Patent Application
20230414565
The invention relates to the mobilization of hematopoietic stem and progenitor cells from a donor, such as a human donor, and to the treatment of patients suffering from various pathologies, such as hematological malignancies, among others.
Despite advances in the medicinal arts, there remains a demand for treating pathologies of the hematopoietic system, such as diseases of a particular blood cell, metabolic disorders, cancers, and autoimmune conditions, among others. While hematopoietic stem cells have significant therapeutic potential, a limitation that has hindered their use in the clinic has been the difficulty associated with releasing hematopoietic stem cells from the bone marrow into the peripheral blood of a donor, from which the hematopoietic stem cells may be isolated for infusion into a patient.
A further limitation is that up to 80% of mobilized peripheral blood (mPB) allogeneic recipients will experience graft-versus-host disease (GVHD). Despite the higher levels of CD3+ T cells in mPB grafts compared to bone marrow transplants, the level of acute GVHD observed following transplant of HLA-matched mPB is comparable to HLA-matched bone marrow. One explanation is that G-CSF mobilized grafts contain myeloid-derived suppressor cells (MDSCs) possessing potent immunosuppressive properties capable of inhibiting T cell proliferation in vitro. The percentage of MDSCs is variable in grafts mobilized with G-CSF, and clinical data suggest that patients transplanted with mPB grafts that contain higher numbers of MDSCs may have better outcomes including lower rates of acute GVHD (Vendramin et al., (2014) BBMT 20(12):2049-2055).
Accordingly, there is currently a need for compositions and methods for promoting the mobilization of hematopoietic stem and progenitor cells, and particularly for methods of identifying populations of mobilized cells that are suitable for therapeutic use. There is also a need for compositions and methods for promoting the mobilization of hematopoietic stem and progenitor cells that consistently produce higher numbers of MDSCs than do prior art methods.
Disclosed herein is a method of mobilizing a population of hematopoietic stem or progenitor cells from the bone marrow of a mammalian donor into peripheral blood, the method comprising administering to the donor a CXCR2 agonist selected from the group consisting of Gro-β, Gro-β T, and variants thereof at a dose of from about 0.001 mg/kg to about 0.1 mg/kg. In various embodiments, the dose is from greater than about 0.015 mg/kg to less than about 0.05 mg/kg. In various embodiments, the dose is about 0.015 mg/kg.
In various embodiments, the CXCR2 agonist comprises Gro-β T. In various embodiments, the CXCR2 agonist is administered intravenously. In various embodiments, method further comprises administering to the donor a CXCR4 antagonist. In various embodiments, the CXCR4 antagonist is plerixafor. In various embodiments, the plerixafor is administered to the donor at a dose of about 240 μg/kg. In various embodiments, the CXCR2 agonist is administered subcutaneously. In various embodiments, the CXCR2 agonist is administered simultaneously with the CXCR4 antagonist. In various embodiments, administration of the CXCR2 agonist and the CXCR4 antagonist is staggered by about 30 minutes to 4 hours. In various embodiments, the CXCR2 agonist and the CXCR4 antagonist are each administered in a single dose. In various embodiments, the CXCR2 agonist and the CXCR4 antagonist are each administered on two consecutive days. In various embodiments, the CXCR2 agonist and the CXCR4 antagonist are each administered once per day on two consecutive days.
Additionally disclosed herein is a method of obtaining hematopoietic stem or progenitor cells, the method comprising obtaining peripheral blood from a donor, wherein the hematopoietic stem or progenitor cells were mobilized according to methods described above. In various embodiments, the peripheral blood is obtained between about 2 and about 10 hours after administration of the CXCR2 agonist. In various embodiments, the peripheral blood is obtained on one or two consecutive days after administration of the CXCR2 agonist.
Additionally disclosed herein is a method of performing apheresis on the peripheral blood of a donor to produce an apheresis product, wherein the donor has been treated according to the methods described above. In various embodiments, about 10 L to about 30 L of peripheral blood is processed. In various embodiments, apheresis occurs over a period of time of from about 3 hours to about 5 hours. In various embodiments, the apheresis product has a volume of about 20 to about 400 mL. In various embodiments, CD34+ cells are present in the apheresis product in an amount of from about 1×106 cells/kg to about 6×106 cells/kg. In various embodiments, CD34+ cells are present in the apheresis product in an amount of at least 2×106 cells/kg. In various embodiments, CD34+ cells are present in the apheresis product in an amount of at least 4×106 cells/kg. In various embodiments, CD34+ cells are present in the apheresis product in an amount of from about 100×106 cells to about 600×106 cells. In various embodiments, the CD34+ cells are viable CD34+ cells. In various embodiments, CD34+CD90+CD45RA− cells are present in the apheresis product in an amount of from about 0.1×106 cells/kg to about 5×106 cells/kg. In various embodiments, the CD34+CD90+CD45RA− cells are viable CD34+CD90+CD45RA− cells. Additionally disclosed herein is a population of hematopoietic stem or progenitor cells, wherein the population of hematopoietic stem or progenitor cells is produced using the methods described above.
Additionally disclosed herein is a population of hematopoietic stem or progenitor cells, the population comprising between about 15 and 30 CD34+CD90+CD45RA− cells per μL. In various embodiments, the population comprises between about 3 and about 15 CD34+CD90+CD45RA− cells per μL. In various embodiments, the population comprises between about 10 and about 15 CD34+CD90+CD45RA− cells per μL. In various embodiments, the population further comprises DMSO or citrate.
Additionally disclosed herein is an apheresis product isolated from a donor comprising CD34+CD90+CD45RA− cells in an amount of from about 0.1×106 cells/kg to about 5×106 cells/kg or at a frequency of about 15 to about 75% of CD34+ cells present in the apheresis product. In various embodiments, the CD34+CD90+CD45RA− cells are viable CD34+CD90+CD45RA− cells. In various embodiments, CD34+ cells are present in an amount of from about 1×106 cells/kg to about 6×106 cells/kg. In various embodiments, CD34+ cells are present in the apheresis product in an amount of from about 100×106 cells to about 600×106 cells. In various embodiments, the CD34+ cells are viable CD34+ cells. In various embodiments, the concentration of white blood cells is higher in the apheresis product than in the peripheral blood of the donor.
In various embodiments, the apheresis product further comprises an anticoagulant. In various embodiments, the anticoagulant is citrate in an amount above physiological levels. In various embodiments, the anticoagulant is heparin.
In various embodiments, the volume of the product is from about 20 to about 400 mL. In various embodiments, the apheresis product prevents, reduces the risk of developing, or reduces the severity of graft versus host disease (GVHD) in a patient in need thereof as compared to an apheresis product obtained from a donor administered G-CSF.
Additionally disclosed herein is a method of treating a stem cell disorder, the method comprising administering any of the population of hemopoietic stem or progenitor cells or the apheresis product described above. Additionally disclosed herein is a method of treating a hematological malignancy, the method comprising administering any of the population of hemopoietic stem or progenitor cells or the apheresis product described above to a patient with the hematological malignancy.
In various embodiments, the hematological malignancy is acute myelogenous leukemia. In various embodiments, the hematological malignancy is acute lymphoblastic leukemia. In various embodiments, the patient is in 1st remission or beyond. In various embodiments, the patient has less than or equal to 5% marrow blasts. In various embodiments, the hematological malignancy is myelodysplasia. In various embodiments, the patient has less than or equal to 10% marrow blasts. In various embodiments, the patient has less than 1% circulating blasts. In various embodiments, the patient has less than 0.1% circulating blasts.
Additionally disclosed herein is a method of preventing, reducing the risk of developing, or reducing the severity of graft versus host disease (GVHD) in a patient in need thereof, the method comprising administering the population of hemopoietic stem or progenitor cells or the apheresis product described above to the patient. In various embodiments, the population of hemopoietic stem or progenitor cells has an increased engraftment rate as compared to hemopoietic stem or progenitor cells mobilized using G-CSF.
In various embodiments, patients receiving the population of hemopoietic stem or progenitor cells or the apheresis product experience increased neutrophil and/or platelet recovery in comparison to patients receiving hematopoietic stem cells or progenitor cells that were mobilized using G-CSF. In various embodiments, a reduced proportion of patients receiving the population of hemopoietic stem or progenitor cells or the apheresis product experiences primary or secondary graft failure in comparison to patients receiving hematopoietic stem cells or progenitor cells that were mobilized using G-CSF. In various embodiments, patients receiving the population of hemopoietic stem or progenitor cells or the apheresis product experience reduced incidence of treatment-related mortality and/or disease relapse and progression in comparison to patients receiving hematopoietic stem cells or progenitor cells that were mobilized using G-CSF. In various embodiments, patients receiving the population of hemopoietic stem or progenitor cells or the apheresis product experience increased progression-free and/or overall survival in comparison to patients receiving hematopoietic stem cells or progenitor cells that were mobilized using G-CSF. In various embodiments, patients receiving the population of hemopoietic stem or progenitor cells or the apheresis product experience reduced adverse events related to allograft in comparison to patients receiving hematopoietic stem cells or progenitor cells that were mobilized using G-CSF.
The present invention provides compositions and methods for mobilizing hematopoietic stem and progenitor cells in a subject. For example, the subject may be a hematopoietic stem and progenitor cell donor (i.e., a donor), such as a mammalian donor (e.g., a human donor). The compositions and methods described herein can additionally be used for the treatment of one or more stem cell disorders in a patient, such as a human patient. Using the compositions and methods described herein, a C-X-C chemokine receptor type 2 (CXCR2) agonist, such as Gro-β or a variant thereof, such as a truncated form of Gro-β (e.g., Gro-β T), as described herein, optionally in combination with a C-X-C chemokine receptor type 4 (CXCR4) antagonist, such as 1,1′-[1,4-phenylenebis(methylene)]-bis-1,4,8,11-tetra-azacyclotetradecane or a variant thereof, may be administered to a donor, as described herein, in amounts sufficient to mobilize hematopoietic stem and progenitor cells. The compositions and methods described herein thus enable the selective mobilization of hematopoietic stem and progenitor cells in a donor, which may then be isolated from a donor for therapeutic use.
The invention is based, in part, on the discovery that administration of a surprisingly low dose of a CXCR2 agonist, such as Gro-β, Gro-β T, or a variant thereof, optionally in combination with a CXCR4 antagonist, such as plerixafor or a pharmaceutically acceptable salt thereof, at particular doses can provide the important clinical benefit of mobilizing hematopoietic stem and progenitor cells. In addition, CD34+CD90+CD45− cells, a population indicative of a stem cell phenotype associated with long term engraftment, are effectively mobilized by the methods of administration as described herein. Thus, the populations of mobilized hematopoietic stem and progenitor cells produced using the compositions and methods described herein are particularly suitable for hematopoietic stem cell transplantation therapy.
Following mobilization, the hematopoietic stem or progenitor cells may be isolated for ex vivo expansion and/or for therapeutic use. In some embodiments, upon collection of the mobilized hematopoietic stem and/or progenitor cells, the withdrawn cells may be infused into a patient, such as the donor or another subject (e.g., a subject that is HLA-matched to the donor) for the treatment of one or more pathologies of the hematopoietic system. Additionally or alternatively, the mobilized cells may be withdrawn and then expanded ex vivo, such as by contacting the cells with an aryl hydrocarbon receptor antagonist, so as to produce a population of hematopoietic stem cells having a sufficient quantity of cells for transplantation.
As described herein, hematopoietic stem cells are capable of differentiating into a multitude of cell types in the hematopoietic lineage, and can thus be administered to a patient in order to populate or repopulate a cell type that is defective or deficient in the patient. The patient may be one, for example, that is suffering from one or more blood disorders, such as an autoimmune disease, cancer, hemoglobinopathy, or other hematopoietic pathology, and is therefore in need of hematopoietic stem cell transplantation. The invention thus provides methods of treating a variety of hematopoietic conditions, such as sickle cell anemia, thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, adenosine deaminase deficiency-severe combined immunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan anemia and Schwachman-Diamond syndrome, human immunodeficiency virus infection, and acquired immune deficiency syndrome, as well as cancers and autoimmune diseases, among others.
The sections that follow provide a description of CXCR4 antagonists and CXCR2 agonists that can be administered to a donor so as to induce mobilization of a population of hematopoietic stem or progenitor cells from a stem cell niche into peripheral blood, from which the cells may subsequently be isolated and infused into a patient for the treatment, for example, of one or more stem cell disorders, such as a cancer, autoimmune disease, of metabolic disorder described herein. The following sections additionally describe methods of determining whether populations of cells mobilized with a CXCR2 agonist and/or a CXCR4 antagonist are suitable for release for ex vivo expansion and/or for therapeutic applications.
As used herein, the term “about” refers to a value that is within 10% above or below the value being described. For example, the term “about 5 nM” indicates a range of from 4.5 nM to 5.5 nM.
As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes polyclonal, monoclonal, genetically engineered, and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen binding fragments of antibodies, including, for example, Fab′, F(ab′)2, Fab, Fv, rIgG, and scFv fragments. Unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules, as well as antibody fragments (including, for example, Fab and F(ab′)2 fragments) that are capable of specifically binding to a target protein. As used herein, the Fab and F(ab′)2 fragments refer to antibody fragments that lack the Fc fragment of an intact antibody. Examples of these antibody fragments are described herein.
The term “antigen-binding fragment,” as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The antibody fragments can be, for example, a Fab, F(ab′)2, scFv, diabody, a triabody, an affibody, a nanobody, i-body, an aptamer, or a domain antibody. Examples of binding fragments encompassed of the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment that consists of a VH domain (see, e.g., Ward et al. (1989) Nature 341:544-546); (vii) a dAb which consists of a VH or a VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more (e.g., two, three, four, five, or six) isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in certain cases, by chemical peptide synthesis procedures known in the art.
As used herein, the term “bispecific antibody” refers to, for example, a monoclonal, often a human or humanized antibody that is capable of binding at least two different antigens or two different epitopes on the same antigen.
As used herein, the term “complementarity determining region” (CDR) refers to a hypervariable region found both in the light chain and the heavy chain variable domains of an antibody. The more highly conserved portions of variable domains are referred to as framework regions (FRs). The amino acid positions that delineate a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art. Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions. The antibodies described herein may contain modifications in these hybrid hypervariable positions. The variable domains of native heavy and light chains each contain four framework regions that primarily adopt a β-sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the framework regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the other antibody chains, contribute to the formation of the target binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, M D., 1987). As used herein, numbering of immunoglobulin amino acid residues is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated.
As used herein, the terms “conservative mutation,” “conservative substitution,” or “conservative amino acid substitution” refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric volume. These properties are summarized for each of the twenty naturally-occurring amino acids in TABLE 1 below.
†based on volume in A3: 50-100 is small, 100-150 is intermediate, 150-200 is large, and >200 is bulky
From this table it is appreciated that the conservative amino acid families include, e.g., (i) G, A, V, L, I, P, and M; (ii) D and E; (iii) C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W. A conservative mutation or substitution is therefore one that substitutes one amino acid for a member of the same amino acid family (e.g., a substitution of Ser for Thr or Lys for Arg).
As used herein, “CRU (competitive repopulating unit)” refers to a unit of measure of long-term engrafting stem cells, which can be detected after in-vivo transplantation.
As used herein, the term “donor” refers to a subject, such as a mammalian subject (e.g., a human subject) from which one or more cells are isolated prior to administration of the cells, or progeny thereof, into a recipient, or, with respect to neutropenia, the term “donor” is used to mean “subject” or “patient,” because in the context of treating neutropenia, cells are not isolated from the subject or patient and donated to a recipient. The one or more cells may be, for example, a population of hematopoietic stem or progenitor cells.
As used herein, the term “diabody” refers to a bivalent antibody containing two polypeptide chains, in which each polypeptide chain includes VH and VL domains joined by a linker that is too short (e.g., a linker composed of five amino acids) to allow for intramolecular association of VH and VL domains on the same peptide chain. This configuration forces each domain to pair with a complementary domain on another polypeptide chain so as to form a homodimeric structure. Accordingly, the term “triabody” refers to trivalent antibodies containing three peptide chains, each of which contains one VH domain and one VL domain joined by a linker that is exceedingly short (e.g., a linker composed of 1-2 amino acids) to permit intramolecular association of VH and VL domains within the same peptide chain. In order to fold into their native structures, peptides configured in this way typically trimerize so as to position the VH and VL domains of neighboring peptide chains spatially proximal to one another (see, for example, Holliger et al. (1993) Proc. Nat. Acad. Sci. USA 90:6444-48).
As used herein, the term “disrupt” with respect to a gene refers to preventing the formation of a functional gene product. A gene product is functional only if it fulfills its normal (wild-type) functions. Disruption of the gene prevents expression of a functional factor encoded by the gene and comprises an insertion, deletion, or substitution of one or more bases in a sequence encoded by the gene and/or a promoter and/or an operator that is necessary for expression of the gene in the animal. The disrupted gene may be disrupted by, e.g., removal of at least a portion of the gene from a genome of the animal, alteration of the gene to prevent expression of a functional factor encoded by the gene, an interfering RNA, or expression of a dominant negative factor by an exogenous gene. Materials and methods of genetically modifying hematopoietic stem/progenitor cells are detailed in U.S. Pat. No. 8,518,701; U.S. 2010/0251395; and U.S. 2012/0222143, the disclosures of each of which are incorporated herein by reference in their entirety (in case of conflict, the instant specification is controlling).
Various techniques known in the art can be used to inactivate genes to make knock-out animals and/or to introduce nucleic acid constructs into animals to produce founder animals and to make animal lines, in which the knockout or nucleic acid construct is integrated into the genome. Such techniques include, without limitation, pronuclear microinjection (U.S. Pat. No. 4,873,191), retrovirus mediated gene transfer into germ lines (Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA, 82:6148-6152), gene targeting into embryonic stem cells (Thompson et al. (1989) Cell, 56:313-321), electroporation of embryos (Lo (1983)Mol. Cell. Biol., 3:1803-1814), sperm-mediated gene transfer (Lavitrano et al. (2002) Proc. Natl. Acad. Sci. USA, 99:14230-14235; Lavitrano et al. (2006) Reprod. Fert. Develop., 18:19-23), and in vitro transformation of somatic cells, such as cumulus or mammary cells, or adult, fetal, or embryonic stem cells, followed by nuclear transplantation (Wilmut et al. (1997) Nature, 385:810-813; and Wakayama et al. (1998) Nature, 394:369-374). Pronuclear microinjection, sperm mediated gene transfer, and somatic cell nuclear transfer are particularly useful techniques. An animal that is genomically modified is an animal wherein all of its cells have the genetic modification, including its germ line cells. When methods are used that produce an animal that is mosaic in its genetic modification, the animals may be inbred and progeny that are genomically modified may be selected. Cloning, for example, may be used to make a mosaic animal if its cells are modified at the blastocyst state, or genomic modification can take place when a single-cell is modified. Animals that are modified so they do not sexually mature can be homozygous or heterozygous for the modification, depending on the specific approach that is used. If a particular gene is inactivated by a knockout modification, homozygosity would normally be required. If a particular gene is inactivated by an RNA interference or dominant negative strategy, then heterozygosity is often adequate.
As used herein, a “dual variable domain immunoglobulin” (“DVD-Ig”) refers to an antibody that combines the target-binding variable domains of two monoclonal antibodies via linkers to create a tetravalent, dual-targeting single agent (see, for example, Gu et al. (2012) Meth. Enzymol., 502:25-41).
As used herein, the term “endogenous” describes a substance, such as a molecule, cell, tissue, or organ (e.g., a hematopoietic stem cell or a cell of hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte) that is found naturally in a particular organism, such as a human patient.
As used herein, the term “engraftment potential” is used to refer to the ability of hematopoietic stem and progenitor cells to repopulate a tissue, whether such cells are naturally circulating or are provided by transplantation. The term encompasses all events surrounding or leading up to engraftment, such as tissue homing of cells and colonization of cells within the tissue of interest. The engraftment efficiency or rate of engraftment can be evaluated or quantified using any clinically acceptable parameter as known to those of skill in the art and can include, for example, assessment of competitive repopulating units (CRU); incorporation or expression of a marker in tissue(s) into which stem cells have homed, colonized, or become engrafted; or by evaluation of the progress of a subject through disease progression, survival of hematopoietic stem and progenitor cells, or survival of a recipient. Engraftment can also be determined by measuring white blood cell counts in peripheral blood during a post-transplant period. Engraftment can also be assessed by measuring recovery of marrow cells by donor cells in a bone marrow aspirate sample.
As used herein, the term “exogenous” describes a substance, such as a molecule, cell, tissue, or organ (e.g., a hematopoietic stem cell or a cell of hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte) that is not found naturally in a particular organism, such as a human patient. Exogenous substances include those that are provided from an external source to an organism or to cultured matter extracted therefrom.
As used herein, the term “framework region” or “FW region” includes amino acid residues that are adjacent to the CDRs of an antibody or antigen-binding fragment thereof. FW region residues may be present in, for example, human antibodies, humanized antibodies, monoclonal antibodies, antibody fragments, Fab fragments, single chain antibody fragments, scFv fragments, antibody domains, and bispecific antibodies, among others.
As used herein, the term “hematopoietic progenitor cells” includes pluripotent cells capable of differentiating into several cell types of the hematopoietic system, including, without limitation, granulocytes, monocytes, erythrocytes, megakaryocytes, B-cells and T-cells, among others. Hematopoietic progenitor cells are committed to the hematopoietic cell lineage and generally do not self-renew. Hematopoietic progenitor cells can be identified, for example, by expression patterns of cell surface antigens, and include cells having the following immunophenotype: Lin− KLS+ Flk2− CD34+. Hematopoietic progenitor cells include short-term hematopoietic stem cells, multi-potent progenitor cells, common myeloid progenitor cells, granulocyte-monocyte progenitor cells, and megakaryocyte-erythrocyte progenitor cells. The presence of hematopoietic progenitor cells can be determined functionally, for example, by detecting colony-forming unit cells, e.g., in complete methylcellulose assays, or phenotypically through the detection of cell surface markers using flow cytometry and cell sorting assays described herein and known in the art.
As used herein, the term “hematopoietic stem cells” (“HSCs”) refers to immature blood cells having the capacity to self-renew and to differentiate into mature blood cells containing diverse lineages including but not limited to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B-cells and T-cells). Such cells may include CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells are believed to include a subpopulation of cells with the stem cell properties defined above, whereas in mice, HSCs are CD34−. In addition, HSCs also refer to long term repopulating HSCs (LT-HSC) and short term repopulating HSCs (ST-HSC). LT-HSCs and ST-HSCs are differentiated, based on functional potential and on cell surface marker expression. For example, human HSCs are CD34+, CD38−, CD45RA−, CD90+, CD49F+, and lin− (negative for mature lineage markers including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A). In mice, bone marrow LT-HSCs are CD34−, SCA-1+, C-kit+, CD135−, Slamfl/CD150+, CD48−, and lin− (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra), whereas ST-HSCs are CD34+, SCA-1+, C-kit+, CD135−, Slamfl/CD150+, and lin− (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra). In addition, ST-HSCs are less quiescent and more proliferative than LT-HSCs under homeostatic conditions. However, LT-HSC have greater self-renewal potential (i.e., they survive throughout adulthood, and can be serially transplanted through successive recipients), whereas ST-HSCs have limited self-renewal (i.e., they survive for only a limited period of time, and do not possess serial transplantation potential). Any of these HSCs can be used in the methods described herein. ST-HSCs are particularly useful because they are highly proliferative and thus, can more quickly give rise to differentiated progeny.
As used herein, the term “hematopoietic stem cell functional potential” refers to the functional properties of hematopoietic stem cells which include 1) multi-potency (which refers to the ability to differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B-cells and T-cells), 2) self-renewal (which refers to the ability of hematopoietic stem cells to give rise to daughter cells that have equivalent potential as the mother cell, and further that this ability can repeatedly occur throughout the lifetime of an individual without exhaustion), and 3) the ability of hematopoietic stem cells or progeny thereof to be reintroduced into a transplant recipient whereupon they home to the hematopoietic stem cell niche and re-establish productive and sustained hematopoiesis.
As used herein, the terms “Major histocompatibility complex antigens” (“MHC”, also referred to as “human leukocyte antigens” (“HLA”) in the context of humans) refer to proteins expressed on the cell surface that confer a unique antigenic identity to a cell. MHC/HLA antigens are target molecules that are recognized by T cells and NK cells as being derived from the same source of hematopoietic stem cells as the immune effector cells (“self”) or as being derived from another source of hematopoietic reconstituting cells (“non-self”). Two main classes of HLA antigens are recognized: HLA class I and HLA class II. HLA class I antigens (A, B, and C in humans) render each cell recognizable as “self,” whereas HLA class II antigens (DR, DP, and DQ in humans) are involved in reactions between lymphocytes and antigen presenting cells. Both have been implicated in the rejection of transplanted organs. An important aspect of the HLA gene system is its polymorphism. Each gene, MHC class I (A, B and C) and MHC class II (DP, DQ and DR) exists in different alleles. For example, two unrelated individuals may carry class I HLA-B, genes B5, and Bw41, respectively. Allelic gene products differ in one or more amino acids in the α and/or β domain(s). Large panels of specific antibodies or nucleic acid reagents are used to type HLA haplotypes of individuals, using leukocytes that express class I and class II molecules. The genes commonly used for HLA typing are the six MHC Class I and Class II proteins, two alleles for each of HLA-A; HLA-B and HLA-DR. The HLA genes are clustered in a “super-locus” present on chromosome position 6p21, which encodes the six classical transplantation HLA genes and at least 132 protein coding genes that have important roles in the regulation of the immune system as well as some other fundamental molecular and cellular processes. The complete locus measures roughly 3.6 Mb, with at least 224 gene loci. One effect of this clustering is that “haplotypes”, i.e. the set of alleles present on a single chromosome, which is inherited from one parent, tend to be inherited as a group. The set of alleles inherited from each parent forms a haplotype, in which some alleles tend to be associated together. Identifying a patient's haplotypes can help predict the probability of finding matching donors and assist in developing a search strategy, because some alleles and haplotypes are more common than others and they are distributed at different frequencies in different racial and ethnic groups.
