Compositions and methods of immunodepletion for the treatment of malignant and non-malignant hematological diseases

Abstract

To provide a method for depleting hematopoietic stem cells of a subject, and a method for treating a subject afflicted with a non-cancerous disorder treatable via genetically edited cell therapy.SOLUTION: This invention provides a method for depleting a subject's hematopoietic stem cells that includes administering to the subject an effective amount of a radiolabeled antibody against CD34, CD117, or CD135, where preferred radiolabels include 131I and 225Ac. This invention also provides a method for treating a subject afflicted with a non-cancerous disorder treatable via genetically edited cell therapy, where the method includes (i) administering to the subject an amount of the radiolabeled antibody effective to deplete the subject's hematopoietic stem cells, and (ii) after a suitable time period, performing the therapy on the subject to treat the subject's disorder. Finally, this invention provides articles of manufacture for performing the subject methods.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 838,589, filed April 25, 2019, which is incorporated herein in its entirety.

[0002] The present invention relates to compositions useful for the selective depletion and destruction of hematopoietic stem cells and their use in methods for the treatment of malignant and non-malignant hematologic disorders. [Background technology]

[0003] Hematopoietic stem cell transplantation (HSCT) is primarily indicated for treating malignancies and requires conditioning of the recipient's tissue (e.g., bone marrow tissue) prior to engraftment. HSCT indications and hemoglobinopathies include sickle cell anemia, beta-thalassemia, Wiskott-Aldrich syndrome, adenosine deaminase-associated severe combined immunodeficiency (ADA-SCID), metachromatic leukodystrophy, and HIV / AIDS, and the list of indications will continue to expand as gene editing technologies improve. In certain cases, engraftment of 20% of transplanted cells can alleviate or cure the disease.

[0004] Gene editing technology has progressed substantially with the emergence of site-specific editing methods such as TALEN, CRISPR / cas9, and zinc finger nuclease (ZFN). These methods have therapeutic potential for patients suffering from non-malignant genetic diseases, such as hemoglobinopathies, congenital immunodeficiencies, and viral disorders such as AIDS. Gene editing technology makes it possible to treat and even cure germline blood disorders, such as severe combined immunodeficiency (SCID), sickle cell disease (SCD), and β-thalassemia.

[0005] Gene editing precisely and permanently alters the sequence of genomic DNA, which remains under endogenous gene regulation, ensuring the correct and appropriate expression of the modified genetic element. Currently, there are four major classes of nucleases for human genome gene editing: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases (MNs), and clustered regularly interspaced short tandem repeats (CRISPR / Cas9). Each of these can recognize and bind to specific target sequences in DNA. Depending on the characteristics of the approach, the target DNA can be cleaved on one or both strands. To correct the mutation, a correction template for homologous recombination repair of the break introduced at the site of the target lesion is used.

[0006] For example, current non-targeted conditioning methods, including irradiation (e.g., total body irradiation or TBI) and DNA alkylating / denaturing agents, are highly toxic to multiple organ systems, hematopoietic and non-hematopoietic cells, and the hematopoietic microenvironment. These harsh conditioning regimens effectively kill immune and niche cells in the host subject and adversely affect multiple organ systems, often resulting in life-threatening complications.

[0007] To fully realize the curative potential of HSCT, it is essential to develop a mild conditioning regimen that avoids undesired toxicity.There is a need for a new, preferably non-myeloablative composition and method that can be used to condition target tissue (for example, bone marrow tissue) while reducing undesired toxicity and minimizing the occurrence of serious adverse reactions.There is also a need for a new such therapy that can selectively destroy endogenous hematopoietic stem cell population in target tissue while minimizing or eliminating the effect of therapy on non-target cells and tissues, such as platelets, white blood cells, and red blood cells.There is also a need for assays and methods for identifying drugs that can selectively deplete or destroy endogenous hematopoietic stem cell population. Summary of the Invention

[0008] The present invention provides methods and compositions useful for targeted depletion of hematopoietic stem cells in a subject, comprising administering to the subject an effective amount of a radiolabeled antibody, wherein the antibody is selected from one or more of anti-CD34, anti-CD117, or anti-CD135. 131 I, 125 I, 123 I, 90 Y, 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 32 P, 225 Ac, 213 Bi, 213 Po, 211 At, 212 Bi, 213 Bi, 223 Ra, 227 Th, 149 Tb, 137 Cs, 212 Pb, and 103 The radiolabel may comprise a radiolabel selected from Pd.

[0009] The present invention also provides methods for targeted depletion of hematopoietic stem cells in a subject to condition the subject's tissue for engraftment or transplantation. Accordingly, the present invention provides a method of treating a subject afflicted with a cancerous disorder treatable with bone marrow transplantation, the method comprising (i) administering to the subject an amount of a radiolabeled antibody effective to substantially deplete or destroy the subject's hematopoietic stem cells, and optionally (ii) after a suitable period of time, performing a bone marrow transplant in the subject.

[0010] The present invention further provides a method for treating a subject afflicted with a non-cancerous disorder treatable via gene-edited cell therapy, the method comprising: (i) administering to the subject an amount of a radiolabeled antibody effective to deplete the subject's hematopoietic stem cells; and, optionally, (ii) after a suitable period of time, administering the therapy to the subject to treat the subject's disorder.

[0011] Finally, the present invention provides an article of manufacture comprising: (a) at least one of the radiolabeled antibodies described above; and (b) a label instructing a user to administer to a subject an amount of the antibody effective to deplete the subject's hematopoietic stem cells.

[0012] According to certain embodiments, the radiolabel on the antibody, i.e., anti-CD34, anti-CD117, or anti-CD135, is 131 I or 225 Ac, 131 An effective amount of an antibody labeled with I can be up to 1200 mCi (e.g., 10-200 mCi, 200-400 mCi, or 400-1,200 mCi), 225 An effective amount of an antibody labeled with Ac can be up to 5.0 μCi / kg (subject body weight) (e.g., 0.1 μCi / kg to 5.0 μCi / kg, 0.1 μCi / kg to 1.0 μCi / kg (subject body weight), 1.0 μCi / kg to 3.0 μCi / kg (subject body weight), 3.0 μCi to 5.0 μCi / kg (subject body weight)). DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention provides radiolabeled antibody-based methods for depleting hematopoietic stem cells in a subject, as well as related methods and articles of manufacture. Where these methods precede certain gene-edited cell-based therapies, they can enhance the outcomes of those therapies while minimizing adverse effects.

[0014] Throughout this application, various publications are cited. The disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0015] definition In this application, certain terms are used which have the meanings set forth below.

[0016] The singular forms "a," "an," "the," etc. include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an" antibody includes both a single antibody and a plurality of different antibodies.

[0017] The term "about" when used before numerical designations, such as temperatures, times, amounts, and concentrations, indicates approximations that may vary by ±10%, ±5%, or ±1%.

[0018] As used herein, "administering" with respect to an antibody means delivering the antibody to the subject's body via any known method suitable for antibody delivery. Specific administration modes include, but are not limited to, intravenous, transdermal, subcutaneous, intraperitoneal, and intrathecal administration. Exemplary antibody administration methods may be substantially as described in International Publication No. WO2016 / 187514, which is incorporated herein by reference.

[0019] Furthermore, in the present invention, antibody can be formulated with one or more commonly used pharmaceutically acceptable carriers.Such carriers are well known to those skilled in the art.For example, injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymer injections, and can include excipients such as soluble modifiers (e.g., ethanol, propylene glycol, and sucrose) and polymers (e.g., polycaprylactone and PLGA).

[0020] As used herein, the term "antibody" includes, but is not limited to, (a) immunoglobulin molecules comprising two heavy chains and two light chains and recognizing an antigen, (b) polyclonal and monoclonal immunoglobulin molecules, (c) monovalent and divalent fragments thereof (e.g., Fab, di-Fab), and (d) bispecific forms thereof. Immunoglobulin molecules can be derived from any of the commonly known classes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. Antibodies can be of both natural and non-natural origin (e.g., IgG-Fc-silent). Furthermore, antibodies include chimeric antibodies, fully synthetic antibodies, single-chain antibodies (e.g., scFv), single- and dual-domain antibodies (e.g., VHH), and fragments thereof. Antibodies can be human, humanized, or non-human.

