Radiolabeling of anti-CD45 immunoglobulin and methods of use thereof

Abstract

Disclosed herein are compositions and methods useful for treating hemoglobinopathies and blood disorders. The compositions comprise an actinium-225-labeled anti-CD45 antibody (BC8) formulated as a single patient dose that can be completely delivered to the patient in a single dose. The actinium-225-labeled anti-CD45 may be administered alone or in combination with additional therapeutic agents, such as other immunotherapeutics or radiosensitizers, or additional therapeutic interventions, such as bone marrow transplantation or adoptive cell therapy.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 901,290, filed September 17, 2019, which is incorporated herein by reference in its entirety.

[0002] Viewing the sequence list This application contains a Sequence Listing showing the same sequences found herein, which was submitted via EFS and is incorporated herein as a Supplementary File in accordance with 37 CFR § 1.52(e)(5) and PCT Rule 13.1(a). The present disclosure relates to methods for radiolabeling monoclonal antibodies to CD45, compositions comprising radiolabeled monoclonal antibodies to CD45, and methods for the use of radiolabeled anti-CD45 antibodies for the treatment of malignant and non-malignant hematological disorders. [Background technology]

[0003] CD45 is a type I transmembrane glycoprotein that is a member of the protein tyrosine phosphatase (PTP) family and plays a key role in T cell receptor and B cell receptor signaling. CD45 regulates the activation of the Src family protein-tyrosine kinases Lck and Fyn. CD45 deficiency results in T and B lymphocyte dysfunction, a form of severe combined immune deficiency. It has also been reported to play a critical role in autoimmune diseases and cancer, as well as infectious diseases, including fungal infections (Penninger et al., 2001, CD45: new jobs for an old acquaintance, Nat. Immunol., 2(5):389-396), and metabolic disorders. Primary ligands described for CD45 include galectin-1, CD1, CD2, CD3, CD4, TCR, CD22, and Thy-1. CD45, also known as leukocyte common antigen (LCA), T200, or Ly-5, consists of two intracellular phosphatase domains, a transmembrane domain, and an extracellular domain. Both intracellular phosphatase domains are required for proper phosphate activity, but only one possesses intrinsic kinase activity (Desai et al., 1994, "The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation," EMBO J. 13:4002-4010).

[0004] Generally, all cells of hematopoietic origin, except for mature erythrocytes and platelets, express at least one isoform of CD45. High expression of CD45 is observed in most acute lymphoblastic and acute myeloid leukemias. Because CD45 is not found in tissues of nonhematopoietic origin, its specific expression in leukemia has made it a good target for developing therapeutics, including immunotherapeutics. For example, CD45 is expressed on circulating leukocytes and malignant B cells at a density of approximately 200,000 to 300,000 sites per cell. A specific anti-CD45 antibody (BC8) has been investigated as a candidate immunotherapeutic agent for the treatment of leukemia, either alone or in combination with chemotherapy or total body irradiation. Anti-CD45 antibody-based lymphodepletion is also known (see, for example, Louis, et al., 2009, Blood, 113:2442-2450). However, this approach has drawbacks. For example, in the study by Louis et al., eight patients underwent lymphodepletion with anti-CD45 antibodies and showed an increase in the frequency of desired T cells in the peripheral blood after infusion. However, only three patients experienced clinical benefit, and only one had a complete response.

[0005] CD45 exists as multiple isoforms due to alternative splicing of three of the 34 exons in its extracellular domain (exons 4, 5, and 6, designated A, B, and C; see Figure 1) (Streuli et al., 1987, Differential usage of three exons generates at least five different mRNAs encoding human leukocyte common antigens, J. Exp. Med. 166:1548-1566; Chang et al., 2016, Initiation of T cell signaling by CD45 segregation at 'close-contacts', Nat. Immunol. 17(5):574-582). These three exons encode multiple sites for O-linked glycosylation and are variably modified with sialic acid. As a result, the various isoforms differ substantially in size (391–552 amino acids, molecular weights ranging from 180–240 kDa), shape, and negative charge. The remaining membrane-proximal extracellular domain is heavily N-glycosylated and contains three fibronectin type III repeats after a cysteine-rich spacer region.

[0006] Eight isoforms of CD45 are possible, but only six have been identified in humans: RO (absence of all three exons), RA (exon A), RB (exon B), RAB (exons A and B), RBC (exons B and C), and RABC (exons A, B, and C). These different isoforms are differentially expressed in subpopulations of B- and T-cell lymphocytes and are specific for the activation and maturation states of the cells. For example, CD45-RA and CD45-RB are expressed in naive T cells, whereas CD45-RO is expressed in activated T cells, some B-cell subsets, activated monocytes / macrophages, and granulocytes, and CD45-RABC is preferentially expressed in B cells (Hermiston et al., 2003, CD45: A critical regulator of signaling thresholds in immune cells, Ann. Rev. Immunol., 21:107-137). Antibodies that selectively recognize the various isoforms of CD45 have been identified. Furthermore, monoclonal antibodies (mAbs) that bind to epitopes common to all the different isoforms have also been identified. For example, the anti-CD45 mouse antibody BC8 recognizes all human isoforms of the CD45 antigen.

[0007] Iodine-131 (I) for the treatment of subjects requiring bone marrow transplantation 131 While the use of BC8 labeled with I) has been explored (see WO 2017 / 155937, incorporated herein by reference in its entirety), there remains a need for compositions and methods of their use for the treatment of malignant and non-malignant hematologic disorders. Specifically, there is a need for therapeutic compositions and methods that (i) employ agents that are more specific than chemotherapeutic drugs, (ii) are sufficiently potent to be effective at low doses, and (iii) avoid significant depletion of at least some types of hematopoietic stem cells. Summary of the Invention

[0008] The present disclosure takes advantage of the pan-specific nature of the BC8 monoclonal antibody to provide compositions and methods useful for depletion, reversible immunosuppression and / or ablation of specific cell populations, and further methods for using these compositions and methods to treat certain malignant and non-malignant hematological disorders. The present disclosure provides compositions and methods of use for treating various disorders of the hematopoietic system, as well as metabolic disorders, cancer, and autoimmune diseases, among others. The present disclosure further features a method for conditioning a patient prior to hematopoietic stem cell transplantation therapy to promote engraftment of the hematopoietic stem cell graft. The patient may be suffering from one or more blood disorders, such as hemoglobinopathies or other hematopoietic conditions. The patient may be in need of a hematopoietic stem cell transplant. As described herein, hematopoietic stem cells are capable of differentiating into many cell types in the hematopoietic lineage and can be administered to a patient to populate or repopulate a cell type that is deficient in the patient. The present disclosure provides methods for (i) directly treating diseases, such as hematological diseases, metabolic diseases, cancer, or autoimmune diseases, among others, as described herein, by selectively depleting, reversibly suppressing, or ablating a population of cells that express CD45, such as ectopic blood cells, cancer cells, or autoimmune cells, and / or (ii) using radiolabeled antibodies, specifically actinium-225 ( 225 Ac) or lutetium-177( 177 The present invention features a method of treating a patient with anti-CD45 immunoglobulin labeled with (Lu).