As used herein, the term “HLA-matched” refers to a donor-recipient pair in which none of the HLA antigens are mismatched between the donor and recipient, such as a donor providing a hematopoietic stem cell graft to a recipient in need of hematopoietic stem cell transplant therapy. HLA-matched (i.e., where all of the 6 alleles are matched) donor-recipient pairs have a decreased risk of graft rejection, as endogenous T cells and NK cells are less likely to recognize the incoming graft as foreign, and are thus less likely to mount an immune response against the transplant.
As used herein, the term “HLA-mismatched” refers to a donor-recipient pair in which at least one HLA antigen, in particular with respect to HLA-A, HLA-B and HLA-DR, is mismatched between the donor and recipient, such as a donor providing a hematopoietic stem cell graft to a recipient in need of hematopoietic stem cell transplant therapy. In some embodiments, one haplotype is matched and the other is mismatched. HLA-mismatched donor-recipient pairs may have an increased risk of graft rejection relative to HLA-matched donor-recipient pairs, as endogenous T cells and NK cells are more likely to recognize the incoming graft as foreign in the case of an HLA-mismatched donor-recipient pair, and such T cells and NK cells are thus more likely to mount an immune response against the transplant.
As used herein, the term “human antibody” refers to an antibody in which substantially every part of the protein (for example, all CDRs, framework regions, CL, CH domains (e.g., CH1, CH2, CH3), hinge, and VL and VH domains) is substantially non-immunogenic in humans, with only minor sequence changes or variations. A human antibody can be produced in a human cell (for example, by recombinant expression) or by a non-human animal or a prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (such as heavy chain and/or light chain) genes. When a human antibody is a single chain antibody, it can include a linker peptide that is not found in native human antibodies. For example, an Fv can contain a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. Such linker peptides are considered to be of human origin. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes (see, for example, PCT Publication Nos. WO 1998/24893; WO 1992/01047; WO 1996/34096; WO 1996/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598).
As used herein, the term “humanized” antibody refers to a non-human antibody that contains minimal sequences derived from non-human immunoglobulin. In general, a humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. All or substantially all of the FW regions may also be those of a human immunoglobulin sequence. The humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Methods of antibody humanization are known in the art and have been described, for example, in Riechmann et al. (1988) Nature 332:323-7; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370.
As used herein, patients that are “in need of” a hematopoietic stem cell transplant include patients that exhibit a defect or deficiency in one or more blood cell types, as well as patients having a stem cell disorder, autoimmune disease, cancer, or other pathology described herein. Hematopoietic stem cells generally exhibit 1) multi-potency, and can thus differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B-cells and T-cells), 2) self-renewal, and can thus give rise to daughter cells that have equivalent potential as the mother cell, and 3) the ability to be reintroduced into a transplant recipient whereupon they home to the hematopoietic stem cell niche and re-establish productive and sustained hematopoiesis. Hematopoietic stem cells can thus be administered to a patient defective or deficient in one or more cell types of the hematopoietic lineage in order to re-constitute the defective or deficient population of cells in vivo. For example, the patient may be suffering from cancer, and the deficiency may be caused by administration of a chemotherapeutic agent or other medicament that depletes, either selectively or non-specifically, the cancerous cell population. Additionally or alternatively, the patient may be suffering from a hemoglobinopathy (e.g., a non-malignant hemoglobinopathy), such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome. The subject may be one that is suffering from adenosine deaminase severe combined immunodeficiency (ADA SCID), HIV/AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome. The subject may have or be affected by an inherited blood disorder (e.g., sickle cell anemia) or an autoimmune disorder. Additionally or alternatively, the subject may have or be affected by a malignancy, such as neuroblastoma or a hematologic cancer. In some embodiments, the subject may have a leukemia, lymphoma, or myeloma. In some embodiments, the subject has acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. In some embodiments, the subject has myelodysplastic syndrome. In some embodiments, the subject has an autoimmune disease, such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, Type 1 diabetes, or another autoimmune pathology described herein. In some embodiments, the subject is in need of chimeric antigen receptor T-cell (CART) therapy. In some embodiments, the subject has or is otherwise affected by a metabolic storage disorder. The subject may suffer or otherwise be affected by a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher Disease, Hurler Disease, sphingolipidoses, metachromatic leukodystrophy, globoid cell leukodystrophy, cerebral adrenoleukodystrophy, or any other diseases or disorders which may benefit from the treatments and therapies disclosed herein and including, without limitation, severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyper immunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis and those diseases, or disorders described in “Bone Marrow Transplantation for Non-Malignant Disease,” ASH Education Book, 1:319-338 (2000), the disclosure of which is incorporated herein by reference in its entirety as it pertains to pathologies that may be treated by administration of hematopoietic stem cell transplant therapy. Additionally or alternatively, a patient “in need of” a hematopoietic stem cell transplant may one that is or is not suffering from one of the foregoing pathologies, but nonetheless exhibits a reduced level (e.g., as compared to that of an otherwise healthy subject) of one or more endogenous cell types within the hematopoietic lineage, such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeoblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes. One of skill in the art can readily determine whether one's level of one or more of the foregoing cell types, or other blood cell type, is reduced with respect to an otherwise healthy subject, for example, by way of flow cytometry and fluorescence activated cell sorting (FACS) methods, among other procedures, known in the art.
As used herein, the term “leukocyte” refers to a heterogeneous group of nucleated blood cell types, and excludes erythrocytes and platelets. Leukocytes can be divided into two general groups: polymorphonucleocytes, which include neutrophils, eosinophils, and basophils, and mononucleocytes, which include lymphocytes and monocytes. Polymorphonucleocytes contain many cytoplasmic granules and a multilobed nucleus and include the following: neutrophils, which are generally amoeboid in shape, phagocytic, and stain with both basic and acidic dyes, and eosinophils and basophils, which contain cytoplasmic granules that stain with acidic dyes and with basic dyes, respectively.
As used herein, the term “lymphocyte” refers to a mononuclear leukocyte that is involved in the mounting of an immune response. In general, lymphocytes include B lymphocytes, T lymphocytes, and NK cells.
As used herein, the terms “mobilize” and “mobilization” refer to processes by which a population of hematopoietic stem or progenitor cells (e.g., a neutrophil) is released from a stem cell niche, such as the bone marrow of a subject, into circulation in the peripheral blood. Mobilization of hematopoietic stem and progenitor cells can be monitored, for example, by assessing the quantity or concentration of hematopoietic stem or progenitor cells in a peripheral blood sample isolated from a subject. For example, the peripheral blood sample may be withdrawn from the subject, and the quantity or concentration of hematopoietic stem or progenitor cells in the peripheral blood sample may subsequently be assessed, following the administration of a hematopoietic stem or progenitor cell mobilization regimen to the subject. The mobilization regimen may include, for example, a CXCR4 antagonist, such as a CXCR4 antagonist described herein (e.g., plerixafor or a variant thereof), and a CXCR2 agonist, such as a CXCR2 agonist described herein (e.g., Gro-R or a variant thereof, such as a truncation of Gro-β, for example, Gro-β T). The quantity or concentration of hematopoietic stem or progenitor cells in the peripheral blood sample isolated from the subject following administration of the mobilization regimen may be compared to the quantity or concentration of hematopoietic stem or progenitor cells in a peripheral blood sample isolated from the subject prior to administration of the mobilization regimen. An observation that the quantity or concentration of hematopoietic stem or progenitor cells has increased in the peripheral blood of the subject following administration of the mobilization regimen is an indication that the subject is responding to the mobilization regimen, and that hematopoietic stem and progenitor cells have been released from one or more stem cell niches, such as the bone marrow, into peripheral blood circulation. In some embodiments, an observation that the quantity or concentration of hematopoietic stem or progenitor cells has increased in the peripheral blood of the subject by 1%, 100%, 1,000%, or more (e.g., by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1,000%, or more) following administration of the mobilization regimen is an indication that the subject is responding to the mobilization regimen, and that hematopoietic stem and progenitor cells have been released from one or more stem cell niches, such as the bone marrow, into peripheral blood circulation. Methods for determining the quantity or concentration of hematopoietic stem or progenitor cells are described herein and known in the art, and include, for example, flow cytometry techniques that quantify hematopoietic stem or progenitor cells on the basis of the antigen expression profile of such cells, which is described herein. For example, human HSCs are CD34+, CD38−, CD45RA−, CD90+, CD49F+, and lin− (negative for mature lineage markers including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A). Additional methods for determining the quantity or concentration of hematopoietic stem or progenitor cells in a peripheral blood sample isolated from a subject include assays that quantify the number of colony-forming units (CFUs) in the sample, which is a measure of the quantity of viable hematopoietic stem or progenitor cells that, upon incubation with an appropriate culture medium, give rise to an individual population of hematopoietic stem or progenitor cells.
As used herein, the term “mobilizing amount” refers to a quantity of one or more agents, such as a quantity of a CXCR4 antagonist and/or a CXCR2 agonist described herein (In some embodiments, a quantity of plerixafor, or a variant thereof, and/or Gro-β, or a variant thereof, such as a truncation of Gro-β, for example, Gro-β T) that mobilizes a population of hematopoietic stem or progenitor cells upon administration to a subject, such as a mammalian subject (e.g., a human subject). Exemplary mobilizing amounts of these agents include amounts sufficient to effectuate the release of a population of, for example, from about 20 to about 40 CD34+ cells/μL of peripheral blood, such as from about 21 to about 39 CD34+ cells/μL of peripheral blood, about 22 to about 38 CD34+ cells/μL of peripheral blood, about 23 to about 37 CD34+ cells/μL of peripheral blood, about 24 to about 36 CD34+ cells/μL of peripheral blood, about 25 to about 35 CD34+ cells/μL of peripheral blood, about 26 to about 34 CD34+ cells/μL of peripheral blood, about 27 to about 33 CD34+ cells/μL of peripheral blood, about 28 to about 32 CD34+ cells/μL of peripheral blood, or about 29 to about 31 CD34+ cells/μL of peripheral blood (e.g., about 20 CD34+ cells/μL of peripheral blood, 21 CD34+ cells/μL of peripheral blood, 22 CD34+ cells/μL of peripheral blood, 23 CD34+ cells/μL of peripheral blood, 24, CD34+ cells/μL of peripheral blood, 25 CD34+ cells/μL of peripheral blood, 26 CD34+ cells/μL of peripheral blood, 27 CD34+ cells/μL of peripheral blood, 28 CD34+ cells/μL of peripheral blood, 29 CD34+ cells/μL of peripheral blood, 30 CD34+ cells/μL of peripheral blood, 31 CD34+ cells/μL of peripheral blood, 32 CD34+ cells/μL of peripheral blood 33 CD34+ cells/μL of peripheral blood, 34 CD34+ cells/μL of peripheral blood, 35 CD34+ cells/μL of peripheral blood, 36 CD34+ cells/μL of peripheral blood, 37 CD34+ cells/μL of peripheral blood, 38 CD34+ cells/μL of peripheral blood, 39 CD34+ cells/μL of peripheral blood, 40 CD34+ cells/μL of peripheral blood, or more. In certain embodiments, mobilizing amounts of these agents include amounts sufficient to effectuate the release of a population of, for example, from about 5 to about 20 CD34+CD90+CD45RA− cells/μL of peripheral blood, such as from about 5 to about 8 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 5 to about 10 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 5 to about 12 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 5 to about 15 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 5 to about 18 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 8 to about 10 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 8 to about 12 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 8 to about 15 CD34+CD90+CD45RA− cells/μL of peripheral blood, or about 8 to about 18 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 8 to about 20 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 10 to about 12 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 10 to about 15 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 10 to about 18 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 10 to about 20 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 12 to about 15 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 10 to about 18 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 10 to about 20 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 12 to about 15 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 12 to about 18 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 12 to about 20 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 15 to about 18 CD34+CD90+CD45RA− cells/μL of peripheral blood, or about 15 to about 20 CD34+CD90+CD45RA− cells/μL of peripheral blood. In certain embodiments, mobilizing amounts of these agents include amounts sufficient to effectuate at least a 2 fold release of a population CD34+CD90+CD45RA− cells/μL of peripheral blood, e.g., at least a 3 fold release, at least a 4 fold release, at least a 5 fold release, at least a 6 fold release at least a 7 fold release, at least an 8 fold release, at least a 9 fold release or at least a 10 fold release of a population CD34+CD90+CD45RA− cells/μL of peripheral blood. In certain embodiments, mobilizing amounts of these agents include amounts sufficient to effectuate a 2 fold release to a 10 fold release, e.g., a 2 fold to 4 fold release, a 2 fold to 6 fold release, a 2 fold to 8 fold release, a 4 fold to 6 fold release, a 4 fold to 8 fold release, a 4 fold to 10 fold release, a 6 fold to 8 fold release, a 6 fold to 10 fold release, or a 8 fold to 10 release of a population CD34+CD90+CD45RA− cells/μL of peripheral blood.
As used herein, the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
As used herein, the term “monocyte” refers to a CD14+ and CD34 peripheral blood mononuclear cell (PBMC), which is generally capable of differentiating into a macrophage and/or dendritic cell upon activation by one or more foreign substances, such as, a microbial product. In particular, a monocyte may express elevated levels of the CD14 surface antigen marker, and may express at least one biomarker selected from CD64, CD93, CD180, CD328 (also known as sialic acid-binding Ig-like lectin 7 or Siglec7), and CD329 (sialic acid-binding Ig-like lectin 9 or Siglec9), as well as the peanut agglutinin protein (PNA).
As used herein, a “peptide” refers to a single-chain polyamide containing a plurality of amino acid residues, such as naturally-occurring and/or non-natural amino acid residues, that are consecutively bound by amide bonds. Examples of peptides include shorter fragments of full-length proteins, such as full-length naturally-occurring proteins.
As used herein, the term “recipient” refers to a patient that receives a transplant, such as a transplant containing a population of hematopoietic stem cells. The transplanted cells administered to a recipient may be, e.g., autologous, syngeneic, or allogeneic cells.
As used herein, the term “sample” refers to a specimen (e.g., blood, blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placental or dermal), pancreatic fluid, chorionic villus sample, and cells) taken from a subject. A sample may be, for example, withdrawn peripheral blood from a donor that is undergoing or has undergone a hematopoietic stem or progenitor cell mobilization regimen described herein.
As used herein, the term “scFv” refers to a single chain Fv antibody in which the variable domains of the heavy chain and the light chain from an antibody have been joined to form one chain. scFv fragments contain a single polypeptide chain that includes the variable region of an antibody light chain (VL) (e.g., CDR-L1, CDR-L2, and/or CDR-L3) and the variable region of an antibody heavy chain (VH) (e.g., CDR-H1, CDR-H2, and/or CDR-H3) separated by a linker. The linker that joins the VL and VH regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids. Alternative linkers can be used to so as to increase the resistance of the scFv fragment to proteolytic degradation (for example, linkers containing D-amino acids), in order to enhance the solubility of the scFv fragment (for example, hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (for example, a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (for example, linkers containing glycosylation sites). It will also be understood by one of ordinary skill in the art that the variable regions of the scFv molecules described herein can be modified such that they vary in amino acid sequence from the antibody molecule from which they were derived. For example, nucleotide or amino acid substitutions leading to conservative substitutions or changes at amino acid residues can be made (e.g., in CDR and/or framework residues) so as to preserve or enhance the ability of the scFv to bind to the antigen recognized by the corresponding antibody.
As used herein, the phrase “stem cell disorder” broadly refers to any disease, disorder, or condition that may be treated or cured by engrafting or transplanting a population of hematopoietic stem or progenitor cells in a target tissue within a patient. For example, Type I diabetes has been shown to be cured by hematopoietic stem cell transplant, along with various other disorders. Exemplary diseases that can be treated by infusion of hematopoietic stem or progenitor cells into a patient are sickle cell anemia, thalassemias, Fanconi anemia, aplastic anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV/AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome. Additional diseases that may be treated by transplantation of hematopoietic stem and progenitor cells as described herein include blood disorders (e.g., sickle cell anemia) and autoimmune disorders, such as scleroderma, multiple sclerosis, ulcerative colitis, and Crohn's disease. Additional diseases that may be treated using hematopoietic stem and progenitor cell transplant therapy include cancer, such as a cancer described herein. Exemplary stem cell disorders are malignancies, such as a neuroblastoma or a hematologic cancer, such as leukemia, lymphoma, and myeloma. In some embodiments, the cancer may be acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. Additional diseases treatable using hematopoietic stem or progenitor cell transplant therapy include myelodysplastic syndrome. In some embodiments, the patient has or is otherwise affected by a metabolic storage disorder. For example, the patient may suffer or otherwise be affected by a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher Disease, Hurler Disease, sphingolipidoses, metachromatic leukodystrophy, globoid cell leukodystrophy, cerebral adrenoleukodystrophy, or any other diseases or disorders which may benefit from the treatments and therapies disclosed herein and including, without limitation, severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyper immunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis and those diseases, or disorders described in “Bone Marrow Transplantation for Non-Malignant Disease,” ASH Education Book, 1:319-338 (2000), the disclosure of which is incorporated herein by reference in its entirety as it pertains to pathologies that may be treated by administration of hematopoietic stem or progenitor cell transplant therapy.
As used herein in the context of hematopoietic stem cell mobilization, the term “stem cell niche” refers to a microenvironment within a donor, such as a mammalian donor (e.g., a human donor) in which endogenous hematopoietic stem or progenitor cells reside. An exemplary stem cell niche is bone marrow tissue.
As used herein, the terms “subject” and “patient” refer to an organism, such as a human, that receives treatment for a particular disease or condition as described herein. In some embodiments, a patient, such as a human patient, that is in need of hematopoietic stem cell transplantation may receive treatment that includes a population of hematopoietic stem cells so as to treat a stem cell disorder, such as a cancer, autoimmune disease, or metabolic disorder described herein. The hematopoietic stem cells that are transplanted into the patient may be, for example, a population of hematopoietic stem cells that has been mobilized and withdrawn from a donor in accordance with the compositions and methods described herein. In some embodiments, the hematopoietic stem cells that are transplanted into the patient may be mobilized within a donor by administration of a CXCR4 antagonist and/or a CXCR2 agonist to the donor. In certain embodiments, the terms “subject” and “patient” refer to an organism, such as a human, that receives treatment for neutropenia.
As used herein, the term “transfection” refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, such as electroporation, lipofection, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
As used herein, the terms “treat” or “treatment” refer to therapeutic treatment, in which the object is to prevent or slow down (lessen) an undesired physiological change or disorder or to promote a beneficial phenotype in the patient being treated. Beneficial or desired clinical results include, but are not limited to, promoting the engraftment of exogenous hematopoietic cells in a patient following hematopoietic stem or progenitor cell transplant therapy. In certain embodiments, the benefits include a more rapid engraftment of transplanted cells, e.g., neutrophils and platelets. For example, in certain embodiments, using the methods described herein, neutrophil recovery occurs within about 5-20 days post-transplant, about 5-15 days post-transplant, about 5-10 days post-transplant, about 7-12 days post-transplant, about 8-12 days post-transplant, about 9-15 days post-transplant, about 10-15 days post-transplant, or about 10 days post-transplant. In certain embodiments, using the methods described herein, platelet recovery occurs within about 10-20 days post-transplant, about 10-15 days post-transplant, about 15-20 days post-transplant, about 12-18 days post-transplant, about 12-17 days post-transplant, about 13-18 days post-transplant, about 12-17 days post-transplant, or about 15 days post-transplant. Additional beneficial results include an increase in the cell count or relative concentration of hematopoietic stem cells in a patient in need of a hematopoietic stem or progenitor cell transplant following administration of an exogenous hematopoietic stem or progenitor cell graft to the patient. Beneficial results of therapy described herein may also include an increase in the cell count or relative concentration of one or more cells of hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte, following and subsequent hematopoietic stem cell transplant therapy. Additional beneficial results may include the reduction in quantity of a disease-causing cell population, such as a population of cancer cells or autoimmune cells. In the context of treating neutropenia, beneficial or desired clinical results include, but are not limited to, increasing the number of neutrophils in the blood and/or preventing or reducing at least one symptom associated with neutropenia.
As used herein, the terms “variant” and “derivative” are used interchangeably and refer to naturally-occurring, synthetic, and semi-synthetic analogues of a compound, peptide, protein, or other substance described herein. A variant or derivative of a compound, peptide, protein, or other substance described herein may retain or improve upon the biological activity of the original material.
As used herein, the term “vector” includes a nucleic acid vector, such as a plasmid, a DNA vector, a plasmid, an RNA vector, virus, or other suitable replicon. Expression vectors described herein may contain a polynucleotide sequence as well as, for example, additional sequence elements used for the expression of proteins and/or the integration of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used for the expression of peptides and proteins, such as those described herein, include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors for expression of peptides and proteins described herein contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements may include, for example, 5′ and 3′ untranslated regions and a polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.
As used herein, the term “alkyl” refers to a straight- or branched-chain alkyl group having, for example, from 1 to 20 carbon atoms in the chain. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and the like.
As used herein, the term “alkylene” refers to a straight- or branched-chain divalent alkyl group. The divalent positions may be on the same or different atoms within the alkyl chain. Examples of alkylene include methylene, ethylene, propylene, isopropylene, and the like.
As used herein, the term “heteroalkyl” refers to a straight or branched-chain alkyl group having, for example, from 1 to 20 carbon atoms in the chain, and further containing one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur, among others) in the chain.
As used herein, the term “heteroalkylene” refers to a straight- or branched-chain divalent heteroalkyl group. The divalent positions may be on the same or different atoms within the heteroalkyl chain. The divalent positions may be one or more heteroatoms.
As used herein, the term “alkenyl” refers to a straight- or branched-chain alkenyl group having, for example, from 2 to 20 carbon atoms in the chain. Examples of alkenyl groups include vinyl, propenyl, isopropenyl, butenyl, tert-butylenyl, hexenyl, and the like.
As used herein, the term “alkenylene” refers to a straight- or branched-chain divalent alkenyl group. The divalent positions may be on the same or different atoms within the alkenyl chain. Examples of alkenylene include ethenylene, propenylene, isopropenylene, butenylene, and the like.
As used herein, the term “heteroalkenyl” refers to a straight- or branched-chain alkenyl group having, for example, from 2 to 20 carbon atoms in the chain, and further containing one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur, among others) in the chain.
As used herein, the term “heteroalkenylene” refers to a straight- or branched-chain divalent heteroalkenyl group. The divalent positions may be on the same or different atoms within the heteroalkenyl chain. The divalent positions may be one or more heteroatoms.
As used herein, the term “alkynyl” refers to a straight- or branched-chain alkynyl group having, for example, from 2 to 20 carbon atoms in the chain. Examples of alkynyl groups include propargyl, butynyl, pentynyl, hexynyl, and the like.
As used herein, the term “alkynylene” refers to a straight- or branched-chain divalent alkynyl group. The divalent positions may be on the same or different atoms within the alkynyl chain.
As used herein, the term “heteroalkynyl” refers to a straight- or branched-chain alkynyl group having, for example, from 2 to 20 carbon atoms in the chain, and further containing one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur, among others) in the chain.
As used herein, the term “heteroalkynylene” refers to a straight- or branched-chain divalent heteroalkynyl group. The divalent positions may be on the same or different atoms within the heteroalkynyl chain. The divalent positions may be one or more heteroatoms.
As used herein, the term “cycloalkyl” refers to a monocyclic, or fused, bridged, or spiro polycyclic ring structure that is saturated and has, for example, from 3 to 12 carbon ring atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[3.1.0]hexane, and the like.
As used herein, the term “cycloalkylene” refers to a divalent cycloalkyl group. The divalent positions may be on the same or different atoms within the ring structure. Examples of cycloalkylene include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and the like.
As used herein, the term “heterocycloalkyl” refers to a monocyclic, or fused, bridged, or spiro polycyclic ring structure that is saturated and has, for example, from 3 to 12 ring atoms per ring structure selected from carbon atoms and heteroatoms selected from, e.g., nitrogen, oxygen, and sulfur, among others. The ring structure may contain, for example, one or more oxo groups on carbon, nitrogen, or sulfur ring members.
As used herein, the term “heterocycloalkylene” refers to a divalent heterocyclolalkyl group. The divalent positions may be on the same or different atoms within the ring structure.
As used herein, the term “aryl” refers to a monocyclic or multicyclic aromatic ring system containing, for example, from 6 to 19 carbon atoms. Aryl groups include, but are not limited to, phenyl, fluorenyl, naphthyl, and the like. The divalent positions may be one or more heteroatoms.
As used herein, the term “arylene” refers to a divalent aryl group. The divalent positions may be on the same or different atoms.
As used herein, the term “heteroaryl” refers to a monocyclic heteroaromatic, or a bicyclic or a tricyclic fused-ring heteroaromatic group. Heteroaryl groups include pyridyl, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadia-zolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl, [2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, isobenzothienyl, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl, quinolizinyl, quinazolinyl, pthalazinyl, quinoxalinyl, cinnolinyl, napthyridinyl, pyrido[3,4-b]pyridyl, pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl, xanthenyl, benzoquinolyl, and the like.
As used herein, the term “heteroarylene” refers to a divalent heteroaryl group. The divalent positions may be on the same or different atoms. The divalent positions may be one or more heteroatoms.