[0021] "Monoclonal antibody" refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope, or, in the case of multispecific monoclonal antibodies, binding specificities for two or more distinct epitopes. Thus, a "monoclonal antibody" refers to an antibody population having a single amino acid composition in each heavy and light chain, except for possible known variations such as removal of the C-terminal lysine from the antibody heavy chain. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, or monovalent, bivalent, or multivalent.

[0022] As used herein, an "anti-CDXX antibody" is an antibody that binds to any available epitope of CDXX, where XX can be 34, 117, or 135 (i.e., CD34, CD117, or CD135). An anti-CDXX antibody can be a bispecific antibody that binds to two different epitopes, which can include one of CD34, CD117, or CD135 and another related epitope (e.g., CD45), or two different epitopes of a single cell surface target (two different epitopes of one of CD34, CD117, or CD135). A bispecific antibody can be a recombinant antibody, monoclonal antibody, chimeric antibody, humanized antibody, human antibody, or antibody fragment.

[0023] As used herein with respect to a subject's hematopoietic stem cells ("HSCs," i.e., pluripotent hematopoietic stem cells, also referred to as hemoblasts), "depletion" refers to reducing the subject's population of HSCs. According to certain embodiments, depleting a subject's HSCs refers to reducing the subject's HSC population by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. According to certain embodiments, depleting a subject's HSCs refers to reducing the subject's HSC population by 100%. Methods for measuring HSC populations are routine. These include, for example, the use of flow cytometry to detect human HSCs in bone marrow samples and staining for various cell surface markers (such as Lin, CD34, CD38, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, and HLA DR). A decrease in the patient's immune cells can also be detected in the peripheral blood, for example, by determining the absolute lymphocyte count (ALC) via detection of CD3, CD4, and CD8-positive cells as an indicator of immunosuppression.

[0024] As used herein with respect to a subject's hematopoietic stem cells (HSCs), the term "targeted depletion" refers to substantially reducing the population of the subject's HSCs, as described above, while leaving the population of mature, differentiated hematopoietic stem cells substantially unaffected. For example, targeted depletion can be taken to mean that non-targeted mature, differentiated hematopoietic stem cells (e.g., lymphoid, myeloid, etc.) are depleted by less than 20%, or less than 10%.

[0025] As used herein, an amount of radiolabeled antibody is "effective" if, when administered, it reduces HSC levels in a subject.

[0026] Radiolabeled antibodies 131 In embodiments labeled with I, the effective amount is, for example, less than 1,200 mCi (i.e., less than 1,200 mCi administered to a subject). 131 1 dose delivers a whole-body radiation dose of less than 1,200 mCi).

[0027] Radiolabeled antibodies 131 According to embodiments labeled with I, the effective amount is less than 1,100 mCi, less than 1,000 mCi, less than 900 mCi, less than 800 mCi, less than 700 mCi, less than 600 mCi, less than 500 mCi, less than 400 mCi, less than 350 mCi, less than 300 mCi, less than 250 mCi, less than 200 mCi, less than 150 mCi, less than 100 mCi, less than 50 mCi, less than 40 mCi, less than 30 mCi, less than 20 mCi, or less than 10 mCi.

[0028] Radiolabeled antibodies 131According to embodiments labeled with I, the effective amount is 1 mCi to 10 mCi, 1 mCi to 200 mCi, 10 mCi to 20 mCi, 10 mCi to 30 mCi, 10 mCi to 40 mCi, 10 mCi to 50 mCi, 10 mCi to 100 mCi, 10 mCi to 150 mCi, 10 mCi to 200 mCi, 20 mCi to 30 mCi, 30 mCi i~40mCi, 40mCi~50mCi, 50mCi~100mCi, 50mCi~150mCi, 50mCi~200mCi, 60mCi~140mCi, 70mCi~130mCi, 80mCi~120mCi, 90mCi~110mCi, 100mCi~150mCi, 150mCi~200mCi, 200mCi ~250mCi, 200mCi~300mCi, 200mCi~350mCi, 200mCi~400mCi, 200mCi~500mCi, 200mCi~6 00mCi, 200mCi~700mCi, 200mCi~800mCi, 200mCi~900mCi, 200mCi~1000mCi, 200mCi~1,1 00mCi, 200mCi to 1,200mCi, 400mCi to 500mCi, 400mCi to 600mCi, 400mCi to 700mCi, 400mCi to 800mCi, 400mCi to 900mCi, 400mCi to 1,000mCi, 400mCi to 1,100mCi, or 400mCi to 1,200mCi.

[0029] Radiolabeled antibodies 131 According to embodiments labeled with I, effective amounts are 1 mCi, 10 mCi, 20 mCi, 30 mCi, 40 mCi, 50 mCi, 60 mCi, 70 mCi, 80 mCi, 90 mCi, 100 mCi, 110 mCi, 120 mCi, 130 mCi, 140 mCi, 150 mCi, 200 mCi, 250 mCi, 300 mCi, 350mCi, 400mCi, 450mCi, 500mCi, 550mCi, 600mCi, 650mCi, 700mCi, 750mCi, 800mCi, 850mCi, 900mCi, 950mCi, 1,000mCi, 1,050mCi, 1,100mCi, 1,150mCi, or 1,200mCi.

[0030] Radiolabeled antibodies 225In embodiments labeled with Ac, the effective amount is, for example, less than 5.0 μCi / kg (i.e., less than 5.0 μCi / kg administered to a subject). 225 The amount of Ac-anti-CDXX delivers a radiation dose of less than 5.0 μCi per kilogram of subject body weight).

[0031] Radiolabeled antibodies 225 According to embodiments labeled with Ac, the effective amount is less than 4.5 μCi / kg, 4.0 μCi / kg, 3.5 μCi / kg, 3.0 μCi / kg, 2.5 μCi / kg, 2.0 μCi / kg, 1.5 μCi / kg, 1.0 μCi / kg, 0.9 μCi / kg, 0.8 μCi / kg, 0.7 μCi / kg, 0.6 μCi / kg, 0.5 μCi / kg, 0.4 μCi / kg, 0.3 μCi / kg, 0.2 μCi / kg, 0.1 μCi / kg, or 0.05 μCi / kg.

[0032] Radiolabeled antibodies 225 According to the embodiment labeled with Ac, the effective amount is 0.05μCi / kg~0.1μCi / kg, 0.1μCi / kg~0.2μCi / kg, 0.2μCi / kg~0.3μCi / kg, 0.3μCi / kg~0.4μC i / kg, 0.4μCi / kg~0.5μCi / kg, 0.5μCi / kg~0.6μCi / kg, 0.6μCi / kg~0.7μCi / kg, 0.7μCi / kg~0.8μCi / kg, 0.8μCi / kg~0. 9μCi / kg, 0.9μCi / kg~1.0μCi / kg, 1.0μCi / kg~1.5μCi / kg, 1.5μCi / kg~2.0μCi / kg, 2.0μCi / kg~2.5μCi / kg, 2.5μCi / kg ~3.0μCi / kg, 3.0μCi / kg~3.5μCi / kg, 3.5μCi / kg~4.0μCi / kg, 4.0μCi / kg~4.5μCi / kg, or 4.5μCi / kg~5.0μCi / kg.

[0033] Radiolabeled antibodies 225According to embodiments labeled with Ac, the effective amount is 0.05 μCi / kg, 0.1 μCi / kg, 0.2 μCi / kg, 0.3 μCi / kg, 0.4 μCi / kg, 0.5 μCi / kg, 0.6 μCi / kg, 0.7 μCi / kg, 0.8 μCi / kg, 0.9 μCi / kg, 1.0 μCi / kg, 1.5 μCi / kg, 2.0 μCi / kg, 2.5 μCi / kg, 3.0 μCi / kg, 3.5 μCi / kg, 4.0 μCi / kg, or 4.5 μCi / kg.

