total concentration ratio of the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 (according to any of the embodiments described herein) to BL-8040 (SEQ ID NO: 1) is no more than about 1 %. In some such embodiments, the total concentration ratio is in a range of from 0.01 % to 1 %. In some embodiments, the total concentration ratio is in a range of from 0.02 % to 1 %.

[0104] In some embodiments, the total concentration ratio is in a range of from 0.03 % to 1 %. In some embodiments, the total concentration ratio IS in a range of from 0.05 % to 1 %. In some embodiments, the total concentration ratio IS in a range of from 0.075 % to 1 %. In some embodiments, the total concentration ratio IS in a range of from 0.1 % to 1 %. In some embodiments, the total concentration ratio IS in a range of from 0.2 % to 1 %. In some embodiments, the total concentration ratio IS in a range of from 0.3 % to 1 %. In some embodiments, the total concentration ratio IS in a range of from 0.4 % to 1 %. In some embodiments, the total concentration ratio is in a range of from 0.5 % to 1 %.

[0105] In some of any of the embodiments described herein, a total concentration ratio of the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 (according to any of the embodiments described herein) to BL-8040 (SEQ ID NO: 1) is no more than about 0.5 %. In some such embodiments, the total concentration ratio is in a range of from 0.01 % to 0.5 %. In some embodiments, the total concentration ratio is in a range of from 0.02 % to 0.5 %. In some embodiments, the total concentration ratio is in a range of from 0.03 % to 0.5 %. In some embodiments, the total concentration ratio is in a range of from 0.05 % to 0.5 %. In some embodiments, the total concentration ratio is in a range of from 0.075 % to 0.5 %. In some embodiments, the total concentration ratio is in a range of from 0.1 % to 0.5 %. In some embodiments, the total concentration ratio is in a range of from 0.2 % to 0.5 %. In some embodiments, the total concentration ratio is in a range of from 0.3 % to 0.5 %.

[0106] In some of any of the embodiments described herein, a total concentration ratio of the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 (according to any of the embodiments described herein) to BL-8040 (SEQ ID NO: 1) is no more than about 0.3 %. In some such embodiments, the total concentration ratio is in a range of from 0.01 % to 0.3 %. In some embodiments, the total concentration ratio is in a range of from 0.02 % to 0.3 %. In some embodiments, the total concentration ratio is in a range of from 0.03 % to 0.3 %. In some embodiments, the total concentration ratio is in a range of from 0.05 % to 0.3 %. In some embodiments, the total concentration ratio is in a range of from 0.075 % to 0.3 %. In some embodiments, the total concentration ratio is in a range of from 0.1 % to 0.3 %. In some embodiments, the total concentration ratio is in a range of from 0.2 % to 0.3 %.

[0107] In some of any of the embodiments described herein, a total concentration ratio of the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 (according to any of the embodiments described herein) to BL-8040 (SEQ ID NO: 1) is no more than about 0.25 %. In some such embodiments, the total concentration ratio is in a range of from 0.01 % to 0.25 %. In some embodiments, the total concentration ratio is in a range of from 0.02 % to 0.25 %. In some embodiments, the total concentration ratio is in a range of from 0.03 % to 0.25 %. In some embodiments, the total concentration ratio is in a range of from 0.05 % to 0.25 %. In some embodiments, the total concentration ratio is in a range of from 0.075 % to 0.25 %. In some embodiments, the total concentration ratio is in a range of from 0.1 % to 0.25 %.

[0108] In some of any of the embodiments described herein, a total concentration ratio of the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 (according to any of the embodiments described herein) to BL-8040 (SEQ ID NO: 1) is no more than about 0.2 %. In some such embodiments, the total concentration ratio is in a range of from 0.01 % to 0.2 %. In some embodiments, the total concentration ratio is in a range of from 0.02 % to 0.2 %. In some embodiments, the total concentration ratio is in a range of from 0.03 % to 0.2 %. In some embodiments, the total concentration ratio is in a range of from 0.05 % to 0.2 %. In some embodiments, the total concentration ratio is in a range of from 0.075 % to 0.2 %.

[0109] In some of any of the embodiments described herein, a total concentration ratio of the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 (according to any of the embodiments described herein) to BL-8040 (SEQ ID NO: 1) is in a range of from about 0.075 % to about 0.225 %. In some such embodiments, the total concentration ratio is in a range of from 0.1 % to 0.2 %.

[0110] In some of any of the embodiments described herein, the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 (according to any of the embodiments described herein) represents at least 20 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter as determined according to absorption at a wavelength at about 226 nm (as defined herein); that is, at least 20 % of all compounds other than BL-8040 (SEQ ID NO: 1) (determined as described herein) are characterized by a relative retention time in the indicated range (e.g., from 0.86 to 0.88). In some such embodiments, the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 represents at least 30 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 represents at least 40 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 represents at least 50 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 represents at least 60 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 represents at least 70 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 represents at least 80 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 represents at least 90 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein).

[0111] In some of any of the embodiments described herein, a total concentration ratio (as defined herein) of the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 (according to any of the embodiments described herein) to BL-8040 (SEQ ID NO: 1) is at least about 0.01 %. In some such embodiments, the total concentration ratio is at least about 0.02 %. In some embodiments, the total concentration ratio is at least about 0.03 %. In some embodiments, the total concentration ratio is at least about 0.05 %. In some embodiments, the total concentration ratio is at least about 0.075 %. In some embodiments, the total concentration ratio is at least about 0.1 %. In some embodiments, the total concentration ratio is at least about 0.2 %. In some embodiments, the total concentration ratio is at least about 0.3 %. In some embodiments, the total concentration ratio is at least about 0.4 %.

[0112] In some of any of the embodiments described herein, a total concentration ratio of the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 (according to any of the embodiments described herein) to BL-8040 (SEQ ID NO: 1) is no more than about 0.5 %. In some such embodiments, the total concentration ratio is in a range of from 0.01 % to 0.5 %. In some embodiments, the total concentration ratio is in a range of from 0.02 % to 0.5 %. In some embodiments, the total concentration ratio is in a range of from 0.03 % to 0.5 %. In some embodiments, the total concentration ratio is in a range of from 0.05 % to 0.5 %. In some embodiments, the total concentration ratio is in a range of from 0.075 % to 0.5 %. In some embodiments, the total concentration ratio is in a range of from 0.1 % to 0.5 %. In some embodiments, the total concentration ratio is in a range of from 0.2 % to 0.5 %. In some embodiments, the total concentration ratio is in a range of from 0.3 % to 0.5 %.

[0113] In some of any of the embodiments described herein, a total concentration ratio of the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 (according to any of the embodiments described herein) to BL-8040 (SEQ ID NO: 1) is no more than about 0.3 %. In some such embodiments, the total concentration ratio is in a range of from 0.01 % to 0.3 %. In some embodiments, the total concentration ratio is in a range of from 0.02 % to 0.3 %. In some embodiments, the total concentration ratio is in a range of from 0.03 % to 0.3 %. In some embodiments, the total concentration ratio is in a range of from 0.05 % to 0.3 %. In some embodiments, the total concentration ratio is in a range of from 0.075 % to 0.3 %. In some embodiments, the total concentration ratio is in a range of from 0.1 % to 0.3 %. In some embodiments, the total concentration ratio is in a range of from 0.2 % to 0.3 %.

[0114] In some of any of the embodiments described herein, a total concentration ratio of the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 (according to any of the embodiments described herein) to BL-8040 (SEQ ID NO: 1) is no more than about 0.15 %. In some such embodiments, the total concentration ratio is in a range of from 0.01 % to 0.15 %. In some embodiments, the total concentration ratio is in a range of from 0.02 % to 0.15 %. In some embodiments, the total concentration ratio is in a range of from 0.03 % to 0.15 %. In some embodiments, the total concentration ratio is in a range of from 0.05 % to 0.15 %. In some embodiments, the total concentration ratio is in a range of from 0.075 % to 0.15 %. In some embodiments, the total concentration ratio is in a range of from 0.1 % to 0.15 %.

[0115] In some of any of the embodiments described herein, the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 (according to any of the embodiments described herein) represents at least 10 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter as determined according to absorption at a wavelength at about 226 nm (as defined herein). In some such embodiments, the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 represents at least 20 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 represents at least 30 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 represents at least 40 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 represents at least 50 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 represents at least 60 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 represents at least 70 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 represents at least 80 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein).

[0116] In some of any of the embodiments described herein, a total concentration of the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 (according to any of the embodiments described herein) and the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 (according to any of the embodiments described herein) represents at least 50 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter as determined according to absorption at a wavelength at about 226 nm (as defined herein). In some such embodiments, the total concentration represents at least 60 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the total concentration represents at least 70 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the total concentration represents at least 80 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the total concentration represents at least 90 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the total concentration represents at least 95 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the total concentration represents at least 98 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein). In some embodiments, the total concentration represents at least 99 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter (determined as described herein).

[0117] In some of any of the embodiments described herein, the composition-of-matter is characterized by a relatively low amount of deamidated BL-8040 (SEQ ID NO: 2) (wherein the C- terminus is a carboxylic acid rather than an amide) and / or D-Lys(Ac) BL-8040 (SEQ ID NO: 3) (wherein the D-Lys residue is acetylated).