Unless otherwise constrained by the definition of the individual substituent, the foregoing chemical moieties, such as “alkyl,” “alkylene,” “heteroalkyl,” “heteroalkylene,” “alkenyl,” “alkenylene,” “heteroalkenyl,” “heteroalkenylene,” “alkynyl,” “alkynylene,” “heteroalkynyl,” “heteroalkynylene,” “cycloalkyl,” “cycloalkylene,” “heterocyclolalkyl,” heterocycloalkylene,” “aryl,” “arylene,” “heteroaryl,” and “heteroarylene” groups can optionally be substituted. As used herein, the term “optionally substituted” refers to a compound or moiety containing one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substituents, as permitted by the valence of the compound or moiety or a site thereof, such as a substituent selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkyl aryl, alkyl heteroaryl, alkyl cycloalkyl, alkyl heterocycloalkyl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, nitro, and the like. The substitution may include situations in which neighboring substituents have undergone ring closure, such as ring closure of vicinal functional substituents, to form, for example, lactams, lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals, aminals, and hemiaminals, formed by ring closure, for example, to furnish a protecting group.
The present invention is based, in part, on the discovery that hematopoietic stem and progenitor cells (e.g., neutrophils) can be mobilized by administering particular doses of a CXCR2 agonist, such as Gro-β, Gro-β T, or a variant thereof, optionally in combination with a CXCR4 antagonist to a mammalian donor (e.g., a human donor).
Exemplary CXCR2 agonists that may be used in conjunction with the compositions and methods described herein are Gro-β and variants thereof. Gro-β (also referred to as growth-regulated protein β, chemokine (C-X-C motif) ligand 2 (CXCL2), and macrophage inflammatory protein 2-α (MIP2-α)) is a cytokine capable of mobilizing hematopoietic stem and progenitor cells, for example, by stimulating the release of proteases, and particularly MMP-9, from peripheral neutrophils. In contrast, TIMP-1 blocks MMP migration. Without being limited by mechanism, MMP-9 may induce mobilization of hematopoietic stem and progenitor cells from stem cell niches, such as the bone marrow, to circulating peripheral blood by stimulating the degradation of proteins such as stem cell factor, its corresponding receptor, CD117, and CXCL12, all of which generally maintain hematopoietic stem and progenitor cells immobilized in bone marrow. Therefore, an increase in MMP-9 or an increase in MMP-9:TIMP-1 ratio can be indicative of increased mobilization of hematopoietic stem and progenitor cells and/or neutrophils.
Accordingly, in certain embodiments, therefore, the method results in an increase in plasma MMP-9 for example, to an amount of about 100 ng/mL to about 300 ng/mL (e.g., about 150 ng/mL to about 250 ng/mL) peripheral blood within 30 minutes of administration of a CXCR2 agonist (e.g., Gro-β or Gro-β T). In certain embodiments, the method results in an increase in the MMP-9:TIMP-1 ratio in peripheral blood, e.g., to a molar ratio of between about 0.2 and about 0.6 (e.g., about 0.25 to about 0.45) MMP-9:TIMP-1.
In addition to Gro-β, exemplary CXCR2 agonists that may be used in conjunction with the compositions and methods described herein are truncated forms of Gro-β, such as those that feature a deletion at the N-terminus of Gro-β of from 1 to 8 amino acids (e.g., peptides that feature an N-terminal deletion of 1 amino acids, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, or 8 amino acids). In some embodiments, CXCR2 agonists that may be used in conjunction with the compositions and methods described herein include Gro-β T, which is characterized by a deletion of the first four amino acids from the N-terminus of Gro-β. Gro-β T exhibits particularly advantageous biological properties, such as the ability to induce hematopoietic stem and progenitor cell mobilization with a potency superior to that of Gro-β by multiple orders of magnitude. Gro-β and Gro-β T are described, for example, in U.S. Pat. No. 6,080,398, the disclosure of which is incorporated herein by reference in its entirety.
In addition, exemplary CXCR2 agonists that may be used in conjunction with the compositions and methods described herein are variants of Gro-β containing an aspartic acid residue in place of the asparagine residue at position 69 of SEQ ID NO: 1. This peptide, is referred to herein as Gro-β N69D. Similarly, CXCR2 agonists that may be used with the compositions and methods described herein include variants of Gro-β T containing an aspartic acid residue in place of the asparagine residue at position 65 of SEQ ID NO: 2. This peptide, referred to herein as Gro-β T N65D, not only retains hematopoietic stem and progenitor cell-mobilizing capacity, but exhibits a potency that is substantially greater than that of Gro-β T. Gro-β N69D and Gro-β T N65D are described, for example, in U.S. Pat. No. 6,447,766, the disclosure of which is incorporated herein by reference in its entirety.
The amino acid sequences of Gro-β, Gro-β T, Gro-β N69D, and Gro-β T N65D are set forth in TABLE 2 below.
Additional CXCR2 agonists that may be used in conjunction with the compositions and methods described herein include other variants of Gro-β, such as peptides that have one or more amino acid substitutions, insertions, and/or deletions relative to Gro-β. In some embodiments, CXCR2 agonists that may be used in conjunction with the compositions and methods described herein include peptides having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1 (e.g., a peptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1). In some embodiments, the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 1 only by way of one or more conservative amino acid substitutions. In some embodiments, in some embodiments, the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 1 by no more than 20, no more than 15, no more than 10, no more than 5, or no more than 1 nonconservative amino acid substitutions. In some embodiments, the CXCR2 agonist is Gro-β. In some embodiments, the Gro-β T is not covalently modified. In some embodiments, the Gro-β is not covalently modified with a polyalkylene glycol moiety, such as a polyethylene glycol moiety.
Additional examples of CXCR2 agonists useful in conjunction with the compositions and methods described herein are variants of Gro-β T, such as peptides that have one or more amino acid substitutions, insertions, and/or deletions relative to Gro-β T. In some embodiments, the CXCR2 agonist may be a peptide having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 2 (e.g., a peptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2). In some embodiments, the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 2 only by way of one or more conservative amino acid substitutions. In some embodiments, in some embodiments, the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 2 by no more than 20, no more than 15, no more than 10, no more than 5, or no more than 1 nonconservative amino acid substitutions.
Additional examples of CXCR2 agonists useful in conjunction with the compositions and methods described herein are variants of Gro-β N69D, such as peptides that have one or more amino acid substitutions, insertions, and/or deletions relative to Gro-β N69D. In some embodiments, the CXCR2 agonist may be a peptide having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 3 (e.g., a peptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3). In some embodiments, the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 3 only by way of one or more conservative amino acid substitutions. In some embodiments, in some embodiments, the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 3 by no more than 20, no more than 15, no more than 10, no more than 5, or no more than 1 nonconservative amino acid substitutions.
Additional examples of CXCR2 agonists useful in conjunction with the compositions and methods described herein are variants of Gro-β T N65D, such as peptides that have one or more amino acid substitutions, insertions, and/or deletions relative to Gro-β T N65D. In some embodiments, the CXCR2 agonist may be a peptide having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 4 (e.g., a peptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4). In some embodiments, the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 4 only by way of one or more conservative amino acid substitutions. In some embodiments, in some embodiments, the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 4 by no more than 20, no more than 15, no more than 10, no more than 5, or no more than 1 nonconservative amino acid substitutions.
In some embodiments, the CXCR2 agonist is an antibody or antigen-binding fragment thereof that binds CXCR2 and activates CXCR2 signal transduction. In some embodiments, the CXCR2 agonist may be an antibody or antigen-binding fragment thereof that binds the same epitope on CXCR2 as Gro-β or a variant or truncation thereof, such as Gro-β T, as assessed, for example, by way of a competitive CXCR2 binding assay. In some embodiments, the CXCR2 agonist is an antibody or an antigen-binding fragment thereof that competes with Gro-β or a variant or truncation thereof, such as Gro-β T, for binding to CXCR2.
In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′)2 molecule, and a tandem di-scFv. In some embodiments, the antibody has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE.
The peptidic CXCR2 agonists described herein, such as Gro-β, Gro-βT, and variants thereof, may be prepared synthetically, for instance, using solid phase peptide synthesis techniques. Systems and processes for performing solid phase peptide synthesis include those that are known in the art and have been described, for instance, in U.S. Pat. Nos. 9,169,287; 9,388,212; 9,206,222; 6,028,172; and 5,233,044, among others, the disclosures of each of which are incorporated herein by reference as they pertain to protocols and techniques for the synthesis of peptides on solid support. Solid phase peptide synthesis is a process in which amino acid residues are added to peptides that have been immobilized on a solid support, such as a polymeric resin (e.g., a hydrophilic resin, such as a polyethylene-glycol-containing resin, or hydrophobic resin, such as a polystyrene-based resin).
Peptides, such as those containing protecting groups at amino, hydroxy, thiol, and carboxy substituents, among others, may be bound to a solid support such that the peptide is effectively immobilized on the solid support. For example, the peptides may be bound to the solid support via their C termini, thereby immobilizing the peptides for subsequent reaction in at a resin-liquid interface.
The process of adding amino acid residues to immobilized peptides can include exposing a deprotection reagent to the immobilized peptides to remove at least a portion of the protection groups from at least a portion of the immobilized peptides. The deprotection reagent exposure step can be configured, for instance, such that side-chain protection groups are preserved, while N-terminal protection groups are removed. For instance, an exemplary amino protecting contains a fluorenylmethyloxycarbonyl (Fmoc) substituent. A deprotection reagent containing a strongly basic substance, such as piperidine (e.g., a piperidine solution in an appropriate organic solvent, such as dimethyl formamide (DMF)) may be exposed to the immobilized peptides such that the Fmoc protecting groups are removed from at least a portion of the immobilized peptides. Other protecting groups suitable for the protection of amino substituents include, for instance, the tert-butyloxycarbonyl (Boc) moiety. A deprotection reagent comprising a strong acid, such as trifluoroacetic acid (TFA) may be exposed to immobilized peptides containing a Boc-protected amino substituent so as to remove the Boc protecting group by an ionization process. In this way, peptides can be protected and deprotected at specific sites, such as at one or more side-chains or at the N- or C-terminus of an immobilized peptide so as to append chemical functionality regioselectively at one or more of these positions. This can be used, for instance, to derivatize a side-chain of an immobilized peptide, or to synthesize a peptide, e.g., from the C-terminus to the N-terminus.
The process of adding amino acid residues to immobilized peptides can include, for instance, exposing protected, activated amino acids to the immobilized peptides such that at least a portion of the activated amino acids are bonded to the immobilized peptides to form newly-bonded amino acid residues. For example, the peptides may be exposed to activated amino acids that react with the deprotected N-termini of the peptides so as to elongate the peptide chain by one amino acid. Amino acids can be activated for reaction with the deprotected peptides by reaction of the amino acid with an agent that enhances the electrophilicity of the backbone carbonyl carbon of the amino acid. For example, phosphonium and uronium salts can, in the presence of a tertiary base (e.g., diisopropylethylamine (DIPEA) and triethylamine (TEA), among others), convert protected amino acids into activated species (for example, BOP, PyBOP, HBTU, and TBTU all generate HOBt esters). Other reagents can be used to help prevent racemization that may be induced in the presence of a base. These reagents include carbodiimides (for example, DCC or WSCDI) with an added auxiliary nucleophile (for example, 1-hydroxy-benzotriazole (HOBt), 1-hydroxy-azabenzotriazole (HOAt), or HOSu) or derivatives thereof. Another reagent that can be utilized to prevent racemization is TBTU. The mixed anhydride method, using isobutyl chloroformate, with or without an added auxiliary nucleophile, can also be used, as well as the azide method, due to the low racemization associated with this reagent. These types of compounds can also increase the rate of carbodiimide-mediated couplings, as well as prevent dehydration of Asn and Gln residues. Typical additional reagents include also bases such as N,N-diisopropylethylamine (DIPEA), triethylamine (TEA) or N-methylmorpholine (NMM). These reagents are described in detail, for instance, in U.S. Pat. No. 8,546,350, the disclosure of which is incorporated herein in its entirety.
During the recombinant expression and folding of Gro-β and Gro-β T in aqueous solution, a particular C-terminal asparagine residue (Asn69 within Gro-β and Asn65 within Gro-β T) is prone to deamidation. This process effectuates the conversion of the asparagine residue to aspartic acid. Without wishing to be bound by any theory, the chemical synthesis of Gro-β and Gro-β T may overcome this problem, for instance, by providing conditions that reduce the exposure of this asparagine residue to nucleophilic solvent. When prepared synthetically (i.e., chemically synthesized), for instance, using, e.g., the solid phase peptide synthesis techniques described above, synthetic Gro-β, Gro-β T, and variants thereof that may be used in conjunction with the compositions and methods described herein may have a purity of, e.g., at least about 95% relative to the deamidated versions of these peptides (i.e., contain less than 5% of the corresponding deamidated peptide). For instance, synthetic Gro-β, Gro-β T, and variants thereof that may be used in conjunction with the compositions and methods described herein may have a purity of about 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or more, relative to the deamidated versions of these peptides (e.g., the Asn69 deamidated version of SEQ ID NO: 1 or the Asn65 deamidated version of SEQ ID NO: 2). For instance, synthetic Gro-β, Gro-β T, and variants thereof may have, for instance, a purity of from about 95% to about 99.99%, such as a purity of from about 95% to about 99.99%, about 96% to about 99.99%, about 97% to about 99.99%, about 98% to about 99.99%, about 99% to about 99.99%, about 99.9% to about 99.99%, about 95% to about 99.5%, about 96% to about 99.5%, about 95% to about 99%, or about 97% to about 99% relative to the deamidated versions of these peptides (e.g., the Asn69 deamidated version of SEQ ID NO: 1 or the Asn65 deamidated version of SEQ ID NO: 2).
Exemplary CXCR4 antagonists for use in conjunction with the compositions and methods described herein are compounds represented by formula (I)
Z-linker-Z′ (I)
or a pharmaceutically acceptable salt thereof, wherein Z is:
—N(R)—(CR2)n—X (IC)
In some embodiments, Z and Z′ may each independently a cyclic polyamine containing from 9 to 32 ring members, of which from 2 to 8 are nitrogen atoms separated from one another by 2 or more carbon atoms. In some embodiments, Z and Z′ are identical substituents. As an example, Z may be a cyclic polyamine including from 10 to 24 ring members. In some embodiments, Z may be a cyclic polyamine that contains 14 ring members. In some embodiments, Z includes 4 nitrogen atoms. In some embodiments, Z is 1,4,8,11-tetraazocyclotetradecane.
In some embodiments, the linker is represented by formula (ID)
wherein ring D is an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted cycloalkyl group, or an optionally substituted heterocycloalkyl group; and
X and Y are each independently optionally substituted alkylene (e.g., optionally substituted C1-C6 alkylene), optionally substituted heteroalkylene (e.g., optionally substituted C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., optionally substituted C2-C6 alkenylene), optionally substituted heteroalkenylene (e.g., optionally substituted C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., optionally substituted C2-C6 alkynylene), or optionally substituted heteroalkynylene (e.g., optionally substituted C2-C6 heteroalkynylene).
As an example, the linker may be represented by formula (IE)
wherein ring D is an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted cycloalkyl group, or an optionally substituted heterocycloalkyl group; and
X and Y are each independently optionally substituted alkylene (e.g., optionally substituted C1-C6 alkylene), optionally substituted heteroalkylene (e.g., optionally substituted C1-C6 heteroalkylene), optionally substituted C2-C6 alkenylene (e.g., optionally substituted C2-C6 alkenylene), optionally substituted heteroalkenylene (e.g., optionally substituted C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., optionally substituted C2-C6 alkynylene), or optionally substituted heteroalkynylene (e.g., optionally substituted C2-C6 heteroalkynylene). In some embodiments, X and Y are each independently optionally substituted C1-C6 alkylene. In some embodiments, X and Y are identical substituents. In some embodiments, X and Y may be each be methylene, ethylene, n-propylene, n-butylene, n-pentylene, or n-hexylene groups. In some embodiments, X and Y are each methylene groups.
The linker may be, for example, 1,3-phenylene, 2,6-pyridine, 3,5-pyridine, 2,5-thiophene, 4,4′-(2,2′-bipyrimidine), 2,9-(1,10-phenanthroline), or the like. In some embodiments, the linker is 1,4-phenylene-bis-(methylene).
CXCR4 antagonists useful in conjunction with the compositions and methods described herein include plerixafor (also referred to herein as “AMD3100” and “Mozobil”), or a pharmaceutically acceptable salt thereof, represented by formula (II), 1,1′-[1,4-phenylenebis(methylene)]-bis-1,4,8,11-tetra-azacyclotetradecane.
Additional CXCR4 antagonists that may be used in conjunction with the compositions and methods described herein include variants of plerixafor, such as a compound described in U.S. Pat. No. 5,583,131, the disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists. In some embodiments, the CXCR4 antagonist may be a compound selected from the group consisting of. 1,1′-[1,3-phenylenebis(methylene)]-bis-1,4,8,11-tetra-azacyclotetradecane; 1,1′-[1,4-phenylene-bis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; bis-zinc or bis-copper complex of 1,1′-[1,4-phenylene-bis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[3,3′-biphenylene-bis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 11,11′-[1,4-phenylene-bis-(methylene)]-bis-1,4,7,11-tetraazacyclotetradecane; 1,11′-[1,4-phenylene-bis-(methylene)]-1,4,8,11-tetraazacyclotetradecane-1, 4,7,11-tetraazacyclotetradecane; 1,1′-[2,6-pyridine-bis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1-[3,5-pyridine-bis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[2,5-thiophene-bis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[4,4′-(2,2′-bipyridine)-bis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[2,9-(1,10-phenanthroline)-bis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[1,3-phenylene-bis-(methylene)]-bis-1,4,7,10-tetraazacyclotetradecane; 1,1′-[1,4-phenylene-bis-(methylene)]-bis-1,4,7,10-tetraazacyclotetradecane; 1′-[5-nitro-1,3-phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane; 1′,1′-[2,4,5,6-tetrachloro-1,3-phenyleneis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[2,3,5,6-tetra-fluoro-1,4-phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[1,4-naphthylene-bis-(methylene)]bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[1,3-phenylenebis-(methylene)]bis-1,5,9-triazacyclododecane; 1,1′-[1,4-phenylene-bis-(methylene)]-1,5,9-triazacyclododecane; 1,1′-[2,5-dimethyl-1,4-phenylenebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[2,5-dichloro-1,4-phenylenebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[2-bromo-1,4-phenylenebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; and 1,1′-[6-phenyl-2,4-pyridinebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane.
In some embodiments, the CXCR4 antagonist is a compound described in U.S. 2006/0035829, the disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists. In some embodiments, the CXCR4 antagonist may be a compound selected from the group consisting of 3,7,11,17-tetraazabicyclo(13.3.1)heptadeca-1(17),13,15-triene; 4,7,10,17-tetraazabicyclo(13.3.1)heptadeca-1(17),13,15-triene; 1,4,7,10-tetraazacyclotetradecane; 1,4,7-triazacyclotetradecane; and 4,7,10-triazabicyclo(13.3.1)heptadeca-1(17),13,15-triene.
The CXCR4 antagonist may be a compound described in WO 2001/044229, the disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists. In some embodiments, the CXCR4 antagonist may be a compound selected from the group consisting of N-[4-(11-fluoro-1,4,7-triazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-(11,11-difluoro-1,4,7-triazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-(1,4,7-triazacyclotetradecan-2-onyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[12-(5-oxa-1,9-diazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-(11-oxa-1,4,7-triazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-(11-thia-1,4,7-triazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-(11-sulfoxo-1,4,7-triazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-(11-sulfono-1,4,7-triazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; and N-[4-(3-carboxo-1,4,7-triazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine.
Additional CXCR4 antagonists useful in conjunction with the compositions and methods described herein include compounds described in WO 2000/002870, the disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists. In some embodiments, the CXCR4 antagonist may be a compound selected from the group consisting of. N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis-(methylene)]-2-(aminomethyl)pyridine; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-N-methyl-2-(aminomethyl)pyridine; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-4-(aminomethyl)pyridine; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-3-(aminomethyl)pyridine; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-(2-aminomethyl-5-methyl)pyrazine; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-2-(aminoethyl) pyridine; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-2-(aminomethyl)thiophene; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-2-(aminomethyl)mercaptan; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-2-amino benzylamine; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-4-amino benzylamine; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-4-(aminoethyl)imidazole; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-benzylamine; N-[4-(1,4,7-triazacyclotetra-decanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[7-(4,7,10,17-tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-trienyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[7-(4,7,10-triazabicyclo[13.3.1]heptadeca-1(17),13,15-trienyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[1-(1,4,7-triazacyclotetra-decanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-[4,7,10,17-tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-trienyl]-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-[4,7,10-triazabicyclo[13.3.1]heptadeca-1(17),13,15-trienyl]-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[1,4,8,11-tetraazacyclotetradecanyl-1,4-phenylenebis(methylene)]-purine; 1-[1,4,8,11-tetraazacyclotetradecanyl-1,4-phenylenebix(methylene)]-4-phenylpiperazine; N-[4-(1,7-diazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; and N-[7-(4,10-diazabicyclo[13.3.1]heptadeca-1(17),13,15-trienyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine.
In some embodiments, the CXCR4 antagonist is a compound selected from the group consisting of: 1-[2,6-dimethoxypyrid-4-yl(methylene)]-1,4,8,11-tetraazacyclotetradecane; 1-[2-chloropyrid-4-yl(methylene)]-1,4,8,11-tetraazacyclotetradecane; 1-[2,6-dimethylpyrid-4-yl(methylene)]-1,4,8,11-tetraazacyclotetradecane; 1-[2-methylpyrid-4-yl(methylene)]-1,4,8,11-tetraazacyclotetradecane; 1-[2,6-dichloropyrid-4-yl(methylene)]-1,4,8,11-tetraazacyclotetradecane; 1-[2-chloropyrid-5-yl(methylene)]-1,4,8,11-tetraazacyclotetradecane; and 7-[4-methylphenyl (methylene)]-4,7,10,17-tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-triene.
In some embodiments, the CXCR4 antagonist is a compound described in U.S. Pat. No. 5,698,546, the disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists. In some embodiments, the CXCR4 antagonist may be a compound selected from the group consisting of: 7,7′-[1,4-phenylene-bis(methylene)]bis-3,7,11,17-tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-triene; 7,7′-[1,4-phenylene-bis(methylene)]bis[15-chloro-3,7,11,17-tetraazabicyclo [13.3.1]heptadeca-1 (17),13,15-triene]; 7,7′-[1,4-phenylene-bis(methylene)]bis[15-methoxy-3,7,11,17-tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-triene]; 7,7′-[1,4-phenylene-bis(methylene)]bis-3,7,11,17-tetraazabicyclo[13.3.1]-heptadeca-13,16-triene-15-one; 7,7′-[1,4-phenylene-bis(methylene)]bis-4,7,10,17-tetraazabicyclo[13.3.1]-heptadeca-1(17),13,15-triene; 8,8′-[1,4-phenylene-bis(methylene)]bis-4,8,12,19-tetraazabicyclo[15.3.1]nonadeca-1(19),15,17-triene; 6,6′-[1,4-phenylene-bis(methylene)]bis-3,6,9,15-tetraazabicyclo[11.3.1]pentadeca-1 (15),11,13-triene; 6,6′-[1,3-phenylene-bis(methylene)]bis-3,6,9,15-tetraazabicyclo[11.3.1]pentadeca-1 (15),11,13-triene; and 17,17′-[1,4-phenylene-bis(methylene)]bis-3,6, 14,17,23,24-hexaazatricyclo[17.3.1.11,12]tetracosa-1(23),8,10,12(24),19,21-hexaene.
In some embodiments, the CXCR4 antagonist is a compound described in U.S. Pat. No. 5,021,409, the disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists. In some embodiments, the CXCR4 antagonist may be a compound selected from the group consisting of: 2,2′-bicyclam, 6,6′-bicyclam; 3,3′-(bis-1,5,9,13-tetraaza cyclohexadecane); 3,3′-(bis-1,5,8,11,14-pentaazacyclohexadecane); methylene (or polymethylene) di-1-N-1,4,8,11-tetraaza cyclotetradecane; 3,3′-bis-1,5,9,13-tetraazacyclohexadecane; 3,3′-bis-1,5,8,11,14-pentaazacyclohexadecane; 5,5′-bis-1,4,8,11-tetraazacyclotetradecane; 2,5′-bis-1,4,8,11-tetraazacyclotetradecane; 2,6′-bis-1,4,8,11-tetraazacyclotetradecane; 11,11′-(1,2-ethanediyl)bis-1,4,8,11-tetraazacyclotetradecane; 11,11′-(1,2-propanediyl)bis-1,4,8,11-tetraazacyclotetradecane; 11,11′-(1,2-butanediyl)bis-1,4,8,11-tetraazacyclotetradecane; 11,11′-(1,2-pentanediyl)bis-1,4,8,11-tetraazacyclotetradecane; and 11,11′-(1,2-hexanediyl)bis-1,4,8,11-tetraazacyclotetradecane.