[0034] An effective amount of radiolabeled antibody can be provided as a single dose. The majority of the antibody administered to a subject typically consists of unlabeled antibody, with a minority of labeled antibody. The ratio of labeled antibody to unlabeled antibody can be adjusted using known methods. Thus, according to certain aspects of the invention, the antibody may be provided in a total protein amount of up to 100 mg, for example, less than 60 mg, or 5 mg to 45 mg, or 0.1 ug / kg to 1 mg / kg of patient weight, for example, 1 ug / kg to 1 mg / kg of patient weight, or 10 ug / kg to 1 mg / kg of patient weight, or 100 ug / kg to 1 mg / kg of patient weight, or 0.1 ug / kg to 100 ug / kg of patient weight, or 0.1 ug / kg to 50 ug / kg of patient weight, or 0.1 ug / kg to 10 ug / kg of patient weight, or 0.1 ug / kg to 40 ug / kg of patient weight, or 1 ug / kg to 40 ug / kg of patient weight, or 0.1 mg / kg to 1.0 mg / kg of patient weight, for example, 0.2 mg / kg to 0.6 mg / kg of patient weight.

[0035] According to certain embodiments of the present invention, the radiolabeled antibody may comprise a labeled fraction and an unlabeled fraction, and the ratio of labeled fraction to unlabeled fraction may be about 0.01:10 to 1:1, e.g., 0.1:10 to 1:1 labeled fraction to unlabeled fraction.

[0036] Additionally, radiolabeled antibodies can be provided as single-dose compositions tailored to a particular patient, i.e., patient-specific therapeutic compositions, where the amount of labeled and unlabeled antibody in the composition can depend on at least the patient's weight, height, body surface area, age, sex, and / or medical or health condition. In this manner, the total volume of the patient-specific therapeutic composition can be provided in a vial configured to be administered entirely to a patient in one treatment session, such that little or no composition remains in the vial after administration.

[0037] "Hematologic malignancies" or "malignant hematologic diseases," also known as blood cancers, are cancers that arise in blood-forming tissues, such as bone marrow or other cells of the immune system. Hematologic malignancies include leukemias (e.g., acute myeloid leukemia (AML), acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), acute mixed lineage leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia (CLL), hairy cell leukemia, and large granular lymphocytic leukemia), myelodysplastic syndromes (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocytosis, primary myelofibrosis, and leukemia), and leukemias (e.g., leukemias of various types, such as leukemias of various types, ... These include, but are not limited to, chronic myelogenous leukemia), lymphoma, multiple myeloma, MGUS and similar disorders, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies.

[0038] "Solid cancer" includes, but is not limited to, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, prostate cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, cancer of the fallopian tubes, endometrial cancer, cancer of the cervix, cancer of the vagina, cancer of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, cancer of the urethra, cancer of the penis, childhood tumors, cancer of the bladder, cancer of the kidney or ureter, cancer of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, and environmentally induced cancers including those induced by asbestos.

[0039] "Non-malignant hematologic diseases" or "non-cancerous disorders" include, but are not limited to, type I diabetes, hemoglobinopathies (e.g., SCD and β-thalassemia), congenital immunodeficiencies (e.g., SCID), and viral infections (e.g., HIV infection). According to certain embodiments, the disorder is SCD and the therapy is gene-edited β-globin hematopoietic stem cell therapy. The stem cell therapy can be, for example, allogeneic or autologous. According to certain embodiments, the disorder is SCID and the therapy is gene-edited hematopoietic stem cell therapy and the edited gene is the common gamma chain (γc) gene, the adenosine deaminase (ADA) gene, and / or the Janus kinase 3 (JAK3) gene. The stem cell therapy can be, for example, allogeneic or autologous.

[0040] As used herein, the term "subject" includes, but is not limited to, mammals such as humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs, rats, and mice. When the subject is human, the subject can be of any age. According to certain embodiments, the subject is an infant. According to further embodiments, the subject is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years old. According to still further embodiments, the subject is 10-15 years old, or 15-20 years old. According to still further embodiments, the subject is 20 years old or older, 25 years old or older, 30 years old or older, 35 years old or older, 40 years old or older, 45 years old or older, 50 years old or older, 55 years old or older, 60 years old or older, 65 years old or older, 70 years old or older, 75 years old or older, 80 years old or older, 85 years old or older, or 90 years old or older.

[0041] As used herein, an "appropriate period" after administering a radiolabeled antibody to a subject and before administering a treatment to the subject is a period during which the administered antibody is sufficient to deplete the subject's HSCs and / or the subject's HSCs remain depleted. According to certain embodiments, the appropriate period is less than 15 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, or less than 3 days. According to certain embodiments, the appropriate period is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or more than 15 days.

[0042] As used herein, a "radioisotope" may be an alpha-, beta-, and / or gamma-emitting isotope. Examples of radioisotopes include: 131 I, 125 I, 123 I, 90 Y, 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 32 P, 225 Ac, 213 Bi, 213 Po, 211At, 212 Bi, 213 Bi, 223 Ra, 227 Th, 149 Tb, 137 Cs, 212 Pb, and 103 Radioisotopes include Pd.Methods for attaching radioisotopes to antibodies (i.e., using radioisotopes to "label" antibodies) are well known.Some of these methods are described, for example, in International Patent Application Publication No. WO2017 / 155937.

[0043] As used herein, "treating" a subject afflicted with a disorder includes, but is not limited to, (i) slowing, halting, or reversing the progression of the disorder; (ii) slowing, halting, or reversing the progression of symptoms of the disorder; (iii) reducing, ideally eliminating, the likelihood of recurrence of the disorder; and / or (iv) reducing, ideally eliminating, the likelihood of recurrence of symptoms of the disorder. According to certain preferred embodiments, treating a subject afflicted with a disorder means (i) reversing the progression of the disorder, ideally until the disorder is eliminated, and / or (ii) reversing the progression of symptoms of the disorder, ideally until the symptoms are eliminated, and / or (iii) reducing or eliminating the likelihood of recurrence. Ideally, treating a subject afflicted with a disorder means curing the disorder by removing or neutralizing its genetic cause.

[0044] Throughout this application, various publications are cited. The disclosures of these publications are incorporated herein by reference into this application in order to more fully describe the prior art to which this invention pertains. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing described herein, suitable methods and materials are described below.

[0045] Aspects of the present invention The present invention addresses an unmet need in the art by providing an unexpectedly superior method for depleting a subject's hematopoietic stem cells, ideally before bone marrow transplantation or gene-edited cell-based therapy, such as gene-edited β-globin hematopoietic stem cell therapy for SCD. The present invention uses radiolabeled antibodies, such as radiolabeled anti-CD34, anti-CD117, or anti-CD135, for this purpose. The antibodies can safely and effectively deplete a subject's hematopoietic stem cells through targeted conditioning. This approach avoids certain adverse effects caused by less specific agents, such as chemotherapeutic agents, or external irradiation.

[0046] CD34 is a glycosylated type I transmembrane protein of 105-120 kD that is specifically expressed on the surface of hematopoietic stem cells / progenitor cells (HSCs / HPCs) in humans and other mammals. Two transcript variants encoding distinct isoforms have been found for the CD34 gene. It functions as an intercellular adhesion factor, mediating stem cell attachment to bone marrow extracellular matrix or direct attachment to stromal cells. CD34 expression gradually decreases with hematopoietic cell maturation. CD34 is also expressed on normal and neoplastic microvascular endothelial cells. Anti-CD34 antibodies that can be used in conjunction with the targeted depletion methods described herein include, but are not limited to, antibodies produced and released from ATCC accession numbers AC133.1 and HB 12346, as disclosed in U.S. Patent No. 5,843,633.

[0047] CD117, or mast / stem cell growth factor receptor (SCFR), or c-Kit, is a receptor tyrosine kinase protein encoded by the KIT gene in humans. Multiple transcript variants encoding different isoforms have been found for this gene. Stem cell factor (SCF) signals via CD117 in a pathway that plays a key role in hematopoiesis. CD117 is expressed on pluripotent hematopoietic stem cells, precursors of mature cells belonging to the lymphoid and erythroid lineages. While other mature hematopoietic cells exhibit reduced or absent CD117 expression, mast cell precursors and mature mast cells retain high levels of CD117 expression. Therefore, SCF signaling through CD117 is essential for mast cell development, function, trafficking, and survival. Exemplary commercially available anti-CD117 antibodies include IMC-CK6, AMG191, KTN0158, A3C6E2, and LMJ729.

[0048] Other anti-CD117 antibodies that can be used in conjunction with the targeted depletion methods described herein include, for example, the antibody produced and released from ATCC Accession No. 10716 (deposited as BA7.3C.9) disclosed in U.S. Patent No. 5,489,516, e.g., the SR-1 antibody.