[0118] In some of any of the embodiments described herein, a concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) in the composition-of-matter is no more than 50 % of a total concentration of the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 according to any of the embodiments described herein. In some such embodiments, the concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) is no more than 30 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) is no more than 20 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) is no more than 10 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) is no more than 5 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) is no more than 3 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) is no more than 2 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) is no more than 1 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88.

[0119] In some of any of the embodiments described herein, a concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) in the composition-of-matter is no more than a total concentration of the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 according to any of the embodiments described herein. In some such embodiments, the concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) is no more than 60 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) is no more than 40 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) is no more than 20 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) is no more than 10 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) is no more than 5 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) is no more than 2 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) is no more than 1 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73.

[0120] In some of any of the embodiments described herein, a concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) in the composition-of-matter is no more than 50 % of a total concentration of the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 according to any of the embodiments described herein. In some such embodiments, the concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 30 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 20 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 10 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 5 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 3 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 2 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 1 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88.

[0121] In some of any of the embodiments described herein, a concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) in the composition-of-matter is no more than a total concentration of the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 according to any of the embodiments described herein. In some such embodiments, the concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 60 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the concentration of cyclic D- Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 40 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 20 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 10 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 5 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 2 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the concentration of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 1 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73.

[0122] In some of any of the embodiments described herein, a total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) in the composition-of-matter is no more than 80 % of a total concentration of the at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 according to any of the embodiments described herein. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 60 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 50 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 40 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 30 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 20 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 10 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 5 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 2 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88.

[0123] In some of any of the embodiments described herein, a total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) in the composition-of-matter is no more than 150 % of a total concentration of the at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 according to any of the embodiments described herein. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 120 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 80 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 60 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 40 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 20 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 10 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73. In some such embodiments, the total concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) and cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is no more than 5 % of a total concentration of the aforementioned at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73.

[0124] Without being bound by any particular theory, it is believed that the cyclic forms of SEQ ID NO: 2 and / or SEQ ID NO: 3 are not necessarily more deleterious than the corresponding non- cyclic forms, but rather the non-cyclic forms will typically be considerably rarer (e.g., under conditions in which the peptide having SEQ ID NO: 1 is predominantly in cyclic form), and thus may optionally be disregarded for convenience.

[0125] The above mentioned peptides - cyclic deamidated BL-8040 (SEQ ID NO: 2) and / or cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) - may optionally be quantified by determining retention times under conditions described herein, e.g., using a reference sample of the peptide to precisely identify the relative retention time (relative to the retention time of BL-8040 (SEQ ID NO: 1), as defined herein). For example, under the HPLC conditions described herein, the relative retention time of cyclic deamidated BL-8040 (SEQ ID NO: 2) is typically about 0.91, and the relative retention time of cyclic D-Lys(Ac) BL-8040 (SEQ ID NO: 3) is about 1.10.

[0126] As exemplified herein, compositions-of-matter such as described herein may optionally be prepared using solid phase synthesis, in which the peptide (in a protected form) is formed attached to a resin and then cleaved from the resin (while removing protecting groups). Intramolecular disulfide bonds, to form the cyclic peptide, may then be formed by oxidation of Cys residues.

[0127] In some of any of the embodiments described herein, the process is a large-scale process.

[0128] Herein, the term “large-scale process” refers to a process for preparation of at least 100 grams (in a single run of the process) of the final product (BL-8040 (SEQ ID NO: 1)).

[0129] In some of any of the embodiments described herein relating to a large-scale process, at least 250 grams of BL-8040 (SEQ ID NO: 1) peptide are prepared, or at least 500 grams of the cyclic peptide, or at least 1 kg of the cyclic peptide, or at least 3 kg of the cyclic peptide, or even at least 10 kg of cyclic peptide are prepared by the process.

[0130] According to some embodiments of the present invention, compositions-of-matter as described herein are obtainable by a process as described herein.

[0131] In some of any of the embodiments described herein relating to a process, the obtained composition-of-matter according to any of the respective embodiments described is associated with a characteristic set of minor components other than BL-8040 (SEQ ID NO: 1), e.g., representing a “signaturion” of the process.

[0132] The sum of all compounds other than BL-8040 (SEQ ID NO: 1), as defined according to any of the respective embodiments described herein, is also referred to herein interchangeably as a “signatory composition”, which characterizes the process as described herein.

[0133] Without being bound by any particular theory, it is believed that the signatory composition is associated with enhanced biological activity described herein (e.g., in the section relating to biological activity).

[0134] According to some embodiments of the present invention, the process as described herein provides a signatory composition-of-matter (a signaturion, as described herein), which comprises BL-8040 (SEQ ID NO: 1) and an advantageous signatory composition of compounds other than BL-8040 (SEQ ID NO: 1), as described herein in any of the respective embodiments.

[0135] As detailed herein, the process preferably comprises sequentially coupling amino acids and 4-fluoro-benzoic acid to a resin (e.g., Rink AMS resin) by solid phase peptide synthesis (according to general procedures known in the art), to obtain a linear peptide coupled to the resin. The order of the sequential coupling is Arg, Cys, Cit (citrulline), Arg, Tyr, Pro, DLys (D-lysine), Lys, Cit, Tyr, Cys, Nal (naphthylalanine), Arg, Arg, 4-fluoro-benzoic acid.

[0136] The process further comprises cleaving the peptide formed by coupling from the resin, thereby obtaining a free peptide having SEQ ID NO: 1 in non-cyclic form, and oxidizing the cysteine residues therein to form an intramolecular disulfide bond, thereby obtaining the cyclic form (in solution).

[0137] Coupling comprises forming an amide bond between a carboxylate group (e.g., of an amino acid or N-terminal carboxylic acid) and an amine group (e.g., of a resin in the first step or an N- terminus in subsequent steps), and is preferably effected using one or more coupling reagents known in the art to be suitable for amide bond formation, for example, a carbodiimide (e.g., 1- ethyl-3 -(3 -dimethylaminoprop yl)carbodiimide, dicyclohexylcarbodiimide and / or diisopropylcarbodiimide), aminium / uronium (e.g., HATU, HBTU, TBTU and / or HCTU) or phosphonium salt (e.g., PyBOP and / or PyAOP) and / or propanephosphonic acid anhydride.

[0138] The one or more coupling reagents optionally comprises a carbodiimide (e.g., diisopropylcarbodiimide) and an additional agent (e.g., in about the same molar concentration as the carbodiimide) such as a hydroxytriazole (e.g., 1-hydroxy-benzotriazole (HOBt) or 1-hydroxy- 7-azabenzotriazole (HOAt) and / or a cyanohydroxyiminoacetate ester (e.g., ethyl cyanohydroxyiminoacetate) .

[0139] Coupling may optionally be effected using a carbodiimide and a cyanohydroxyiminoacetate ester, for example, wherein the carbodiimide and / or the cyanohydroxyiminoacetate ester are used in a molar excess of about two-fold (relative to the amino acid or other carboxylic acid being coupled).

[0140] Coupling may optionally be effected using a carbodiimide with ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole, for example, wherein the carbodiimide and / or the ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole are used in a molar excess of about two-fold (relative to the amino acid or other carboxylic acid being coupled).

[0141] Coupling may optionally be effected using diisopropylcarbodiimide (DIC) with ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole, for example, wherein the DIC and the ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole are used in a molar excess of about two-fold (relative to the amino acid or other carboxylic acid being coupled).

[0142] At each amino acid coupling step, the N-terminus of the amino acid is protected by a readily removable group, preferably fluorenylmethyloxycarbonyl (Fmoc), which may optionally be cleaved by a mild base such as piperidine (e.g., 20-50 % piperidine in DMF). An exemplary concentration of piperidine in DMF is about 20 %.

[0143] For example, Fmoc groups may optionally be removed by washing twice in piperidine solution (according to any of the embodiments described herein), optionally once for 5-10 minutes and then optionally once for 25-30 minutes; followed by washing of the resin (e.g., in DMF without piperidine) to remove the base (which may optionally be ascertained by determining pH).

[0144] In addition, side chains of certain amino acids may be protected by a readily removable group (which is not cleaved by the abovementioned mild base), such as t-butoxy. Thus, side chain hydroxy groups in Tyr may preferably be protected by t-butyl (t-Bu) (to form a t-butoxy group), and side chain amine groups in (L or D) Lys may be protected by t-butoxycarbonyl (Boc) (which comprises a t-butoxy group); and in both cases, cleavage (e.g., in an acidic environment) may regenerate the unprotected hydroxy or amine group. Similarly, side chain thiohydroxy groups of Cys may preferably be protected with trityl (Trt), and / or side chain guanidinium groups of Arg may preferably be protected with 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf); each of which may optionally be cleaved in an acidic environment. The aforementioned side chain protective groups are known for being particularly compatible with the use of Fmoc. The skilled person will be aware of additional protective groups suitable for specific groups in amino acid side chains, and their compatibility with each other and with various N-terminal protective groups.

[0145] The resin to which the amino acids are sequentially coupled is optionally a Rink aminomethylstyrene (AMS) resin (aminomethylstyrene resin substituted with a Rink linker). The Rink AMS resin is optionally protected by Fmoc groups, which may be removed prior to coupling so as to expose amine groups (to which the first amino acid is coupled).

[0146] The degree of substitution of the resin (optionally a Rink AMS resin) to which the amino acids are sequentially coupled is optionally at least 0.3 milliequivalents per gram resin (e.g., from 0.3 to 0.11 milliequivalents per gram resin), optionally at least 0.6 milliequivalents per gram resin, optionally from 0.6 to 1.1 milliequivalents per gram resin, and optionally from 0.6 to 0.9 milliequivalents per gram resin.