[0009] The former activity allows for the direct treatment of a wide range of disorders associated with cells of the hematopoietic lineage, such as leukemia or lymphoma cells of the B-cell or T-cell lineage, autoimmune lymphocytes, such as T cells expressing T-cell receptors that cross-react with self-antigens, among other cell types. The latter activity, i.e., selective depletion, reversible suppression, or ablation of hematopoietic stem cells, in turn, creates a void that can be subsequently filled by transplantation of an exogenous (e.g., autologous, allogeneic, or syngeneic) hematopoietic stem cell graft. Thus, the present disclosure provides methods for treating various non-cancerous hematopoietic conditions, such as hemoglobinopathies (e.g., sickle cell disease or SCD, and β-thalassemia), congenital immune deficiencies (e.g., severe combined immunodeficiency or SCID, Fanconi anemia, Wiskott-Aldrich syndrome, Diamond-Blackfan anemia and Shwachman-Diamond syndrome, adenosine deaminase deficiency), and viral infections (e.g., HIV infection and acquired immune deficiency syndrome). The present disclosure further provides methods for treating cancerous disorders, such as blood cancers or solid tumors. Exemplary blood cancers include acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma.

[0010] Therefore, the present disclosure provides that actinium-225( 225 Ac) or lutetium-177( 177 The present invention relates to a stabilized composition comprising an isolated anti-CD45 immunoglobulin (e.g., the BC8 mAb clone) in a form radioactively labeled with Lu, and its therapeutic use for the treatment of malignant and non-malignant hematological diseases and disorders. Due to its ability to bind to all isoforms of the CD45 antigen in humans, the BC8 antibody is expected to specifically and preferentially accumulate therapeutically high radiation doses in high-density CD45 antigen-bearing cells. Thus, the present disclosure: 225 Ac or 177The present invention also relates to a method for radiolabeling an anti-CD45 immunoglobulin, such as the BC8 antibody, with a radionuclide, such as Lu. According to a specific embodiment, the BC8 antibody is conjugated to a chelating agent, such as S-2-(4-isothiocyanatobenzyl)-1,4,7,10 tetraazacyclododecanetetraacetic acid (p-SCN-Bn-DOTA; referred to as DOTA), to form DOTA-BC8; 225 Radioactively labeled with radionuclides such as Ac, 225 Ac-DOTA-BC8 (i.e., 225 Ac-BC8) or 177 Lu is formed, 177 Lu-DOTA-BC8 (i.e., 177 Lu-BC8).

[0011] 225 Ac-BC8 or 177 Lu-BC8 may be provided as a stabilized formulation containing one or more pharmaceutically acceptable carriers, salts, or excipients. Certain exemplary carriers or excipients include saline, phosphate-buffered saline (e.g., 50 mM PBS buffer, pH 7), and / or 0.5% to 5.0% (w / v) of one or more of ascorbic acid, polyvinylpyrrolidone (PVP), human serum albumin (HSA), water-soluble salts of HSA, and mixtures thereof.

[0012] The BC8 antibody may comprise a light chain variable domain having the amino acid sequence as set forth in SEQ ID NO: 1 and a heavy chain variable domain having the N-terminal amino acid sequence as set forth in SEQ ID NO: 9. The BC8 antibody may comprise a light chain variable domain having at least one complementarity determining region (CDR) having the amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The BC8 antibody may comprise a light chain having an amino acid sequence as set for the in SEQ ID NO: 12 or SEQ ID NO: 13. The BC8 antibody may comprise a heavy chain variable domain having the amino acid sequence as set forth in SEQ ID NO: 2, or a heavy chain variable domain having the N-terminal amino acid sequence as set forth in SEQ ID NO: 10. The BC8 antibody may comprise a heavy chain variable domain having at least one complementarity determining region (CDR) having the amino acid sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. The BC8 antibody may comprise a heavy chain having the amino acid sequence as set forth in SEQ ID NO: 15 or SEQ ID NO: 16.

[0013] According to certain embodiments, the BC8 antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:15 or SEQ ID NO:16, wherein the amino acid at position 141 (relative to the N-terminal amino acid) is ASP or ASN. The ratio of ASP:ASN at position 141 within a population of BC8 proteins may be in the range of 1:99 to 99:1, such as 10:90 to 90:10. According to certain embodiments, the BC8 antibody comprises a heavy chain variable domain having the amino acid sequence as set forth in SEQ ID NO:2, or a heavy chain variable domain having the N-terminal amino acid sequence as set forth in SEQ ID NO:10, wherein the amino acid at position 141 (relative to the N-terminal amino acid) of the heavy chain is ASP or ASN, and the ratio of ASP:ASN within the population of BC8 proteins is in the range of 1:99 to 99:1, such as 10:90 to 90:10.

[0014] According to certain embodiments, any of the BC8 antibodies described above, i.e., those comprising one or more of SEQ ID NOs: 1-10, may be a chimeric or humanized antibody, i.e., BC8c. The BC8c antibody may comprise a human IgG1 heavy chain constant region, a human IgG2 heavy chain constant region, or a human IgG4 heavy chain constant region having the amino acid sequence as set forth in SEQ ID NOs: 17-19, respectively, a human IgG4 heavy chain constant region having the amino acid sequence as set forth in SEQ ID NO: 20 (including the mutation S228P), and / or a human kappa light chain constant region having the amino acid sequence as set forth in SEQ ID NO: 21.

[0015] The present disclosure provides a method for directly treating a subject suffering from a CD45-positive hematological malignancy, comprising administering an effective amount of 225 Ac-BC8 or 177 The method includes administering Lu-BC8 to the subject as a low-dose monotherapy, alone or in combination with other treatments. The present disclosure provides a method for directly treating a subject suffering from a CD45-positive hematological malignancy, comprising administering an effective amount of 225 Ac-BC8 or 177 The method includes administering Lu-BC8 to the subject as a low-dose monotherapy, either alone or in combination with other therapies via a stem cell carrier.

[0016] The present disclosure provides a method for depleting, reversibly suppressing, or ablating hematopoietic stem cells in a subject, comprising administering an effective amount of 225 Ac-BC8 or 177 The method comprises administering Lu-BC8 to the subject. The present disclosure provides an effective amount of erythromycin at a dose that does not myeloablatively and therefore irreversibly deplete hematopoietic stem cells. 225 Ac-BC8 or 177 The present invention provides a method for depleting or reversibly suppressing circulating tumor cells (e.g., as found in leukemia, lymphoma, myeloma, and MDS) by administering Lu-BC8 to the subject. Such cells may include at least regulatory T cells, myeloid-derived suppressor cells, tumor-sensitized macrophages, activated macrophages secreting IL-1 and / or IL-6, and any combination thereof. The present disclosure provides a method for depleting, reversibly suppressing, or ablating lymphocytes in a subject, comprising administering an effective amount of 225 Ac-BC8 or 177 Further provided is a method comprising administering Lu-BC8 to said subject.

[0017] The present disclosure provides a method for treating a subject suffering from a non-cancerous disorder, comprising administering to said subject an amount of a compound effective to deplete, reversibly suppress, or ablate hematopoietic stem cells.225 Ac-BC8 or 177 Also provided is a method comprising administering Lu-BC8 to the subject. According to certain embodiments, the disorder is treatable via gene editing cell therapy, and the method comprises: 225 Ac-BC8 or 177 The method further comprises administering the treatment to the subject after administration of Lu-BC8 to treat the subject's disorder. According to certain embodiments, the disorder is SCD, and the treatment is gene-edited β-globin hematopoietic stem cell therapy. According to certain embodiments, the disorder is SCID, and the treatment is gene-edited hematopoietic stem cell therapy, wherein the edited gene is a common gamma chain (γc) gene, an adenosine deaminase (ADA) gene, and / or a Janus kinase 3 (JAK3) gene. The stem cell therapy can be, for example, allogeneic or autologous.