[0049] CD135, also known as Ly72, Flk-2, Flt-3, or B230315G04, is a type I transmembrane cytokine receptor belonging to the receptor tyrosine kinase class III. CD135 is a receptor for the cytokine Flt3 ligand (FLT3L). CD135 signaling is important for the normal development of hematopoietic stem and progenitor cells. It is expressed on the surface of many hematopoietic progenitor cells and is found on the majority of malignant hematopoietic cells (e.g., AML, ALL). CD135 expression is usually lost upon differentiation of hematopoietic stem cells (HSCs), with the exception of mature dendritic cells (DCs), which show persistent CD135 expression. Exemplary commercially available anti-CD135 antibodies include LY3012218 (IMC-EB10).

[0050] Other anti-CD135 antibodies that can be used in conjunction with the targeted depletion methods described herein include, for example, the antibodies produced and released by American Type Culture Collection (ATCC) Accession No. ATCC HB 11,557, as described in U.S. Pat. No. 5,635,388, or the antibodies produced and released by ATCC Accession No. FTA-4089, as described in U.S. Pat. No. 7,183,385, or the antibodies described in U.S. Pat. No. 5,548,065 (including, for example, anti-CD135 antibodies, antigen-binding fragments thereof, and ligands produced and released by ATCC Accession Nos. CRL 10907, CRL 10935, CRL 10936, and CRL 11005).

[0051] Each of these targets is differentially expressed on hematopoietic stem cells and hematopoietic progenitor cells, and generally shows reduced or absent expression on mature, differentiated hematopoietic stem cells. Thus, the compositions and methods of the present invention provide novel methods for targeting hematopoietic stem cells and depleting hematopoietic stem cell populations while minimizing effects on other cells / tissues. In certain embodiments, the present invention provides methods for depleting hematopoietic stem cells in a subject, comprising administering to the subject an effective amount of a radiolabeled antibody, e.g., anti-CD34, anti-CD117, or anti-CD135.

[0052] An effective amount of radiolabeled antibody may be the maximum tolerated dose (MTD) or may be an amount sufficient to induce bone marrow conditioning or even myeloablation. Such treatment may be an effective conditioning treatment for transplantation using allogeneic or autologous stem cells and may provide improved therapeutic outcomes for categories of patients who have poor prognosis with standard prior art therapies (i.e., radiation and / or chemotherapy).

[0053] According to certain embodiments of the invention, the methods involve administering an effective amount of a radiolabeled antibody, such as anti-CD34, anti-CD117, or anti-CD135, for the treatment of a proliferative disease or hematological malignancy.

[0054] This depletion method (also referred to herein as the conditioning method) is also useful for treating subjects with cancerous disorders treatable through HSCT, e.g., bone marrow transplantation. For example, hematopoietic stem cells can be depleted or destroyed to replace them after standard cancer treatments using high-dose chemotherapy or radiation that damage the bone marrow, or to provide new stem cells that can replace diseased or damaged bone marrow or directly kill cancer cells. Thus, the present invention provides a method for treating a subject with a cancerous disorder, comprising administering to the subject an amount of a radiolabeled antibody effective to deplete or destroy the subject's hematopoietic stem cells. According to certain embodiments, the method may further comprise, after an appropriate period of time, performing a bone marrow transplant to treat the subject's disorder.

[0055] This depletion method is also useful for improving the outcome of subsequent gene-edited cell-based therapies in which hematopoietic stem cell depletion is desired. According to certain preferred aspects of this method, the subject is suffering from a non-cancer disorder treatable via gene-edited cell therapy and is undergoing such therapy to treat the disorder. Thus, the present invention also provides a method for treating a subject suffering from a non-cancer disorder treatable via gene-edited cell therapy, the method comprising administering to the subject an amount of a radiolabeled antibody effective to deplete the subject's hematopoietic stem cells. According to certain aspects, the method may further comprise, after an appropriate period of time, administering a therapy to the subject to treat the subject's disorder.

[0056] According to certain preferred aspects of the subject method, the radiolabeled antibody comprises: 131 I or 225 Radiolabeled antibody is radiolabeled with Ac. 131 When labeled with I, an effective amount can be, for example, 10 mCi to 200 mCi, 200 mCi to 400 mCi, or 400 mCi to 1200 mCi. 225When labeled with Ac, the effective amount can be, for example, 0.1 μCi / kg to 1.0 μCi / kg, 1.0 μCi / kg to 3.0 μCi / kg, 3.0 μCi / kg to 5.0 μCi / kg, or 0.1 μCi / kg to 5.0 μCi / kg.

[0057] The present invention provides, inter alia, seven specific embodiments of a subject method for treating a human subject suffering from a cancerous disorder treatable via bone marrow transplantation. The first embodiment comprises administering (i) 10 mCi to 200 mCi of 131 A second embodiment includes (i) administering 200 mCi to 400 mCi of an antibody labeled with I to a subject; and (ii) 6, 7, or 8 days later, performing a bone marrow transplant on the subject to treat the cancer in the subject. 131 A third embodiment includes (i) administering 400 mCi to 1,200 mCi of an antibody labeled with I to a subject; and (ii) 8, 9, 10, 11, or 12 days later, performing a bone marrow transplant on the subject to treat the cancer in the subject. 131 A fourth embodiment includes (i) administering to a subject an antibody labeled with I; and (ii) 10, 11, 12, 13, or 14 days later, performing a bone marrow transplant in the subject to treat the cancer in the subject. 225 A fifth embodiment includes (i) administering an Ac-labeled antibody to a subject, and (ii) 6, 7, or 8 days later, performing a bone marrow transplant on the subject to treat the cancer in the subject. 225 A sixth embodiment includes (i) administering an Ac-labeled antibody to a subject, and (ii) 6, 7, or 8 days later, performing a bone marrow transplant on the subject to treat the cancer in the subject. 225 A seventh embodiment includes (i) administering an Ac-labeled antibody to a subject, and (ii) 6, 7, or 8 days later, performing a bone marrow transplant on the subject to treat the cancer in the subject. 225administering an Ac-labeled antibody to a subject; and (ii) 6, 7, or 8 days later, performing a bone marrow transplant on the subject to treat the cancer in the subject.

[0058] Exemplary cancers treated by these methods include at least lymphoma and / or leukemia. For example, the cancer can be lymphoblastic leukemia, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, or a combination thereof.

[0059] The present invention provides, inter alia, seven specific embodiments of a subject method for treating a human subject suffering from a non-cancer disorder treatable via gene-edited allogeneic or autologous cell therapy. The first embodiment comprises administering (i) 10 mCi to 200 mCi of 131 A second embodiment includes (i) administering 200 mCi to 400 mCi of an antibody labeled with I to a subject; and (ii) 6, 7, or 8 days later, administering therapy to the subject to treat the disorder. 131 A third embodiment includes (i) administering 400 mCi to 1,200 mCi of an antibody labeled with I to a subject; and (ii) 8, 9, 10, 11, or 12 days later, administering a therapy to the subject to treat the disorder. 131 A fourth embodiment includes (i) administering to a subject an antibody labeled with I; and (ii) 10, 11, 12, 13, or 14 days later, administering a therapy to the subject to treat the disorder. 225 A fifth embodiment includes (i) administering an antibody labeled with Ac to a subject, and (ii) 6, 7, or 8 days later, administering a therapy to the subject to treat the disorder. 225 A sixth embodiment includes (i) administering an antibody labeled with Ac to a subject, and (ii) 6, 7, or 8 days later, administering a therapy to the subject to treat the disorder. 225A seventh embodiment includes (i) administering an Ac-labeled antibody to a subject, and (ii) 6, 7, or 8 days later, administering a therapy to the subject to treat the disorder. 225 administering an Ac-labeled antibody to a subject; and (ii) 6, 7, or 8 days later, administering a therapy to the subject to treat the disorder.

[0060] The present invention further provides an article of manufacture comprising (a) a radiolabeled antibody and (b) a label instructing a user to administer to a subject, preferably a human, an amount of the antibody effective to deplete the subject's hematopoietic stem cells.

[0061] According to certain preferred embodiments of the subject article, the radiolabeled antibody comprises: 131 I or 225 Radiolabeled antibodies such as Ac or CD34, CD117, or CD135. 131 When labeled with I, an effective amount can be, for example, 10 mCi to 200 mCi, 200 mCi to 400 mCi, or 400 mCi to 1200 mCi. 225 When labeled with Ac, the effective amount can be, for example, 0.1 μCi / kg to 5.0 μCi / kg.