[0147] The resin is optionally washed (e.g., once or twice) with a solvent such as dimethylformamide (DMF) prior to each coupling step, for example, to facilitate swelling of the resin. In some exemplary embodiments, the resin is washed for 20-30 minutes prior to deprotection of the resin (e.g., removal of Fmoc groups according to any of the respective embodiments described herein) and coupling of the first amino acid, and washed (e.g., twice) for about 2 minutes prior to each subsequent coupling step. Cleavage of the peptide (formed by sequential coupling) from the resin, and cleavage of the protecting groups from the amino acid side chains, is preferably effected by contacting the peptide (coupled to the resin) with a liquid comprising an acid such as trifluoroacetic acid (TFA). The concentration of TFA in the liquid is optionally at least about 80 %, or at least about 90 %, or at least about 95 % by weight (e.g., wherein the balance is primarily water).

[0148] The liquid (e.g., acidic liquid) used for cleavage optionally further comprises a scavenger (e.g., a scavenger of reactive cationic species which may form during cleavage), such as thiols (e.g., ethanedithiol, dithioerythritol, dithio threitol), trialkylsilanes, (e.g., triisopropylsilane), phenols (e.g., m-cresol) and / or water.

[0149] Dithiothreitol (DTT) and / or dithioerythritol (DTE) are examples of a suitable scavenger, optionally wherein the liquid is a solution comprising TFA (e.g., at a concentration described herein) and DTT and / or DTE, preferably in combination with water. The (total) concentration of DTT and / or DTE in a solution used for cleavage is optionally at least 10 mg / mL (e.g., from 10 to 500 mg / mL, or from 10 to 200 mg / mL, or from 10 to 100 mg / mL, or from 10 to 50 mg / mL), optionally at least 25 mg / mL (e.g., from 25 to 500 mg / mL, or from 25 to 200 mg / mL, or from 25 to 100 mg / mL, or from 25 to 50 mg / mL), optionally at least 50 mg / mL (e.g., from 50 to 500 mg / mL, or from 50 to 200 mg / mL, or from 50 to 100 mg / mL), and optionally about 50 mg / mL.

[0150] Upon cleavage of the peptide from the resin, the free peptide is preferably precipitated, for example, by addition of a liquid in which the peptide is insoluble. Precipitation is optionally preceded by concentrating the free peptide by evaporation of a portion of solvent of the solution, thus reducing a volume of the solution (e.g., by about 65 % to about 70 %). Alternatively, no concentration of free peptide is performed prior to precipitation.

[0151] In some of any of the embodiments described herein, precipitation of the free peptide is performed without concentrating the free peptide prior to precipitation.

[0152] Precipitation of the peptide is optionally effected by addition of a dialkyl ether, such as tert-butyl methyl ether (MTBE).

[0153] The MTBE is optionally admixed with hexane. In some embodiments, the mixture of MTBE and hexane (optionally chilled, e.g., to a temperature in a range of about 5 °C to about 15 °C) is added to the peptide at a volume of at least 40 mL mixture per gram of resin, and optionally about 45 mL mixture per gram of resin. Exemplary mixtures of MTBE and hexane are composed of MTBE and hexane at a 60:40 (MTBE: hexane) volume ratio.

[0154] The precipitated peptide is optionally dried, for example, by lyophilization or by vacuum. As discussed herein, an intramolecular disulfide bond may be formed (resulting in cyclization of the peptide) by oxidizing cysteine residues of the obtained peptide. Any suitable technique known in the art may optionally be used.

[0155] Such oxidizing is optionally effected by contacting the peptide with hydrogen peroxide, for example, by gradual (e.g., dropwise) addition of a hydrogen peroxide solution (e.g., about 1.5 % by weight hydrogen peroxide in water). The molar ratio of hydrogen peroxide to pair of Cys residues (e.g., to peptide, wherein the peptide comprises exactly two Cys residues, such as in SEQ ID NO: 1) is preferably at least 1:1 (i.e., there is at least one hydrogen peroxide molecule per pair of Cys residues), optionally at least 2:1, optionally at least 3:1, and optionally at least 4:1. An exemplary molar ratio of hydrogen peroxide to pair of Cys residues (e.g., to peptide) is at about 5:1. Alternatively or additionally, hydrogen peroxide may optionally be added until no thiohydroxy groups remain (e.g., as determined by a suitable assay, such as an Ellman test).

[0156] Oxidizing is optionally effected by contacting an aqueous solution comprising the peptide at a concentration of at least 5 mg / mL (e.g., from 5 to 20 mg / mL, or from 5 to 15 mg / mL, or from 5 to 10 mg / mL, or about 10 mg / mL) with hydrogen peroxide (e.g., according to any of the respective embodiments described herein). Optionally the peptide concentration is at least 10 mg / mL (e.g., from 10 to 20 mg / mL, or from 10 to 15 mg / mL). The aqueous solution is optionally mildly alkaline, for example, an aqueous solution of ammonium bicarbonate (NH4HCO3). An exemplary concentration of ammonium bicarbonate is about 0.1 M.

[0157] The process preferably further comprises isolating the obtained BL-8040 (SEQ ID NO: 1). As the isolation procedure will not be 100 % perfect, some additional compounds (as described herein) remain. Any suitable technique known in the art may optionally be used.

[0158] Isolation of the peptide optionally comprises chromatography, for example, at least one step (e.g., optionally one or two steps) of preparative high performance liquid chromatography (HPLC), for example, using a C18 column. Isolation optionally comprises loading the obtained peptide (e.g., in at least one step of HPLC described herein) on a reverse phase chromatography column (e.g., a C18 column) at a concentration of no more than 40 grams peptide per kg of the column (by weight of resin of the column); and / or optionally at a concentration of at least 4 grams (e.g., from 4 to 40 grams or from 4 to 30 grams or from 4 to 25 grams) peptide per kg of the column (by weight of resin of the column), optionally of at least 10 grams (e.g., from 10 to 40 grams or from 10 to 30 grams or from 10 to 25 grams) peptide per kg of the column, and optionally of at least 20 grams (e.g., from 20 to 40 grams or from 20 to 30 grams or from 20 to 25 grams) peptide per kg of the column. The peptide loaded on a column (according to any of the respective embodiments described herein) is eluted, e.g., using a buffer solution (e.g., in combination with an acetonitrile gradient, according to any of the respective embodiments described herein). The pH of the buffer solution is optionally in a range of from 1.5 to 3, optionally from 2.0 to 2.5, and optionally about 2.25. Triethylammonium phosphate buffer may optionally be used to elute the peptide from the column, for example, at a concentration of at least about 0.01 M, or at least about 0.03 M, or at least about 0.1 M. In exemplary embodiments, the concentration of triethylammonium phosphate is about 0.1 M.

[0159] Elution from an HPLC column is optionally effected with an acetonitrile gradient. The gradient optionally comprises increasing a concentration of acetonitrile by a rate (which is optionally varied over time) in a range of from 0.1 % acetonitrile per minute to 2 % acetonitrile per minute, e.g., about 1 % per 5-6 minutes and / or about 1 % per minute. Alternatively or additionally, the gradient optionally comprises increasing a concentration of acetonitrile (e.g., from 0 %) to a concentration in a range of from 10 % to 40 %, or from 20 % to 30 %, or to about 25 %.

[0160] An exemplary gradient (e.g., using triethylammonium phosphate buffer according to any of the respective embodiments described herein) comprises 0 % acetonitrile for about 8 minutes, from 0 % to about 5 % acetonitrile in about 5 minutes (e.g., a rate of about 1 % per minute), and from about 5 % to about 25 % acetonitrile in about 120 minutes (e.g., a rate of about 0.17 % per minute).

[0161] Optionally, eluted peptide is loaded on a column (according to any of the respective embodiments described herein) a second time and eluted, e.g., using a buffer solution (e.g., in combination with an acetonitrile gradient, according to any of the respective embodiments described herein). Acetic acid buffer (e.g., acetic acid / ammonium acetate) may optionally be used to elute the peptide from the column (e.g., in order to obtain the peptide as an acetate salt), for example, at a concentration of at least about 5 mM, or at least about 20 mM, or at least about 35 mM. In exemplary embodiments, the concentration of acetate is about 35 mM.

[0162] An exemplary gradient (e.g., using acetic acid buffer according to any of the respective embodiments described herein) for a second elution comprises 0 % acetonitrile for 8 minutes, and from 0 % to 22 % acetonitrile in 110 minutes (a rate of 0.2 % per minute).

[0163] The process optionally further comprises lyophilizing the product obtained by chromatography (e.g., according to any of the respective embodiments described herein).