[0018] The present disclosure provides a method for treating a subject afflicted with a cancerous disorder treatable via gene editing cell therapy, comprising administering to said subject (i) an amount of a compound effective to deplete, reversibly suppress, or ablate hematopoietic stem cells, comprising administering to said subject ... 225 Ac-BC8 or 177 Also provided is a method comprising administering Lu-BC8 to the subject, and (ii) after a suitable period of time, administering the treatment to the subject to treat the subject's disorder. According to certain embodiments, the treatment suitable for treating the subject's disorder can be bone marrow transplantation or adoptive cell therapy. Finally, the present disclosure provides: (a) 225 Ac-BC8 or 177 An article of manufacture is provided that includes a radiolabeled anti-CD45 antibody, such as Lu-BC8, and (b) a label instructing a user to administer to a subject an amount of the antibody effective to deplete hematopoietic stem cells in said subject.

[0019] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 shows a schematic representation of exon usage in the various isoforms of CD45 generated by differential splicing of the human CD45 gene. [Figure 2] The protein sequences of the complementarity-determining regions (CDRs), framework regions, and variable domain sequences of the light (VL) and heavy (VH) chains of anti-CD45 mAb BC8 are provided. The CDRs are bolded and underlined (SEQ ID NO: 1 and SEQ ID NO: 2). [Figure 3] 1 provides the light and heavy chain CDR and N-terminal protein sequences of anti-CD45 mAb BC8 (SEQ ID NO: 3 to SEQ ID NO: 10). [Figure 4A] The complete nucleotide (SEQ ID NO: 11) and amino acid (SEQ ID NO: 12) sequences of the light chain of anti-CD45 mAb BC8 are provided. [Figure 4B] 1 provides the amino acid sequence of the light chain of anti-CD45 mAb BC8 without the leader sequence (SEQ ID NO: 13). [Figure 5A] Provided are the full nucleotide (SEQ ID NO: 14) and amino acid (SEQ ID NO: 15) sequences of the heavy chain of anti-CD45 mAb BC8, in which the asparagine at position 141 (from the n-terminus of the protein sequence) is found to be deaminated to aspartic acid in at least a portion of the protein population. [Figure 5B] Provided is the amino acid sequence (SEQ ID NO: 16) of the heavy chain of anti-CD45 mAb BC8 without the leader sequence, in which the asparagine at position 141 (from the n-terminus of the protein sequence) is found to be deaminated to aspartic acid in at least a portion of the protein population. [Figure 6A]SEQ ID NOS: 17 to 19 are provided, respectively, which are the amino acid sequences of the human heavy chain constant regions. [Figure 6B] SEQ ID NOS: 17 to 19 are provided, respectively, which are the amino acid sequences of the human heavy chain constant regions. [Figure 6C] SEQ ID NOS: 17 to 19 are provided, respectively, which are the amino acid sequences of the human heavy chain constant regions. [Figure 6D] 1 provides the amino acid sequence of the human heavy chain constant region containing the mutation S228P (SEQ ID NO: 20). [Figure 6E] 1 provides the amino acid sequence of the human kappa light chain constant region (SEQ ID NO:21). [Figure 7] 1 illustrates a method for lymphodepleting a subject prior to administering adoptive cell therapy according to certain aspects of the present disclosure. [Figure 8] 1 shows pharmacokinetic data illustrating exemplary clearance and administration times for lymphodepletion protocols according to the present disclosure. [Figure 9A] 9A and 9B provide a schematic diagram of a method for radiolabeling anti-CD45 mAb BC8 with actinium (225Ac), where FIG. 9A shows the attachment of the bifunctional chelator S-2-(4-isothiocyanatobenzyl)-1,4,7,10 tetraazacyclododecanetetraacetic acid (p-SCN-Bn-DOTA; referred to as DOTA in the figure) to a monoclonal antibody against CD45, and FIG. 9B shows the radiolabeling of the DOTA-anti-CD45 conjugate with 225Ac to provide 225Ac-DOTA-anti-CD45. [Figure 9B] 9A and 9B provide a schematic diagram of a method for radiolabeling anti-CD45 mAb BC8 with actinium (225Ac), where FIG. 9A shows the attachment of the bifunctional chelator S-2-(4-isothiocyanatobenzyl)-1,4,7,10 tetraazacyclododecanetetraacetic acid (p-SCN-Bn-DOTA; referred to as DOTA in the figure) to a monoclonal antibody against CD45, and FIG. 9B shows the radiolabeling of the DOTA-anti-CD45 conjugate with 225Ac to provide 225Ac-DOTA-anti-CD45. [Figure 10A]Elution profiles for BC8 standard and 225Ac-DOTA-BC8 from SEC-HPLC (size exclusion chromatography high performance liquid chromatography) are provided; Figure 10A shows the elution of BC8 standard, and Figure 10B shows the elution of 225Ac-DOTA-BC8 (the peak at 13 minutes is HSA added to stabilize the conjugated antibody). [Figure 10B] Elution profiles for BC8 standard and 225Ac-DOTA-BC8 from SEC-HPLC (size exclusion chromatography high performance liquid chromatography) are provided; Figure 10A shows the elution of BC8 standard, and Figure 10B shows the elution of 225Ac-DOTA-BC8 (the peak at 13 minutes is HSA added to stabilize the conjugated antibody). [Figure 11] 1 provides a graph showing the stability of 225Ac-DOTA-BC8 at various storage dilutions and temperatures as a function of time. [Figure 12] 1 provides a graph showing 225Ac-DOTA-BC8 immunoreactivity on Ramos cells (CD45 positive cells) and EL4 cells (Cd45 negative cells). [Figure 13A] Graphs are provided showing the binding of various antibody samples to Cytotrol cells as measured by flow cytometry, with FIG. 13A comparing the binding of native BC8 antibody and native 18B7 (non-specific control) antibody to Cytotrol cells, and FIG. 13B comparing the binding of native BC8 antibody and DOTA-BC8 antibody to Cytotrol cells. [Figure 13B] Graphs are provided showing the binding of various antibody samples to Cytotrol cells as measured by flow cytometry, with FIG. 13A comparing the binding of native BC8 antibody and native 18B7 (non-specific control) antibody to Cytotrol cells, and FIG. 13B comparing the binding of native BC8 antibody and DOTA-BC8 antibody to Cytotrol cells. [Figure 14A]1 provides a graph comparing the binding of naive BC8 and DOTA-BC8 to Cytotrol cells as measured by flow cytometry. [Figure 14B] A bar graph is provided showing the binding of DOTA-BC8 samples from Figure 14A immediately after labeling with 225Ac (i.e., 225Ac-DOTA-BC8) compared to the binding of 225Ac-18B7 (binding to Cytotrol cells) as measured by the fractional radioactivity retained on the cells after washing. [Figure 15A] Graphs comparing the binding of DOTA-BC8 to different human multiple myeloma cell lines, namely H929 and U266, measured after labeling with 225Ac (i.e., 225Ac-DOTA-BC8; FIG. 15B), as measured by flow cytometry (FIG. 15A) or by the fractional radioactivity retained on the cells after washing. [Figure 15B] Graphs comparing the binding of DOTA-BC8 to different human multiple myeloma cell lines, namely H929 and U266, measured after labeling with 225Ac (i.e., 225Ac-DOTA-BC8; FIG. 15B), as measured by flow cytometry (FIG. 15A) or by the fractional radioactivity retained on the cells after washing. [Figure 16A] 16A and 16B show bar graphs comparing the biodistribution of 225Ac-DOTA-BC8 (FIG. 16A) and 225Ac-DOTA-18B7 (FIG. 16B) antibodies in control mice at 1 hour, 4 hours, 24 hours, 48 ​​hours, and 96 hours. [Figure 16B] 16A and 16B show bar graphs comparing the biodistribution of 225Ac-DOTA-BC8 (FIG. 16A) and 225Ac-DOTA-18B7 (FIG. 16B) antibodies in control mice at 1 hour, 4 hours, 24 hours, 48 ​​hours, and 96 hours. [Figure 17A]17A and 17B show bar graphs comparing the biodistribution of 225Ac-DOTA-18B7 (control; FIG. 17A) and 225Ac-DOTA-BC8 (FIG. 17B) antibodies in U266 SCID-NOD tumor-bearing mice and H929 SCID-NOD tumor-bearing mice at 1 hour, 4 hours, 24 hours, 48 ​​hours, and 96 hours. [Figure 17B] 17A and 17B show bar graphs comparing the biodistribution of 225Ac-DOTA-18B7 (control; FIG. 17A) and 225Ac-DOTA-BC8 (FIG. 17B) antibodies in U266 SCID-NOD tumor-bearing mice and H929 SCID-NOD tumor-bearing mice at 1 hour, 4 hours, 24 hours, 48 ​​hours, and 96 hours. [Figure 18A] 1 shows a graph comparing tumor volumes in H929 multiple myeloma xenograft-bearing SCID-NOD mice after radioimmunotherapy treatment with 225Ac-DOTA-BC8 or 225Ac-DOTA-18B7 (control). [Figure 18B] 1 shows a graph comparing tumor volumes in SCID-NOD mice bearing U266 multiple myeloma xenografts after radioimmunotherapy treatment with 225Ac-DOTA-BC8 or 225Ac-DOTA-18B7 (control). [Figure 19] Figure 19 shows histological analysis of tumors excised from U266 multiple myeloma xenograft-bearing SCID-NOD mice and H929 multiple myeloma xenograft-bearing SCID-NOD mice, where Figure 19A shows an untreated H929 tumor, Figure 19B shows a 225Ac-DOTA-BC8-treated H929 tumor, Figure 19C shows an untreated U266 tumor, and Figure 19D shows a 225Ac-DOTA-BC8-treated U266 tumor. [Figure 20] Shown are microSPEC / CT scans of C57B1 / 6 mice injected (ip) with 111Ln-anti-CD45 obtained 1 hour, 24 hours, 48 ​​hours, 72 hours, 96 hours and 6 days after injection. [Figure 21A] Bar graphs showing the depletion of various immune cell subpopulations in non-tumor-bearing C57B1 / 6 mice after treatment with (A) 177Lu-anti-CD45 or (B) 131I-anti-CD45. [Figure 21B] Bar graphs showing the depletion of various immune cell subpopulations in non-tumor-bearing C57B1 / 6 mice after treatment with (A) 177Lu-anti-CD45 or (B) 131I-anti-CD45. [Figure 22] Bar graphs showing the depletion of various immune cell populations in the spleens of non-tumor-bearing C57B1 / 6 mice after treatment with (A) 177Lu-anti-CD45 or (B) 131I-anti-CD45. [Figure 23A] Graphs showing that 177Lu-anti-CD45 lymphodepletion and 131I-anti-CD45 lymphodepletion enable tumor control in the OT I adoptive cell therapy model. (A) Targeted pretreatment mediated by 177Lu-anti-CD45 and 131I-anti-CD45 prior to adoptively transferred OT I cells enabled control of EG.7 tumor growth. (B) Tumor size for individual mice in each group. (C) Survival rates of control mice that received no treatment or OT I cells treatment, and mice pretreated with 177Lu-anti-CD45 and 131I-anti-CD45. [Figure 23B] Graphs showing that 177Lu-anti-CD45 lymphodepletion and 131I-anti-CD45 lymphodepletion enable tumor control in the OT I adoptive cell therapy model. (A) Targeted pretreatment mediated by 177Lu-anti-CD45 and 131I-anti-CD45 prior to adoptively transferred OT I cells enabled control of EG.7 tumor growth. (B) Tumor size for individual mice in each group. (C) Survival rates of control mice that received no treatment or OT I cells treatment, and mice pretreated with 177Lu-anti-CD45 and 131I-anti-CD45. [Figure 23C]Graphs showing that 177Lu-anti-CD45 lymphodepletion and 131I-anti-CD45 lymphodepletion enable tumor control in the OT I adoptive cell therapy model. (A) Targeted pretreatment mediated by 177Lu-anti-CD45 and 131I-anti-CD45 prior to adoptively transferred OT I cells enabled control of EG.7 tumor growth. (B) Tumor size for individual mice in each group. (C) Survival rates of control mice that received no treatment or OT I cells treatment, and mice pretreated with 177Lu-anti-CD45 and 131I-anti-CD45.