In some embodiments, the CXCR4 antagonist is a compound described in WO 2000/056729, the disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists. In some embodiments, the CXCR4 antagonist may be a compound selected from the group consisting of N-(2-pyridinylmethyl)-N′-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(6,7-dihydro-5H-cyclopenta[b]pyridin-7-yl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(1,2,3,4-tetrahydro-1-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(1-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-[(2-pyridinylmethyl)amino]ethyl]-N′-(1-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-[(1H-imidazol-2-ylmethyl)amino]ethyl]-N′-(1-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(1,2,3,4-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-[(1H-imidazol-2-ylmethyl)amino]ethyl]-N′-(1,2,3,4-tetrahydro-1-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(2-phenyl-5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N,N′-bis(2-pyridinylmethyl)-N′-(2-phenyl-5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;
N-(2-pyridinylmethyl)-N′-(5,6,7,8-tetrahydro-5-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(1H-imidazol-2-ylmethyl)-N′-(5,6,7,8-tetrahydro-5-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(1H-imidazol-2-ylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[(2-amino-3-phenyl)propyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(1H-imidazol-4-ylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(2-quinolinylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(2-(2-naphthoyl)aminoethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[(S)-(2-acetylamino-3-phenyl)propyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[(S)-(2-acetylamino-3-phenyl)propyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[3-((2-naphthalenylmethyl)amino)propyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-(S)-pyrollidinylmethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-(R)-pyrollidinylmethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[3-pyrazolylmethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-pyrrolylmethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-thiopheneylmethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-thiazolylmethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-furanylmethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-[(phenylmethyl)amino]ethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(2-aminoethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-3-pyrrolidinyl-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-4-piperidinyl-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-[(phenyl)amino]ethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(7-methoxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(6-methoxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(1-methyl-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(7-methoxy-3,4-dihydronaphthalenyl)-1-(aminomethyl)-4-benzamide; N-(2-pyridinylmethyl)-N′-(6-methoxy-3,4-dihydronaphthalenyl)-1-(aminomethyl)-4-benzamide; N-(2-pyridinylmethyl)-N′-(1H-imidazol-2-ylmethyl)-N′-(7-methoxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(8-hydroxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(1H-imidazol-2-ylmethyl)-N′-(8-hydroxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(8-Fluoro-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(1H-imidazol-2-ylmethyl)-N′-(8-Fluoro-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(5,6,7,8-tetrahydro-7-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(1H-imidazol-2-ylmethyl)-N′-(5,6,7,8-tetrahydro-7-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-[(2-naphthalenylmethyl)amino]ethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-(isobutylamino)ethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-[(2-pyridinylmethyl)amino]ethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-[(2-furanylmethyl)amino]ethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(2-guanidinoethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-[bis-[(2-methoxy)phenylmethyl]amino]ethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-[(1H-imidazol-4-ylmethyl)amino]ethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-[(1H-imidazol-2-ylmethyl)amino]ethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-(phenylureido)ethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[[N″-(n-butyl)carboxamido]methyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(carboxamidomethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[(N″-phenyl)carboxamidomethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(carboxymethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(phenylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(1H-benzimidazol-2-ylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(5,6-dimethyl-1H-benzimidazol-2-ylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine (hydrobromide salt); N-(2-pyridinylmethyl)-N′-(5-nitro-1H-benzimidazol-2-ylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[(1H)-5-azabenzimidazol-2-ylmethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N-(4-phenyl-1H-imidazol-2-ylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-(2-pyridinyl)ethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(2-benzoxazolyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(trans-2-aminocyclohexyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(2-phenylethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(3-phenylpropyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(trans-2-aminocyclopentyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-glycinamide; N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-(L)-alaninamide; N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahy dro-8-quinolinyl)-(L)-aspartamide; N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-pyrazinamide; N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-(L)-prolinamide; N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-(L)-lysinamide; N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahy dro-8-quinolinyl)-benzamide; N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-picolinamide; N′-Benzyl-N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-urea; N′-phenyl-N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-urea; N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-yl)-4-[[(2-pyridinylmethyl)amino]methyl]benzamide; N-(5,6,7,8-tetrahydro-8-quinolinyl)-4-[[(2-pyridinylmethyl)amino]methyl]benzamide; N,N′-bis(2-pyridinylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N,N′-bis(2-pyridinylmethyl)-N′-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-yl)-1,4-benzenedimethanamine; N,N′-bis(2-pyridinylmethyl)-N′-(6,7-dihydro-5H-cyclopenta[bacteriapyridin-7-yl)-1,4-benzenedimethanamine; N,N′-bis(2-pyridinylmethyl)-N′-(1,2,3,4-tetrahydro-1-naphthalenyl)-1,4-benzenedimethanamine; N,N′-bis(2-pyridinylmethyl)-N′-[(5,6,7,8-tetrahydro-8-quinolinyl)methyl]-1,4-benzenedimethanamine; N,N′-bis(2-pyridinylmethyl)-N′[(6,7-dihydro-5H-cyclopenta[bacteriapyridin-7-yl)methyl]-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N-(2-methoxyethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N-[2-(4-methoxyphenyl)ethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N,N′-bis(2-pyridinylmethyl)-1,4-(5,6,7,8-tetrahydro-8-quinolinyl)benzenedimethanamine; N-[(2,3-dimethoxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N,N′-bis(2-pyridinylmethyl)-N-[1-(N″-phenyl-N″-methylureido)-4-piperidinyl]-1,3-benzenedimethanamine; N,N′-bis(2-pyridinylmethyl)-N-[N″-p-toluenesulfonylphenylalanyl)-4-piperidinyl]-1,3-benzenedimethanamine; N,N′-bis(2-pyridinylmethyl)-N-[1-[3-(2-chlorophenyl)-5-methyl-isoxazol-4-oyl]-4-piperidinyl]-1,3-benzenedimethanamine; N-[(2-hydroxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-yl)-1,4-benzenedimethanamine; N-[(4-cyanophenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-yl)-1,4-benzenedimethanamine; N-[(4-cyanophenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(4-acetamidophenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(4-phenoxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-yl)-1,4-benzenedimethanamine; N-[(1-methyl-2-carboxamido)ethyl]-N,N′-bis(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[(4-benzyloxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-yl)-1,4-benzenedimethanamine; N-[(thiophene-2-yl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-yl)-1,4-benzenedimethanamine; N-[1-(benzyl)-3-pyrrolidinyl]-N,N′-bis(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[[1-methyl-3-(pyrazol-3-yl)]propyl]-N,N′-bis(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[1-(phenyl)ethyl]-N,N′-bis(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[(3,4-methylenedioxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-[1-benzyl-3-carboxymethyl-4-piperidinyl]-N,N′-bis(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[(3,4-methylenedioxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(3-pyridinylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-[[1-methyl-2-(2-tolyl)carboxamido]ethyl]-N,N′-bis(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[(1,5-dimethyl-2-phenyl-3-pyrazolinone-4-yl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(4-propoxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-(1-phenyl-3,5-dimethylpyrazolin-4-ylmethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[H-imidazol-4-ylmethyl]-N,N′-bis(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[(3-methoxy-4,5-methylenedioxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-[(3-cyanophenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-[(3-cyanophenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(5-ethylthiophene-2-ylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-(5-ethylthiophene-2-ylmethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(2,6-difluorophenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-[(2,6-difluorophenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(2-difluoromethoxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-(2-difluoromethoxyphenylmethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(1,4-benzodioxan-6-ylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N,N′-bis(2-pyridinylmethyl)-N-[1-(N″-phenyl-N″-methylureido)-4-piperidinyl]-1,4-benzenedimethanamine; N,N′-bis(2-pyridinylmethyl)-N-[N″-p-toluenesulfonylphenylalanyl)-4-piperidinyl]-1,4-benzenedimethanamine; N-[1-(3-pyridinecarboxamido)-4-piperidinyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[1-(cyclopropylcarboxamido)-4-piperidinyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[1-(1-phenylcyclopropylcarboxamido)-4-piperidinyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-(1,4-benzodioxan-6-ylmethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[1-[3-(2-chlorophenyl)-5-methyl-isoxazol-4-carboxamido]-4-piperidinyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[1-(2-thiomethylpyridine-3-carboxamido)-4-piperidinyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[(2,4-difluorophenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(1-methylpyrrol-2-ylmethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(2-hydroxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(3-methoxy-4,5-methylenedioxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(3-pyridinylmethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[2-(N″-morpholinomethyl)-1-cyclopentyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[(1-methyl-3-piperidinyl)propyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-(1-methylbenzimidazol-2-ylmethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[1-(benzyl)-3-pyrrol idinyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[[(1-phenyl-3-(N″-morpholino)]propyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[1-(iso-propyl)-4-piperidinyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[i-(ethoxycarbonyl)-4-piperidinyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(1-methyl-3-pyrazolyl)propyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[1-methyl-2-(N″,N″-diethylcarboxamido)ethyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[(1-methyl-2-phenylsulfonyl)ethyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(2-chloro-4,5-methylenedioxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[1-methyl-2-[N″-(4-chlorophenyl)carboxamido]ethyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(1-acetoxyindol-3-ylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-[(3-benzyloxy-4-methoxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-(3-quinolylmethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(8-hydroxy)-2-quinolylmethyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-(2-quinolylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-[(4-acetamidophenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-[1H-imidazol-2-ylmethyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-(3-quinolylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-(2-thiazolylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-(4-pyridinylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-[(5-benzyloxy)benzo[b]pyrrol-3-ylmethyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-(1-methylpyrazol-2-ylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-[(4-methyl)-1H-imidazol-5-ylmethyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[[(4-dimethylamino)-1-napthalenyl]methyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[1,5-dimethyl-2-phenyl-3-pyrazolinone-4-ylmethyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[1-[(1-acetyl-2-(R)-prolinyl]-4-piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[1-[2-acetamidobenzoyl-4-piperidinyl]-4-piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[(2-cyano-2-phenyl)ethyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-[(N″-acetyltryptophanyl)-4-piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[(N″-benzoylvalinyl)-4-piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[(4-dimethylaminophenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-(4-pyridinylmethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(1-methylbenzimadazol-2-ylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-[1-butyl-4-piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[1-benzoyl-4-piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[1-(benzyl)-3-pyrrolidinyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[(1-methyl)benzo[b]pyrrol-3-ylmethyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[1H-imidazol-4-ylmethyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[1-(benzyl)-4-piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[1-methylbenzimidazol-2-ylmethyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[(2-phenyl)benzo[b]pyrrol-3-ylmethyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[(6-methylpyridin-2-yl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(3-methyl-1H-pyrazol-5-ylmethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,3-benzenedimethanamine; N-[(2-methoxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,3-benzenedimethanamine; N-[(2-ethoxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,3-benzenedimethanamine; N-(benzyloxyethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,3-benzenedimethanamine; N-[(2-ethoxy-1-naphthalenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,3-benzenedimethanamine; N-[(6-methylpyridin-2-yl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,3-benzenedimethanamine; 1-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]guanidine; N-(2-pyridinylmethyl)-N-(8-methyl-8-azabicyclo[3.2.1]octan-3-yl)-1,4-benzenedimethanamine; 1-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]homopiperazine; 1-[[3-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]homopiperazine; trans and cis-1-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-3,5-piperidinediamine; N,N′-[1,4-Phenylenebis(methylene)]bis-4-(2-pyrimidyl)piperazine; 1-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-1-(2-pyridinyl)methylamine; 2-(2-pyridinyl)-5-[[(2-pyridinylmethyl)amino]methyl]-1,2,3,4-tetrahydroisoquinoline; 1-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-3,4-diaminopyrrolidine; 1-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-3,4-diacetylaminopyrrolidine; 8-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-2,5,8-triaza-3-oxabicyclo [4.3.0]nonane; and 8-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-2,5,8-triazabicyclo[4.3.0]nonane.
Additional CXCR4 antagonists that may be used to in conjunction with the compositions and methods described herein include those described in WO 2001/085196, WO 1999/050461, WO 2001/094420, and WO 2003/090512, the disclosures of each of which are incorporated herein by reference as they pertain to compounds that inhibit CXCR4 activity or expression.
Additional CXCR4 antagonists that may be used to in conjunction with the compositions and methods described herein include those described in WO 2015/063768, for example, analog 4F-benzoyl TN14003 (4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH2; wherein Nal=naphthylalanine, Cit=citrulline, DLys=D-lysine), also known as BL-8040 (BioLineRx, Modi'in, Israel).
Additional CXCR4 antagonists that may be used to in conjunction with the compositions and methods described herein include anti-CXCR4 antibodies (including modified forms of antibodies fragments, as described above). Anti-CXCR4 antibodies that may be used to in conjunction with the compositions and methods described herein include ulocuplumab (F7 in WO 2008/060367; also referred to as BMS-936564 or MDX-1338; Bristol-Myers Squibb), and the antibodies, including modified forms and fragments, provided in TABLE 3.
Cancer Res 19(2): 357-366)
Rep 7(1): 7305)
Oncotarget 9(35): 23890-23899)
Peptides and proteins described herein (e.g., CXCR2 agonists, such as Gro-β, Gro-β T, Gro-β N69D, Gro-β T N65D, and variants thereof) can be expressed in host cells, for example, by delivering to the host cell a nucleic acid encoding the corresponding peptide or protein. The sections that follow describe a variety of techniques that can be used for the purposes of introducing nucleic acids encoding peptides and proteins described herein to a host cell for the purposes of recombinant expression.
Techniques that can be used to introduce a polynucleotide, such as nucleic acid encoding a CXCR2 agonist, such as Gro-β, Gro-β T, Gro-β N69D, Gro-β T N65D, or a variant thereof, into a cell (e.g., a mammalian cell, such as a human cell) are known in the art. In some embodiments, electroporation can be used to permeabilize mammalian cells (e.g., human cells) by the application of an electrostatic potential to the cell of interest. Mammalian cells, such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, e.g., in Chu et al. (1987) Nucleic Acids Research 15:1311, the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection™, utilizes an applied electric field in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. Nucleofection™ and protocols useful for performing this technique are described in detail, e.g., in Distler et al. (2005) Experimental Dermatology 14:315, as well as in U.S. 2010/0317114, the disclosures of each of which are incorporated herein by reference.
Additional techniques useful for the transfection of host cells for the purposes of recombinant peptide and protein expression include the squeeze-poration methodology. This technique induces the rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membranous pores that form in response to the applied stress. This technology is advantageous in that a vector is not required for delivery of nucleic acids into a cell, such as a human cell. Squeeze-poration is described in detail, e.g., in Sharei et al. (2013) Journal of Visualized Experiments 81:e50980, the disclosure of which is incorporated herein by reference.
Lipofection represents another technique useful for transfection of cells. This method involves the loading of nucleic acids into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that exploit ionic interactions with the cell membrane to provoke the uptake of foreign nucleic acids include contacting a cell with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides so as to impart a positive charge favorable for interaction with the cell membrane are activated dendrimers (described, e.g., in Dennig (2003) Topics in Current Chemistry 228:227, the disclosure of which is incorporated herein by reference) and diethylaminoethyl (DEAE)-dextran, the use of which as a transfection agent is described in detail, for example, in Gulick et al. (1997) Current Protocols in Molecular Biology 40:I:9.2:9.2.1, the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of nucleic acids. This technology is described in detail, for example, in U.S. 2010/0227406, the disclosure of which is incorporated herein by reference.
Another useful tool for inducing the uptake of exogenous nucleic acids by cells is laserfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al. (2007) Methods in Cell Biology 82:309, the disclosure of which is incorporated herein by reference.
Microvesicles represent another potential vehicle that can be used to introduce a nucleic acid encoding a peptide or protein described herein into a host cell for the purpose of recombinant expression. In some embodiments, microvesicles that have been induced by the co-overexpression of the glycoprotein VSV-G with, e.g., a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins into a cell that subsequently catalyze the site-specific cleavage of an endogenous polynucleotide sequence so as to prepare the genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also referred to as Gesicles, for the genetic modification of eukaryotic cells is described in detail, e.g., in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract]. In: Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.
Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous nucleic acids encoding peptides and proteins described herein, such as CXCR2 agonists, including Gro-β, Gro-β T, Gro-β N69D, Gro-β T N65D, and variants thereof, into host cells for the purpose of recombinant expression. Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes may be incorporated into the genome of a cell, for example, by way of generalized or specialized transduction. These processes may occur as part of the natural replication cycle of a viral vector, and may not require added proteins or reagents in order to induce gene integration. Examples of viral vectors that may be used to introduce a nucleic acid molecule encoding a peptide or protein described herein into a host cell for recombinant expression include parvovirus, such as adeno-associated virus (AAV), retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picomavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding peptides and proteins described herein to host cells for recombinant expression purposes include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in U.S. Pat. No. 5,801,030, the disclosure of which is incorporated herein by reference as it pertains to viral vectors for use in gene delivery and recombinant protein and peptide expression.
As described herein, hematopoietic stem cell transplant therapy can be administered to a subject in need of treatment so as to populate or repopulate one or more blood cell types, such as a blood cell lineage that is deficient or defective in a patient suffering from a stem cell disorder. Hematopoietic stem and progenitor cells exhibit multi-potency, and can thus differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B-cells and T-cells). Hematopoietic stem cells are additionally capable of self-renewal, and can thus give rise to daughter cells that have equivalent potential as the mother cell, and also feature the capacity to be reintroduced into a transplant recipient whereupon they home to the hematopoietic stem cell niche and re-establish productive and sustained hematopoiesis. Thus, hematopoietic stem and progenitor cells represent a useful therapeutic modality for the treatment of a wide array of disorders in which a patient has a deficiency or defect in a cell type of the hematopoietic lineage. The deficiency or defect may be caused, for example, by depletion of a population of endogenous cells of the hematopoietic system due to administration of a chemotherapeutic agent (e.g., in the case of a patient suffering from a cancer, such as a hematologic cancer described herein). The deficiency or defect may be caused, for example, by depletion of a population of endogenous hematopoietic cells due to the activity of self-reactive immune cells, such as T lymphocytes or B lymphocytes that cross-react with self antigens (e.g., in the case of a patient suffering from an autoimmune disorder, such as an autoimmune disorder described herein). Additionally or alternatively, the deficiency or defect in cellular activity may be caused by aberrant expression of an enzyme (e.g., in the case of a patient suffering from various metabolic disorders, such as a metabolic disorder described herein).
Thus, hematopoietic stem cells can be administered to a patient defective or deficient in one or more cell types of the hematopoietic lineage in order to re-constitute the defective or deficient population of cells in vivo, thereby treating the pathology associated with the defect or depletion in the endogenous blood cell population. Hematopoietic stem and progenitor cells can be used to treat, e.g., a non-malignant hemoglobinopathy (e.g., a hemoglobinopathy selected from the group consisting of sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome). In these cases, for example, a CXCR4 antagonist and/or a CXCR2 agonist may be administered to a donor, such as a donor identified as likely to exhibit release of a population of hematopoietic stem and progenitor cells from a stem cell niche, such as the bone marrow, into circulating peripheral blood in response to such treatment. The hematopoietic stem and progenitor cells thus mobilized may then be withdrawn from the donor and administered to a patient, where the cells may home to a hematopoietic stem cell niche and re-constitute a population of cells that are damaged or deficient in the patient.
Additionally or alternatively, hematopoietic stem and progenitor cells can be used to treat an immunodeficiency, such as a congenital immunodeficiency. Additionally or alternatively, the compositions and methods described herein can be used to treat an acquired immunodeficiency (e.g., an acquired immunodeficiency selected from the group consisting of HIV and AIDS). In these cases, for example, a CXCR4 antagonist and/or a CXCR2 agonist may be administered to a donor, such as a donor identified as likely to exhibit release of a population of hematopoietic stem and progenitor cells from a stem cell niche, such as the bone marrow, into circulating peripheral blood in response to such treatment. The hematopoietic stem and progenitor cells thus mobilized may then be withdrawn from the donor and administered to a patient, where the cells may home to a hematopoietic stem cell niche and re-constitute a population of immune cells (e.g., T lymphocytes, B lymphocytes, NK cells, or other immune cells) that are damaged or deficient in the patient.
Hematopoietic stem and progenitor cells can also be used to treat a metabolic disorder (e.g., a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher Disease, Hurler Disease, sphingolipidoses, metachromatic leukodystrophy, globoid cell leukodystrophy, and cerebral adrenoleukodystrophy). In these cases, for example, a CXCR4 antagonist and/or a CXCR2 agonist may be administered to a donor, such as a donor identified as likely to exhibit release of a population of hematopoietic stem and progenitor cells from a stem cell niche, such as the bone marrow, into circulating peripheral blood in response to such treatment. The hematopoietic stem and progenitor cells thus mobilized may then be withdrawn from the donor and administered to a patient, where the cells may home to a hematopoietic stem cell niche and re-constitute a population of hematopoietic cells that are damaged or deficient in the patient.
Additionally or alternatively, hematopoietic stem or progenitor cells can be used to treat a malignancy or proliferative disorder, such as a hematologic cancer or myeloproliferative disease. In the case of cancer treatment, for example, a CXCR4 antagonist and/or a CXCR2 agonist may be administered to a donor, such as a donor identified as likely to exhibit release of a population of hematopoietic stem and progenitor cells from a stem cell niche, such as the bone marrow, into circulating peripheral blood in response to such treatment. The hematopoietic stem and progenitor cells thus mobilized may then be withdrawn from the donor and administered to a patient, where the cells may home to a hematopoietic stem cell niche and re-constitute a population of cells that are damaged or deficient in the patient, such as a population of hematopoietic cells that is damaged or deficient due to the administration of one or more chemotherapeutic agents to the patient. In some embodiments, hematopoietic stem or progenitor cells may be infused into a patient in order to repopulate a population of cells depleted during cancer cell eradication, such as during systemic chemotherapy. Exemplary hematological cancers that can be treated by way of administration of hematopoietic stem and progenitor cells in accordance with the compositions and methods described herein are acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma, myelodysplasia (MDS) as well as other cancerous conditions, including neuroblastoma.
In various embodiments, hematopoietic stem or progenitor cells can be used to treat a hematologic cancer or myeloproliferative disease that is in remission. Such a remission can be a 1st remission, 2nd remission, 3rd remission, 4th remission, or 5th remission. For example, hematopoietic stem or progenitor cells can be used to treat acute myelogenous leukemia (AML) in 1st remission, 2nd remission, 3rd remission, 4th remission, or 5th remission. As another example, hematopoietic stem or progenitor cells can be used to treat acute lymphoblastic leukemia (ALL) in 1st remission, 2nd remission, 3rd remission, 4th remission, or 5th remission. In various embodiments, hematopoietic stem or progenitor cells can be used to treat a hematologic cancer or myeloproliferative disease with less than a threshold percentage of marrow blasts. For example, hematopoietic stem or progenitor cells can be used to treat acute myelogenous leukemia (AML) in a patient with less than a threshold percentage of marrow blasts. As another example, hematopoietic stem or progenitor cells can be used to treat acute lymphoblastic leukemia (ALL) in a patient with less than a threshold percentage of marrow blasts. As another example, hematopoietic stem or progenitor cells can be used to treat myelodysplasia (MDS) in a patient with less than a threshold percentage of marrow blasts. In various embodiments, the threshold percentage of marrow blasts is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. In particular embodiments, the threshold percentage of marrow blasts is 5%. In particular embodiments, the threshold percentage of marrow blasts is 10%.
In various embodiments, hematopoietic stem or progenitor cells can be used to treat a hematologic cancer or myeloproliferative disease in a patient with less than a threshold percentage of circulating blasts. For example, hematopoietic stem or progenitor cells can be used to treat acute myelogenous leukemia (AML) in a patient with less than a threshold percentage of circulating blasts. As another example, hematopoietic stem or progenitor cells can be used to treat acute lymphoblastic leukemia (ALL) in a patient with less than a threshold percentage of circulating blasts. As another example, hematopoietic stem or progenitor cells can be used to treat myelodysplasia (MDS) in a patient with less than a threshold percentage of circulating blasts. In various embodiments, the threshold percentage of circulating blasts is 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. In particular embodiments, the threshold percentage of marrow blasts is 1%. In various embodiments, hematopoietic stem or progenitor cells can be used to treat a hematologic cancer or myeloproliferative disease in a patient with no circulating blasts. For example, hematopoietic stem or progenitor cells can be used to treat any of acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), or myelodysplasia (MDS) in a patient with no circulating blasts.
Hematopoietic stem or progenitor cells mobilized to the peripheral blood of a subject may be withdrawn (e.g., harvested or collected) from the subject by any suitable technique. For example, the hematopoietic stem or progenitor cells may be withdrawn by a blood draw. In some embodiments, hematopoietic stem or progenitor cells mobilized to a subject's peripheral blood as contemplated herein may be harvested (i.e., collected) using apheresis. In some embodiments, apheresis may be used to enrich a donor's blood with mobilized hematopoietic stem or progenitor cells.
Additional diseases that can be treated by the administration of hematopoietic stem and progenitor cells to a patient include, without limitation, adenosine deaminase deficiency and severe combined immunodeficiency, hyper immunoglobulin M syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis.
In addition, administration of hematopoietic stem and progenitor cells can be used to treat autoimmune disorders. In certain embodiments, mobilization of hematopoietic stem and progenitor cells in a subject with an autoimmune disorder using the methods disclosed herein lessens or avoids the autoimmune disorder flares that can occur during mobilization with G-CSF. Accordingly, provided herein is a method of mobilizing HSCs in a patient having an autoimmune disorder, e.g., multiple sclerosis, by administering a CXCR2 agonist, optionally in combination with a CXCR4 antagonist, wherein the risk of inducing a flare of the patient's autoimmune disorder is reduced or eliminated, e.g., as compared to mobilizing HSCs in a patient having an autoimmune disorder by administering G-CSF.