[0164] Optionally, the process is characterized by at least one, or at least two, or at least three, or at least four, or at least five, or at least six of the following features (i)-(vii): (i) coupling is effected using diisopropylcarbodiimide (DIC) in combination with ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzo triazole (e.g., according to any of the respective embodiments described herein);

[0165] (ii) cleavage of the peptide from the resin is effected by contacting the linear peptide coupled to the resin with a solution comprising trifluoroacetic acid (TFA) and a scavenger which is dithiothreitol (DTT) and / or dithioerythritol (DTE) (e.g., according to any of the respective embodiments described herein);

[0166] (iii) the process further comprises precipitating the free linear peptide after cleaving without concentrating the free linear peptide by evaporation prior to precipitation of the peptide (e.g., according to any of the respective embodiments described herein);

[0167] (iv) oxidation comprises contacting an aqueous solution comprising the linear peptide at a concentration of at least 5 mg / mL with hydrogen peroxide (e.g., according to any of the respective embodiments described herein);

[0168] (v) isolating the peptide comprises loading the cyclic peptide on a reverse phase chromatography column at a concentration of no more than 40 grams cyclic peptide per kg of the column, and eluting the cyclic peptide from the column (e.g., according to any of the respective embodiments described herein);

[0169] (vi) isolating the cyclic peptide comprises lyophilization, and the process further comprises grinding the cyclic peptide following lyophilization (e.g., according to any of the respective embodiments described herein); and / or

[0170] (vii) a degree of substitution of the resin used for peptide synthesis is at least 0.3 milliequivalents per gram and / or the resin is a Rink AMS resin (e.g., according to any of the respective embodiments described herein).

[0171] Biological activity:

[0172] In some of any of the respective embodiments, a BL-8040 (SEQ ID NO: l)-containing composition-of-matter according to any of the respective embodiments described herein exhibits an enhanced biological activity as compared to a corresponding BL-8040 (SEQ ID NO: 1)- containing composition-of-matter (e.g., with the same BL-8040 (SEQ ID NO: 1) concentration, counter-ions and solvent) without a compound having a relative retention rate of 0.71 to 0.73 and / or 0.86 to 0.88 (according to any of the respective embodiments described herein).

[0173] In some of any of the respective embodiments, the biological activity of the composition- of-matter is characterized by promotion of in vivo neutrophil migration to peripheral blood (which may also be regarded as inhibition of migration to bone marrow), e.g., in mice. As exemplified herein, a composition-of-matter according to embodiments of the invention (e.g., with a signatory composition comprising compounds having relative retention times of about 0.72 and about 0.87) was associated with an almost two-fold enhancement of the increase in neutrophils in peripheral blood, relative to a control composition-of-matter comprising the same amount of BL-8040 (SEQ ID NO: 1) without the aforementioned additional compounds.

[0174] In some of any of the respective embodiments, promotion of in vivo neutrophil migration to peripheral blood is determined by subcutaneously injecting mice with 5 mg / kg of BL-8040 (SEQ ID NO: 1), reconstituted in 200 pl of PBS, then bleeding the mice 2 hours after injection (e.g., by cardiac puncture into tubes with heparin) and quantifying the number of mature neutrophils in the peripheral blood (e.g., by flow cytometry) relative to control (untreated) mice.

[0175] In some of any of the respective embodiments, the biological activity of the composition- of-matter is characterized by one or more of the following:

[0176] (i) promotion of in vivo neutrophil migration to peripheral blood (which may also be regarded as inhibition of migration to bone marrow), e.g., in mice;

[0177] (ii) inhibition of in vitro CXCL12-induced migration of breast cancer cells;

[0178] (iii) inhibition of in vitro CXCL12-induced migration of T-cell-derived leukemia cells;

[0179] (iv) inhibition of metastatic breast cancer cell migration to lungs in an animal (e.g., mouse) model;

[0180] (v) inhibition of delayed-type hypersensitivity in an animal (e.g., mouse) model;

[0181] (vi) inhibition of collagen-induced arthritis in an animal (e.g., mouse) model; and

[0182] (vii) inhibition of binding of CXCL12 to CXCR4 in cells (e.g., Jurkat cells) in vitro (e.g., increasing an IC50 of the CXCL12).

[0183] In some of the respective embodiments, the biological activity of the composition-of-matter is characterized by at least two of activities (i)-(vii). In some embodiments, the biological activity of the composition-of-matter is characterized by at least three of activities (i)-(vii).

[0184] An enhanced biological activity according to any of the respective embodiments described herein is optionally characterized by an increase or decrease in a relevant quantitative parameter (e.g., a fold-change in the parameter relative to control) of at least 10 %, optionally at least 20 %, optionally at least 30 %, and optionally at least 50 % (e.g., as determined according to procedures described herein). For example, an enhanced promotion of in vivo neutrophil migration to peripheral blood according to any of the respective embodiments described herein is optionally characterized by an increase in neutrophil levels in the peripheral blood of at least 10 %, optionally at least 20 %, optionally at least 30 %, and optionally at least 50 % (e.g., as determined according to procedures described herein). Activities (i)-(vii) may optionally be evaluated using procedures such as described in U.S. Patent No. 7,423,007 and / or as described in the Examples section herein.

[0185] In some of any of the respective embodiments, activities (ii) and (iii) (inhibition of in vitro CXCL12-induced migration) is determined by evaluating migration of cells through a suitable microporous membrane and / or insert separating two compartments (e.g., of a Transwell® plate). CXCL12 (100 nM for breast cancer cells and 30 nM for leukemia cells) and cell growth medium (optionally 600 pl of DMEM with 0.1 % bovine serum albumin) with the BL-8040 (SEQ ID NO: 1) composition is added to a first (e.g., upper) compartment, and the tested BL-8040 (SEQ ID NO: 1) composition and MDA-MB-231 breast cancer cells or SUP-T1 leukemia cells (e.g., at 2 x 106cells / ml) and cell growth medium (optionally 100 pl of DMEM with 0.1 % bovine serum albumin) are added to the second (e.g., lower) compartment. The amount of cells which migrated through the membrane and / or insert upon incubation (e.g., for 15 hours for breast cancer cells and 4 hours for leukemia cells) under conditions suitable for cell growth and activity (e.g., at 37 °C) are quantified, optionally by flow cytometry and / or by fixing and staining (e.g., with 25 % methanol and 0.5 % crystal violet), and compared to a control sample with no BL-8040 (SEQ ID NO: 1).

[0186] In some of any of the respective embodiments, activity (iv) (inhibition of metastatic breast cancer cell migration to lungs) is determined by implanting intravenously 106MDA-MB-231 human breast cancer cells into the tails of mice (optionally five week-old female CB-17 SCID mice). A BL-8040 (SEQ ID NO: 1) composition comprising physiological saline, adjusted to a BL-8040 (SEQ ID NO: 1) concentration of 80 mg / ml is administered gradually over the course of four weeks to the mice (optionally using sustained-release osmotic pumps) to administer about 18.2 mg / kg / day). The amount of cancer cells in the lungs of the mice is then evaluated, optionally using Evan’s Blue solution (e.g., 0.2 %, 2 ml) injected through the windpipes followed by soaking the lungs in Bouin’s liquid, staining and fixing.

[0187] In some of any of the respective embodiments, activity (v) (inhibition of delayed-type hypersensitivity) is determined according to the degree of inflammation (represented by change in ankle thickness) generated by injection of sheep red blood cells into ankles of sensitized mice. To sensitize the mice, sheep red blood cells suspended in physiological saline solution (2 x 107cells per 50 pl) are administered subcutaneously to the ankles of the left hind limbs of mice (optionally 6 week-old male BALB / c mice), and after 5 days, 108cells per 50 pl sheep red blood cells are administered subcutaneously to the ankles of the right hind limbs. Immediately prior to and about 24 hours after the induction of antigens, the ankle thickness is determined. A BL-8040 (SEQ ID NO: 1) composition comprising PBS is administered gradually to the mice (optionally using sustained-release osmotic pumps) to administer about 4, about 8, about 24 and / or about 120 pg / day BL-8040 (SEQ ID NO: 1), beginning on the day before sensitization.

[0188] In some of any of the respective embodiments, activity (vi) (inhibition of collagen-induced arthritis) is determined by generating collagen-induced arthritis in mice (optionally 6 week-old male DBA / 1JN mice) via intradermal injection (e.g., into the base of the tail) of an emulsion comprising 1 mg / ml bovine type II collagen, 0.025 M acetic acid, and 50 % (by volume) Freund’s complete adjuvant, followed by a similar immunization 21 days after the injection. For two weeks after the second immunization, body weight and / or hind limb thickness is optionally measured and arthritis scoring is optionally performed. Arthritis is optionally scored at 0-3 points of each limb, and evaluated by the total of the same (12 points being the full points; 0 = normal; 1 = mild swelling or swelling of a single digit; 2 = moderate swelling or swelling of plural digits; 3 = severe swelling). Type II collagen- specific IgG2a antibodies are optionally quantified (at the end of the two week period) using any suitable technique. Inhibition of collagen-induced arthritis may optionally be manifested as inhibition of body weight loss, reduction in arthritis score and / or reduction in Type II collagen- specific antibodies.

[0189] In some of any of the respective embodiments, activity (vii) (inhibition of binding of CXCE12 to CXCR4 in cells) is optionally determined using radioactively labeled CXCE12 in Jurkat human T-cell leukemia cells (e.g., increasing an IC50 of the CXCE12). 200 pM of125I- CXCE12 is incubated with Jurkat cells (optionally at 6 x 106cells / ml) suspended in buffer at a pH of about 7 (e.g., Dulbecco’s PBS solution, pH 7.0, containing 0.5 % bovine serum albumin and 20 mM HEPES) and the tested BL-8040 (SEQ ID NO: 1) composition-of-matter diluted by buffer, at a volume ratio of 1:2:1 (e.g., 25 pl radio-labeled125I-CXCL12, 50 pl Jurkat cell solution, and 25 pl BL-8040 (SEQ ID NO: 1) composition in buffer). The samples are subjected to fixation reaction for 1 hour at room temperature, followed by determining each sample’s radioactivity.