[0021] A brief description of arrays SEQ ID NO: 1 is the amino acid sequence of the variable domain of the light chain of anti-CD45 mouse immunoglobulin BC8. SEQ ID NO: 2 is the amino acid sequence of the variable domain of the heavy chain of anti-CD45 mouse immunoglobulin BC8. SEQ ID NO: 3 is the amino acid sequence of CDR1 of the light chain of anti-CD45 mouse immunoglobulin BC8.

[0022] SEQ ID NO: 4 is the amino acid sequence of CDR2 of the light chain of anti-CD45 mouse immunoglobulin BC8. SEQ ID NO: 5 is the amino acid sequence of the CDR3 of the light chain of anti-CD45 mouse immunoglobulin BC8. SEQ ID NO: 6 is the amino acid sequence of CDR1 of the heavy chain of anti-CD45 mouse immunoglobulin BC8.

[0023] SEQ ID NO: 7 is the amino acid sequence of CDR2 of the heavy chain of anti-CD45 mouse immunoglobulin BC8. SEQ ID NO: 8 is the amino acid sequence of CDR3 of the heavy chain of anti-CD45 mouse immunoglobulin BC8. SEQ ID NO: 9 is the N-terminal amino acid sequence of the light chain of anti-CD45 mouse immunoglobulin BC8.