[0062] The present invention provides, inter alia, seven specific embodiments of the subject article. The first embodiment comprises: (a) 131 I-labeled antibodies and (b) 10 mCi–200 mCi 131 and a label instructing the user to administer the antibody labeled with I to a human subject. 131 I-labeled antibody and (b) 200 mCi–400 mCi 131 and a label instructing the user to administer the antibody labeled with I to a human subject. 131 I-labeled antibodies and (b) 400 mCi to 1,200 mCi 131and a label instructing a user to administer the antibody labeled with I to a human subject. 225 (b) 0.1 μCi / kg to 5.0 μCi / kg of Ac-labeled antibody 225 and a label instructing a user to administer the Ac-labeled antibody to a human subject. 225 (b) 0.1 μCi / kg to 1.0 μCi / kg of Ac-labeled antibody 225 and a label instructing a user to administer the Ac-labeled antibody to a human subject. 225 (b) 1.0 μCi / kg to 3.0 μCi / kg of Ac-labeled antibody 225 and a label instructing a user to administer the Ac-labeled antibody to a human subject. 225 (b) 3.0 μCi / kg to 5.0 μCi / kg of Ac-labeled antibody 225 and a label instructing the user to administer the Ac-labeled antibody to a human subject.

[0063] Accordingly, the following aspects are disclosed in the present application:

[0064] Embodiment 1. A method for targeted depletion of hematopoietic stem cells in a subject, the method comprising administering to the subject an effective amount of a radiolabeled antibody against CD34, CD117, CD135, or a combination thereof.

[0065] Embodiment 2. The radiolabeled antibody is 131 I, 125 I, 123 I, 90 Y, 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 32 P, 225 Ac, 213 Bi, 213 Po, 211 At, 212 Bi, 213 Bi, 223 Ra,227 Th, 149 Tb, 137 Cs, 212 Pb, and 103 2. The method of embodiment 1, wherein the compound is labeled with Pd.

[0066] Embodiment 3. The radiolabeled antibody is 131 It is labeled with I, 131 3. The method of embodiment 2, wherein the effective amount of the antibody labeled with I is 10 mCi to 200 mCi, or 200 mCi to 400 mCi, or 400 mCi to 1200 mCi.

[0067] Embodiment 4. The radiolabeled antibody is 225 It is labeled with Ac, 225 The method of embodiment 2, wherein the effective amount of the Ac-labeled antibody is 0.1 μCi / kg to 5.0 μCi / kg (subject body weight), or 0.1 μCi / kg to 1.0 μCi / kg (subject body weight), or 1.0 μCi / kg to 3.0 μCi / kg (subject body weight), or 3.0 μCi / kg to 5.0 μCi / kg (subject body weight).

[0068] Embodiment 5. The method of any one of embodiments 1-4, wherein the subject is afflicted with a non-cancer disorder treatable via gene-edited cell therapy and is undergoing such therapy to treat the disorder, and wherein an effective amount of the radiolabeled antibody is administered as a single dose.

[0069] Embodiment 6. The method of embodiment 5, wherein the disorder is selected from the group consisting of a hemoglobinopathy, a congenital immunodeficiency, and a viral infection.

[0070] Embodiment 7. The method of embodiment 5, wherein the disorder is selected from the group consisting of sickle cell disease (SCD), severe combined immunodeficiency (SCID), and β-thalassemia.

[0071] Embodiment 8. The method of embodiment 7, wherein the disorder is SCD and the therapy is gene-edited β-globin hematopoietic stem cell therapy.

[0072] Embodiment 9. The method of embodiment 7, wherein the disorder is SCID, the therapy is gene-edited hematopoietic stem cell therapy, and the edited gene is selected from the group consisting of the common gamma chain (γc) gene, the adenosine deaminase (ADA) gene, and the Janus kinase 3 (JAK3) gene.

[0073] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the hematopoietic stem cells are depleted by at least 50%, or at least 70%, or at least 90%.

[0074] Embodiment 11 The method of embodiment 10, wherein mature differentiated hematopoietic stem cells are depleted by less than 20%, or less than 10%.

[0075] Embodiment 12. The method of any one of embodiments 1 to 4, wherein the subject is suffering from a cancerous disorder treatable by bone marrow transplantation.

[0076] Embodiment 13 The method of embodiment 12, wherein the disorder is leukemia or lymphoma.

[0077] Embodiment 14. The method of embodiment 12, wherein the acute disorder is lymphoblastic leukemia, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, or a combination thereof.

[0078] Embodiment 15. A method for treating a subject afflicted with a cancerous disorder treatable via bone marrow transplantation, comprising: (i) administering to the subject an amount of a radiolabeled antibody effective to deplete or destroy the subject's hematopoietic stem cells; and (ii) after a suitable period of time, performing a bone marrow transplant on the subject to treat the subject's disorder, wherein the antibody comprises anti-CD34, anti-CD117, anti-CD135, or a combination thereof.

[0079] Embodiment 16 The method of embodiment 15, wherein the disorder is leukemia or lymphoma.

[0080] Embodiment 17. The method of embodiment 15 or 16, wherein the acute disorder is lymphoblastic leukemia, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, or a combination thereof.

[0081] Embodiment 18. The method of any one of embodiments 15 to 17, wherein hematopoietic stem cells are depleted, or at least 70%, or at least 90%, and mature differentiated hematopoietic stem cells are depleted by less than 20%, or less than 10%.

[0082] Embodiment 19. The radiolabeled antibody is 131 I, 125 I, 123 I, 90 Y, 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 32 P, 225 Ac, 213 Bi, 213 Po, 211 At, 212 Bi, 213 Bi, 223 Ra, 227 Th, 149 Tb, 137 Cs, 212 Pb, or 103 19. The method of any one of embodiments 15 to 18, wherein the compound is labeled with Pd.

[0083] 20. The radiolabeled antibody is 131 It is labeled with I, 131 20. The method of any one of aspects 15-19, wherein the effective amount of the antibody labeled with I is 10 mCi to 200 mCi administered 6, 7, or 8 days prior to bone marrow transplantation.

[0084] 21. The radiolabeled antibody is 131 It is labeled with I, 131 20. The method of any one of aspects 15-19, wherein the effective amount of the antibody labeled with I is 200 mCi to 400 mCi administered 8, 9, 10, 11, or 12 days prior to bone marrow transplantation.

[0085] 22. The radiolabeled antibody is 131 It is labeled with I, 131 20. The method of any one of aspects 15-19, wherein the effective amount of the I-labeled antibody is 400 mCi to 1,200 mCi administered 10, 11, 12, 13, or 14 days prior to bone marrow transplantation.

[0086] 23. The radiolabeled antibody is 225 It is labeled with Ac, 225 20. The method of any one of aspects 15 to 19, wherein the effective amount of the Ac-labeled antibody is 0.1 μCi / kg to 5.0 μCi / kg (subject body weight) administered 6, 7, 8, 9, 10, 11, or 12 days prior to bone marrow transplantation.

[0087] Embodiment 24. A method for treating a subject afflicted with a non-cancerous disorder treatable via gene-edited cell therapy, comprising: (i) administering to the subject an amount of a radiolabeled antibody effective to deplete the subject's hematopoietic stem cells; and (ii) after a suitable period of time, administering therapy to the subject to treat the subject's disorder, wherein the antibody comprises anti-CD34, anti-CD117, anti-CD135, or a combination thereof.

[0088] Embodiment 25. The method of embodiment 24, wherein the subject is afflicted with a non-cancer disorder treatable via gene-edited cell therapy and is undergoing such therapy to treat the disorder, and wherein an effective amount of the radiolabeled antibody is administered as a single dose.

[0089] Embodiment 26 The method of embodiment 24 or 25, wherein the disorder is selected from the group consisting of hemoglobinopathy, congenital immunodeficiency, and viral infection.

[0090] Embodiment 27. The method of any one of embodiments 24 to 26, wherein the disorder is selected from the group consisting of sickle cell disease (SCD), severe combined immunodeficiency (SCID), and β-thalassemia.