[0190] The composition-of-matter comprising BL-8040 (SEQ ID NO: 1) (and compounds other than BL-8040 (SEQ ID NO: 1)) according to any of the respective embodiments described herein may optionally be for use in treatment of a condition treatable by BL-8040 (SEQ ID NO: 1).

[0191] According to an aspect of some embodiments of the invention, there is provided a use of a composition-of-matter according to any of the respective embodiments described herein in the manufacture of a medicament for treating a condition treatable by BL-8040 (SEQ ID NO: 1).

[0192] According to an aspect of some embodiments of the invention, there is provided a method of treating a condition treatable by BL-8040 (SEQ ID NO: 1), the method comprising administering a composition-of-matter according to any of the respective embodiments described herein to a subject in need thereof. In some of any of the embodiments described herein relating to treatment, the treatment may be any treatment in which inhibiting CXCR4 is advantageous.

[0193] As used herein the phrase "subject in need thereof" refers to a mammalian male or female subject (e.g., human being) who is diagnosed with a CXCR4-related condition, e.g., cancer. In a specific embodiment, this term encompasses individuals who are at risk to develop the condiditon, e.g., cancer. Veterinary uses are also contemplated. The subject may be of any gender or at any age including neonatal, infant, juvenile, adolescent, adult and elderly adult.

[0194] The terms “treating” or "treatment" refers to inhibiting, preventing or arresting the development of a pathology (e.g. cancer) and / or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.

[0195] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0196] Cancers which can be treated by the method of this aspect of some embodiments of the invention can be any solid or non-solid cancer and / or cancer metastasis.

[0197] According to a specific embodiment, the cancer is a solid tumor. According another specific embodiment, the cancer is a non-solid tumor.

[0198] Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non- small-cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), melanoma cancer, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; acute myeloblastic leukemia; Multiple Myeloma; and posttransplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome. Preferably, the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non- small cell lung cancer, non-Hodgkins lymphoma (NHL), acute lymphoblastic leukemia (ALL); chronic myeloblastic leukemia (CML); acute myeloblastic leukemia (AML); renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. The cancerous conditions amenable for treatment of the invention include metastatic cancers.

[0199] According to specific embodiments the cancer is selected from the group consisting of lung cancer, glioma, colon cancer, ovarian cancer, renal cancer, melanoma cancer, hepatocellular cancer, gastric or stomach cancer, glioblastoma, cervical cancer, bladder cancer, breast cancer, colorectal cancer, prostate cancer, thyroid cancer, head and neck and pancreatic cancer.

[0200] According to specific embodiments, the cancer is selected from the group consisting of lung cancer, glioma, colon cancer and pancreatic cancer.

[0201] According to a specific embodiment, the subject is diagnosed with metastatic pancreatic adenocarcinoma.

[0202] According to a specific embodiment, the subject is newly diagnosed with previously untreated metastatic pancreatic adenocarcinoma.

[0203] In some embodiments, the individual has measurable disease (for example, according to RECIST criteria).

[0204] In some embodiments, the individual has one or more metastatic tumors measurable, for example, by CT scan (or MRI).

[0205] In some embodiments, the pancreatic cancer is unresectable pancreatic cancer. In some embodiments, the pancreatic cancer is a resectable pancreatic cancer.

[0206] In some embodiments, the pancreatic cancer is borderline resectable.

[0207] In some embodiments, the primary location of the pancreatic cancer is the head of the pancreas. In some embodiments, the primary location of the pancreatic cancer is the body of the pancreas. In some embodiments, the primary location of the pancreatic cancer is the tail of the pancreas.

[0208] According to a specific embodiment, the treatment is a first-line treatment against the metastatic pancreatic adenocarcinoma.

[0209] Generally, lines of therapy” refer to the order in which different therapies are given to people as their disease progresses. As used herein “first-line treatment” refers to a treatment that is the first choice for treating a particular condition, in this case metastatic pancreatic adenocarcinoma, generally, because it is considered a very effective treatment for that condition with the least likelihood of causing side effects.

[0210] According to a specific embodiment, the subject is diagnosed with metastatic pancreatic adenocarcinoma.

[0211] According to a specific embodiment the metastatic pancreatic adenocarcinoma is histologically confirmed (based on a tissue biopsy).

[0212] According to a specific embodiment, the metastatic pancreatic adenocarcinoma is cytologically confirmed (based on biopsy) .

[0213] According to a specific embodiment, the subject has a measurable disease (> 1 measurable lesion) based on Response Evaluation Criteria In Solid Tumors (RECIST) vl.l.

[0214] According to other specific embodiments, the cancer is selected from the group consisting of multiple myeloma and leukemia.

[0215] Other examples of conditions treatable (according to any of the aspects described herein) by BL-8040 (SEQ ID NO: 1) include, without limitation, retinoblastoma and / or neuroectodermal derived tumors, e.g., as described in International Patent Application Publication WO 2012 / 095849 (the contents of which are incorporated herein by reference, particularly contents regarding treatment of conditions by the aforementioned peptide); large cell lung cancer, e.g., as described in WO 2013 / 160895 (the contents of which are incorporated herein by reference, particularly contents regarding treatment of conditions by the aforementioned peptide); multiple myeloma, microglioma and / or glioma, e.g., as described in International Patent Application Publication WO 2008 / 075370 (the contents of which are incorporated herein by reference, particularly contents regarding treatment of conditions by the aforementioned peptide); breast cancer and / or pancreatic cancer, e.g., as described in U.S. Patent No. 7,423,007; thrombocytopenia, e.g., as described in International Patent Application Publication WO 2010 / 146578 (the contents of which are incorporated herein by reference, particularly contents regarding treatment of conditions by the aforementioned peptide); risk of bone marrow suppression, e.g., as described in International Patent Application Publication WO 2008 / 075369 (the contents of which are incorporated herein by reference, particularly contents regarding treatment of conditions by the aforementioned peptide); and HIV infection, e.g., as described in U.S. Patent No. 8,435,939 (the contents of which are incorporated herein by reference, particularly contents regarding treatment of conditions by the aforementioned peptide). According to specific embodiments, the CXCR4 antagonistic peptide is administered to the subject in combination with one or more white blood cell mobilizing agents. For example, the peptide may be administered in sequential or concomitant combination with one or more other growth factors or cytokines that affect mobilization such as, but not limited to, G-CSF, GM-CSF and SCF.

[0216] As used herein the term "immune-check point regulator" or “immune-check point modulator" refers to a molecule that modulates the activity of one or more immune-check point proteins in an agonistic or antagonistic manner resulting in recruitment of an immune cell to elicit an immune activity against a cancer cell.

[0217] According to specific embodiments, the immune-check point regulator modulates the activity of a specific immune-check point protein with no cross reactivity with other immune- check point proteins.

[0218] According to other specific embodiments, the immune-check point regulator modulates the activity of at least 2, at least 3, at least 4 immune-check point proteins.

[0219] According to specific embodiments the immune-check point regulator binds directly the immune-check point protein.

[0220] According to other specific embodiments, the immune-check point regulator indirectly binds the immune-check point protein through an intermediary molecule.

[0221] As used herein the term "activation" refers to the process of stimulating an immune cell (e.g. T cell, NK cell, B cell) that results in cellular proliferation, maturation, cytokine production and / or induction of regulatory or effector functions.

[0222] As used herein the term "immune-check point protein" refers to an antigen independent protein that modulates an immune cell response (i.e. activation or function). Immune-check point proteins can be either co- stimulatory proteins [i.e. positively regulating an immune cell activation or function by transmitting a co- stimulatory secondary signal resulting in activation of an immune cell] or inhibitory proteins (i.e. negatively regulating an immune cell activation or function by transmitting an inhibitory signal resulting in suppressing activity of an immune cell).

[0223] According to specific embodiments, the immune-check point protein regulates activation or function of a T cell. Numerous checkpoint proteins are known in the art and include, but not limited to, PD1, PDL-1, CTLA-4, CD80, LAG-3, TIM-3, KIR, IDO, 0X40, OX40L, CD 137 (4- 1BB), 4-1BBL, CD27, CD70, CD40, CD40L, GITR, CD28, CD86, and ICOS (CD278), ICOSL.

[0224] Methods of determining signaling of a stimulatory or inhibitory signal are well known in the art and include, but are not limited to, binding assay using e.g. BiaCore, HPLC or flow cytometry, enzymatic activity assays such as kinase activity assays, and expression of molecules involved in the signaling cascade using e.g. PCR, Western blot, immunoprecipitation and immunohistochemistry. Additionally or alternatively, determining transmission of a signal (costimulatory or inhibitory) can be effected by evaluating immune cell activation or function. Methods of evaluating immune cell activation or function are well known in the art and include, but are not limited to, proliferation assays such as BRDU and thymidine incorporation, cytotoxicity assays such as chromium release, cytokine secretion assays such as intracellular cytokine staining ELISPOT and ELISA, expression of activation markers such as CD25, CD69 and CD69 using flow cytometry.

[0225] According to specific embodiments, determining the signaling activity is effected in-vitro or ex-vivo e.g. in a mixed lymphocyte reaction (MLR).

[0226] For the same culture conditions the signaling activity or the immune cell activation or function are generally expressed in comparison to the signaling, activation or function in a cell of the same species but not contacted with the immune-check point regulator or contacted with a vehicle control, also referred to as control.