[0024] SEQ ID NO: 10 is the N-terminal amino acid sequence of the heavy chain of anti-CD45 mouse immunoglobulin BC8. SEQ ID NO: 11 is the nucleotide sequence of the light chain of anti-CD45 mouse immunoglobulin BC8. SEQ ID NO: 12 is the amino acid sequence of the light chain of anti-CD45 mouse immunoglobulin BC8, including the leader sequence. SEQ ID NO: 13 is the amino acid sequence of the light chain of anti-CD45 mouse immunoglobulin BC8, starting at the N-terminus of the protein (i.e., without the leader sequence). SEQ ID NO: 14 is the nucleotide sequence of the heavy chain of anti-CD45 mouse immunoglobulin BC8. SEQ ID NO: 15 is the amino acid sequence of the heavy chain of anti-CD45 mouse immunoglobulin BC8, including the leader sequence. SEQ ID NO: 16 is the amino acid sequence of the heavy chain of anti-CD45 mouse immunoglobulin BC8, starting at the N-terminus of the protein (i.e., without the leader sequence). SEQ ID NO: 17 is the amino acid sequence of the human IgG1 heavy chain constant region. SEQ ID NO: 18 is the amino acid sequence of the human IgG2 heavy chain constant region. SEQ ID NO: 19 is the amino acid sequence of the human IgG4 heavy chain constant region. SEQ ID NO: 20 is the amino acid sequence of the human IgG4 heavy chain constant region containing the mutation S228P. SEQ ID NO: 21 is the amino acid sequence of the human kappa light chain constant region. DETAILED DESCRIPTION OF THE INVENTION

[0025] Definitions and Abbreviations 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 disclosure pertains. In this application, certain terms are used that have the meanings indicated below. The singular forms "a," "an," "the," and the like include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an" antibody includes a single antibody as well as a plurality of different antibodies. The term "about," when used before numerical designations, such as temperature, time, amount, and concentration (including ranges), indicates an approximation that may vary by ±10%, ±5%, or ±1%.

[0026] As used herein, "administration" in reference to an antibody refers to 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 administration, transdermal administration, subcutaneous administration, intraperitoneal administration, and intrathecal administration. Exemplary administration methods for antibodies may be substantially as described in International Publication No. 2016 / 187514, the entire contents of which are incorporated herein by reference. Furthermore, according to aspects of the present disclosure, antibodies can be formulated using one or more routinely 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 polymeric injections, and can contain excipients such as solubility-altering agents (e.g., ethanol, propylene glycol, and sucrose) and polymers (e.g., polycaprylactones and PLGA).

[0027] As used herein, the term "antibody" includes, but is not limited to, (a) immunoglobulin molecules comprising two heavy chains and two light chains that recognize an antigen, (b) polyclonal and monoclonal immunoglobulin molecules, (c) monovalent and bivalent fragments thereof (e.g., di-Fab), and (d) bispecific forms thereof. Immunoglobulin molecules may 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, human IgG2, human IgG3, and human IgG4. Antibodies can be both naturally occurring and non-naturally occurring (e.g., IgG-Fc-silent). Furthermore, antibodies include chimeric antibodies, fully synthetic antibodies, single-chain antibodies, and fragments thereof. Antibodies may be human, humanized, or non-human.

[0028] A "humanized" antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody have been replaced with corresponding amino acids derived from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids derived from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains the same antigen specificity as the original antibody. "Chimeric antibody" refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species, such as an antibody whose variable region is derived from a mouse antibody and whose constant region is derived from a human antibody.

[0029] As used herein, "non-cancerous disorder" or "non-malignant disorder" includes, but is not limited to, hemoglobinopathies (e.g., SCD), congenital immune deficiencies (e.g., SCID), autoimmune disorders (e.g., multiple sclerosis, rheumatoid arthritis, scleroderma, systemic lupus, type 1 diabetes, myasthenia gravis, Sjogren's disease, polymyositis, etc.), and viral infections (e.g., HIV infection). Non-cancerous disorders do not include, for example, solid cancers (e.g., tumors) and hematological malignancies.

[0030] As used herein, "cancer" or "malignant disorder" includes, but is not limited to, solid cancers (e.g., tumors) and hematological malignancies. "Hematological malignancies," also known as blood cancers, are cancers that originate in blood-forming tissues, such as the bone marrow or other cells of the immune system. Hematological malignancies include, but are not limited to, leukemia (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 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.

[0031] "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, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma 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, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, pediatric tumors, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, tumors of the spinal axis, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, and environmentally induced cancers including those induced by asbestos.

[0032] As used herein, the term "burden" refers to the amount when used in relation to cancerous cells. Thus, cancerous cell "burden" refers to the amount of cancerous cells. Cancerous cells have a burden related to their tissue of origin (i.e., the primary site of disease), for example, in the case of AML, "bone marrow blast burden." Cancerous cells also have a burden related to one or more tissues other than their origin, for example, in the case of AML, blast burden in the blood, liver, and spleen. The term "peripheral burden" refers to such cells. The peripheral burden of cancerous cells, such as blasts in the case of AML, can be measured in different ways with different outcomes. For example, in the case of AML, "peripheral blast burden" can be measured as the total blast population outside the bone marrow, or the total blast population in the blood, spleen, and liver combined, or simply the blast population in the blood as measured per unit volume. As used herein in connection with AML and other cancers originating in the bone marrow, unless otherwise specified, the term "peripheral cancerous cell burden" (e.g., peripheral blast burden) refers to the cancerous cell population of the blood as measured in cells per unit volume (e.g., cells / μL). This blood-based measurement is a useful surrogate for more cumbersome measurements of, for example, spleen burden and liver burden.

[0033] As used herein, the peripheral cancerous cell burden in a subject is "high" if, when administered to a subject at the maximum safe dose, an agent targeting a hematologic malignancy-associated antigen, such as a radiolabeled anti-CD45 antibody of the present disclosure, does not reach the primary site of disease in an amount sufficient to bind to more than 90% of its target antigen at that site. Conversely, the peripheral cancerous cell burden in a subject is "low" if, when administered to a subject at the maximum safe dose, the agent reaches the primary site of disease in an amount sufficient to bind to more than 90% of its target antigen at that site. In the case of AML, examples of low peripheral blast burdens are those that result in a blood blast load of 1,000 cells / μL or less, 500 cells / μL or less, 400 cells / μL or less, 300 cells / μL or less, 200 cells / μL or less, 100 cells / μL or less, and 50 cells / μL or less.

[0034] As used herein, a "low dose" of a radiolabeled anti-CD45 antibody of the present disclosure is one that is subsaturating, and thus introduces fewer target antigen binding sites (i.e., CD45 binding sites on the administered antibody) into the subject's body than there are target antigens (i.e., CD45 molecules). According to certain embodiments, a low dose of a radiolabeled anti-CD45 antibody is one in which the ratio of CD45 binding sites to CD45 molecules is 9:10 or less, e.g., 1:2 or less, or 1:5 or less, or 1:10 or less, or 1:20 or less, or 1:100 or less. As used herein, the terms "subject" and "patient" are interchangeable and include, but are 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 other embodiments, the subject is 10-15 years old, or 15-20 years old. According to still other 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.

[0035] As used herein, "treating" a subject suffering from a disorder includes, but is not limited to, (i) slowing, stopping, or reversing the progression of the disorder; (ii) slowing, stopping, 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 suffering from a disorder means (i) reversing the progression of the disorder, ideally to the point of eliminating the disorder, and / or (ii) reversing the progression of symptoms of the disorder, ideally to the point of eliminating the symptoms, and / or (iii) reducing or eliminating the likelihood of recurrence. Ideally, treating a subject suffering from a disorder means curing the disorder by removing or otherwise disabling its genetic cause. As used herein, "depleting" with respect to a specific cell type in a subject means reducing that cell population in the subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. As used herein, "ablating" with respect to a specific cell type in a subject means reducing that cell population in the subject by more than 95%, for example, by at least 96%, or 97%, or 98%, or 99%, or even 100%.