In some embodiments, upon infusion into a patient, transplanted hematopoietic stem and progenitor cells may home to a stem cell niche, such as the bone marrow, and establish productive hematopoiesis. This, in turn, can re-constitute a population of cells depleted during autoimmune cell eradication, which may occur due to the activity of self-reactive lymphocytes (e.g., self-reactive T lymphocytes and/or self-reactive B lymphocytes). Autoimmune diseases that can be treated by way of administering hematopoietic stem and progenitor cells to a patient include, without limitation, psoriasis, psoriatic arthritis, Type 1 diabetes mellitus (Type 1 diabetes), rheumatoid arthritis (RA), human systemic lupus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), lymphocytic colitis, acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, coeliac sprue-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus, dysautonomia, endometriosis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome (GBS), Hashimoto's thyroiditis, Hidradenitis suppurativa, idiopathic and/or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis, Kawasaki's disease, lichen planus, Lyme disease, Meniere disease, mixed connective tissue disease (MCTD), myasthenia gravis, neuromyotonia, opsoclonus myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyglandular syndromes, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff person syndrome, Takayasu's arteritis, temporal arteritis (also known as “giant cell arteritis”), ulcerative colitis, collagenous colitis, uveitis, vasculitis, vitiligo, vulvodynia (“vulvar vestibulitis”), and Wegener's granulomatosis.
In some embodiments, a method of harvesting hematopoietic stem cells from a human subject is provided. The method comprises administering a CXCR2 agonist and a CXCR4 antagonist to the human subject and harvesting the hematopoietic stem cells from peripheral blood of the human subject.
In some embodiments, a method of transplanting hematopoietic stem cells into a human patient in need thereof is provided. The method comprises administering a CXCR2 agonist and optionally a CXCR4 antagonist to a hematopoietic stem cell donor, harvesting the hematopoietic stem cells from peripheral blood of the donor, and transplanting the harvested hematopoietic stem cells into the patient.
In addition, the disclosure relates to a method of preventing, reducing the risk of developing, or reducing the severity of graft versus host disease (GVHD) in a patient in need thereof, wherein the method includes infusing into the patient a therapeutically effective amount of hematopoietic stem cells, wherein the hematopoietic stem cells were mobilized from bone marrow of a mammalian donor into peripheral blood by the methods described herein, e.g., including administering to the mammalian donor a CXCR2 agonist and a CXCR4 antagonist.
For example, in some embodiments, a method of transplanting hematopoietic stem cells into a human patient in need thereof is provided, where the method prevents, reduces the risk of developing, or reduces the severity of graft versus host disease (GVHD) in the patient, wherein the method includes infusing into the patient a therapeutically effective amount of hematopoietic stem cells, wherein the hematopoietic stem cells were mobilized from bone marrow of a mammalian donor into peripheral blood by the methods described herein, e.g., including administering to the mammalian donor a CXCR2 agonist and a CXCR4 antagonist. In certain embodiments, the hematopoietic stem cells infused into the patient were obtained from the donor by apheresis. (e.g., leukapheresis) after being mobilized into the peripheral blood of the donor.
In certain embodiments, the number and/or proportion of CD8+ T-cells mobilized by the methods described herein is less than the number and/or proportion of CD8+ T-cells mobilized using G-CSF or plerixafor alone. For example, the number of CD8+ T-cell mobilized can be from about 0 to about 0.6×108/kg (e.g., from about 0 to about 1×108/kg, about 0 to about 2×108/kg, about 0 to about 3×108/kg, about 0 to about 4×108/kg, about 0 to about 5×108/kg, about 1×108/kg to about 2×108/kg, about 1×108/kg to about 3×108/kg, about 1×108/kg to about 4×108/kg, about 1×108/kg to about 5×108/kg, about 1×108/kg to about 6×108/kg, about 2×108/kg to about 3×108/kg, about 2×108/kg to about 4×108/kg, about 2×108/kg to about 5×108/kg, about 2×108/kg to about 6×108/kg, about 3×108/kg to about 4×108/kg, about 3×108/kg to about 5, about 3×108/kg to about 6×108/kg, about 4×108/kg to about 5×108/kg, about 4×108/kg to about 6×108/kg, or about 5×108/kg to about 6×108/kg). In certain embodiments, CD8+ T-cells constitute from about 0.5 to about 5% of the graft (e.g., from about 0.5% to about 1%, about 0.5% to about 2%, about 0.5% to about 3%, about 0.5% to about 4%, about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 3% to about 4%, about 3% to about 5%, or about 4% to about 5%).
In certain embodiments, a method of treating a subject who has been exposed to radiation is provided. The method comprises administering a CXCR2 agonist and optionally a CXCR4 antagonist to the subject to prevent or reduce at least one symptom of acute radiation syndrome (ARS). Initial symptoms of ARS include nausea, vomiting, diarrhea, loss of appetite, fatigue, headache, fever, and skin reddening and itching. Hours or weeks later, the subject who has been exposed to radiation may develop infections, bleeding, dehydration, confusion and/or death.
Depending upon the amount of radiation to which the subject has been exposed, they may experience bone marrow syndrome (0.7 to 10 Gy); gastrointestinal syndrome (10-50 Gy) and/or neurovascular syndrome (>50 Gy). Bone marrow syndrome is characterized by aplastic anemia, which may result in infections due to low numbers of white blood cells, bleeding due to a lack of platelets, and anemia due to low numbers of red blood cells. Gastrointestinal syndrome is characterized by nausea, vomiting, diarrhea, and loss of appetite, and death with this dose of radiation is common. Neurovascular syndrome presents with neurological symptoms such as dizziness, headache or decreased level of consciousness occurring within minutes to a few hours.
Accordingly, in certain embodiments, administering a CXCR2 agonist and optionally a CXCR4 antagonist to a subject who has been exposed to radiation prevents or reduces at least one of the symptoms selected from nausea, vomiting, diarrhea, loss of appetite, fatigue, headache, fever, skin reddening and itching, infections, bleeding, anemia, dehydration, dizziness, confusion and death.
In certain embodiments, the CXCR2 agonist and optionally a CXCR4 antagonist is administered in combination with G-CSF; interleukin-3; an interleukin-3 receptor agonist; an Flt-3 ligand; WR-2721; an adenosine receptor agonist (e.g., IB-MECA); a vitamin E analog (e.g., tocopherol succinate or gamma-tocotrienol); a phytochemical; (e.g., genistein); a derivative of Salmonella fagellin (e.g., CBLB502); an adrenocortical steroid hormone (e.g., 5-androstenediol); a cyclooxygenase-2 inhibitor (meloxicam); and/or an immunomodulator (e.g., glucan).
In certain embodiments, the CXCR2 agonist and optionally a CXCR4 antagonist is administered within about 24 hours of radiation exposure, e.g., within about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 12 hours, or about 18 hours. In certain embodiments, administration is repeated daily, weekly, every two weeks, or monthly, for example, until symptoms of ARS have diminished or are resolved.
The present invention further provides methods and compositions for treating neutropenia in a patient, such as a mammalian patient (e.g., a human patient) in need thereof. Patients that are “in need” of treatment include patients that have been diagnosed by a physician as having neutropenia, e.g., exhibiting mild neutropenia (about 1000 to about 1500 neutrophils/μl of blood); moderate neutropenia (about 500 to about 1000 neutrophils/μl of blood); or severe neutropenia (below about 500 neutrophils/μl of blood). Patients “in need” of treatment also include patients that will or are currently undergoing a therapeutic regimen expected to induce neutropenia, e.g., chemotherapy.
Using the compositions and methods described herein, a C-X-C chemokine receptor type 2 (CXCR2) agonist, such as Gro-β or a variant thereof, such as a truncated form of Gro-β (e.g., Gro-β T), as described herein, optionally in combination with a C-X-C chemokine receptor type 4 (CXCR4) antagonist, such as 1,1′-[1,4-phenylenebis(methylene)]-bis-1,4,8,11-tetra-azacyclotetradecane or a variant thereof, may be administered to a patient, as described herein, in amounts sufficient in to mobilize a population of neutrophils from the bone marrow of the patient into peripheral blood.
The invention is further based, in part, on the discovery that administration of a surprisingly low dose of a CXCR2 agonist, such as Gro-β, Gro-β T, or a variant thereof, optionally in combination with a CXCR4 antagonist, such as plerixafor or a pharmaceutically acceptable salt thereof, at particular doses can provide the important clinical benefit of mobilizing neutrophils.
The CXCR4 antagonists and CXCR2 agonists described herein, using the methods described herein for the mobilization of stem and progenitor cells, likewise can be administered to a patient so as to induce mobilization of a population of neutrophils from bone marrow into peripheral blood, thereby to treat neutropenia.
In some embodiments, administration of a CXCR2 agonist, optionally in combination with a CXCR4 antagonist, may decrease the incidence of infection (e.g., as manifested by febrile neutropenia) in patients with cancer (e.g., nonmyeloid malignancies receiving) anticancer drugs (e.g., myelosuppressive drugs) associated with neutropenia (e.g., a significant incidence of severe neutropenia).
In some embodiments, administration of a CXCR2 agonist, optionally in combination with a CXCR4 antagonist, may reduce the time to neutrophil recovery following chemotherapy treatment (e.g., induction or consolidation chemotherapy) of patients suffering from cancer (e.g., a blood cancer, e.g., acute myeloid leukemia). In some embodiments, administration of a CXCR2 agonist, optionally in combination with a CXCR4 antagonist, may reduce the duration of fever following chemotherapy treatment (e.g., induction or consolidation chemotherapy) of patients suffering from cancer (e.g., a blood cancer, e.g., acute myeloid leukemia). In certain embodiments, a CXCR2 agonist, optionally in combination with a CXCR4 antagonist, may be administered to a patient in combination with G-CSF to reduce the number of days to neutrophil recovery.
In some embodiments, administration of a CXCR2 agonist, optionally in combination with a CXCR4 antagonist, may reduce the duration of neutropenia and neutropenia-related clinical sequelae, e.g., febrile neutropenia, in patients with nonmyeloid malignancies undergoing myeloablative chemotherapy followed by hematopoietic stem cell transplantation.
In some embodiments, administration of a CXCR2 agonist, optionally in combination with a CXCR4 antagonist, may reduce the incidence and duration of sequelae of neutropenia (e.g., fever, infections, oropharyngeal ulcers) in symptomatic patients with congenital neutropenia, cyclic neutropenia, or idiopathic neutropenia.
As used herein with respect to neutropenia, the term “mobilizing amount” refers to a quantity of one or more agents, such as a quantity of a CXCR4 antagonist and/or a CXCR2 agonist described herein (In some embodiments, a quantity of plerixafor, or a variant thereof, and/or Gro-β, or a variant thereof, such as a truncation of Gro-3, for example, Gro-β T) that mobilizes a population of neutrophils upon administration to a subject, such as a mammalian subject (e.g., a human subject). Exemplary mobilizing amounts of these agents include amounts sufficient to effectuate the release of a population of, for example, from about 5×103 to about 20×103 neutrophils/μL of peripheral blood, such as from about 5 to about 8 neutrophils/μL of peripheral blood, about 5 to about 10 neutrophils/μL of peripheral blood, about 5 to about 12 neutrophils/μL of peripheral blood, about 5 to about 15 neutrophils/μL of peripheral blood, about 5 to about 18 neutrophils/μL of peripheral blood, about 8 to about 10 neutrophils/μL of peripheral blood, about 8 to about 12 neutrophils/μL of peripheral blood, about 8 to about 15 neutrophils/μL of peripheral blood, or about 8 to about 18 neutrophils/μL of peripheral blood, about 8 to about 20 neutrophils/μL of peripheral blood, about 10 to about 12 neutrophils/μL of peripheral blood, about 10 to about 15 neutrophils/μL of peripheral blood, about 10 to about 18 neutrophils/μL of peripheral blood, about 10 to about 20 neutrophils/μL of peripheral blood, about 12 to about 15 neutrophils/μL of peripheral blood, about 10 to about 18 neutrophils/μL of peripheral blood, about 10 to about 20 neutrophils/μL of peripheral blood, about 12 to about 15 neutrophils/μL of peripheral blood, about 12 to about 18 neutrophils/μL of peripheral blood, about 12 to about 20 neutrophils/μL of peripheral blood, about 15 to about 18 neutrophils/μL of peripheral blood, or about 15 to about 20 neutrophils/μL of peripheral blood.
In some embodiments, the patient is the donor. In such cases, withdrawn hematopoietic stem or progenitor cells may be re-infused into the patient, such that the cells may subsequently home hematopoietic tissue and establish productive hematopoiesis, thereby populating or repopulating a line of cells that is defective or deficient in the patient (e.g., a population of megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeoblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes). In this scenario, the transplanted hematopoietic stem or progenitor cells are least likely to undergo graft rejection, as the infused cells are derived from the patient and express the same HLA class I and class II antigens as expressed by the patient.
Alternatively, the patient and the donor may be distinct. In some embodiments, the patient and the donor are related, and may, for example, be HLA-matched. In some embodiments, the patient and the donor are matched for the (i.e., carry the same) alleles for HLA-A, -B, -C, -DRB1, -DQB1, -DQA1, -DPB1, and -DPA1. As described herein, HLA-matched donor-recipient pairs have a decreased risk of graft rejection, as endogenous T cells and NK cells within the transplant recipient are less likely to recognize the incoming hematopoietic stem or progenitor cell graft as foreign, and are thus less likely to mount an immune response against the transplant. Exemplary HLA-matched donor-recipient pairs are donors and recipients that are genetically related, such as familial donor-recipient pairs (e.g., sibling donor-recipient pairs).
In some embodiments, the patient and the donor are HLA-mismatched, which occurs when at least one HLA antigen, in particular with respect to HLA-A, HLA-B and HLA-DR, is mismatched between the donor and recipient. To reduce the likelihood of graft rejection, for example, one haplotype may be matched between the donor and recipient, and the other may be mismatched.
In certain embodiments, the donor exhibits a serum creatine level that is less than 1.5 times the institution upper limit of normal (ULN) or an estimated creatine clearance (CRCL) greater than 50 mL/min using the Modification of Diet in Renal Disease Study (MDRD) equation or similar method.
In certain embodiments, the donor has a mild or moderate reduction in glomerular filtration rate (GFR). Patients with chronic kidney disease are classified into stages based upon their glomerular filtration rate (GFR). The GFR for stage 1 patients is ≥90 mL/minute/1.73 m2 (considered normal), stage 2 is 60-89 mL/minute/1.73 m2 (considered mild), stage 3 is 30-59 mL/minute/1.73 m2 (considered mild to moderate), stage 4 is 15-29 mL/minute/1.73 m2 (considered severe), and stage 5 is <15 mL/minute/1.73 m2 (kidney failure).
In certain embodiments, the patient exhibits acceptable cardiac function, defined as having a left ventricular ejection fraction of at least 45% based on most recent echocardiogram or multigated acquisition (MUGA) scan results. In certain embodiments, the patient exhibits a normal glomerular filtration rate (GFR). In certain embodiments, the patient exhibits acceptable pulmonary function defined as a diffusing capacity of the lungs for carbon monoxide (DLCO) corrected for hemoglobin that is at least 50% and having forced expiratory volume in first second (FEV1) predicted at least 50% based on most recent DLCO results. In certain embodiments, the patient exhibits a Karnofsky performance status (KPS) of 70% or greater. In certain embodiments, the patient exhibits a Hematopoietic Cell Transplantation-Comorbidity Index (HCT-CI) score of 4 or less.
In certain embodiments, the patient has not received an allogeneic transplant previously. In certain embodiments, the patient does not have an active, uncontrolled infection. In certain embodiments, the patient does not have clinical evidence of active central nervous system (CNS) tumor involvement as evidenced by documented disease on examination of spinal fluid or MRI within 45 days of the start of conditioning for receiving a graft.
Prior to infusion into a patient, such as a patient having one or more stem cell disorders described herein, hematopoietic stem cells obtained from a donor (or progeny thereof) may be genetically modified, for example, by editing (e.g., correcting, disrupting, etc.) an endogenous gene. This strategy can be used, for example, to silence the expression of one or more major histocompatibility complex genes in a hematopoietic stem cell that is allogeneic with respect to the patient, thereby reducing the likelihood of graft rejection upon transplantation.
A wide array of methods has been established for the disruption of target genes in a population of cells. In some embodiments, one such method is through the use of a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, a system that originally evolved as an adaptive defense mechanism in bacteria and archaea against viral infection. The CRISPR/Cas system includes palindromic repeat sequences within plasmid DNA and an associated Cas9 nuclease. This ensemble of DNA and protein directs site specific DNA cleavage of a target sequence by first incorporating foreign DNA into CRISPR loci. Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are in turn transcribed in a host cell to create a guide RNA, which can subsequently anneal to a target sequence and localize the Cas9 nuclease to this site. In this manner, highly site-specific Cas9-mediated DNA cleavage can be engendered in a foreign polynucleotide because the interaction that brings Cas9 within close proximity of the target DNA molecule is governed by RNA:DNA hybridization. As a result, one can theoretically design a CRISPR/Cas system to cleave any target DNA molecule of interest. This technique has been exploited in order to edit eukaryotic genomes (Hwang et al. (2013) Nature Biotechnology 31:227, the disclosure of which is incorporated herein by reference) and can be used as an efficient means of site-specifically editing hematopoietic stem cell genomes in order to cleave DNA, for example, prior to the incorporation of a gene encoding a target protein. The use of CRISPR/Cas to modulate gene expression has been described in, e.g., U.S. Pat. No. 8,697,359, the disclosure of which is incorporated herein by reference. Alternative methods for site-specifically cleaving genomic DNA prior to the incorporation of a gene of interest in a hematopoietic stem cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR/Cas system, these enzymes do not contain a guiding polynucleotide to localize to a specific target sequence. Target specificity is instead controlled by DNA binding domains within these enzymes. The use of ZFNs and TALENs in genome editing applications is described, e.g., in Urnov et al. (2010) Nature Reviews Genetics 11:636; and in Joung et al. (2013) Nature Reviews Molecular Cell Biology 14:49, the disclosure of both of which are incorporated herein by reference.
Additional genome editing techniques that can be used to incorporate polynucleotides encoding target genes into the genome of a hematopoietic stem cell include the use of ARCUS™ meganucleases that can be rationally designed so as to site-specifically cleave genomic DNA. The use of these enzymes for the incorporation of genes encoding target genes into the genome of a mammalian cell is advantageous in view of the defined structure-activity relationships that have been established for such enzymes. Single chain meganucleases can be modified at certain amino acid positions in order to create nucleases that selectively cleave DNA at desired locations, enabling the site-specific incorporation of a target gene into the nuclear DNA of a hematopoietic stem cell. These single-chain nucleases have been described extensively in, e.g., U.S. Pat. Nos. 8,021,867 and 8,445,251, the disclosures of each of which are incorporated herein by reference.
Prior to infusion into a patient, hematopoietic stem and progenitor cells may be expanded ex vivo, for example, by contacting the cells with an aryl hydrocarbon receptor antagonist. Aryl hydrocarbon receptor antagonists useful in conjunction with the compositions and methods described herein include those described in U.S. Pat. Nos. 9,580,426 and 10,351,572, the disclosures of each of which are incorporated herein by reference in their entirety.
In some embodiments, hematopoietic stem and progenitor cells mobilized by the methods contemplated herein and collected (e.g., by apheresis) from a patient for use in an autologous transplant may be expanded (e.g., by culturing the collected, mobilized hematopoietic stem and progenitor cells in the presence of an aryl hydrocarbon receptor antagonist) and subsequently infused into the same patient.
In some embodiments, hematopoietic stem and progenitor cells mobilized by the methods contemplated herein and collected (e.g., by apheresis) from a donor for use in an allogeneic transplant may be expanded (e.g., by culturing the collected, mobilized hematopoietic stem and progenitor cells in the presence of an aryl hydrocarbon receptor antagonist) and subsequently infused into a recipient patient. For example, in instances where additional hematopoietic stem and progenitor cells are desired from a particular hematopoietic stem cell donor, the mobilized hematopoietic stem and progenitor cells collected from the donor may be expanded to yield a higher dose of mobilized hematopoietic stem and progenitor cells for a recipient.
In some embodiments, aryl hydrocarbon receptor antagonists include those represented by formula (III)
In some embodiments, aryl hydrocarbon receptor antagonists useful in conjunction with the compositions and methods described herein include SR-1, represented by formula (1), below, or a salt thereof.
In some embodiments, the aryl hydrocarbon receptor antagonist is Compound 2, represented by formula (2), below, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.
In some embodiments, the aryl hydrocarbon receptor antagonist is Compound 2-ent, represented by formula (2-ent), below, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.
In some embodiments, the aryl hydrocarbon receptor antagonist is Compound 2-rac, represented by formula (2-rac), below, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.
In some embodiments, the aryl hydrocarbon receptor antagonist is a compound represented by formula (IV) or (V)
wherein:
In some embodiments, the aryl hydrocarbon receptor antagonist is compound (3), compound (4), compound (5), compound (6), compound (7), compound (8), compound (9), compound (10), compound (11), compound (12), compound (13), compound (25), compound (27), or compound (28)
or a salt thereof.
In some embodiments, the aryl hydrocarbon receptor antagonist is compound (14), compound (15), compound (16), compound (17), compound (18), compound (19), compound (20), compound (21), compound (22), compound (23), compound (24), compound (26), compound (29), or compound (30)
or a salt thereof.
The methods described herein can give rise to a population of hematopoietic stem and/or progenitor cells useful for transplant to a donor. In certain embodiments, the population comprises between about 5 to about 30 CD34+CD90+CD45RA− cells/μL of peripheral blood, such as from about 5 to about 8 CD34+CD90+CD45RA− cells/μL, about 5 to about 10 CD34+CD90+CD45RA− cells/μL, about 5 to about 12 CD34+CD90+CD45RA− cells/μL, about 5 to about 15 CD34+CD90+CD45RA− cells/μL, about 5 to about 18 CD34+CD90+CD45RA− cells/μL, about 5 to about 20 CD34+CD90+CD45RA− cells/μL, about 5 to about 22 CD34+CD90+CD45RA− cells/μL, about 5 to about 25 CD34+CD90+CD45RA− cells/μL, about 5 to about 28 CD34+CD90+CD45RA− cells/μL, about 8 to about 10 CD34+CD90+CD45RA− cells/μL, about 8 to about 12 CD34+CD90+CD45RA− cells/μL, about 8 to about 15 CD34+CD90+CD45RA− cells/μL, or about 8 to about 18 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 8 to about 20 CD34+CD90+CD45RA− cells/μL, about 8 to about 22 CD34+CD90+CD45RA− cells/μL, about 8 to about 25 CD34+CD90+CD45RA− cells/μL, about 8 to about 28 CD34+CD90+CD45RA− cells/μL, about 8 to about 30 CD34+CD90+CD45RA− cells/μL, about 10 to about 12 CD34+CD90+CD45RA− cells/μL, about 10 to about 15 CD34+CD90+CD45RA− cells/μL, about 10 to about 18 CD34+CD90+CD45RA− cells/μL, about 10 to about 20 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 12 to about 15 CD34+CD90+CD45RA− cells/μL, about 10 to about 18 CD34+CD90+CD45RA− cells/μL, about 10 to about 20 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 10 to about 22 CD34+CD90+CD45RA− cells/μL, about 10 to about 25 CD34+CD90+CD45RA− cells/μL, about 10 to about 28 CD34+CD90+CD45RA− cells/μL, about 10 to about 30 CD34+CD90+CD45RA− cells/μL of peripheral blood, about 12 to about 15 CD34+CD90+CD45RA− cells/μL, about 12 to about 18 CD34+CD90+CD45RA− cells/μL, about 12 to about 15 CD34+CD90+CD45RA− cells/μL, about 12 to about 18 CD34+CD90+CD45RA− cells/μL, about 12 to about 20 CD34+CD90+CD45RA− cells/μL, about 12 to about 22 CD34+CD90+CD45RA− cells/μL, about 12 to about 25 CD34+CD90+CD45RA− cells/μL, about 12 to about 28 CD34+CD90+CD45RA− cells/μL, about 12 to about 30 CD34+CD90+CD45RA− cells/μL, about 15 to about 18 CD34+CD90+CD45RA− cells/μL, about 15 to about 20 CD34+CD90+CD45RA− cells/μL, about 15 to about 22 CD34+CD90+CD45RA− cells/μL, or about 15 to about 25 CD34+CD90+CD45RA− cells/μL, about 15 to about 28 CD34+CD90+CD45RA− cells/μL, about 15 to about 30 CD34+CD90+CD45RA− cells/μL, about 18 to about 20 CD34+CD90+CD45RA− cells/μL, about 18 to about 22 CD34+CD90+CD45RA− cells/μL, or about 18 to about 25 CD34+CD90+CD45RA− cells/μL, about 18 to about 28 CD34+CD90+CD45RA− cells/μL, about 18 to about 30 CD34+CD90+CD45RA− cells/μL, about 20 to about 22 CD34+CD90+CD45RA− cells/μL, or about 20 to about 25 CD34+CD90+CD45RA− cells/μL, about 20 to about 28 CD34+CD90+CD45RA− cells/μL, about 20 to about 30 CD34+CD90+CD45RA− cells/μL, about 22 to about 25 CD34+CD90+CD45RA− cells/μL, about 22 to about 28 CD34+CD90+CD45RA− cells/μL, about 22 to about 30 CD34+CD90+CD45RA− cells/μL, about 25 to about 28 CD34+CD90+CD45RA− cells/μL, about 25 to about 30 CD34+CD90+CD45RA− cells/μL, or about 28 to about 30 CD34+CD90+CD45RA− cells/μL.
In certain embodiments, the population of hemopoietic stem or progenitor cells further comprises DMSO or citrate, and optionally has been frozen, e.g., using liquid nitrogen.