[0227] Depending on the immune-check point protein (i.e. co-stimulatory or inhibitory) the immune-check point regulator can be an agonist or antagonist.

[0228] According to specific embodiment the immune-check point regulator is an antagonist.

[0229] As used herein the term “antagonist” refers to a molecule that prevents and / or inhibits the biological function and / or expression of an immune-check point protein.

[0230] According to specific embodiments, the antagonist prevents and / or inhibits the suppressive effect of an immune-check point protein on an immune cell (e.g. T cells).

[0231] According to specific embodiments, the antagonist prevents and / or inhibits signaling to an immune cell (e.g. T cell) by an immune-check point protein.

[0232] The molecule may be a reversible or an irreversible antagonist.

[0233] According to specific embodiments, the antagonist completely prevents the biological function (e.g. signal transduction) of the immune-check point protein.

[0234] According to other specific embodiments, the antagonist inhibits the biological function (e.g. signal transduction) of the immune-check point protein e.g., as detected by e.g. kinase activity, proliferation assay, cytotoxicity assay or cytokine secretion assay. The reduction may be by at least a 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % as compared to same in the absence of the antagonist.

[0235] Preventing and / or inhibiting the biological function of an immune-check point protein can be effected at the protein level (e.g., antibodies, small molecules, inhibitory peptides, enzymes that cleave the polypeptide, aptamers and the like) but may also be effected at the genomic (e.g. homologous recombination and site specific endonucleases) and / or the transcript level using a variety of molecules which interfere with transcription and / or translation (e.g., RNA silencing agents) of an inhibitory immune-check point protein.

[0236] Non limiting examples of agents that can function as antagonists are described in details in W02017 / 009842, which is hereby incorporated by reference in its enitrety.

[0237] For example, the CXCR4 antagonistic peptides of the present invention may be used in combination with an immune-check point regulator for the treatment of cancer. According to specific embodiments, wherein the immune-check protein is an inhibitory protein, the CXCR4 antagonistic peptides of the present invention are administered in combination with an immune- check point antagonist. For example, there is provided a method of treating cancer in a subject in need thereof, the method comprising:

[0238] (a) intravenously administering to the subject a therapeutically effective amount of a peptide having an amino acid sequence as set forth in SEQ ID NO: 1; and

[0239] (b) administering to the subject a therapeutically effective amount of a PD1 antagonist, thereby treating the cancer in the subject.

[0240] According to an aspect of the present invention there is provided an article of manufacture identified for use in treating cancer, comprising a packaging material packaging a peptide set forth in BL8040 formulated for intravenous administration and a PD1 antagonist.

[0241] PD1 (Programmed Death 1, also known as CD279) is a 55 kDa type I transmembrane protein that is part of the Ig gene superfamily, which is expressed on the surface of several immune cells such as activated T cells, B cells, NK cells and myeloid cells. PD-1 contains a membrane proximal immunoreceptor tyrosine inhibitory motif (ITIM) and a membrane distal tyrosine-based switch motif (ITSM).

[0242] The presence of an ITIM on PD- 1 indicates that this molecule functions to attenuate antigen receptor signaling by recruitment of cytoplasmic phosphatases.

[0243] According to a specific embodiment, the PD1 protein refers to the human protein, such as provided in the following GenBank Number NP_005009. Two ligands for PD-1 have been identified, PD-L1 and PD-L2 (also known as B7-DC).

[0244] According to a specific embodiment, the PD-L1 protein refers to the human protein, such as provided in the following GenBank Number NP_001254635 and NP_054862. According to a specific embodiment, the PD-L2 protein refers to the human protein, such as provided in the following GenBank Number NP_079515.

[0245] PD1 pattern of expression and function is dependent on the cell type. PD1 expression is induced following effector T cells activation. Upon ligand binding, PD1 inhibits kinases that are involved in T cell activation through e.g. the phosphatase SHP2, thereby transmits an inhibitory signal. Conversely, PD1 is highly expressed on regulatory T cells, where it may enhance their proliferation upon ligand binding. PD-1 is also induced on other activated non-T lymphocyte subsets, including B cells and NK cells, where upon ligand binding it transmits an inhibitory signal which limits their antibody production and lytic activity, respectively [Pardoll (2012) Nature Reviews Cancer 12, 252-264].

[0246] Thus, PD1 blockade may enhance the activity of effector T cells, NK cells and antibody production in tissues and in the tumor microenvironment. Because many tumors are highly infiltrated with regulatory T cells that probably further suppress effector immune responses, blockade of the PD1 pathway may also enhance antitumor immune responses by diminishing the number and / or suppressive activity of intra-tumoral regulatory T cells.

[0247] As used herein, the term "PD1 antagonist" refers to an antagonistic agent that prevents and / or inhibits the biological function and / or expression of PD1.

[0248] According to specific embodiments, the PD1 antagonist prevents and / or inhibits signaling to an immune cell (e.g. T cells, B cells, NK cells) by PD1; thereby suppresses PD1 immune- suppressive activity.

[0249] According to specific embodiments, the PD1 antagonist promotes immune response of an effector T cell following TCR activating signal.

[0250] According to specific embodiments, the PD1 antagonist of the present invention binds directly PD1 and / or binds a ligand of PD1 and interferes with and / or inhibits the binding of the ligands to PD1.

[0251] According to other specific embodiments, the PD1 antagonist indirectly binds PD1 by acting through an intermediary molecule, for example the antagonist binds to or modulates a molecule that in turn binds to or modulates PD1.

[0252] According to specific embodiments, the PD1 antagonist binds PD1. According to other specific embodiments, the PD1 antagonist binds at least one of the PD-1 ligands (e.g. PDL-1 or PDL-2), as further described hereinbelow.

[0253] In certain embodiments, the PD1 antagonist exhibits one or more desirable functional properties, such as high affinity binding to PD1, e.g., binding to human PD1 with a KD of 10’7M, 10’8M, 10’9M, IO10M or less; lack of significant cross-reactivity to other immune-check point proteins, e.g., CD28, CTLA-4 and ICOS; the ability to stimulate T cell proliferation; the ability to increase IFN-y and / or IL-2 secretion; the ability to inhibit binding of one or more PD1 ligands (e.g., PD-L1 and / or PD-L2) to PD1; the ability to stimulate antigen- specific memory responses; the ability to stimulate antibody responses and / or the ability to inhibit growth of tumor cells. According to a specific embodiment, the PD1 antagonist is an antibody.

[0254] According to specific embodiments, the PD1 antagonist is an anti-PDl antibody. Anti- PD1 antibodies suitable for use in the invention can be generated using methods well known in the art especially in light of the detailed description hereinabove. Alternatively, art recognized anti-PDl antibodies can be used. Examples of anti-PDl antibodies are disclosed for example in Topalian, et al. NEJM 2012, U.S. Patent Nos. 7,488,802; 8,008,449; 8,609,089; 6,808,710; 7,521,051; and 8168757, U.S. Patent Application Publication Nos. 20140227262; 20100151492; 20060210567; and 20060034826 and International Patent Application Publication Nos. WO2008156712; W02010089411; W02010036959; WO2011159877; WO2013 / 019906; WO2014159562; WO2011109789; WOOl / 14557; W02004 / 004771 ; and W02004 / 056875, which are hereby incorporated by reference in their entirety.

[0255] Specific anti-PDl antibodies that can be used according to some embodiments of the present invention include, but are not limited to:

[0256] Nivolumab (also known as MDX1106, BMS-936558, ONO-4538), marketed by BMY as Opdivo, a fully human IgG4 antibody with the structure described in WHO Drug Information, Vol. 27, No. 1, pages 68-69 (2013) that binds to and blocks the activation of PD-1 by its ligands PD-L1 and PD-L2;

[0257] Pembrolizumab (also known as MK-3475, Keytruda, SCH 900475, produced by Merck), a humanized monoclonal IgG4 antibody with the structure described in WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013) that binds to and blocks the activation of PD1 by its ligands;

[0258] Pidilizumab (also known as CT-011, hBAT, hBAT-1, produced by CureTech), a humanized monoclonal IgGl antibody that binds PD-1;

[0259] AMP-514 (also known as MEDI-0680, produced by AZY and Medlmmune), a humanized monoclonal IgG4 antibody that binds PD-1.

[0260] Humanized antibodies h409Al 1, h409A16 and h409A17, which are described in PCT Patent Application No. WO2008 / 156712.

[0261] According to a specific embodiment, the antibody is Zimberelimab® (Gloria Biosciences,

[0262] According to a specific embodiment, the antibody competes with any of the above- mentioned antibodies for binding to PD1.

[0263] According to a specific embodiment, the antibody competes for binding with and / or binds to the same epitope on PD1 as the above-mentioned antibodies.

[0264] According to another specific embodiment, the antibody has at least about 90 % variable region amino acid sequence identity with the above-mentioned antibodies. According to a specific embodiment, the PD1 antagonist is an anti-PDL-1 antibody, as further described hereinbelow.

[0265] According to a specific embodiment, the PD1 antagonist is an anti-PDL-2 antibody.

[0266] Anti-PDL-2 antibodies suitable for use in the invention can be generated using methods well known in the art especially in light of the detailed description hereinabove. Alternatively, art recognized anti-PDL-2 antibodies can be used. Examples of anti-PDl antibodies are disclosed for example in International Application Publication Nos. W003 / 042402 and W002 / 00730, which are hereby incorporated by reference in their entirety.