[0036] Specific cell types depleted using the compositions and methods of the present disclosure include at least hematopoietic stem cells (i.e., multipotent hematopoietic stem cells, also called hemoblasts) and lymphocytes, such as peripheral blood lymphocytes or bone marrow lymphocytes. Hematopoietic stem cells ("HSCs") are multipotent, self-renewing progenitor cells that arise from all differentiated blood cell types during the process of hematopoiesis. HSCs are thought to differentiate into two lineage-restricted lymphoid oligopotent progenitors and lineage-restricted myeloid erythroid oligopotent progenitors, although an alternative "bone marrow-based" model of blood lineage development describes a novel intermediate myeloid lymphoid progenitor cell that has the ability to generate progeny from both lineages.

[0037] As used herein, the term "hematopoietic stem cells" ("HSC") refers to immature blood cells that have the ability to self-renew and differentiate into mature blood cells, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), platelets (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+ cytoplasmic cells. CD34+ cells are immature cells that express the CD34 cell surface marker.

[0038] 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 (e.g., Lin, CD34, CD38, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, and HLA DR). Immune cell depletion in patients may also be detected in peripheral blood. Methods for measuring peripheral blood lymphocyte populations are routine. These include, for example, flow cytometry on whole blood samples to determine lymphocyte counts based on labeling with fluorescent antibodies against specific cell surface markers, such as CD45, CD3, CD4, or CD8. Methods for measuring peripheral blood neutrophil populations are also routine. These include, for example, flow cytometry on whole blood samples to determine neutrophil counts based on labeling with fluorescent antibodies against specific cell surface markers, such as Ly6G.

[0039] According to a specific embodiment of the present disclosure, the reduction of lymphocytes in a subject is determined by measuring the subject's peripheral blood lymphocyte level. As used herein, the "peripheral blood lymphocytes" of a subject refer to mature lymphocytes circulating in the subject's blood. Examples of peripheral blood lymphocytes include, but are not limited to, peripheral blood T cells, peripheral blood NK cells, and peripheral blood B cells. Thus, for example, if the population of at least one type of peripheral blood lymphocyte in a subject is reduced by 95% or less, the subject's lymphocyte population is depleted. For example, if the subject's peripheral blood T cell level is reduced by 50%, the subject's peripheral blood NK cell level is reduced by 40%, and / or the subject's peripheral blood B cell level is reduced by 30%, the subject's lymphocytes are depleted. In this example, the subject's lymphocytes are depleted even if the level of another immune cell type, such as neutrophils, is not reduced. According to certain embodiments, lymphodepleting a subject is reflected by a reduction in the peripheral blood lymphocyte population of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

[0040] As used herein, patients "in need of" hematopoietic stem cell transplantation include those who exhibit a defect or deficiency of one or more blood cell types, and those with stem cell disorders, autoimmune diseases, cancer, or other conditions described herein. Hematopoietic stem cells generally exhibit: 1) multipotency (therefore, they can differentiate into multiple different blood lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), platelets (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, lymphocytes (e.g., NK cells, B cells, and T cells)); 2) self-renewal (therefore, they can give rise to daughter cells with equivalent potential as the parent cells); and 3) the ability to be reintroduced into the transplant recipient, so that they can home to the hematopoietic stem cell niche and reestablish proliferative and sustained hematopoiesis.

[0041] Additionally or alternatively, a patient "in need of" hematopoietic stem cell transplant may be a patient who may or may not be afflicted with a disease state, but who nevertheless exhibits reduced levels (e.g., compared to levels in otherwise healthy subjects) of one or more endogenous cell types within the hematopoietic lineage, such as megakaryocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes.

[0042] Anti-CD45 antibody As used herein, an "anti-CD45 antibody" or "anti-CD45 immunoglobulin" is an antibody that binds to an epitope of CD45. According to a specific embodiment, the anti-CD45 antibody can bind to the epitope recognized by the monoclonal antibody "BC8." BC8 is known as a method for producing it. These methods are described, for example, in International Publication No. 2017 / 155937, the entire contents of which are incorporated herein by reference, and in the Examples provided herein.

[0043] The BC8 monoclonal antibody may comprise a light chain having the amino acid sequence set forth in SEQ ID NO: 12, including the leader sequence (FIG. 4A), or the amino acid sequence set forth in SEQ ID NO: 13, without the leader sequence (FIG. 4B). The BC8 monoclonal antibody may comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 (FIG. 2). The BC8 monoclonal antibody may comprise a light chain having an N-terminal amino acid sequence set forth in SEQ ID NO: 9 (FIG. 3). According to certain embodiments, the light chain comprises at least one complementarity determining region having an amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 (FIG. 3). According to certain embodiments, the light chain comprises the N-terminal amino acid sequence set forth in SEQ ID NO: 9 and at least one complementarity determining region having an amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 (FIG. 3). The BC8 monoclonal antibody may comprise a heavy chain having the amino acid sequence set forth in SEQ ID NO: 15, including the leader sequence (FIG. 5A), or the amino acid sequence set forth in SEQ ID NO: 16, without the leader sequence (FIG. 5B). The BC8 monoclonal antibody may comprise a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 2 (FIG. 2). The BC8 monoclonal antibody may comprise a heavy chain having an N-terminal amino acid sequence set forth in SEQ ID NO: 10 (FIG. 3). According to certain embodiments, the heavy chain comprises at least one complementarity determining region having an amino acid sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 (FIG. 3). According to certain embodiments, the heavy chain comprises a heavy chain having an N-terminal amino acid sequence set forth in SEQ ID NO: 10 and at least one complementarity determining region having an amino acid sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 (FIG. 3).

[0044] According to certain embodiments, the BC8 monoclonal antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16, in which the amino acid at position 141 (relative to the N-terminal amino acid) is ASP or ASN. The ratio of ASP:ASN at position 141 in a population of BC8 proteins may be in the range of 1:99 to 99:1, such as 10:90 to 90:10. According to certain embodiments, the BC8 monoclonal antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:2, in which the amino acid at position 141 of the constant region (relative to the N-terminal amino acid) is ASP or ASN. The ratio of ASP:ASN at position 141 in a population of BC8 proteins may be in the range of 1:99 to 99:1, such as 10:90 to 90:10.

[0045] According to certain embodiments, antibodies against CD45 (anti-CD45 antibodies) may be chimeric or humanized. For example, the BC8 monoclonal antibody may include a humanized BC8 antibody or a chimeric BC8 antibody (referred to herein as "BC8c"). For example, a humanized BC8c monoclonal antibody may include a parent mouse variable (V) region grafted onto a human IgG1, IgG2, or IgG4 constant region for the heavy chain, or onto a human kappa region for the light chain. IgG4 antibodies can exchange Fab arms by replacing the heavy chain and additional light chain (half molecules) with heavy-light chain pairs from other molecules, thereby generating bispecific antibodies. This process, referred to herein as "Fab arm exchange," has been shown to occur in mice under reducing conditions in vitro and in vivo. The ability of IgG4 antibodies to undergo Fab arm exchange has been attributed to an unstable core-hinge sequence in combination with sequence determinants in the IgG4 CH3 domain. Substitution of the core-hinge residue Ser228 with Pro(S228P) results in partial stabilization of the IgG4 molecule in vitro and in vivo. Thus, according to certain embodiments, an IgG4 can contain an S or P at position 228, where the mutation S228P may help to stabilize the Ab and prevent Fab arm exchange.