In certain embodiments, the disclosure relates to a method of performing apheresis on the peripheral blood of a donor to produce an apheresis product, wherein the donor has been administered a CXCR2 agonist according to the methods described herein. In certain embodiments, about 10 L to about 30 L of peripheral blood is processed. In certain embodiments, apheresis occurs over a period of time of from about 3 hours to about 5 hours. In certain embodiments, the apheresis product has a volume of about 20 to about 400 mL. In certain embodiments, CD34+ cells are present in the apheresis product in an amount of from about 100×106 cells to 600×106 cells. For example, CD34+ cells can be present in the apheresis product in an amount of from about 100×106 cells to 600×106 cells. In some embodiments, CD34+ cells are present in the apheresis product in an amount of from about 200×106 cells to 550×106 cells, from about 300×106 cells to 500×106 cells, from about 350×106 cells to 450×106 cells, or from about 375×106 cells to 400×106 cells. In some embodiments, CD34+ cells are present in the apheresis product in an amount of from about 275×106 cells to 375×106 cells, from about 300×106 cells to 350×106 cells, or from about 310×106 cells to 330×106 cells.
In some embodiments, the apheresis product described herein possesses advantageous properties as compared to an apheresis product obtained from a donor mobilized using G-CSF. For example, in some embodiments, the apheresis product described herein may prevent, reduce the risk of developing, or reduce the severity of graft versus host disease (GVHD) in a patient in need thereof as compared to an apheresis product obtained from a donor administered G-CSF, where the apheresis product obtained from the donor administered G-CSF comprises hematopoietic stem cells that were mobilized into the peripheral blood of the donor following administration to the donor of a therapeutically effective amount of G-CSF. In certain embodiments, use of an apheresis product described herein in a patient undergoing a hematopoietic stem cell transplant may provide an increased engraftment rate for the hematopoietic stem cell transplant in the patient as compared to an apheresis product comprising hematopoietic stem cells that were mobilized into the peripheral blood of a donor administered a therapeutically effective amount of G-CSF.
In certain embodiments, CD34+ cells are present in the apheresis product in an amount of from about 1×106 cells/kg body weight of the recipient to about 6×106 cells/kg body weight of the recipient. As used herein, unless indicated otherwise, units expressed as cells/kg refers to cells/kg body weight of a recipient. In certain embodiments, the recipient is about 70 kg. In certain embodiments, the recipient is about 5 kg to about 150 kg. In certain embodiments, CD34+ cells are present in an amount of from about 1×106 cells/kg to about 6×106 cells/kg. In certain embodiments, CD34+ cells are present in the apheresis product in an amount of at least about 2×106 cells/kg or at least about 4×106 cells/kg. In certain embodiments, CD34+ cells are present in an amount of from about 2×106 cells/kg to about 5.75×106 cells/kg, from about 3×106 cells/kg to about 5.5×106 cells/kg, from about 3.5×106 cells/kg to about 5.25×106 cells/kg, from about 4.0×106 cells/kg to about 5.0×106 cells/kg, from about 4.25×106 cells/kg to about 4.75×106 cells/kg, or from about 4.4×106 cells/kg to about 4.6×106 cells/kg. In certain embodiments, the CD34+ cells are viable CD34+ cells. In certain embodiments, the apheresis product was collected in one apheresis collection. In certain embodiments, the apheresis product was collected in two apheresis collections.
In certain embodiments, CD34+CD90+CD45RA− cells are present in the apheresis product in an amount of from about 0.1×106 cells/kg to about 5×106 cells/kg. In certain embodiments, CD34+CD90+ cells are present in the apheresis product in an amount of from about 0.1×106 cells/kg to about 5×106 cells/kg.
In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising CD34+CD90+CD45RA− cells in an amount of from about 0.1×106 cells/kg body weight of the recipient to about 5×106 cells/kg body weight or at a frequency of about 15 to about 75% of CD34+ cells present in the apheresis product. For example, the apheresis product isolated from a donor can comprise CD34+CD90+CD45RA− cells in an amount from about 0.2×106 cells/kg to about 4×106 cells/kg, from about 0.5×106 cells/kg to about 3×106 cells/kg, from about 1.0×106 cells/kg to about 3×106 cells/kg, from about 1.2×106 cells/kg to about 2×106 cells/kg, from about 1.4×106 cells/kg to about 1.8×106 cells/kg, or from about 1.5×106 cells/kg to about 1.7×106 cells/kg. In some embodiments, the apheresis product isolated from a donor comprises CD34+CD90+CD45RA− cells at a frequency of about 15 to about 50% of CD34+ cells present in the apheresis product. In some embodiments, the apheresis product isolated from a donor comprises CD34+CD90+CD45RA− cells at a frequency of about 20 to about 45% of CD34+ cells present in the apheresis product. In some embodiments, the apheresis product isolated from a donor comprises CD34+CD90+CD45RA− cells at a frequency of about 25 to about 40%, 30 to about 35%, about 31 to about 34%, or about 32 to about 33% of CD34+ cells present in the apheresis product. In certain embodiments, the CD34+CD90+CD45RA− cells are viable CD34+CD90+CD45RA− cells.
In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising CD34+ cells in an amount of from about 1.0×106 cells/kg body weight of the recipient to about 8.0×106 cells/kg body weight. In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising CD34+ cells in an amount of from about 1.5×106 cells/kg body weight of the recipient to about 7.0×106 cells/kg body weight. For example, the apheresis product can comprise CD34+ cells in an amount from about 1.5×106 cells/kg to about 7.0×106 cells/kg, from about 2.0×106 cells/kg to about 6.0×106 cells/kg, from about 2.5×106 cells/kg to about 5.0×106 cells/kg, from about 3.0×106 cells/kg to about 4.5×106 cells/kg, from about 3.5×106 cells/kg to about 4.0×106 cells/kg, or from about 3.6×106 cells/kg to about 3.8×106 cells/kg.
In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising CD3+ cells in an amount of from about 3.0×108 cells/kg body weight of the recipient to about 6.5×108 cells/kg body weight or at a frequency of about 35 to about 55% of CD45+ cells present in the apheresis product. In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising CD3+ cells in an amount of from about 3.3×108 cells/kg body weight of the recipient to about 6.2×108 cells/kg body weight or at a frequency of about 31.7 to about 51.1% of CD45+ cells present in the apheresis product. For example, the apheresis product can comprise CD3+ cells in an amount from about 3.5×108 cells/kg to about 6.0×108 cells/kg, from about 3.75×108 cells/kg to about 5.5×108 cells/kg, from about 4.0×108 cells/kg to about 5.0×108 cells/kg, from about 4.25×108 cells/kg to about 4.75×108 cells/kg, from about 4.35×108 cells/kg to about 4.65×108 cells/kg, or from about 4.45×108 cells/kg to about 4.55×108 cells/kg. In some embodiments, the apheresis product comprises CD3+ cells at a frequency of about 32 to about 51% of CD45+ cells present in the apheresis product. In some embodiments, the apheresis product comprises CD3+ cells at a frequency of about 34 to about 48% of CD45+ cells present in the apheresis product. In some embodiments, the apheresis product comprises CD3+ cells at a frequency of about 36 to about 46%, 38 to about 44%, about 39 to about 42%, or about 40 to about 41% of CD45+ cells present in the apheresis product.
In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising CD4+ cells in an amount of from about 3.0×108 cells/kg body weight of the recipient to about 5.0×108 cells/kg body weight or at a frequency of about 25 to about 50% of CD45+ cells present in the apheresis product. In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising CD4+ cells in an amount of from about 3.0×108 cells/kg body weight of the recipient to about 5.0×108 cells/kg body weight or at a frequency of about 27.2 to about 48.1% of CD45+ cells present in the apheresis product. For example, the apheresis product can comprise CD4+ cells in an amount from about 3.2×108 cells/kg to about 4.8×108 cells/kg, from about 3.3×108 cells/kg to about 4.6×108 cells/kg, from about 3.4×108 cells/kg to about 4.4×108 cells/kg, from about 3.5×108 cells/kg to about 4.2×108 cells/kg, from about 3.6×108 cells/kg to about 4.0×108 cells/kg, or from about 3.7×108 cells/kg to about 3.8×108 cells/kg. In some embodiments, the apheresis product comprises CD4+ cells at a frequency of about 28 to about 48% of CD45+ cells present in the apheresis product. In some embodiments, the apheresis product comprises CD4+ cells at a frequency of about 30 to about 45% of CD45+ cells present in the apheresis product. In some embodiments, the apheresis product comprises CD4+ cells at a frequency of about 31 to about 40%, 32 to about 36%, about 32 to about 35%, or about 33 to about 34% of CD45+ cells present in the apheresis product.
In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising CD8+ cells in an amount of from about 0.0×108 cells/kg body weight of the recipient to about 1.0×108 cells/kg body weight or at a frequency of about 0.5 to about 5% of CD45+ cells present in the apheresis product. In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising CD8+ cells in an amount of from about 0.0×108 cells/kg body weight of the recipient to about 0.6×108 cells/kg body weight or at a frequency of about 0.5 to about 4.8% of CD45+ cells present in the apheresis product. For example, the apheresis product can comprise CD8+ cells in an amount from about 0.1×108 cells/kg to about 0.55×108 cells/kg, from about 0.12×108 cells/kg to about 0.50×108 cells/kg, from about 0.14×108 cells/kg to about 0.40×108 cells/kg, from about 0.16×108 cells/kg to about 0.30×108 cells/kg, from about 0.18×108 cells/kg to about 0.25×108 cells/kg, or from about 0.20×108 cells/kg to about 0.22×108 cells/kg. In some embodiments, the apheresis product comprises CD8+ cells at a frequency of about 0.5 to about 4.8% of CD45+ cells present in the apheresis product. In some embodiments, the apheresis product comprises CD8+ cells at a frequency of about 0.6 to about 4.0% of CD45+cells present in the apheresis product. In some embodiments, the apheresis product comprises CD8+ cells at a frequency of about 0.8 to about 3.5%, 1.2 to about 3.0%, about 1.4 to about 2.5%, or about 1.6 to about 2.0% of CD45+ cells present in the apheresis product.
In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising CD19+ cells in an amount of from about 1.0×108 cells/kg body weight of the recipient to about 2×108 cells/kg body weight or at a frequency of about 10 to about 20% of CD45+ cells present in the apheresis product. In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising CD19+ cells in an amount of from about 1.1×108 cells/kg body weight of the recipient to about 1.9×108 cells/kg body weight or at a frequency of about 12.3 to about 19.7% of CD45+ cells present in the apheresis product. For example, the apheresis product can comprise CD19+ cells in an amount from about 1.2×108 cells/kg to about 1.9×108 cells/kg, from about 1.3×108 cells/kg to about 1.9×108 cells/kg, from about 1.4×108 cells/kg to about 1.9×108 cells/kg, from about 1.5×108 cells/kg to about 1.9×108 cells/kg, from about 1.6×108 cells/kg to about 1.9×108 cells/kg, from about 1.7×108 cells/kg to about 1.9×108 cells/kg, or from about 1.8×108 cells/kg to about 1.9×108 cells/kg. In some embodiments, the apheresis product comprises CD19+ cells at a frequency of about 0.2 to about 1.0% of CD45+ cells present in the apheresis product. In some embodiments, the apheresis product comprises CD19+ cells at a frequency of about 12.5 to about 19.5% of CD45+ cells present in the apheresis product. In some embodiments, the apheresis product comprises CD19+ cells at a frequency of about 13.0 to about 18%, 13.5 to about 17.0%, about 14.0 to about 16.0% of CD45+ cells, or about 14.5 to about 15.0% of CD45+ cells present in the apheresis product.
In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising CD56+ cells in an amount of from about 0.2×108 cells/kg body weight of the recipient to about 1.0×108 cells/kg body weight or at a frequency of about 2 to about 9% of CD45+ cells present in the apheresis product. In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising CD56+ cells in an amount of from about 0.2×108 cells/kg body weight of the recipient to about 1.0×108 cells/kg body weight or at a frequency of about 2.1 to about 8.3% of CD45+ cells present in the apheresis product. For example, the apheresis product can comprise CD56+ cells in an amount from about 0.3×108 cells/kg to about 0.9×108 cells/kg, from about 0.35×108 cells/kg to about 0.8×108 cells/kg, from about 0.40×108 cells/kg to about 0.7×108 cells/kg, from about 0.45×108 cells/kg to about 0.6×108 cells/kg, or from about 0.475×108 cells/kg to about 0.55×108 cells/kg. In some embodiments, the apheresis product comprises CD56+ cells at a frequency of about 2.5 to about 8.0% of CD45+ cells present in the apheresis product. In some embodiments, the apheresis product comprises CD56+ cells at a frequency of about 3.0 to about 7.0% of CD45+ cells present in the apheresis product. In some embodiments, the apheresis product comprises CD56+ cells at a frequency of about 3.5 to about 6.5%, 4.0 to about 6.0%, or about 4.5 to about 5.5% of CD45+ cells present in the apheresis product.
In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising Treg cells at a frequency of about 0.5 to about 6% of CD4+ T cells. In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising Treg cells at a frequency of about 0.7 to about 5.5% of CD4+ T cells. In some embodiments, the apheresis product comprises Treg at a frequency of about 1.0 to about 5.0% of CD4+ T cells present in the apheresis product. In some embodiments, the apheresis product comprises Tregs at a frequency of about 1.5 to about 4.5%, 1.75 to about 4.0%, about 2.0 to about 3.5%, about 2.25 to about 3.0%, or about 2.5 to about 2.75% of CD4+ T cells present in the apheresis product.
In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising NKT cells at a frequency of about 0.5 to about 3% of CD3+ T cells. In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising NKT cells at a frequency of about 0.6 to about 2.7% of CD3+ T cells. In some embodiments, the apheresis product comprises NKT cells at a frequency of about 0.7 to about 2.5% of CD3+ T cells present in the apheresis product. In some embodiments, the apheresis product comprises NKT cells at a frequency of about 0.8 to about 2.4%, 0.9 to about 2.3%, about 1.0 to about 2.2%, about 1.1 to about 2.1%, about 1.2 to about 2.0%, about 1.3 to about 1.9%, about 1.4 to about 1.8%, or about 1.5 to about 1.7% of CD3+ T cells present in the apheresis product.
In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising iNKT cells at a frequency of about 0.00 to about 0.1% of CD3+ T cells. In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising iNKT cells at a frequency of about 0.00 to about 0.03% of CD3+ T cells. In some embodiments, the apheresis product comprises iNKT cells at a frequency of about 0.001 to about 0.025%, 0.005 to about 0.020%, about 0.007 to about 0.015%, about 0.008 to about 0.012%, or about 0.009 to about 0.011% of CD3+ T cells present in the apheresis product.
In certain embodiments, the disclosure relates to an apheresis product isolated from a donor comprising CD14+ monocytes. CD14+ monocytes possess immunosuppressive effects such that when the apheresis product is administered, the CD14+ monocytes exert the immunosuppressive effects, thereby preventing or reducing the severity of GvHD. In various embodiments, CD14+ cells are present in the apheresis product in an amount of from about 1×106 cells/kg body weight of the recipient to 1000×106 cells/kg body weight. For example, CD34+ cells can be present in the apheresis product in an amount of from about 1×106 cells/kg body weight to 1000×106 cells/kg body weight. In some embodiments, CD34+ cells are present in the apheresis product in an amount of from about 10×106 cells/kg body weight to 900×106 cells/kg body weight, from about 100×106 cells/kg body weight to 800×106 cells/kg body weight, from about 250×106 cells/kg body weight to 750×106 cells/kg body weight, from about 375×106 cells/kg body weight to 600×106 cells/kg body weight, or from about 425×106 cells/kg body weight to 550×106 cells/kg body weight.
In certain embodiments, the concentration of white blood cells is higher in the apheresis product than in the peripheral blood of the donor. In certain embodiments, the apheresis product further comprises an anticoagulant. In certain embodiments, citrate in an amount above physiological levels. In certain embodiments, the anticoagulant is heparin. In certain embodiments, the volume of the product is from about 20 to about 400 mL.
In certain embodiments, the disclosure relates to a method of treating a stem cell disorder, the method comprising administering the apheresis product or population of hemopoietic stem or progenitor cells described herein. It has been surprisingly discovered that the composition of apheresis products obtained using the methods described herein are unexpectedly potent. In certain embodiments, apheresis products obtained from a donor in which apheresis yields are lower than 2×106 CD34+ cells/kg body weight (e.g., five times lower, 10 times lower) are still efficacious.
For cases in which the donor is administered both a CXCR4 antagonist and a CXCR2 agonist, the two agents may be administered to the donor substantially simultaneously (e.g., at the same time or one immediately after the other). In some embodiments, the CXCR4 antagonist and the CXCR2 agonist may be co-formulated with one another and administered in the same pharmaceutical composition as a single dose. Alternatively, the CXCR4 antagonist and the CXCR2 agonist may be formulated in distinct pharmaceutical compositions and administered separately but substantially simultaneously to the donor.
In some embodiments, the CXCR2 agonist is administered to the donor after administration of the CXCR4 antagonist. In some embodiments, the CXCR2 agonist is administered to the donor within about 12 hours (e.g., within about 10, 8, 6, 4, 2, or 1 hour) of administration of the CXCR4 antagonist. In some embodiments, the CXCR2 agonist is administered to the donor from about 30 minutes to about 180 minutes after administration of the CXCR4 antagonist, such as from about 40 minutes to about 160 minutes, about 50 minutes to about 150 minutes, about 60 minutes to about 140 minutes, about 70 minutes to about 130 minutes, about 60 minutes to about 120 minutes, about 70 minutes to about 110 minutes, or about 80 minutes to about 100 minutes (e.g., about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, about 150 minutes, about 155 minutes, about 160 minutes, about 165 minutes, about 170 minutes, about 175 minutes, or about 180 minutes after administration of the CXCR4 antagonist). In some embodiments, the CXCR2 agonist is administered about 2 hours after the CXCR4 antagonist.
In certain embodiments, peripheral blood containing a population of hematopoietic stem or progenitor cells is isolated from the donor from about 10 minutes to about 60 minutes following completion of the administration of the CXCR4 antagonist and the CXCR2 agonist (e.g., about 10 minutes to about 1.9 hours, about 20 minutes to about 1.8 hours, about 25 minutes to about 1.7 hours, about 30 minutes to about 1.6 hours, about 40 minutes to about 1.5 hours (e.g., about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or about 120 minutes following completion of the administration of the CXCR4 antagonist and the CXCR2 agonist). In certain embodiments, peripheral blood containing a population of hematopoietic stem or progenitor cells is isolated from the donor from about 10 minutes to about 20 minutes following completion of the administration of the CXCR4 antagonist and the CXCR2 agonist (e.g., about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20 minutes following completion of the administration of the CXCR4 antagonist and the CXCR2 agonist). In some embodiments, isolation of the population of hematopoietic stem or progenitor cells commences about 15 minutes following completion of the administration of the CXCR4 antagonist and the CXCR2 agonist.
In certain embodiments, peripheral blood containing a population of hematopoietic stem or progenitor cells is isolated from the donor between about 2 hours to about 10 hours after administration of the CXCR2 agonist and/or the CXCR4 antagonist, e.g., between about 2 hours to about 3 hours, between about 2 hours to about 4 hours, between about 2 hours to about 5 hours, between about 2 hours to about 6 hours, between about 2 hours to about 7 hours, between about 2 hours about 8 hours, between about 2 hours to about 9 hours, between about 3 hours to about 4 hours, between about 3 hours to about 5 hours, between about 3 hours to about 6 hours, between about 3 hours to about 7 hours, between about 3 hours about 8 hours, between about 3 hours to about 9 hours, between about 3 hours to about 10 hours, between about 4 hours to about 5 hours, between about 4 hours to about 6 hours, between about 4 hours to about 7 hours, between about 4 hours about 8 hours, between about 4 hours to about 9 hours, between about 4 hours to about 10 hours, between about 5 hours to about 6 hours, between about 5 hours to about 7 hours, between about 5 hours about 8 hours, between about 5 hours to about 9 hours, between about 5 hours to about 10 hours, between about 6 hours to about 7 hours, between about 6 hours about 8 hours, between about 6 hours to about 9 hours, between about 6 hours to about 10 hours, between about 7 hours to 8 hours, between about 7 hours to about 9 hours, between about 7 hours to about 10 hours, between about 8 hours to about 9 hours, between about 8 hours to about 10 hours, or between about 9 hours to about 10 hours.
In some embodiments, the population of hematopoietic stem or progenitor cells is isolated from the donor over a period of from about 15 minutes to about 3 hours, such as from about 20 minutes to about 4.5 hours, about 30 minutes to about 4 hours, about 40 minutes to about 3.5 hours, about 50 minutes to about 3 hours, or about 1 hour to about 2 hours (e.g., over a period of about 15 minutes, about 20 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, or about 360 minutes). In some embodiments, the population of hematopoietic stem and progenitor cells may be isolated from the donor over a period of from about 30 minutes to about 1 hour (e.g., over a period of about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes).
In some embodiments, the hematopoietic stem or progenitor cells may be harvested by apheresis. In some embodiments, the hematopoietic stem or progenitor cells may be harvested by drawing peripheral blood from the donor (i.e., subject).
In certain embodiments, the apheresis product is collected in one apheresis collection. In certain embodiments, the apheresis product is collected in two apheresis collections. In certain embodiments, the time from the administration of a CXCR2 agonist or a CXCR4 antagonist to the time of completion of the apheresis collection is no more than about 72 hours, no more than about 60 hours, no more than about 48 hours, no more than about 36 hours, no more than about 24 hours, no more that about 18 hours, no more than about 16 hours, no more than about 14 hours, not more than about 12 hours, no more than about 8 hours, no more than about 6 hours.
The CXCR4 antagonists and CXCR2 agonists described herein may be administered to a patient by a variety of routes, such as intravenously, subcutaneously, intramuscularly, or parenterally. The most suitable route for administration in any given case will depend on the particular agent administered, the patient, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the patient's age, body weight, sex, severity of the diseases being treated, the patient's diet, and the patient's excretion rate.
The CXCR2 agonists and CXCR4 antagonists contemplated herein may each be formulated into a pharmaceutical composition for administration to a subject, such as a mammalian subject (e.g., a human subject). For instance, contemplated herein are pharmaceutical compositions comprising a CXCR2 agonist and/or a CXCR4 antagonist, in admixture with one or more suitable diluents, carriers, and/or excipients. Pharmaceutical compositions may include sterile aqueous suspensions. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington: The Science and Practice of Pharmacy (2012, 22nd ed.) and in The United States Pharmacopeia: The National Formulary (2015, USP 38 NF 33), the disclosure of which is incorporated herein by reference in its entirety.
A pharmaceutical composition may be administered to a subject, such as a human subject, alone or in combination with pharmaceutically acceptable carriers, the proportion of which may be determined by the quantity of active pharmaceutical ingredient (i.e., CXCR2 agonist and/or a CXCR4 antagonist), chosen route of administration, and standard pharmaceutical practice.
Administration and Dosing of CXCR2 Agonists and/or CXCR4 Antagonists
Contemplated CXCR2 agonists and CXCR4 antagonists, may be administered to a subject, such as a mammalian subject (e.g., a human subject), by one or more routes of administration. For instance, contemplated CXCR2 agonists and CXCR4 antagonists may be administered to a subject by intravenous, intraperitoneal, intramuscular, intraarterial, or subcutaneous infusion, among others.
Contemplated CXCR2 agonists can be administered in an amount of between about 0.001 mg/kg to about 0.1 mg/kg body weight of the subject, for example, between about 0.05 mg/kg and about 0.1 mg/kg, between about 0.05 mg/kg about 0.07 mg/kg, and between about 0.07 mg/kg and about 0.1 mg/kg.
Contemplated CXCR2 agonists can be administered in an amount of between about 0.001 mg/kg and less than about 0.05 mg/kg body weight of the subject, for example, between about 0.0015 mg/kg and less than about 0.05 mg/kg, between about 0.002 mg/kg and less than about 0.05 mg/kg, between about 0.025 mg/kg and less than about 0.05 mg/kg, between about 0.003 mg/kg and less than about 0.05 mg/kg, between about 0.0035 mg/kg and less than about 0.05 mg/kg, between about 0.004 mg/kg and less than about 0.05 mg/kg, between about 0.0045 mg/kg and less than about 0.05 mg/kg, between about 0.005 mg/kg and less than about 0.05 mg/kg, between about 0.0055 mg/kg and less than about 0.05 mg/kg, between about 0.006 mg/kg and less than about 0.05 mg/kg, between about 0.0065 mg/kg and less than about 0.05 mg/kg, between about 0.007 mg/kg and less than about 0.05 mg/kg, between about 0.075 mg/kg and less than about 0.05 mg/kg, between about 0.008 mg/kg and less than about 0.05 mg/kg, between about 0.0085 mg/kg and less than about 0.05 mg/kg, between about 0.009 mg/kg and less than about 0.05 mg/kg, between about 0.0095 mg/kg and less than about 0.05 mg/kg, between about 0.01 mg/kg and less than about 0.05 mg/kg, between about 0.015 mg/kg and less than about 0.05 mg/kg, between about 0.02 and less than about 0.05 mg/kg, between about 0.025 mg/kg and less than about 0.05 mg/kg; between about 0.03 mg/kg and less than about 0.05 mg/kg, between about 0.035 mg/kg and less than about 0.05 mg/kg, between about 0.04 mg/kg and less than about 0.05 mg/kg, and between about 0.045 mg/kg and less than about 0.05 mg/kg.