[0267] Other PD1 antagonists include an immunoadhesin that specifically binds to PD-1 or any one of its ligands, such as a fusion protein containing the extracellular or PD1 binding portion of PD-L1 or PD-L2 fused to a constant region such as an Fc region of an immunoglobulin molecule. Examples of immunoadhesin molecules that specifically bind to PD1 are described in International Patent Application Publication Nos. W02010 / 027827 and WO2011 / 066342, which are hereby incorporated by reference in their entirety.

[0268] A specific fusion protein that can be used according to some embodiments of the present invention is AMP-224 (also known as B7-DCIg, produced by AZY and GSK), an engineered recombinant fusion protein comprised of human PD-L2 and the Fc domain of human IgGl.

[0269] Other PD1 antagonists that can be used according to some embodiments of the present invention including nucleotides, expression vectors, small molecules, peptides, fusion proteins and fragments targeting PD1, PDL-1 or PDL-2, non-functional PD1, soluble PD1 or fragments thereof that bind to PD1 ligands and prevent binding to the endogenous PD1 receptor, are disclosed for examples in U.S. Patent Nos. 8,609,089 and 6,808,710, U.S. Patent Application Publication Nos. 20140227262; 20100151492; 20040137577; 20030232323; 20030044768; 20030039653; 20020164600; 20020110836; 20020107363; 20020106730; 20090305950 and 20140271677, International Patent Application Publication Nos. W003042402; W02010036959; WO201 1066342; WO2011082400; WO2011161699; WO2014012479; WO2011109789; and WO2013132317, which are hereby incorporated by reference in their entirety.

[0270] According to specific embodiments the CXCR4 antagonistic peptide of the invention or the pharmaceutical composition comprising same is administered in a dose ranging between 0.05 mg / kg-0.75 mg / kg, 0.5 mg / kg-0.75 mg / kg, 0.1 mg / kg-0.75 mg / kg, 0.2 mg / kg-0.75 mg / kg, 0.1 mg / kg-0.5 mg / kg, 0.1 mg / kg-0.35 mg / kg, 0.1 mg / kg-0.4 mg / kg, 0.2 mg / kg-0.65 mg / kg, 0.2 mg / kg- 0.6 mg / kg, 0.2 mg / kg-0.4 mg / kg of body weight. According to a specific embodiment, the range is 0.25-0.5mg / kg. According to a specific embodiment, the concentration of the peptide is 0.1-3 mg / ml, 0.1- 1 mg / ml, 0.2-1 mg / ml, 0.3-2.5 mg / ml (e.g., 0.3 mg / ml to 0.7mg / mL or 0.8-1.2 mg / ml e.g., Img / mL).

[0271] In the unit dosage form or methods, as described herein, the amount of the peptide is 5-50 mg, 5-45 mg, 5-40 mg, 5-35 mg, 10-50 mg, 10-40 mg, 10-30 mg, 10-20 mg, e.g., 15-35 mg. As mentioned the formulations described herein are for intravenous administration such as by way of infusion.

[0272] According to a specific embodiment, the infusion is over a time period of 0.5-2 hours (e.g., 20 min to 45 min).

[0273] According to a specific embodiment, the amount of BL-8040 in the method or the dosage unit is 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg.

[0274] According to a specific embodiment, the concentration of BL-8040 in the dosage unit or in the method or use is ranging between 0.1 mg / ml-3mg / ml.

[0275] According to a specific embodiment, the amount of BL-8040 administered to the subject is 0.05, O.lmg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg or any number in-between the range provided herein and up to the dose of 0.75 mg / kg.

[0276] As mentioned, treatment may be augmented by the use of an additional agent (e.g., chemotherapy and / or checkpoint modulator).

[0277] Examples of chemotherapy, such as for the treatment of pancreatic cancer (e.g., smetastatic) include but are not limited to, gemtabicine, FOLFIRINOX, erlotinib, 5-fluorouracil, paclitaxel, nab-paclitaxel, docetaxel, capecitabine, oxaliplatin cisplatin, FOLFOXIRI, abraxane, an anti-CD40 antibody, oregovomab, Nelfinavir, cetuximab, tegafur, leucovorin, irinotecan and combinations thereof.

[0278] According to a specific embodiment the chemotherapy is irinotecan, a topoiseomarase inhibitor.

[0279] Irinotecan is converted by esterase enzymes into the more active metabolite, SN-38. The chemical name of irinotecan is (S)-4,l l-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxolH- pyrano [4', 4'- : 6,7] -indolizino [ 1 ,2-b] quinolin- 9-yl- [ 1 ,4'bipiperidine] - 1 '-carboxylate. Irinotecan hydrochloride trihydrate is also referred to by the name CPT-11 and by the trade name CAMPTOSAR®.

[0280] The topoisomerase inhibitor can be camptothecin conjugated to a biocompatible polymer such as a cyclodextrin or cyclodextrin analog (e.g., sulfonated cyclodextrins). For example, the topoisomerase inhibitor can be a cyclodextrin-containing polymer chemically bound to a camptothecin, irinotecan, SN-38 or other topoisomerase 1 inhibitor compound. A cyclodextrin- camptothecin conjugated topoisomerase 1 inhibitor can be administered at a pharmaceutically acceptable dose including 6, 12, or 18 mg / m2 weekly administration, or 12, 15 or 18 mg / m2 biweekly administration. Examples of camptothecin-cyclodextrin conjugate topoisomerase 1 inhibitors (e.g., the cyclodextrin-containing polymer conjugate with camptothecin designated "CRLX101"), and related intermediates for preparing the same, are disclosed, for example, in Greenwald et al., Bioorg. Med. Chem., 1998, 6, 551-562, as well as United States Patent Application 2010 / 0247668, United States Patent Application 2011 / 0160159 and United States Patent Application 2011 / 0189092.

[0281] The topoisomerase inhibitor can also be a liposomal formulation of a topoisomerase inhibitor such as irinotecan, camptothecin or topotecan. Liposomal irinotecan (e.g., MM-398, also called "nal-IRI") is a highly stabilized liposomal formulation of irinotecan that provides for sustained exposure of irinotecan, and the active metabolite SN-38 in the tumor to a higher proportion of cells during the more sensitive S-phase of the cell cycle. MM-398 is a liposomal irinotecan that has shown promising preclinical and clinical activity in a range of cancer types, and was recently approved in the United States in combination with 5-FU / LV for patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy. Compared with free irinotecan, nal-IRI has an extended PK profile with prolonged local tumor exposure of MM-398 and SN-38. Since SN-38 is cleared more quickly from normal tissues than from tumor, it is hypothesized that delayed dosing of veliparib relative to MM-398 will allow for the expected window of maximum irinotecan-induced toxicity to pass in the absence of concurrent veliparib toxicity. However, the tumor levels of SN-38 are predicted to be sustained upon subsequent veliparib dosing, therefore maintaining the ability of both drugs to act on tumor tissue simultaneously and maintain synergy.

[0282] One suitable liposomal Topi inhibitor formulation is liposomal irinotecan available under the brand name ONIVYDE®. (irinotecan liposome injection) (Merrimack Pharmaceuticals, Inc. Cambridge, Mass.), previously designated "MM-398" prior to FDA approval, and liposomal irinotecan products that are bioequivalent to ONIVYDE. The ONIVYDE / MM-398 (irinotecan liposome injection) includes irinotecan as an irinotecan sucrosofate salt encapsulated in liposomes for intravenous use. The drug product liposome is a small unilamellar lipid bilayer vesicle, approximately 110 nm in diameter, which encapsulates an aqueous space which contains irinotecan in a gelated or precipitated state, as the sucrosofate salt. The liposome carriers are composed of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 6.81 mg / mL; cholesterol, 2.22 mg / mL; and methoxy-terminated polyethylene glycol (MW 2000)- distearoylphosphatidylethanolamine (MPEG-2000-DSPE), 0.12 mg / mL. Each mL also contains 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES) as a buffer, 4.05 mg / mL; sodium chloride as isotonicity reagent, 8.42 mg / mL, ONIVYDE / MM-398 is believed to include about 80,000 molecules of irinotecan in a gelated or precipitated state as a sucrosofate salt, encapsulated in a liposome of about 100 nm in diameter.

[0283] Gemcitabine (e.g., Gemzar™) or Gemcitabine-based chemotherapy e.g., combined with other drugs such as albumin-bound paclitaxel (Abraxane), erlotinib (Tarceva), or capecitabine (Xeloda) or combined with radiation (this is called chemoradiation).

[0284] As used herein “leucovorin” refers to folinic acid that is typically administered in combination with Onivyde(R) and 5-FU.

[0285] 5-FU is a thymidylate synthase (TS) inhibitor. Interrupting the action of this enzyme blocks synthesis of the pyrimidine thymidine, which is a nucleoside required for DNA replication. Thymidylate synthase methylates deoxyuridine monophosphate (dUMP) to form thymidine monophosphate (dTMP). Administration of 5-FU causes a scarcity in dTMP, so rapidly dividing cancerous cells undergo cell death via thymineless death. Calcium folinate provides an exogenous source of reduced folinates and hence stabilises the 5-FU-TS complex, hence enhancing 5-FU's cytotoxicity. 5-FU is sold under the brand name Adrucil, among others.

[0286] According to a specific embodiment, the chemotherapy comprises a plurality of chemotherapeutic agents e.g., at least 2, or at least 3, e.g., 2, 3, 4, 5 chemotherapeutic agents).