[0046] Such chimerization, i.e., humanizing BC8 to produce BC8c, can be achieved by methods known in the art, for example, by cloning DNA encoding the BC8 murine heavy chain V region and BC8 murine light chain V region in frame and the endogenous murine signal sequence into mammalian expression vectors for heavy and light chains that already contain a human heavy chain constant region (IgG1, IgG2, or IgG4) or human Ckappa. Thus, according to certain embodiments, the BC8 monoclonal antibody may be chimeric BC8, i.e., BC8c, and may comprise a human IgG1 heavy chain constant region having the amino acid sequence as set forth in SEQ ID NO: 17, or a human IgG2 heavy chain constant region having the amino acid sequence as set forth in SEQ ID NO: 18, or a human IgG4 heavy chain constant region having the amino acid sequence as set forth in SEQ ID NO: 19, or a human IgG4 heavy chain constant region having the amino acid sequence as set forth in SEQ ID NO: 20, or a human kappa light chain constant region having the amino acid sequence as set forth in SEQ ID NO: 21 (Figures 6A-6E).

[0047] According to a particular embodiment, the BC8 monoclonal antibody may be a chimera (BC8c) comprising a human IgG1 heavy chain constant region, a human IgG2 heavy chain constant region, or a human IgG4 heavy chain constant region having the amino acid sequence as set forth in any one of SEQ ID NOs: 17 to 20, and a human kappa light chain constant region having the amino acid sequence as set forth in SEQ ID NO: 21 (Figures 6A to 6E).

[0048] According to certain embodiments, a chimeric BC8c monoclonal antibody may comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 (Figure 2). A BC8c monoclonal antibody may comprise a light chain having an N-terminal amino acid sequence set forth in SEQ ID NO: 9 (Figure 3). A BC8c monoclonal antibody may comprise a light chain having at least one complementarity determining region having an amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 (Figure 3). According to certain embodiments, a chimeric BC8c monoclonal antibody may comprise a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 2 (Figure 2). A BC8c monoclonal antibody may comprise a heavy chain having an N-terminal amino acid sequence set forth in SEQ ID NO: 10 (Figure 3). A BC8c monoclonal antibody may comprise a heavy chain having at least one complementarity determining region having an amino acid sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 (Figure 3). According to certain embodiments, the heavy chain of the BC8 or BC8c monoclonal antibody comprises a C-terminal lysine, a C-terminal glycine (G) missing a C-terminal lysine (K), or missing both G and K. When referring to antibodies comprising a modified heavy chain constant region described herein, the antibody may comprise the provided sequence with a C-terminal GK or a C-terminal K, or alternatively, missing a GK or K.

[0049] Patient-specific composition As used herein, 225 The composition containing Ac-labeled BC8 contains both actinium-225 labeled and unlabeled antibodies, with a minority being actinium-225 labeled antibodies. 177 For Lu-labeled BC8, the composition includes both a labeled and an unlabeled antibody population. The ratio of labeled to unlabeled antibody can be adjusted using known methods. Thus, according to certain embodiments of the present disclosure, the anti-CD45 antibody can be provided at a total protein amount of up to 100 mg, e.g., up to 60 mg, e.g., 5 mg to 45 mg, or at a total protein amount of 0.001 mg / kg (of the subject's body weight) to 3.0 mg / kg (of the subject's body weight), e.g., 0.005 mg / kg (of the subject's body weight) to 2.0 mg / kg (of the subject's body weight), or 0.01 mg / kg (of the subject's body weight) to 1 mg / kg (of the subject's body weight), or 0.1 mg / kg (of the subject's body weight) to 0.6 mg / kg (of the subject's body weight), or 0.3 mg / kg (of the subject's body weight), or 0.4 mg / kg (of the subject's body weight), or 0.5 mg / kg (of the subject's body weight), or 0.6 mg / kg (of the subject's body weight).

[0050] According to certain aspects of the present disclosure, a radiolabeled anti-CD45 antibody (i.e., 225 Ac-labeled BC8 or 177Lu-labeled BC8) may comprise a labeled fraction and an unlabeled fraction, wherein the labeled:unlabeled ratio may be about 0.01:10 to 1:10, e.g., 0.01:5 to 0.1:5, or 0.01:3 to 0.1:3, or 0.01:1 to 0.1:1. Furthermore, the radiolabeled anti-CD45 antibody may be provided as a single-dose composition tailored to a specific patient, wherein the amounts of labeled and unlabeled anti-CD45 antibody in the composition may depend at least on the patient's mass, age, sex, and / or disease status or health condition. See, for example, the administration methods disclosed in International Publication No. WO 2016 / 187514, the entire contents of which are incorporated herein by reference. According to certain embodiments, the radiolabeled anti-CD45 antibody may be provided in multiple doses, where each dose in the regimen may comprise a composition tailored to a particular patient, where the amount of labeled and unlabeled anti-CD45 antibody in the composition may depend at least on the mass, age, sex, and / or disease state or health condition of the patient.

[0051] The present combination of labeled and unlabeled fractions of an anti-CD45 antibody allows the composition to be tailored to a specific patient, with each radiation dose and protein dose of the monoclonal antibody being individualized to the patient based on at least one patient-specific parameter. Thus, each vial of the composition may be created for a specific patient, with the entire contents of the vial being delivered to the patient in a single dose. If a treatment regimen requires multiple doses, each dose may be formulated as a patient-specific dose in a vial that is administered to the patient as a "single dose" (i.e., the entire contents of the vial administered at one time). Subsequent doses may be formulated in a similar manner, such that each dose in the regimen provides a patient-specific dose in a single-dose container. One advantage of the compositions of the present disclosure is that there is no residual radiation that medical personnel need to discard or handle, e.g., no dilution or other manipulation to obtain a dose for the patient. When provided in single-dose containers, the containers are simply placed in line in an infusion tubing set for infusion into a patient, and the amount can be standardized to greatly reduce the likelihood of medical error (i.e., delivery of an incorrect dose when the entire amount of the composition is to be administered in one infusion).

[0052] Treatment of blood disorders The majority of malignancies of hematological origin, whether of myeloid or lymphoid origin, express CD45 to different degrees on the surface of tumor cells. This includes 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 chronic myeloid leukemia), lymphomas, 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. Thus, when administered to a patient, a certain amount of radiolabeled anti-CD45 antibody is effective as a direct anti-tumor treatment to reduce the number of tumor blasts in the periphery and immune cell compartments such as the bone marrow, spleen, and lymph nodes. Direct treatment with radiolabeled anti-CD45 antibodies may be used as a low-dose single agent required to reduce tumor blast numbers, but reversibly spare hematopoietic stem cells, or in combination with other therapeutic agents, such as chemotherapeutic or targeted therapeutic agents (e.g., but not limited to, HDAC inhibitors, BCL2 inhibitors, monoclonal antibodies, or tyrosine kinase receptor inhibitors - TKIs).