[0091] Embodiment 28 The method of embodiment 27, wherein the disorder is SCD and the therapy is gene-edited β-globin hematopoietic stem cell therapy.

[0092] Embodiment 29. The method of embodiment 27, wherein the disorder is SCID, the therapy is gene-edited hematopoietic stem cell therapy, and the edited gene is selected from the group consisting of the common gamma chain (γc) gene, the adenosine deaminase (ADA) gene, and the Janus kinase 3 (JAK3) gene.

[0093] Embodiment 30 The method of embodiment 28 or 29, wherein the stem cell therapy is an allogeneic stem cell therapy, or wherein the stem cell therapy is an autologous stem cell therapy.

[0094] 31. The radiolabeled antibody is 131 I, 125 I, 123 I, 90 Y, 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 32 P, 225 Ac, 213 Bi, 213 Po, 211 At, 212 Bi, 213 Bi, 223 Ra, 227 Th, 149 Tb, 137 Cs, 212 Pb, or 103 31. The method of any one of aspects 24 to 30, wherein the compound is labeled with Pd.

[0095] 32. The radiolabeled antibody is 131 It is labeled with I, 131 32. The method of any one of embodiments 24-31, wherein the effective amount of the antibody labeled with I is between 10 mCi and 200 mCi administered 6, 7, or 8 days before administering a therapy to the subject to treat the disorder.

[0096] 33. The radiolabeled antibody is 131 It is labeled with I, 131 32. The method of any one of aspects 24-31, wherein the effective amount of the antibody labeled with I is 200 mCi to 400 mCi administered 8, 9, 10, 11, or 12 days before administering a therapy to the subject to treat the subject's disorder.

[0097] 34. The radiolabeled antibody is 131 It is labeled with I, 131 32. The method of any one of embodiments 24-31, wherein the effective amount of the antibody labeled with I is 400 mCi to 1,200 mCi administered 10, 11, 12, 13, or 14 days prior to administering to the subject a therapy to treat the subject's disorder.

[0098] 35. The radiolabeled antibody is 225 It is labeled with Ac, 225 The method of any one of aspects 24 to 31, wherein the effective amount of the antibody labeled with Ac is 0.1 μCi / kg to 5.0 μCi / kg of subject body weight administered 6, 7, 8, 9, 10, 11, or 12 days before administering a therapy to the subject to treat the subject's disorder.

[0099] Embodiment 36. An article of manufacture comprising: (a) a radiolabeled antibody; and (b) a label instructing a user to administer to a subject an amount of the antibody effective to deplete hematopoietic stem cells in the subject, wherein the antibody comprises anti-CD34, anti-CD117, anti-CD135, or a combination thereof.

[0100] 37. The radiolabeled antibody is 131 It is labeled with I, 131 37. The article of embodiment 36, wherein the effective amount of the antibody labeled with I is 10 mCi to 200 mCi, or 200 mCi to 400 mCi, or 400 mCi to 1,200 mCi.

[0101] 38. The radiolabeled antibody is 225 It is labeled with Ac, 22537. The article of embodiment 36, wherein the effective amount of the antibody labeled with Ac is 0.1 μCi / kg to 5.0 μCi / kg, or 0.1 μCi / kg to 1.0 μCi / kg (of the subject's body weight), or 1.0 μCi / kg to 3.0 μCi / kg (of the subject's body weight), or 3.0 μCi / kg to 5.0 μCi / kg (of the subject's body weight).

[0102] The present invention will be better understood by reference to the following examples, although those skilled in the art will readily appreciate that the specific examples detailed are merely illustrative of the invention as more fully described in the claims that follow thereafter. [Example]

[0103] Example 1 - Radioiodination of antibodies Commercially available anti-CDXX antibodies, as well as antibodies generated by methods known in the art from CDXX positive cell lines for each of CD34, CD117, and CD135 available from the ATCC as detailed herein, were immunized with iodine-131 ( 131 I).

[0104] 131 A specific example for labeling an anti-CDXX antibody with I includes the following: 1 mg of an anti-CDXX immunoglobulin described herein is labeled with 20-30 mCi of I in the presence of chloramine-T (23 micrograms) in PBS buffer (pH 7.2). 131 The reaction can be labeled with 1000 mCi of I-Na (30 mCi). The reaction can then be quenched by the addition of aqueous sodium thiosulfate (69 micrograms) and diluted with cold NaI (1 mg). Immediately after, a concentrated ascorbic acid solution made in 50 mM PBS (pH 7) can be added to achieve an ascorbic acid strength of 2.5% (w / v) in the quenched reaction mixture. Labeling of up to 3,000 mCi of reaction per batch can be successfully performed using this method.

[0105] The labeled product can be purified by gel filtration on a sterile, pre-packaged, commercially available Sephadex G25 column (GE HiPrep 26 / 10 column, 53 mL bed volume) using a mobile phase of PBS (50 mM, pH 7) supplemented with 2.5% (w / v) ascorbic acid to stabilize the radiolabeled product. Reaction volumes up to 1,000 mCi can be purified on a single column, with product recovery in elution volumes of 5 mL to 35 mL.

[0106] Radioiodinated reaction batches of less than 200 mCi can be purified in a similar manner on a smaller desalting column (GE PD10 column, 8.6 mL bed volume).

[0107] Example 2 - Actinium Labeling of Antibodies Commercially available anti-CDXX antibodies, as well as antibodies generated by methods known in the art from CDXX positive cell lines for each of CD34, CD117, and CD135 available from the ATCC as detailed herein, were used in combination with actinium-225 ( 225 Isotype control human IgG can be purchased from Creative Diagnostics (New York, USA). Actinium-225 (Ac) in dry nitrate form can be used. 225 Ac) can be obtained from Oak Ridge National Laboratory, USA. The bifunctional chelator p-SCN-Bn-DOTA (referred to as DOTA in these examples) can be purchased from Macrocyclics (Texas, USA).

[0108] DOTA can be conjugated to anti-CDXX in an excess, such as 5M, in ammonium acetate buffer for 1.5 hours at 37° C. The anti-CDXX-DOTA conjugate can then be incubated at 37° C. or room temperature for 60 minutes with 1 uCi / ug of anti-CDXX in 0.01 M HCl (pH 6.5). 225 Label with Ac, 0.3 μg 225 It can provide a specific radioactivity of approximately 100 nCi to 500 nCi for Ac-anti-CDXX. 225Ac-anti-CDXX can be diluted with unlabeled anti-CDXX to adjust the total antibody dose and radiation (radiolabel) dose. Samples can be purified to 99±1% purity with disposable spin columns.

[0109] A specific labeling example for conjugating anti-CDXX with the chelator DOTA may include the following: Antibody (2 mg) may be equilibrated with conjugation buffer (Na Carbonate buffer with 1 mM EDTA, pH=8.5-9.0) by four ultrafiltration spins on Centricon filters or Vivaspin ultrafiltration tubes with an appropriately sized MW cutoff (e.g., 50,000). A volume of 1.5 ml of conjugation buffer may be used per spin. For each spin, the antibody may be spun at 53,000 RPM and 4°C for 5-20 minutes until the residual retention volume is 100-200 μl. The antibody may be incubated at 4°C for 30 minutes after the second and third spins to allow equilibration. For DOTA conjugation, a 3 mg / ml solution of S-2-(4-isothiocyanatobenzyl)-1,4,7,10 tetra-azacyclododecanetetraacetic acid (p-SCN-Bz-DOTA, MW = 687) in 0.15 M NHOAc can be prepared by dissolving and vortexing. DOTA-Bz-pSCN and anti-CDXX antibody (>5 mg / ml) can be mixed together in an Eppendorf tube at a 7.5 molar ratio (DOTA:antibody) and incubated at room temperature for 15 hours. For purification of the DOTA-antibody conjugate, unreacted DOTA-Bz-pSCN can be removed by seven rounds of ultrafiltration using 1.5 ml of 0.15 M NHOAc buffer (pH = 6.5) to a volume of approximately 100 μl. After the final wash, 0.15 M NHOAc buffer was added to bring the material to a final concentration of approximately 1 mg / ml. Using this method, the number of DOTA molecules conjugated to an anti-CDXX antibody is generally 1.2 to 1.5 DOTA per antibody.