In certain embodiments, the CXCR2 agonists can be administered in an amount of between about 0.001 mg/kg and about 0.049 mg/kg, for example, between about 0.001 mg/kg and about 0.045 mg/kg, between about 0.001 mg/kg and about 0.04 mg/kg, between about 0.001 mg/kg and about 0.035 mg/kg, between about 0.001 mg/kg and about 0.03 mg/kg, between about 0.001 mg/kg and about 0.025 mg/kg, between about 0.001 mg/kg and about 0.02 mg/kg, between about 0.001 mg/kg and about 0.015 mg/kg, between about 0.001 mg/kg and about 0.01 mg/kg.
In certain embodiments, the CXCR2 agonists can be administered in an amount of between about 0.01 mg/kg and less than about 0.05 mg/kg, between about 0.01 mg/kg and about 0.049 mg/kg, between about 0.01 mg/kg and about 0.045 mg/kg, between about 0.01 mg/kg and about 0.04 mg/kg, between about 0.01 mg/kg and about 0.035 mg/kg, between about 0.01 mg/kg and about 0.03 mg/kg, between about 0.01 mg/kg and about 0.025 mg/kg, between about 0.01 mg/kg and about 0.02 mg/kg, and between about 0.01 mg/kg and about 0.015 mg/kg.
In certain embodiments, the CXCR2 agonists can be administered in an amount of between about 0.02 mg/kg and less than about 0.05 mg/kg, between about 0.02 mg/kg and about 0.049 mg/kg, between about 0.02 mg/kg and about 0.045 mg/kg, between about 0.02 mg/kg and about 0.04 mg/kg, between about 0.02 mg/kg and about 0.035 mg/kg, between about 0.02 mg/kg and about 0.03 mg/kg, and between about 0.02 mg/kg and about 0.025 mg/kg.
In certain embodiments, the CXCR2 agonist is administered at a dose of about 0.03 mg/kg.
In certain embodiments, the CXCR2 agonist is administered at a fixed dose of from about 1 mg to about 8 mg. For example, the CXCR2 agonist can be administered at a fixed dose of from about 1 mg to about 1.5 mg, about 1 mg to about 2 mg, about 1 mg to about 2.5 mg, about 1 mg to about 3 mg, about 1 mg to about 3.5 mg, about 1 mg to about 4 mg, about 1 mg to about 4.5 mg, about 1 mg to about 5 mg, about 1 mg to about 5.5 mg, about 1 mg to about 6 mg, about 1 mg to about 6.5 mg, about 1 mg to about 7 mg, about 1 mg to about 7.5 mg, about 1.5 mg to about 2 mg, about 1.5 mg to about 2.5 mg, about 1.5 mg to about 3 mg, about 1.5 mg to about 3.5 mg, about 1.5 mg to about 4 mg, about 1.5 mg to about 4.5 mg, about 1.5 mg to about 5 mg, about 1.5 mg to about 5.5 mg, about 1.5 mg to about 6 mg, about 1.5 mg to about 6.5 mg, about 1.5 mg to about 7 mg, about 1.5 mg to about 7.5 mg, about 1.5 mg to about 8 mg, about 2 mg to about 2.5 mg, about 2 mg to about 3 mg, about 2 mg to about 3.5 mg, about 2 mg to about 4 mg, about 2 mg to about 4.5 mg, about 2 mg to about 5 mg, about 2 mg to about 5.5 mg, about 2 mg to about 6 mg, about 2 mg to about 6.5 mg, about 2 mg to about 7 mg, about 2 mg to about 7.5 mg, about 2 mg to about 8 mg, about 2.5 mg to about 3 mg, about 2.5 mg to about 3.5 mg, about 2.5 mg to about 4 mg, about 2.5 mg to about 4.5 mg, about 2.5 mg to about 5 mg, about 2.5 mg to about 5.5 mg, about 2.5 mg to about 6 mg, about 2.5 mg to about 6.5 mg, about 2.5 mg to about 7 mg, about 2.5 mg to about 7.5 mg, about 2.5 mg to about 8 mg, about 3 mg to about 3.5 mg, about 3 mg to about 4 mg, about 3 mg to about 4.5 mg, about 3 mg to about 5 mg, about 3 mg to about 5.5 mg, about 3 mg to about 6 mg, about 3 mg to about 6.5 mg, about 3 mg to about 7 mg, about 3 mg to about 7.5 mg, about 3 mg to about 8 mg, about 3.5 mg to about 4 mg, about 3.5 mg to about 4.5 mg, about 3.5 mg to about 5 mg, about 3.5 mg to about 5.5 mg, about 3.5 mg to about 6 mg, about 3.5 mg to about 6.5 mg, about 3.5 mg to about 7 mg, about 3.5 mg to about 7.5 mg, about 3.5 mg to about 8 mg, about 4 mg to about 4.5 mg, about 4 mg to about 5 mg, about 4 mg to about 5.5 mg, about 4 mg to about 6 mg, about 4 mg to about 6.5 mg, about 4 mg to about 7 mg, about 4 mg to about 7.5 mg, about 4 mg to about 8 mg, about 4.5 mg to about 5 mg, about 4.5 mg to about 5.5 mg, about 4.5 mg to about 6 mg, about 4.5 mg to about 6.5 mg, about 4.5 mg to about 7 mg, about 4.5 mg to about 7.5 mg, about 4.5 mg to about 8 mg, about 5 mg to about 5.5 mg, about 5 mg to about 6 mg, about 5 mg to about 6.5 mg, about 5 mg to about 7 mg, about 5 mg to about 7.5 mg, about 5 mg to about 8 mg, about 5.5 mg to about 6 mg, about 5.5 mg to about 6.5 mg, about 5.5 mg to about 7 mg, about 5.5 mg to about 7.5 mg, about 5.5 mg to about 8 mg, about 6 mg to about 6.5 mg, about 6 mg to about 7 mg, about 6 mg to about 7.5 mg, about 6 mg to about 8 mg, about 6.5 mg to about 7 mg, about 6.5 mg to about 7.5 mg, about 6.5 mg to about 8 mg, about 7 mg to about 7.5 mg, about 7 mg to about 8 mg, about 7.5 mg to 8 mg. In certain embodiments, the CXCR2 agonist is administered at a fixed dose of about 1.3 mg, 2.5 mg or 5.5 mg.
In certain embodiments, the CXCR2 agonists can be administered in an amount of about 0.001 mg/kg per day, about 0.0015 mg/kg per day, about 0.002 mg/kg per day, about 0.0025 mg/kg per day, about 0.003 mg/kg per day, about 0.0035 mg/kg per day, about 0.004 mg/kg per day, about 0.0045 mg/kg per day, about 0.005 mg/kg per day, about 0.0055 mg/kg per day, about 0.006 mg/kg per day, about 0.0065 mg/kg per day, about 0.007 mg/kg per day, about 0.0075 mg/kg per day, about 0.008 mg/kg per day, about 0.0085 mg/kg per day, about 0.009 mg/kg per day, about 0.0095 mg/kg per day, about 0.01 mg/kg per day, about 0.015 mg/kg per day, about 0.02 mg/kg per day, about 0.025 mg/kg per day, about 0.03 mg/kg per day, about 0.035 mg/kg per day, about 0.04 mg/kg per day, about 0.045 mg/kg per day, about 0.049 mg/kg per day, or less than about 0.05 mg/kg per day. In certain embodiments, the CXCR2 agonist is administered at a fixed dose of about 1.3 mg per day, 2.5 mg per day, or 5.5 mg per day.
In certain embodiments, the CXCR2 agonist is administered at a fixed dose of from about 1 mg to about 8 mg per day. For example, the CXCR2 agonist can be administered at a fixed dose of from about 1 mg per day, about 1.5 mg per day, about 2 mg per day, about 2.5 mg per day, about 3.5 mg per day, about 4 mg per day, about 5 mg per day, about 5.5 mg per day, about 6 mg per day, about 6.5 mg per day, about 7 mg per day, about 7.5 mg per day, or about 8 mg per day.
In some embodiments, the CXCR4 antagonist is plerixafor or a pharmaceutically acceptable salt thereof. In some embodiments, the CXCR4 antagonist (e.g., plerixafor or a pharmaceutically acceptable salt thereof) is administered subcutaneously to the donor. In some embodiments, the CXCR4 antagonist (e.g., plerixafor or a pharmaceutically acceptable salt thereof) is administered to the donor at a dose of from about 50 μg/kg to about 500 μg/kg body weight of the donor, such as a dose of about 50 μg/kg, 55 μg/kg, 60 μg/kg, 65 μg/kg, 70 μg/kg, 75 μg/kg, 80 μg/kg, 85 μg/kg, 90 μg/kg, 95 μg/kg, 100 μg/kg, 105 μg/kg, 110 μg/kg, 115 μg/kg, 120 μg/kg, 125 μg/kg, 130 μg/kg, 135 μg/kg, 140 μg/kg, 145 μg/kg, 150 μg/kg, 155 μg/kg, 160 μg/kg, 165 μg/kg, 170 μg/kg, 175 μg/kg, 180 μg/kg, 185 μg/kg, 190 μg/kg, 195 μg/kg, 200 μg/kg, 205 μg/kg, 210 μg/kg, 215 μg/kg, 220 μg/kg, 225 μg/kg, 230 μg/kg, 235 μg/kg, 240 μg/kg, 245 μg/kg, 250 μg/kg, 255 μg/kg, 260 μg/kg, 265 μg/kg, 270 μg/kg, 275 μg/kg, 280 μg/kg, 285 μg/kg, 290 μg/kg, 295 μg/kg, 300 μg/kg, 305 μg/kg, 310 μg/kg, 315 μg/kg, 320 μg/kg, 325 μg/kg, 330 μg/kg, 335 μg/kg, 340 μg/kg, 345 μg/kg, 350 μg/kg, 355 μg/kg, 360 μg/kg, 365 μg/kg, 370 μg/kg, 375 μg/kg, 380 μg/kg, 385 μg/kg, 390 μg/kg, 395 μg/kg, 400 μg/kg, 405 μg/kg, 410 μg/kg, 415 μg/kg, 420 μg/kg, 425 μg/kg, 430 μg/kg, 435 μg/kg, 440 μg/kg, 445 μg/kg, 450 μg/kg, 455 μg/kg, 460 μg/kg, 465 μg/kg, 470 μg/kg, 475 μg/kg, 480 μg/kg, 485 μg/kg, 490 μg/kg, 495 μg/kg, or 500 μg/kg. In some embodiments, the CXCR4 antagonist (e.g., plerixafor or a pharmaceutically acceptable salt thereof) is administered to the donor at a dose of from about 200 μg/kg to about 300 μg/kg, such as a dose of about 240 μg/kg.
For example, in some embodiments, the CXCR4 antagonist (e.g., plerixafor or a pharmaceutically acceptable salt thereof) is administered to the donor at a dose of from about 50 μg/kg per day to about 500 μg/kg per day, such as a dose of about 50 μg/kg per day, 55 μg/kg per day, 60 μg/kg per day, 65 μg/kg per day, 70 μg/kg per day, 75 μg/kg per day, 80 μg/kg per day, 85 μg/kg per day, 90 μg/kg per day, 95 μg/kg per day, 100 μg/kg per day, 105 μg/kg per day, 110 μg/kg per day, 115 μg/kg per day, 120 μg/kg per day, 125 μg/kg per day, 130 μg/kg per day, 135 μg/kg per day, 140 μg/kg per day, 145 μg/kg per day, 150 μg/kg per day, 155 μg/kg per day, 160 μg/kg per day, 165 μg/kg per day, 170 μg/kg per day, 175 μg/kg per day, 180 μg/kg per day, 185 μg/kg per day, 190 μg/kg per day, 195 μg/kg per day, 200 μg/kg per day, 205 μg/kg per day, 210 μg/kg per day, 215 μg/kg per day, 220 μg/kg per day, 225 μg/kg per day, 230 μg/kg per day, 235 μg/kg per day, 240 μg/kg per day, 245 μg/kg per day, 250 μg/kg per day, 255 μg/kg per day, 260 μg/kg per day, 265 μg/kg per day, 270 μg/kg per day, 275 μg/kg per day, 280 μg/kg per day, 285 μg/kg per day, 290 μg/kg per day, 295 μg/kg per day, 300 μg/kg per day, 305 μg/kg per day, 310 μg/kg per day, 315 μg/kg per day, 320 μg/kg per day, 325 μg/kg per day, 330 μg/kg per day, 335 μg/kg per day, 340 μg/kg per day, 345 μg/kg per day, 350 μg/kg per day, 355 μg/kg per day, 360 μg/kg per day, 365 μg/kg per day, 370 μg/kg per day, 375 μg/kg per day, 380 μg/kg per day, 385 μg/kg per day, 390 μg/kg per day, 395 μg/kg per day, 400 μg/kg per day, 405 μg/kg per day, 410 μg/kg per day, 415 μg/kg per day, 420 μg/kg per day, 425 μg/kg per day, 430 μg/kg per day, 435 μg/kg per day, 440 μg/kg per day, 445 μg/kg per day, 450 μg/kg per day, 455 μg/kg per day, 460 μg/kg per day, 465 μg/kg per day, 470 μg/kg per day, 475 μg/kg per day, 480 μg/kg per day, 485 μg/kg per day, 490 μg/kg per day, 495 μg/kg per day, or 500 μg/kg per day. In some embodiments, the CXCR4 antagonist (e.g., plerixafor or a pharmaceutically acceptable salt thereof) is administered to the donor at a dose of from about 200 μg/kg per day to about 300 μg/kg per day, such as a dose of about 240 μg/kg per day. In some embodiments, the CXCR4 antagonist may be administered as a single dose. In other embodiments, the CXCR4 antagonist may be administered as two or more doses.
Contemplated CXCR2 agonists and optionally CXCR4 antagonists may be administered to a subject in one or more doses. For example, a CXCR2 agonist and optionally a CXCR4 antagonist may be administered as a single dose or in two, three, four, five, or more doses. When multiple doses are administered, subsequent doses may be provided during the same day or one or more days, weeks, months, or years following the initial dose. For instance, the contemplated CXCR2 agonists and optionally CXCR4 antagonists described herein may be administered to a subject, such as a human subject one or more times daily, weekly, monthly, or yearly, depending on such factors as, for instance, the subject's age, body weight, sex, the subject's diet, and the subject's excretion rate.
In certain embodiments, the contemplated CXCR2 agonists and optionally CXCR4 antagonists are each administered in a single dose once per day. In certain embodiments, the contemplated CXCR2 agonists and optionally CXCR4 antagonists are each administered on two consecutive days. In certain embodiments, the contemplated CXCR2 agonists and CXCR4 antagonists are each administered in a single dose once per day on two consecutive days. In certain embodiments, administration of the contemplated CXCR2 agonists and optionally CXCR4 antagonists on two consecutive days improves the yield of CD34+ cells from the donor. In certain embodiments, administration of the contemplated CXCR2 agonists and optionally CXCR4 antagonists on two consecutive days improves the yield of neutrophils from the patient. In certain embodiments, administration of the contemplated CXCR2 agonists and optionally CXCR4 antagonists on two consecutive days allows for sufficient numbers of CD34+ cells to be obtained from a donor for transplantation, where administration on one day is insufficient. In certain embodiments, the donor may have a condition which results in insufficient mobilization of stem cells from the bone marrow.
In certain embodiments, administration of the CXCR2 agonists and CXCR4 antagonists results in a minimal change in neutrophil activation markers. Examples of neutrophil activation markers include L-selectin, CD11b, CD18, and CD66. In contrast G-CSF, the traditional therapy of choice for mobilization of neutrophils, enhances neutrophil activation, which is problematic, for example, in patients with sickle cell disease where activated neutrophils adhere to the endothelium, thereby increasing the risk of severe and life-threatening complications such as vaso-occlusive crises. Accordingly, in certain embodiments, administration of a CXCR2 agonist and/or CXCR4 antagonists results in less than a 2.5 fold change (e.g., less than a 2 fold change, less than a 1.5 fold change or less than a 1 fold change) in a neutrophil activation marker, such as L-selectin, CDT Tb, CD18, or CD66.
Hematopoietic stem or progenitor cells and pharmaceutical compositions described herein may be administered to a subject in one or more doses. When multiple doses are administered, subsequent doses may be provided one or more days, weeks, months, or years following the initial dose. For instance, the hematopoietic stem cells and pharmaceutical compositions described herein may be administered to a subject, such as a human subject suffering from one or more diseases, conditions, or disorders described herein, one or more times daily, weekly, monthly, or yearly, depending on such factors as, for instance, the subject's age, body weight, sex, severity of the diseases being treated, the subject's diet, and the subject's excretion rate.
The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of the invention.
Mobilized peripheral blood grafts are currently the predominant source of hematopoietic stem and progenitor cells (HSPC) for both autologous and allogeneic transplantation. The most common clinical hematopoietic stem cell mobilization protocol is five days of Filgrastim (G-CSF). This regimen requires daily injections, has been associated with bone pain and often results in unpredictably low yields. In this example, data are presented to demonstrate particular treatment protocols using MGTA-145 and plerixafor, which induces increased mobilization of CD34+ cells, CD34+CD90+CD45RA− cells, neutrophils, and white blood cells (WBCs).
Patients were treated with their respective treatments (e.g., placebo, MGTA-145, plerixafor, or MGTA-145+plerixafor). Patients treated with the combination therapy of MGTA-145 and plerixafor either underwent simultaneous dosing of the combination therapy or staggered dosing (MGTA-145 given two hours after plerixafor).
A peripheral blood sample was collected from patients immediately prior to and 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after administration of both agents (for simultaneous dosing) and after administration of MGTA-145 (staggered dosing) and analyzed by multicolor flow cytometry to quantitate mobilization of CD34+ cells, CD34+CD40+CD45RA− cells, T cells, B cells, and MDSCs. Mobilization of white blood cells (WBCs), neutrophils, lymphocytes, basophils, and eosinophils was quantified by automated hematology analyzer. Additionally, blood samples were analyzed by multicolor flow cytometry to quantify changes in expression of neutrophil activation markers such as L-selectin, CD11b, CD18, and CD66. Additionally, matrix metalloproteinase 9 (MMP-9), a neutrophil protease believed to mediate mobilization in response to MGTA-145, as well as its cognate inhibitor, TIMP-1, were quantified in plasma samples collected at the same time points by sandwich immunoassay.
Pharmacokinetics of MGTA-145 was also assessed by determining the plasma drug concentration immediately prior to and at 1, 2, 3, and 4 hours post-administration. Plasma concentrations of MGTA-145 following single dose administration (0.0075-0.3 mg/kg) as monotherapy are shown in
Patients were monitored for emergent adverse events. TABLE 5 shows the emergent adverse events exhibited by patients enrolled in Parts A-C of the MGTA-145 Phase 1 clinical trial.
1All adverse events (AEs) were grade 1 except for grade 2 abdominal pain (1), nausea (1), and back pain (1) in the plerixafor + MGTA-145 0.075 mg/kg 2h stagger cohort (Part B) and grade 2 headache (1) in the plerixafor + MGTA-145 0.015 mg/kg cohort (Part D). There was no dose response in AEs, so data are aggregated.
2Back pain was associated with MGTA-145 infusion, lasted < 20 minutes in most cases and did not require medical therapy.
Additionally,
TABLE 6 shows the quantified CD34+ cell levels in response to plerixafor alone versus simultaneous MGTA-145+plerixafor treatment across three different MGTA-145 doses (0.03 mg/kg, 0.075 mg/kg, and 0.15 mg/kg). Specifically, TABLE 6 shows the median peak CD34+ cells/μL level and the median fold change in peak CD34+ cells/μL over baseline for patients receiving each treatment. Generally, patients that received the combination of MGTA-145 and plerixafor exhibited higher peak CD34+ levels and higher fold change over baseline in comparison to patients that received plerixafor alone. In particular, patients that received 0.03 mg/kg MGTA-145+plerixafor exhibited a 1.3 fold increase in median peak CD34+ levels in comparison to patients that received plerixafor alone.
TABLE 7 shows the breakdown of peak CD34+ mobilization across patients that received plerixafor alone versus simultaneous MGTA-145+plerixafor treatment across three different MGTA-145 doses (0.03 mg/kg, 0.075 mg/kg, and 0.15 mg/kg). Specifically, the CD34+ levels for each patient were analyzed to determine the percentage of patients in each dose group to achieve ≥20 and/or ≥40 CD34+ cells/μL at peak. Generally, a higher percentage of patients that received the combination of plerixafor and MGTA-145 (any of 0.03 mg/kg, 0.075 mg/kg, or 0.15 mg/kg) achieved a peak CD34+ count of ≥20 or ≥40 cells/μL in comparison to patients that received plerixafor alone. In particular, of the 18 patients receiving combination plerixafor and MGTA-145 (any of 0.03 mg/kg, 0.075 mg/kg, or 0.15 mg/kg), 15 patients achieved a CD34+ count of at least 20 cells/μL at peak. This represents a 1.7 fold increase in comparison to patients that received plerixafor alone. Similarly, of the 10 patients that received the combination plerixafor and MGTA-145 (any of 0.03 mg/kg, 0.075 mg/kg, or 0.15 mg/kg), 3 achieved a peak CD34+ count of at least 40 cells per microliter. This represents a 1.2 fold increase in comparison to patients that received plerixafor alone.
1Devine et al., Blood. 2008.
TABLE 8 shows the quantified CD34+ cell levels in response to simultaneous or staggered administration of MGTA-145+plerixafor. Specifically, TABLE 8 shows the median peak CD34+ cells/μL level, median CD34+ area under the curve (AUC) between 2-8 hours post plerixafor dose administration, and median time (Tmax) of the peak CD34+ level across patients that received one of 1) plerixafor alone, 2) simultaneous plerixafor and 0.03 mg/kg MGTA-145, 3) simultaneous plerixafor and 0.075 mg/kg MGTA-145, 4) simultaneous plerixafor and 0.15 mg/kg MGTA-145, 5) staggered plerixafor and 0.03 mg/kg MGTA-145, 6) staggered plerixafor and 0.070 mg/kg MGTA-145, and 7) staggered plerixafor and 0.07 mg/kg MGTA-145. Generally, patients that received staggered plerixafor and MGTA-145 exhibited higher peak CD34+ levels and higher CD34+ AUC in comparison to patients that received simultaneous administration of plerixafor and MGTA-145 and in comparison to patients that received plerixafor alone. In particular, patients that received staggered plerixafor and MGTA-145 (any of 0.03 mg/kg, 0.07 mg/kg, or 0.075 mg/kg) exhibited a 1.8 fold increase in median peak CD34+ levels in comparison to patients that received plerixafor alone. Additionally, patients that received staggered plerixafor and MGTA-145 (any of 0.03 mg/kg, 0.07 mg/kg, or 0.075 mg/kg) exhibited a 1.7 fold increase in AUC in comparison to patients that received plerixafor alone.
TABLE 9 shows the breakdown of CD34+ mobilization across patients that received either simultaneous or staggered administration of MGTA-145+plerixafor. Specifically, the CD34+ levels for each patient were analyzed to determine the percentage of patients in each dose group to achieve ≥20 and/or ≥40 CD34+ cells/μL at peak. Generally, a higher percentage of patients that received staggered plerixafor and MGTA-145 (any of 0.03 mg/kg, 0.07 mg/kg, or 0.075 mg/kg) achieved a peak CD34+ count of ≥20 or ≥40 per μL in comparison to patients that received plerixafor alone or simultaneous plerixafor and MGTA-145. In particular, of the 22 patients receiving staggered plerixafor and MGTA-145 (any of 0.03 mg/kg, 0.07 mg/kg, or 0.075 mg/kg), 18 (83% o) exhibited a peak of at least 20 CD34+ cells/μL in response to treatment. This represents a 1.7 fold increase in comparison to patients that received plerixafor alone. Similarly, of the 22 patients receiving staggered plerixafor and MGTA-145 (any of 0.03 mg/kg, 0.07 mg/kg, or 0.075 mg/kg), 9 exhibited a peak of at least 40 CD34+ cells/μL in response to treatment. This represents a 2.9 fold increase in comparison to patients that received plerixafor alone.
1Devine et al., Blood. 2008.
As shown in
Part C of the Phase 1 trial of Example 1 involved administering a combination dose of MGTA-145 or placebo and plerixafor on two consecutive days. The dose of MGTA-145 was one of 0.03 mg/kg, 0.075 mg/kg, or 0.15 mg/kg. The dose of plerixafor was 240 μg/kg (0.24 mg/kg). The dose of MGTA-145 was administered 2 hours after plerixafor (i.e., staggered administration). Cells were collected on the second day.
As shown in
As shown in
Without wishing to be bound by theory, it is believed that fewer cells are mobilized on day 2 because CXCR2 expression becomes downregulated following the initial administration of MGTA-145.