[0287] According to a specific embodiment, the chemotherapy comprises irinotecan (e.g., liposome-encapsulated e.g., Onivyde(R)), 5-FU and Leucovorin.

[0288] According to a specific embodiment treatment does not include oxaliplatin.

[0289] According to a specific embodiment, the chemotherapy comprises Gemcitabine and Nab- Paclitaxel.

[0290] According to a specific embodiment, the treatment comprises a monotherapy period with the peptide followed by a combination therapy with the peptide, the anti PD-1 and the chemotherapy.

[0291] As used herein “monotherapy” refers to a use of a single drug to treat the cancer. The term does not exclude using other medications for treating symptoms that are not associated with the disease per se. For instance, the use of analgesics and / or anti-histamine, and / or anti-nausea medications is still contemplated during the period of monotherapy. The term excludes the use of anti-cancer drugs (which are defined as such) during the time of monotherapy.

[0292] According to a specific embodiment, the time period of monotherapy is up to 10 days.

[0293] According to a specific embodiment, the time period of monotherapy is up to 9 days.

[0294] According to a specific embodiment, the time period of monotherapy is up to 8 days. According to a specific embodiment, the time period of monotherapy is up to 7 days.

[0295] According to a specific embodiment, the time period of monotherapy is 5 days (e.g., Daysl, 3 and 5).

[0296] As used herein “combination therapy” refers to the use of a plurality of anti-cancer drugs (peptide, chemotherapy, checkpoint modulator e.g., anti PD-1) such as in an embodiment of the invention.

[0297] According to a specific embodiment, the combination therapy with the chemotherapy and the checkpoint modulator is initiated on Day 6-10.

[0298] According to a specific embodiment, the combination therapy with the chemotherapy and the checkpoint modulator is initiated on Day 8.

[0299] According to a specific embodiment, the combination therapy is once a week with the chemotherapy and once every 2 weeks with the checkpoint modulator.

[0300] According to a specific embodiment, the peptide is administered at the monotherapy period, followed by administration of the antibody and chemotherapy.

[0301] According to a specific embodiment the monotherapy is performed for 1 week.

[0302] According to a specific embodiment the monotherapy is performed on Days 1-5, daily. According to a specific embodiment, the monotherapy is performed on days 1, 3, 5.

[0303] According to a specific embodiment, the combination therapy continues until progression of the disease.

[0304] Imagining, pathology and / or molecular markers are used for assessment of response. For example, CT or MRI may be used. The same imaging method should be used throughout the treatment for each subject. Notwithstanding, clinical assessment for the patients well being can be used too.

[0305] Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

[0306] Examples of excipients, carriers or diluents for intravenous infustions are well known in the art. For example, with sterile saline (e.g., 0.9% sodium hydroxide) or with sterile solution of glucose e.g., 5% glucose.

[0307] Antioxidant excipients can be used such as citric acid, ascorbic acid, a, Vitamin E, Calcium Stearate, Sodium salt of ethylenediaminetetraacetic acid, Butylated Hydroxytoluene, Sodium bisulfite and other sulfurous acid salts. Preservatives excipients can be used such as phenol, benzyl alcohol, phenoxyethanol. Tonicity agents can be added such as mannitol, glucose, dextrose, glycerin, potassium chloride and sodium chloride. pH adjustments can be done as needed, such as with HC1, acetic acid or NaOH.

[0308] As used herein the term “about” refers to ± 10 %.

[0309] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0310] The term “consisting of’ means “including and limited to”.

[0311] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0312] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0313] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0314] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0315] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0316] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0317] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0318] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0319] It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A unit dosage form comprising a peptide as set forth in SEQ ID NO: 1 (BL-8040) and a carrier, wherein an amount of said peptide is 5-50 mg, at a concentration of 0.1-3 mg / ml formulated for intravenous administration.

2. A method of treating a CXCR4-related condition in a subject in need thereof, the method comprising intravenously administering to the subject a therapeutically effective amount of SEQ ID NO: 1 (BL-8040) wherein said therapeutically effective amount of BL-8040 is 0.05 mg / kg-0.75 mg / kg at a concentration of 0.1-3 mg / ml, thereby treating the CXCR4-related condition.

3. The unit dosage form of claim 1 for use in treating a CXCR4-related condition.

4. The unit dosage form of any one of claims 1 and 3, wherein said amount comprises 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg.

5. The unit dosage form of any one of claims 1 and 3, wherein said amount comprises 5-50 mg (e.g., 15-35 mg).

6. The method or unit dosage form of any one of claims 1-5, wherein said intravenous administration is by way of infusion.

7. The method or unit dosage form of claim 6, wherein said infusion is over a time period of 0.5-2 hours.

8. The method of any one of claims 2, 6-7, wherein said therapeutically effective amount comprises 0.05 mg / kg, O.lmg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.30 mg / kg, 0.35 mg / kg, 0.40 mg / kg, 0.45 mg / kg, 0.50 mg / kg, 0.55 mg / kg, 0.60 mg / kg, 0.65 mg / kg or 0.70 mg / kg (e.g., 0.25-0.5mg / kg).

9. The method or unit dosage form of any one of claims 2-8, wherein said CXCR4- related condition is selected from the group consisting of cancer, thrombocytopenia, risk of bone marrow suppression, and HIV infection.

10. The method or unit dosage form of claim 9, wherein said cancer is a solid tumor.

11. The method or unit dosage form of claim 9, wherein said cancer is acute myeloid leukemia (AML).

12. The method or unit dosage form of claim 9, wherein said condition is cancer and further comprising at least one of chemotherapy and an immune modulator.

13. The method or unit dosage form of claim 12, wherein said immune modulator is a checkpoint inhibitor.

14. The method or unit dosage form of claim 13, wherein said checkpoint is PD-1 or PD-L1.

15. The method or unit dosage form of any one of claims 9-14, wherein said cancer is metastatic cancer.

16. The method or unit dosage form of any one of claims 9-15, wherein said cancer is pancreatic cancer optionally pancreatic adenocarcinoma.

17. The method or unit dosage form of any one of claims 9-15, wherein said treating or treatment is first-line treatment.

18. The method or unit dosage form of any one of claims 9-15, wherein said cancer is a chemotherapy-resistant cancer.

19. The method or unit dosage form of any one of claims 1-18, wherein said peptide is in a composition-of-matter comprising said BL-8040 (SEQ ID NO: 1) and at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88, wherein said relative retention time is determined using a first mobile phase which is an aqueous solution of 0.017 M perchlorate at a pH in a range of from about 3.0 to about 3.3, a second mobile phase which is acetonitrile, a gradient whereby a concentration of said second mobile phase increases by about 10 % in about 50 minutes from an initial concentration of about 20 %, a C18 reverse phase column, an injection volume in a range of from about 5 to about 20 pl, and a flowrate of about 1 ml per minute, at a temperature of about 40 °C, and wherein a relative retention time of said BL-8040 (SEQ ID NO: 1) is defined as 1.

20. The method or unit dosage form of claim 19, wherein said at least one compound represents at least 20 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition- of-matter as determined according to absorption at a wavelength at about 226 nm.

21. The method or unit dosage form of claim 19, wherein said at least one compound represents at least 50 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition- of-matter as determined according to absorption at a wavelength at about 226 nm.

22. The method or unit dosage form of any one of claims 19-21, wherein said at least one compound comprises a compound having the same molecular weight as BL-8040 (SEQ ID NO: 1) and / or a compound having a molecular weight which is higher than the molecular weight of BL-8040 (SEQ ID NO: 1) by about 80 Da.

23. The method or unit dosage form of any one of claims 19-22, wherein a total concentration ratio of said at least one compound to said BL-8040 (SEQ ID NO: 1) is at least about 0.01 %.

24. The method or unit dosage form of any one of claims 19-23, wherein a total concentration ratio of said at least one compound to said BL-8040 (SEQ ID NO: 1) is no more than about 1 %.

25. The method or unit dosage form of any one of claims 19-24, wherein a total concentration ratio of said at least one compound to said BL-8040 (SEQ ID NO: 1) is in a range of from about 0.075 % to about 0.225 %.

26. The method or unit dosage form of any one of claims 19-25, wherein said composition-of-matter further comprises at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73.

27. The method or unit dosage form of claim 26, wherein said at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 represents at least 10 %of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter as determined according to absorption at a wavelength at about 226 nm.

28. The method or unit dosage form of claim 26 or 27, wherein a total concentration ratio of said at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 to said BL-8040 (SEQ ID NO: 1) is at least about 0.01 %.

29. The method or unit dosage form of claim 28, wherein said total concentration ratio of said at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 to said BL-8040 (SEQ ID NO: 1) is in a range of from about 0.03 % to about 0.15 %.

30. The method or unit dosage form of any one of claims 26-29, wherein a total concentration of said at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88 and said at least one compound characterized by a relative retention time in a range of from 0.71 to 0.73 represents at least 50 % of all compounds other than BL-8040 (SEQ ID NO: 1) in the composition-of-matter as determined according to absorption at a wavelength at about 226 nm.

31. The method or unit dosage form of any one of claims 19 to 30, wherein a concentration of cyclic deamidated BL-8040 (SEQ ID NO: 2) in the composition-of-matter is no more than 50 % of a total concentration of said at least one compound characterized by a relative retention time in a range of from 0.86 to 0.88.

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