[0110] DOTA-antibody conjugates 225A specific labeling example for radiolabeling with Ac may involve mixing 15 μL of 0.15 M NHOAC buffer (pH=6.5) with 2 μL (10 μg) of DOTA-CDXX (5 mg / ml) in 0.05 M HCl in an Eppendorf reaction tube. 225 Ac (10 μCi) can be added and mixed, and the reaction mixture can be incubated for 90 minutes at 37° C. with shaking at 100 rpm. At the end of the incubation period, 3 μL of 1 mM DTPA solution can be added to the reaction mixture and incubated at room temperature for 20 minutes to remove uncomplexed DTPA. 225 Instant thin layer chromatography (ITLC) was performed using 10 cm silica gel strips and a 10 mM EDTA / normal saline mobile phase. 225 Determine the radiochemical purity of Ac-anti-CDXX 225 Ac-labeled antibody 225 After separation from Ac-DTPA, sections can be counted in a gamma counter equipped with a multichannel analyzer. Radiolabeling efficiency across several runs has been determined to be greater than 80% for standard antibodies.

[0111] Example 3 - Patient-specific treatment Radiolabeled anti-CDXX ("drug product") may be supplied for patient administration as a sterile formulation contained in a container closure system consisting of a depyrogenated Type 1 50 mL glass vial, a sterile gray chlorobutyl rubber stopper, and an open-top style aluminum seal. Each dose vial may contain a drug product fill volume of 45 mL in a 50 mL vial. Similarly, the drug product may be provided as a single-use dose for complete infusion during intravenous administration and may contain patient-specific radioactivity. 131 For I-anti-CDXX, patient-specific doses range from 1 mCi to 1200 mCi, as described herein. 131 I and 1-60 mg of protein (total anti-CDXX). 225 For Ac-anti-CDXX, patient-specific doses range from 0.1 μCi / kg to 5.0 μCi / kg of patient body weight, as described herein. 225The formulation may contain Ac and 1-60 mg of protein (total anti-CDXX). The dose of the anti-CDXX antibody is determined according to ideal body weight at a level of 0.1 mg / kg to 1.0 mg / kg, e.g., 0.5 mg / kg. In certain embodiments, the pharmaceutical product may be co-administered to a patient with 0.9% sodium chloride injection, USP (normal saline), at a 1:9 ratio of pharmaceutical product to saline. The infusion rate depends on the amount of anti-CDXX antibody in the 45 mL pharmaceutical fill volume, so a total pharmaceutical product and saline infusion volume of approximately 430-450 mL is administered over a variable period of time.

[0112] Example 4 - SCD This example describes HSC destruction (i.e., 100% depletion) prior to transplantation with gene-edited HSCs in SCD patients.

[0113] SCD is the most common hemoglobinopathy worldwide. The incidence of SCD in African Americans is approximately 1 in 500. In the United States, an estimated 100,000 people are affected.

[0114] SCD is caused by a single-nucleotide mutation in the beta-globin gene, which produces sickle hemoglobin. Patients with SCD can present with anemia, vaso-occlusive crisis (VOC), hemolysis, chronic organ dysfunction, and premature death. The mortality rate for children with SCD is 0.5 per 100,000. However, the adult mortality rate is greater than 2.5 per 100,000, and the median life expectancy is less than 50 years for both men and women with SCD.

[0115] Currently, the only curative treatment for SCD is hematopoietic stem cell transplantation (HSCT). Unfortunately, HSCT for SCD is not without problems. According to the Center for International Blood and Marrow Transplant Research, only 1,089 patients with SCD underwent HSCT between 1991 and April 2017. Risks associated with HSCT include complications arising from the use of allogeneic donor stem cells, such as graft-versus-host disease.

[0116] With the advent of gene editing technology, there is now an opportunity to cure SCD patients using autologous stem cells in which the beta-globin gene mutation responsible for SCD has been corrected. ZFN, TALEN, CRISPR / cas9, and other nuclease-mediated editing approaches can be used to repair or remove and replace stem cells from SCD patients. For example, Sun and Zhao (Biotech. and Bioeng., 2014, 111(5)) demonstrated successful repair of a human beta-globin gene mutation in a patient's pluripotent HSCs using TALEN. Furthermore, Dever et al. (Nature, 2016, 539:384-389) demonstrated efficient repair of a Glu6Val mutation responsible for SCD in a patient's HSCs using CRISPR / cas9. Clinical trials using this approach for SCD are currently being initiated.

[0117] Unfortunately, standard myeloablative conditioning regimens (i.e., 100% HSC-depleting regimens) using high-dose chemotherapy or total body irradiation are currently used for transplants, including autologous gene-edited cell transplants. There is a need for safer and more effective conditioning methods for these patients. Radiolabeled antibodies would better spare the patient's normal tissue. In particular, older SCD patients may already have organ damage as a result of their disease, and exposure to nonspecific radiation or chemotherapy as myeloablative conditioning regimens may make stem cell transplantation more risky. A radiolabeled anti-CDXX approach offers a better option for these patients.

[0118] Furthermore, due to the genetic nature of the disease, it is preferable to correct the disease through transplantation of gene-edited HSCs as early as possible, because the complications of the disease may be irreversible and may have a negative impact on the long-term survival of the patient.Therefore, it is envisioned that gene-edited HSCs are used to treat infants or young children suffering from SCD.For this purpose, it is preferable to 225 Radiolabeled antibodies such as anti-CD34, anti-CD117, or anti-CD135 labeled with alpha-emitting radionuclides such as Ac would be ideal because of their very short, high-energy radiation pathlength. 225 The use of alpha-emitting radionuclides, such as Ac, concentrates radiation in CD34, CD117, or CD135 positive cells, resulting in ablative doses. 131 This allows for effective destruction without the need to isolate the patient being treated (as is required for conditioning with I-labeled antibodies).

[0119] Example 5 - SCID This example describes HSC destruction prior to transplantation with gene-edited HSCs in SCID patients.

[0120] SCID is a germline genetic disorder in which affected patients exhibit a severe T-cell deficiency, with or without a concomitant B-cell deficiency. SCID is associated with a defective adaptive immune response, preventing patients from mounting an effective antibody response against pathogens. SCID is the most severe form of primary immunodeficiency, with at least nine known genes whose mutations lead to SCID. Because SCID patients are unable to mount an adaptive immune response, they are susceptible to infection and have a high premature mortality rate. SCID is also known as "bubble boy" syndrome because patients must be kept in a sterile environment to avoid life-threatening infections.

[0121] The most common genetic defect in SCID is in the common gamma chain (γc), a protein shared by the receptors for interleukins IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Other mutated genes that can lead to SCID are ADA and JAK3. As with SCD, only stem cell transplantation is potentially curative for SCID. However, delayed immune recovery and GVHD are significant risks for these patients. Also, as with SCD patients, SCID patients are young, so effective and safe treatments, including better pre-transplant conditioning regimens, are needed.

[0122] Gene editing technology can precisely repair defects in SCID patients' own HSCs. Once reintroduced into the body, these engineered HSCs can produce normal lymphocytes, establish a functional adaptive immune response, and protect against infection. Recently, Chang et al. (Cell Reports, 2015, 12:1668-1677) reported that normal lymphocyte development was effectively restored through CRISPR / cas9-mediated repair of a mutation in the JAK3 gene in mice. Furthermore, Alzubi et al. (Nature, Scientific Reports, 2017, 7:12475) recently demonstrated the use of TALEN technology to precisely repair a genetic defect in IL2RG (common gamma chain), a gene involved in X-SCID, in mice.

[0123] It is important that safer and more effective methods for conditioning human SCID patients be developed. For example, 225 Alpha-emitting radioimmunotherapy using Ac-labeled anti-CD34, or anti-CD117, or anti-CD135 is needed to safely condition these primarily young patients.

[0124] Example 6 - Treatment Overview Table I summarizes selected treatment regimens using gene-edited stem cell administration preceded by HSC depletion via administration of radiolabeled antibodies (i.e., conditioning agents). [Table 1]

Claims

1. 1. A method for targeted depletion of hematopoietic stem cells in a subject, said method comprising: administering to the subject an effective amount of a radiolabeled antibody to CD34, CD117, CD135, or a combination thereof; the radiolabeled antibody 131 I, 125 I, 123 I, 90 Y. 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 32 P. 225 Ac, 213 Bi, 213 Po, 211 At, 212 Bi, 213 Bi, 223 Ra, 227 Th, 149 Tb, 137 Cs, 212 Pb, and 103 Pd.