A summary of CD34+ levels for one day/first day treatment is shown in TABLES 10A and 10B. TABLE 10B shows peak CD34+ cell levels in response to MGTA-145 alone across 5 different MGTA-145 doses (0.003 mg/kg, 0.075 mg/kg, 0.15 mg/kg, 0.3 mg/kg and 0.015 mg/kg, and 0.0075 mg/kg) administered to patients enrolled in Part A. TABLES 10A and 10B provide peak CD34+ cell levels in response to plerixafor alone or a MGTA-145+ plerixafor treatment across three different MGTA-145 doses (0.015 mg/kg, 0.03 mg/kg and 0.075 mg/kg (or 0.070 mg/kg)) administered to patients enrolled in Part B. During Part B, two patients were dosed with 0.075 mg/kg, and then the remaining 6 patients were dosed down at 0.07 mg/kg. These data are pooled in TABLE 10A and shown separately in TABLE 10B. TABLES 10A and 10B also provide peak CD34+ cell levels in response to plerixafor alone or a MGTA-145+plerixafor treatment across two different MGTA-145 doses (0.03 mg/kg and 0.075 mg/kg) administered to patients enrolled in Part C of the Phase 1 clinical trial. For Part C patients, only the first day data is included in the table. In addition to the peak CD34+ levels, the CD34+ levels for each patient in Parts B and C were analyzed to determine the percentage of patients in each dose group to achieve ≥20 and/or ≥40 CD34+ cells/μL at peak, and these data are also provided in TABLES 10A and 10B. Generally, a higher percentage of patients that received the combination of plerixafor and MGTA-145 (any of 0.03 mg/kg or 0.075 mg/kg) achieved a peak CD34+ count of ≥20 or ≥40 cells/μL in comparison to patients that received plerixafor alone. In particular, for 20 of the patients in Part B who received combination plerixafor and MGTA-145 (0.015 mg/kg, 0.03 mg/kg, or 0.075 mg/kg/0.070 mg/kg), 17 patients achieved a CD34+ count of at least 20 cells/μL at peak. This represents a 1.3 fold increase in comparison to patients that received plerixafor alone. Additionally, 8 of the 20 patients achieved a CD34+ count of at least 40 cells/μL at peak. This represents a 1.9 fold increase in comparison to patients that received plerixafor alone. For the 8 patients in Part C who received the combination plerixafor and MGTA-145 (any of 0.03 mg/kg or 0.075 mg/kg), 6 achieved a peak CD34+ count of at least 20 cells per microliter whereas none of the patients in Part C who received plerixafor alone achieved a peak CD34+ count of at least 20 cells per microliter. Additionally, 3 of the 8 patients achieved a peak CD34+ count of at least 40 cells per microliter whereas none of the patients in Part C who received plerixafor alone achieved a peak CD34+ count of at least 40 cells per microliter.
aPulsiper et al. (2009) Blood 113(15): 3604-3611.
bHolig (2013) Transfus Med Hemother 40: 225-235.
TABLE 11 shows a comparison of the features of MGTA-145+plerixafor-based mobilization and G-CSF-based mobilization. As compared to G-CSF, which causes mobilization via bone remodeling, MGTA-145+plerixafor stimulate chemokine cell migration. MGTA-145+plerixafor requires much less time for mobilization and collection (less than 1 day, vs. 5+ days for G-CSF). MGTA-145+plerixafor is more tolerable, with subjects showing fewer side effects as referenced in TABLE 11. Further, MGTA-145+plerixafor is more efficacious, with 88% of subjects tested mobilizing ≥2×106 CD34+ cells/kg (as compared with 78% of subjects administered G-CSF, reported by Holig, supra), and the quality of cells is superior, with 35% of CD34+ cells being CD90+, compared with only 10% for G-CSF. The superiority of cells mobilized using MGTA-145+plerixafor is also demonstrated in
In addition, as described in Example 7 below, MGTA-145+plerixafor-mobilized cells provide 5× increased engraftment in mice.
TABLE 12A shows CD34+ and CD34+CD90+CD45RA− cell yields from four subjects (labeled as subjects 801, 807, 817, and 821) who underwent apheresis procedures following treatment with a combination of 0.03 mg/kg MGTA-145 and plerixafor (staggered 2 hour protocol). The table documents the body weight of each subject, the total CD34+ cells that were obtained through the apheresis procedure, the normalized CD34+ cells/kg of body weight, and the percentage of CD34+ cells that were CD90+CD45RA−. Of note, one of the subjects (subject 821) completed only 13 L (65%) of the planned 20 L of apheresis, thereby explaining the lower overall CD34+ cell yield. The total CD34+ cells that were obtained for each patient through the apheresis procedure ranged from 239×106 cells up to 500×106 cells. The normalized CD34+ cells/kg of body weight ranged from 2.7×106 cells/kg up to 5.3×106 cells/kg, with a median of 4.3×106 cells/kg, higher than the clinical threshold for transplant. The percentage of CD34+CD90+CD45− cells amongst the total CD34+ cell population ranged from 19% to 41%.
aSubject 821 completed 13L (65%) of the planned 20L apheresis
bCD90+ (%) represents the percentage of collected CD34+ cells that were CD90+ CD45RA−
TABLE 12B shows CD34+ and CD34+CD90+CD45RA− cell yields from four subjects (labeled as subjects 837, 838, 847, and 850) who underwent apheresis procedures following treatment with combination of 0.015 mg/kg MGTA-145 and plerixafor (staggered 2 hour protocol). The table documents the total CD34+ cells that were obtained through the apheresis procedure, the normalized CD34+ cells/kg of body weight, and the percentage of CD34+ cells that were CD90+CD45RA−. The total CD34+ cells that were obtained for each patient through the apheresis procedure ranged from 118×106 cells up to 525×106 cells. The normalized CD34+ cells/kg of body weight ranged from 1.5×106 cells/kg up to 7.0×106 cells/kg, with a median of 3.7×106 cells/kg, higher than the clinical threshold for transplant. The percentage of CD34+CD90+CD45− cells amongst the total CD34+ cell population ranged from 28% to 41%.
aCD90+ (%) represents the percentage of collected CD34+ cells that were CD90+ CD45RA−
TABLE 12C shows CD34+ and CD34+CD90+CD45RA− cell yields from three subjects (labeled as subjects G-CSF837, 838, 847, and 850) who underwent apheresis procedures following treatment with combination of 0.015 mg/kg MGTA-145 and plerixafor (staggered 2 hour protocol). The table documents the body weight of each subject, the total CD34+ cells that were obtained through the apheresis procedure, the normalized CD34+ cells/kg of body weight, and the percentage of CD34+ cells that were CD90+CD45RA−. The total CD34+ cells that were obtained for each patient through the apheresis procedure ranged from 118×106 cells up to 525×106 cells. The normalized CD34+ cells/kg of body weight ranged from 1.5×106 cells/kg up to 7.0×106 cells/kg, with a median of 3.7×106 cells/kg, higher than the clinical threshold for transplant. The percentage of CD34+CD90+CD45− cells amongst the total CD34+ cell population ranged from 28% to 41%.
TABLE 13 provides apheresis collection yield at the 0.015 mg/kg dose and 0.03 mg/kg dose. At the 0.015 mg/kg dose, a mean of 4.0 CD34+(×106) cells and median of 3.7 CD34+(×106) cells were collected, with a mean of 1.4 CD90+(×106) cells and median of 1.2 CD90+(×106) cells. Accordingly, at the 0.015 mg/kg dose, 37% of CD34+ cells collected were CD90+. At the 0.03 mg/kg dose, a mean of 4.1 CD34+(×106) cells and median of 4.3 CD34+(×106) cells were collected, with a mean of 1.3 CD90+(×106) cells and median of 1.5 CD90+(×106) cells. Accordingly, at the 0.015 mg/kg dose, 31% of CD34+ cells collected were CD90+.
aCD90+ cells defined as CD34+ CD90+ CD45RA− cells.
A summary of these findings is provided in
Flow cytometry was additionally performed to identify yield of different populations of cells (e.g., B, T, and NK cells) following mobilization by MGTA-145+plerixafor. Representative flow plots for mice transplanted with MGTA-145+plerixafor-mobilized CD34+ cells are shown in
In particular, while CD3+ T-cell numbers were comparable between MGTA-145+plerixafor and plerixafor alone, MGTA-145+plerixafor mobilized 0.2 (0.0-0.6)×108/kg CD8+ T-cells, constituting 1.8 (0.5-4.8)% of the graft, a number and proportion significantly lower than that mobilized by either G-CSF or plerixafor alone. As higher numbers CD8+ T cells are associated with higher rates of GvHD, having lower numbers of CD8+ T cells in the MGTA-145+plerixafor graft may be beneficial.
a Data are pooled for donors mobilized with 0.03 or 0.015 mg/kg MGTA-145 + plerixafor.
bDevine dataset reflects median yield of 1 LP for plerixafor and G-CSF grafts.
cChen dataset reflects median yield of 2 LP for plerixafor and 1 LP for G-CSF grafts.
a Data represent 40 healthy donors mobilized with G-CSF as described previously (Singhal et al, BMT. 2000).
bFrequencies reflect % of CD45+ except for NKT/iNKT cells (% of CD3+) and Tregs (% of CD4+ T cells).
cData represent 78 healthy donors mobilized with G-CSF as described previously (Chaidos et al, Blood. 2012).
CD34+ cells from MGTA-145+plerixafor-mobilized donors (Part D, n=2 donors at 0.03 mg/kg dose level) or G-CSF-mobilized peripheral blood (n=3) were transplanted into sublethally irradiated (200 cGy) NSG mice at limit dilution (3 cell doses per donor). Engraftment of human CD45+(hCD45+frequency) and lineage composition (CD3, CD19, CD33) in peripheral blood was measured at week 4 and week 12 post-transplant by flow cytometry. Representative flow plots for mice transplanted with MGTA-145+plerixafor-mobilized CD34+ cells are shown in
This example demonstrates that MGTA-145+plerixafor is a rapid, reliable, and G-CSF free method to obtain high numbers of HSCs with durable engraftment potential and a graft with immunosuppressive properties. The example suggests that MGTA-145+plerixafor is an effective single-day mobilization/collection regimen for both autologous and allogeneic stem cell transplantation resulting in enhanced engraftment and reduced GvHD.
The following examples demonstrates that MGTA-145+plerixafor mobilizes higher numbers of HSCs with durable primary and secondary engraftment in mice relative to other mobilization regimens.
Mice (CD45.1) were mobilized according to the scheme shown in
As shown in
Hematopoietic stem and progenitor cells (CD34+ cells) mobilized in Part D of the study were pre-stimulated for 1 day prior to electroporation with gRNA targeting beta-2 microglobulin (B2M) and Cas9. After electroporation (EP), cells were cultured for 1 or 7 days in the presence or absence of an aryl hydrocarbon receptor antagonist (“AHR;” 500 nM). Controls included were:
Mock Control: These cells were not electroporated and were used to assess growth capabilities of MGTA-145/p mobilized blood±AHR.
Mock EP Control: No gRNA or Cas9 was included in this group which was used to assess cell number and viability after EP.
Minimally Manipulated Control: 1-day culture to approximate culture conditions used by most gene therapy groups for CRISPR-Cas9 based editing strategies.
G-CSF mobilized blood was used as a comparator, because HSCs from G-CSF mobilized blood have been previously shown to be capable of gene-modification and expansion with AHR (Hoban et al. ASGCT 2019).
Number of CD34+ and CD34+CD90+ cells and frequency of editing (beta-2-microglobulin negative) was assessed by flow cytometry.
This example shows that high gene editing rates are achieved (˜90%) across all samples, indicating that MGTA-145/plerixafor mobilized blood can be edited to similar levels as G-CSF mobilized blood. Further, a 15-fold expansion of MGTA-145/plerixafor CD34+CD90+CD45RA− mobilized blood cells was observed using AHR over minimally-manipulated cultures, indicating that MGTA-145/plerixafor-mobilized blood can be expanded by AHR to similar levels as G-CSF-mobilized blood.
This example demonstrates that MGTA-145+plerixafor CD34+ cells from humans can be efficiently gene modified and engrafted in NSG mice.
Mobilized blood cells (MGTA-145/p or G-CSF) were pre-stimulated for 1 day prior to electroporation with gRNA B2M. After electroporation, cells were cultured for 1 or 7 days±AHR, using the same control arms as assessed in vitro (see Example 9). An “uncultured” arm was transplanted to assess engraftment capabilities of cells that were not cultured.
As shown in
This clinical trial will have three arms: donors with normal kidney function, donors with a mild decrease in GFR, and donors with a moderate decrease in GFR. Donors will be administered a single dose of MGTA-145 at 0.07 mg/kg intravenously.
Samples of peripheral blood will be taken at intervals following administration of MGTA-145 and Pharmacokinetics Biomarkers will be determined, including area under the curve (AUC), maximum plasma concentration (Cmax), clearance (CL), volume of distribution at steady state (Vdss), half-life, and renal clearance of MGTA-145. Safety and tolerability of MGTA-145 will be assessed by determining the occurrence of adverse events and administering clinical laboratory test, and taking vital signs and ECGs.
The following describes the inclusion criteria for donors used in this example. Donors:
Specific inclusion criteria for donors with normal renal function includes:
Specific inclusion criteria for donors with renal impairment includes:
Selected donors do not have any clinically significant laboratory value outside the normal range at screening. Selected donors do not have a history of alcoholism or drug abuse within the past 3 years. Selected donors do not have a history of kidney transplantation or requiring dialysis or anticipated to initiate dialysis during the study period. Selected donors have not donated more than 500 mL of blood or plasma within 12 weeks prior to dosing. Subject does not smoke more than 10 cigarettes per day and has not done so within 6 months prior to the screening visit. Selected donors have not had acute illness, infection (requiring medical treatment [e.g., antibiotics]), or surgery within 4 weeks of dosing. Selected donors are not seropositive for hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV) antibody, or human immunodeficiency virus (HIV). Selected donors have not received another investigational drug or participated in an investigational drug or device study within 12 weeks prior to dosing. Selected donors do not have a history of anaphylaxis or clinically important reaction to any drug including plerixafor.
Selected donors having normal renal function will not have any clinically significant laboratory value outside the normal range at Screening. Selected donors with normal renal function do not have any clinically significant laboratory value outside the normal range at screening nor any hematologic, cardiovascular, pulmonary, central nervous system, metabolic, renal, hepatic, or gastrointestinal conditions that may put subject at risk or interfere with study results. Selected donors with normal renal function have not used any prescription drugs within 14 days prior to dosing or any dietary supplements or non-prescription drugs within 7 days prior to dosing unless deemed acceptable.
Selected donors having renal impairment will not have acute kidney injury, clinically significant laboratory abnormalities excluding those associated with renal impairment or the underlying cause of renal disease, an unstable medical condition or underlying medical condition that has changed within the past 90 days, presence of laboratory abnormalities or clinically significant medical condition that may place the subject at an unacceptable risk as a participant in this study or may interfere with the interpretation of the study results, or changes in prescription medications within 14 days prior to dosing or anticipated changes during the study period.
It is expected that administration according to the Example will rapidly give rise to populations of cells that are enriched in CD34+CD90+CD45RA− cells, as described in more detail herein. Furthermore, it is expected that the administration of MGTA-145 will be safe and effective, even for donors with mild and moderate decreases in GFR.
The goal of this example was to determine whether monocytes contribute to immunosuppressive properties of MGTA-145+plerixafor graft in vivo. CD14+ cells were depleted from MGTA-145+plerixafor peripheral blood mononuclear cells (PBMCs) by bead-based separation approaches and transplanted into sublethally-irradiated (200 cGy) NSG mice. Greater than 90% depletion of CD14+ cells were achieved (data not shown).
Survival was compared to non-depleted PBMCs (CD14−+CD14+). Input number was normalized to T cell content.
Altogether, this suggests that CD14 myeloid cells contribute towards immunosuppression, and more specifically contributes towards GvHD prevention and immunosuppression.
This research study tests a composition for mobilizing stem cells within a patient such that the mobilized stem cells can be collected and used for allogeneic stem cell transplant for treatment of hematological malignancies. Here, the composition includes a combination of MGTA-145 and plerixafor. In particular, this research study is a Phase II, open-label, multicenter, prospective study of MGTA-145+plerixafor mobilized HLA-matched sibling and matched unrelated donor allografts for myeloablative hematopoietic stem cell transplantation (HSCT) in recipients with hematological malignancies. Donors will undergo 1 or 2 days of mobilization and apheresis.
Here, the conditions involved in the study include related donors donating peripheral blood stem cells (PBSC) to a family member, healthy donors, patients with acute myelogenous leukemia, patients with acute lymphoblastic leukemia, and patients with myelodysplastic syndrome. In particular, donors donating peripheral blood stem cells (PBSC) to a family member and healthy donors are administered combination MGTA-145+plerixafor for mobilizing stem cells, followed by apheresis and collection of the stem cells. The collected stem cells are administered to a recipient, such as any one of patients with acute myelogenous leukemia, patients with acute lymphoblastic leukemia, and patients with myelodysplastic syndrome. A total of 56 patients are enrolled in this study.
The experimental arm involves donors who receive a single dose MGTA-145 plus plerixafor followed by apheresis on one or two consecutive days. MGTA-145 is administered intravenously at a dose of 0.015 mg/kg. Plerixafor, also referred to as Mozobil, is administered subcutaneously at a dose of 240 μg/kg.
Eligible patients for enrollment are between ages of 18-65 years old. Donor inclusion criteria include:
Recipient inclusion criteria include:
Donor exclusion criteria include:
Recipient exclusion criteria include:
Donors and recipients are enrolled according to the inclusion and exclusion criteria specified in Example 13. Donors are treated with 0.015 mg/kg intravenous (IV) MGTA-145 and 240 μg/kg subcutaneous plerixafor followed by apheresis.
Quantities of stem cells from donors are analyzed to determine the proportion of donors from whom stem cells were successfully mobilized. Specifically, a donor who receives MGTA-145 and plerixafor is characterized as a successful mobilization if a sufficient CD34+ stem cell dose is obtained. Here, the sufficient CD34+ stem cell dose is ≥2.0×106 CD34+ cells/kg in one apheresis setting.
The proportion of donors who are characterized as successful mobilizations is greater than successful mobilizations in donors who receive conventional mobilization treatments (e.g., donors treated with granulocyte colony-stimulating factor (G-CSF) e.g., 10 μg/kg subcutaneous G-CSF, plerixafor alone, or G-CSF in combination with plerixafor).
Donors and recipients are enrolled according to the inclusion and exclusion criteria specified in Example 13. Donors are treated with 0.015 mg/kg intravenous (IV) MGTA-145 and 240 μg/kg subcutaneous plerixafor followed by apheresis.
Quantities of stem cells from donors are analyzed to determine the proportion of donors from whom stem cells were successfully mobilized. Specifically, a donor who receives MGTA-145 and plerixafor is characterized as a successful mobilization if a sufficient CD34+ stem cell dose is obtained. Here, the sufficient CD34+ stem cell dose is ≥4.0×106 CD34+ cells/kg in one apheresis setting.
The proportion of donors who are characterized as successful mobilizations is greater than successful mobilizations in donors who receive conventional mobilization treatments (e.g., donors treated with granulocyte colony-stimulating factor (G-CSF) e.g., 10 μg/kg subcutaneous G-CSF, plerixafor alone, or G-CSF in combination with plerixafor).
Donors and recipients are enrolled according to the inclusion and exclusion criteria specified in Example 13. Donors are treated with 0.015 mg/kg intravenous (IV) MGTA-145 and 240 μg/kg subcutaneous plerixafor followed by apheresis.
Donors are evaluated following treatment for at least 180 days to determine the incidence and severity of acute toxicities before and during apheresis experienced by the donors.
The number of donors who experience severe acute toxicities is reduced in comparison to donors who receive conventional mobilization treatments (e.g., donors treated with granulocyte colony-stimulating factor (G-CSF) e.g., 10 μg/kg subcutaneous G-CSF, plerixafor alone, or G-CSF in combination with plerixafor).
Donors and recipients are enrolled according to the inclusion and exclusion criteria specified in Example 13. Donors are treated with 0.015 mg/kg intravenous (IV) MGTA-145 and 240 μg/kg subcutaneous plerixafor followed by apheresis.
Donors are evaluated following treatment for at least 180 days to determine the incidence and severity of adverse events before and during apheresis experienced by the donors. The number of donors receiving MGTA-145+plerixafor who experience severe adverse events is reduced in comparison to experiences of donors who receive conventional mobilization treatments (e.g., donors treated with granulocyte colony-stimulating factor (G-CSF) e.g., 10 μg/kg subcutaneous G-CSF, plerixafor alone, or G-CSF in combination with plerixafor).
Donors and recipients are enrolled according to the inclusion and exclusion criteria specified in Example 13. Donors are treated with 0.015 mg/kg intravenous (IV) MGTA-145 and 240 μg/kg subcutaneous plerixafor followed by apheresis.
Cells obtained through apheresis are transplanted to recipients. Recipients are evaluated following transplantation at days 28, 100, 180, and 365 to determine engraftment of transplanted cells including neutrophils and platelets. Recipients, when evaluated across the 4 different timepoints (e.g., days 28, 100, 180, and 365), exhibit increased neutrophil and platelet recovery across the timepoints or additionally or alternatively, exhibit more rapid engraftment of neutrophils and platelets within the recipients in comparison to recipients who receive cells from donors receiving conventional mobilization treatments (e.g., donors treated with granulocyte colony-stimulating factor (G-CSF) e.g., 10 μg/kg subcutaneous G-CSF, plerixafor alone, or G-CSF in combination with plerixafor).
Donors and recipients are enrolled according to the inclusion and exclusion criteria specified in Example 13. Donors are treated with 0.015 mg/kg intravenous (IV) MGTA-145 and 240 μg/kg subcutaneous plerixafor followed by apheresis.
Cells obtained through apheresis are transplanted to recipients. Recipients are evaluated following transplantation at days 28, 100, 180, and 365 to determine post-transplantation graft durability. Recipients, when evaluated across the 4 different timepoints (e.g., days 28, 100, 180, and 365), exhibit reduced incidences of graft failure (e.g., primary or secondary graft failure) in comparison to recipients who receive cells from donors receiving conventional mobilization treatments (e.g., donors treated with granulocyte colony-stimulating factor (G-CSF) e.g., 10 μg/kg subcutaneous G-CSF, plerixafor alone, or G-CSF in combination with plerixafor).
Donors and recipients are enrolled according to the inclusion and exclusion criteria specified in Example 13. Donors are treated with 0.015 mg/kg intravenous (IV) MGTA-145 and 240 μg/kg subcutaneous plerixafor followed by apheresis.
Cells obtained through apheresis are transplanted to recipients. Recipients are evaluated following transplantation at days 21, 28, 56, 100, 180, and 365 to determine incidence and severity of acute and chronic graft-versus host disease (GVHD) post-transplantation. Recipients, when evaluated across the 6 different timepoints (e.g., days 21, 28, 56, 100, 180, and 365), exhibit reduced incidences of acute and chronic GVHD and/or reduced severity of acute and chronic GVHD in comparison to recipients who receive cells from donors receiving conventional mobilization treatments (e.g., donors treated with granulocyte colony-stimulating factor (G-CSF) e.g., 10 μg/kg subcutaneous G-CSF, plerixafor alone, or G-CSF in combination with plerixafor).
Donors and recipients are enrolled according to the inclusion and exclusion criteria specified in Example 13. Donors are treated with 0.015 mg/kg intravenous (IV) MGTA-145 and 240 μg/kg subcutaneous plerixafor followed by apheresis.
Cells obtained through apheresis are transplanted to recipients. Recipients are evaluated following transplantation at days 21, 28, 56, 100, 180, and 365 to determine incidence of treatment related mortality and disease relapse/progression post-transplantation. Recipients, when evaluated across the 6 different timepoints (e.g., days 21, 28, 56, 100, 180, and 365), exhibit reduced incidences of treatment related mortality in comparison to recipients who receive cells from donors receiving conventional mobilization treatments (e.g., donors treated with granulocyte colony-stimulating factor (G-CSF) e.g., 10 μg/kg subcutaneous G-CSF or plerixafor alone). Additionally, recipients, when evaluated across the 6 different timepoints (e.g., days 21, 28, 56, 100, 180, and 365), exhibit reduced disease relapse/progression in comparison to recipients who receive cells from donors receiving conventional mobilization treatments (e.g., donors treated with granulocyte colony-stimulating factor (G-CSF) e.g., 10 μg/kg subcutaneous G-CSF, plerixafor alone, or G-CSF in combination with plerixafor).
Donors and recipients are enrolled according to the inclusion and exclusion criteria specified in Example 13. Donors are treated with 0.015 mg/kg intravenous (IV) MGTA-145 and 240 μg/kg subcutaneous plerixafor followed by apheresis.
Cells obtained through apheresis are transplanted to recipients. Recipients are evaluated following transplantation at days 21, 28, 56, 100, 180, and 365 to determine probability of progression-free and overall survival post-transplantation. Recipients, when evaluated across the 6 different timepoints (e.g., days 21, 28, 56, 100, 180, and 365), exhibit improved progression-free survival in comparison to recipients who receive cells from donors receiving conventional mobilization treatments (e.g., donors treated with granulocyte colony-stimulating factor (G-CSF) e.g., 10 μg/kg subcutaneous G-CSF or plerixafor alone). Additionally, recipients, when evaluated across the 6 different timepoints (e.g., days 21, 28, 56, 100, 180, and 365), exhibit improved overall survival in comparison to recipients who receive cells from donors receiving conventional mobilization treatments (e.g., donors treated with granulocyte colony-stimulating factor (G-CSF) e.g., 10 μg/kg subcutaneous G-CSF, plerixafor alone, or G-CSF in combination with plerixafor).
Donors and recipients are enrolled according to the inclusion and exclusion criteria specified in Example 13. Donors are treated with 0.015 mg/kg intravenous (IV) MGTA-145 and 240 μg/kg subcutaneous plerixafor followed by apheresis.
Cells obtained through apheresis are transplanted to recipients. Recipients are evaluated following transplantation at days 21, 28, 56, 100, 180, and 365 to determine probability of progression-free and overall survival post-transplantation. A reduced proportion of recipients, when evaluated across the 6 different timepoints (e.g., days 21, 28, 56, 100, 180, and 365), experience adverse events related to allograft in comparison to the proportion of recipients who receive cells from donors receiving conventional mobilization treatments (e.g., donors treated with granulocyte colony-stimulating factor (G-CSF) e.g., 10 μg/kg subcutaneous G-CSF, plerixafor alone, or G-CSF in combination with plerixafor).
All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
Other embodiments are within the claims.
This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/310,460, filed on Feb. 15, 2022, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63310460 | Feb 2022 | US |