2. the radiolabeled antibody 131 I-radiolabeled 131 3. The method of claim 2, wherein the effective amount of I-radiolabelled is 10 mCi to 200 mCi, or 200 mCi to 400 mCi, or 400 mCi to 1,200 mCi.

3. the radiolabeled antibody 225 Ac-radiolabeled, 225 3. The method of claim 2, wherein the effective amount of Ac-radiolabelled is 0.1 μCi / kg to 5.0 μCi / kg of the subject's body weight, or 0.1 μCi / kg to 1.0 μCi / kg of the subject's body weight, or 1.0 μCi / kg to 3.0 μCi / kg of the subject's body weight, or 3.0 μCi / kg to 5.0 μCi / kg of the subject's body weight.

4. 10. The method of claim 1, wherein the subject is suffering from a non-cancer disorder treatable via gene-edited cell therapy and is undergoing such therapy to treat the non-cancer disorder, and wherein the effective amount of the radiolabeled antibody is administered as a single dose.

5. 5. The method of claim 4, wherein the non-cancerous disorder is selected from the group consisting of a hemoglobinopathy, a congenital immunodeficiency, and a viral infection.

6. 5. The method of claim 4, wherein the non-cancerous disorder is selected from the group consisting of sickle cell disease (SCD), severe combined immunodeficiency (SCID), and β-thalassemia.

7. 7. The method of claim 6, wherein the non-cancerous disorder is SCD and the therapy is genetically edited β-globin hematopoietic stem cell therapy.

8. 7. The method of claim 6, wherein the disorder is SCID, the therapy is gene-edited hematopoietic stem cell therapy, and the edited gene is selected from the group consisting of the common gamma chain (γc) gene, the adenosine deaminase (ADA) gene, and the Janus kinase 3 (JAK3) gene.

9. 2. The method of claim 1, wherein the hematopoietic stem cells are depleted by at least 50%, or at least 70%, or at least 90%, and mature differentiated hematopoietic stem cells are depleted by less than 20%, or less than 10%.

10. 10. The method of claim 1, wherein the subject is suffering from a cancerous disorder treatable by bone marrow transplantation.

11. 11. The method of claim 10, wherein the cancerous disorder is leukemia or lymphoma.

12. 11. The method of claim 10, wherein the acute cancerous disorder is lymphoblastic leukemia, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, or a combination thereof.

13. 1. A method for treating a subject suffering from a cancerous disorder treatable via bone marrow transplantation, said method comprising: (i) administering to the subject an amount of a radiolabeled antibody effective to deplete or destroy hematopoietic stem cells in the subject; (ii) after a suitable period of time, performing the bone marrow transplant in the subject to treat the disorder in the subject; the cancerous disorder is leukemia or lymphoma, and the antibody comprises anti-CD34, anti-CD117, anti-CD135, or a combination thereof; the radiolabeled antibody 131 I, 125 I, 123 I, 90 Y. 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 32 P. 225 Ac, 213 Bi, 213 Po, 211 At, 212 Bi, 213 Bi, 223 Ra, 227 Th, 149 Tb, 137 Cs, 212 Pb, or 103 Pd-labeled, methods.

14. 14. The method of claim 13, wherein the acute cancerous disorder is lymphoblastic leukemia, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, or a combination thereof.

15. 14. The method of claim 13, wherein the hematopoietic stem cells are depleted by at least 70%, or at least 90%, and mature differentiated hematopoietic stem cells are depleted by less than 20%, or less than 10%.

16. the radiolabeled antibody 131 I-radiolabeled 131 the effective amount of I-radiolabelled is 10 mCi to 200 mCi administered 6, 7, or 8 days prior to said bone marrow transplant; or 131 the effective amount of I-radiolabelled is 200 mCi to 400 mCi administered 8, 9, 10, 11, or 12 days prior to said bone marrow transplant; or 131 14. The method of claim 13, wherein the effective amount of I-radiolabelled is 400 mCi to 1,200 mCi administered 10, 11, 12, 13, or 14 days prior to the bone marrow transplant.

17. the radiolabeled antibody 225 Ac-radiolabeled, 225 14. The method of claim 13, wherein the effective amount of Ac-radiolabel is 0.1 μCi / kg to 5.0 μCi / kg of subject body weight administered 6, 7, 8, 9, 10, 11, or 12 days prior to said bone marrow transplant.

18. 1. A method for treating a subject suffering from a non-cancer disorder treatable via gene-edited cell therapy, comprising: (i) administering to the subject an amount of a radiolabeled antibody effective to deplete hematopoietic stem cells in the subject; (ii) after a suitable period of time, administering the therapy to the subject to treat the subject's disorder; the non-cancerous disorder is selected from the group consisting of a hemoglobinopathy, a congenital immunodeficiency, and a viral infection, and the antibody comprises anti-CD34, anti-CD117, anti-CD135, or a combination thereof; the radiolabeled antibody 131 I, 125 I, 123 I, 90 Y. 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 32 P. 225 Ac, 213 Bi, 213 Po, 211 At, 212 Bi, 213 Bi, 223 Ra, 227 Th, 149 Tb, 137 Cs, 212 Pb, or 103 Pd-labeled, methods.

19. 19. The method of claim 18, wherein the subject is afflicted with a non-cancer disorder treatable via gene-edited cell therapy and is undergoing such therapy to treat the disorder, and wherein the effective amount of the radiolabeled antibody is administered as a single dose.

20. 20. The method of claim 19, wherein the non-cancerous disorder is selected from the group consisting of sickle cell disease (SCD), severe combined immunodeficiency (SCID), and β-thalassemia.

21. 21. The method of claim 20, wherein the non-cancerous disorder is SCD and the therapy is gene-edited β-globin hematopoietic stem cell therapy.

22. 21. The method of claim 20, wherein the non-cancerous disorder is SCID, the therapy is gene-edited hematopoietic stem cell therapy, and the edited gene is selected from the group consisting of the common gamma chain (γc) gene, the adenosine deaminase (ADA) gene, and the Janus kinase 3 (JAK3) gene.

23. 23. The method of claim 21 or 22, wherein the stem cell therapy is an allogeneic stem cell therapy, or wherein the stem cell therapy is an autologous stem cell therapy.

24. the radiolabeled antibody 131 I-radiolabeled 131 the effective amount of I-radiolabelled is 10 mCi to 200 mCi administered 6, 7, or 8 days prior to administering said therapy to said subject to treat said disorder; or 131 the effective amount of I-radiolabelled is 200 mCi to 400 mCi administered 8, 9, 10, 11, or 12 days prior to administering said therapy to said subject to treat said disorder; or 131 19. The method of claim 18, wherein the effective amount of I-radiolabelled is 400 mCi to 1,200 mCi administered 10, 11, 12, 13, or 14 days prior to administering said therapy to said subject to treat said disorder.

25. the radiolabeled antibody 225 Ac-radiolabel, 225 19. The method of claim 18, wherein the effective amount of Ac-radiolabelled is 0.1 μCi / kg to 5.0 μCi / kg of subject body weight administered 6, 7, 8, 9, 10, 11, or 12 days prior to administering said therapy to said subject to treat said disorder.

26. 1. An article of manufacture comprising: (a) a pharmaceutical composition comprising a radiolabeled antibody; and (b) a label instructing the user to administer to the subject an amount of the antibody effective to deplete hematopoietic stem cells in the subject, The article of manufacture, wherein the radiolabeled antibody comprises anti-CD34, anti-CD117, anti-CD135, or a combination thereof.

27. the radiolabeled antibody 131 an antibody radiolabeled with I, 131 27. The article of claim 26, wherein the effective amount of the antibody radiolabeled with I is 10 mCi to 200 mCi, or 200 mCi to 400 mCi, or 400 mCi to 1,200 mCi.

28. the radiolabeled antibody 225 Ac-labeled, 225 27. The article of claim 26, wherein the effective amount of the antibody radiolabeled with Ac is 0.1 μCi / kg to 5.0 μCi / kg, or 0.1 μCi / kg to 1.0 μCi / kg of the subject's body weight, or 1.0 μCi / kg to 3.0 μCi / kg of the subject's body weight, or 3.0 μCi / kg to 5.0 μCi / kg of the subject's body weight.

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