[0053] Effective in controlling tumor growth and reducing blast counts without irreversibly depleting hematopoietic stem cells 225The dose of Ac radiolabeled anti-CD45 antibody delivers radiation exposure to the bone marrow below a threshold level. A dose of 2 Gy is considered a non-myeloablative radiation dose. The ideal dose delivers a dose of at least 2 Gy, high enough to eliminate leukemia or lymphoma tumor cells and provide transient but reversible bone marrow suppression. Dose levels above 2 Gy but below myeloablative doses are expected to be effective in controlling tumor burden in lymphoma and leukemia. Furthermore, when a single low-dose radiolabeled anti-CD45 antibody treatment is combined with another targeted agent, a lower dose of the antibody radioconjugate can be used to achieve potent antitumor activity, yet avoiding hematopoietic stem cell depletion.

[0054] An exemplary low dose is less than 150 μCi, such as 10 μCi to 100 μCi. 225 The dose of Ac-BC8 may be less than 2 μCi / kg, such as 0.01 μCi / kg to 1.5 μCi / kg, or 0.1 μCi / kg to 1.0 μCi / kg.

[0055] Depletion of circulating tumor blasts and circulating myeloblasts Hematological malignancies, including but not limited to leukemias such as acute myeloid leukemia, acute lymphocytic leukemia, and multiple myeloma, present a unique set of challenges to effective treatment. The high burden of circulating tumor cells often associated with leukemia can be toxic to patients if killed too quickly. Cytoreductive therapy is the process by which the number of circulating blast cells is reduced. According to certain embodiments, cytoreductive therapy may be used to treat hematological malignancies and generally involves the administration of a low dose of radiolabeled anti-CD45, such as a dose that depletes circulating tumor cells (e.g., leukemia, lymphoma, myeloma, MDS) but is not myeloablative and therefore does not irreversibly deplete HSCs. An exemplary low dose is less than 150 μCi, such as 10 μCi to 100 μCi. 225The dose of Ac-BC8 may be less than 2 μCi / kg, such as 0.01 μCi / kg to 1.5 μCi / kg, or 0.1 μCi / kg to 1.0 μCi / kg.

[0056] hematopoietic stem cell therapy Hematopoietic stem cell therapy involves the administration of hematopoietic stem cells, such as bone marrow transplantation (BMT). Hematopoietic stem cells may be administered to patients with a deficiency or deficiency in one or more cell types of the hematopoietic lineage to reconstitute a deficient or deficient population of cells in vivo. For example, the patient may suffer from cancer or a hemoglobinopathy (e.g., non-malignant hemoglobinopathy), such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome. The subject may also suffer from adenosine deaminase severe combined immunodeficiency (ADA SCID), HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome. The subject may have or be suffering from an inherited blood disease (e.g., sickle cell anemia) or an autoimmune disorder, such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, type 1 diabetes, or other autoimmune condition.

[0057] The cancer may be neuroblastoma or blood cancer.For example, the subject may have leukemia, lymphoma or myeloma.In some embodiments, the subject has acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma or non-Hodgkin's lymphoma.The subject may have myelodysplastic syndrome (MDS).

[0058] Gene editing Gene editing technology has advanced substantially with the advent of site-specific editing methods such as TALEN, CRISPR / cas9, and zinc finger nuclease (ZFN) that have therapeutic potential for patients with malignant and non-malignant genetic diseases. Gene editing precisely and permanently alters the sequence of genomic DNA that remains under endogenous gene regulation and endogenous gene control for proper and appropriate expression of modified genetic elements. 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 palindromic repeats (CRISPR / Cas9). Each of these can recognize and bind to specific target sequences of DNA. Depending on the approach, the target DNA can be cleaved in one or both strands. To correct the mutation, a correction template is used for homology-directed repair of the introduced break at the site of the target lesion. This technology can also be used to silence or ablate specific genes by incorporating mutational insertions or deletions. Additionally, gene editing techniques can be used to alter T cell specificity, for example, by functionally replacing one gene with another within the T-cell receptor alpha constant locus (TRAC) (Eyquem, et. al., 2017, Nature. 543:113-117).

[0059] Adoptive Cell Therapy (“ACT”) Adoptive cell therapy may involve the administration of cells expressing chimeric antigen receptors (CARs) or T cell receptors (TCRs), or may involve tumor-infiltrating lymphocytes (TILs). The population of cells expressing CAR / TCRs may include a population of activated T cells, natural killer (NK) cells, or dendritic cells that express CAR / TCRs that recognize antigens. Dendritic cells are capable of antigen presentation and direct tumor killing. The population of cells expressing CAR / TCRs may include a population of gene-edited cells.

[0060] As used herein, the term "gene-edited" CAR T cells is synonymous with the terms "genetically engineered" CAR T cells and "engineered" CAR T cells. Gene-edited CAR T cells that are "incapable of properly expressing" a checkpoint receptor (e.g., PD1, Lag3, or TIM3) do not express a full-length functional checkpoint receptor. For example, without limitation, a gene-edited CAR T cell that is unable to properly express PD1 may be unable to do so because (i) the PD1 gene of the cell has been ablated or (ii) the PD1 gene of the cell has otherwise been modified so that it does not produce a functional PD1 product, fully or even partially. That is, according to certain embodiments, a gene-edited CAR T cell that is unable to properly express PD1 may be unable to do so because the PD1 gene of the cell has been modified to reduce PD1 expression. Similarly, a gene-edited CAR T cell that is "incapable of properly expressing" a T cell receptor does not express a full-length functional T cell receptor.

[0061] According to certain embodiments, a functional endogenous T cell receptor is replaced by editing an exogenously transduced CAR or recombinant TCR into the native TCR locus by "knock-in." Gene-edited CAR T cells can include, but are not limited to, the following: (i) allogeneic gene-edited CAR T cells that cannot properly express PD1 but properly express all other checkpoint receptors and T cell receptors, (ii) allogeneic gene-edited CAR T cells that cannot properly express a particular T cell receptor but properly express all checkpoint receptors and all other T cell receptors, and (iii) allogeneic gene-edited CAR T cells that cannot properly express PD1 and cannot properly express a particular T cell receptor but properly express all other checkpoint receptors and all other T cell receptors.

[0062] An example of T cell gene editing to generate allogeneic universal CAR T cells is the work of Eyquem et al. (Eyquem, et al., 2017, Nature. 543:113-117). In that study, the endogenous T cell receptor alpha constant locus (TRAC) was effectively replaced with a recombinant CAR gene construct. This method effectively placed the recombinant CAR under the control of the cell's natural TCR regulatory signals. Using this same strategy, a CAR or recombinant TCR can be effectively inserted by knock-in into the T cell receptor beta constant locus (TRBC) or beta 2 microglobulin (B2M) MHC-I-associated locus, which are known to be expressed in all T cells. Another example is the work of Ren et al. (Ren, et al., 2017, Clin. Cancer Res 23:2255-2266). Recognizing that checkpoint receptors may be immunosuppressive and impair the stimulation of exogenous autologous or allogeneic CAR T cells, this group utilized CRISPR / cas9 technology to ablate the endogenous TCRα and TCRβ loci (TRAC and TRBC) and B2M genes, but also silenced the endogenous PD1 gene. With this approach, the engineered cells did not induce graft-versus-host disease but resisted immune checkpoint receptor suppression.

[0063] Lymphodepletion and bone marrow destruction It is common to lymphodeplete patients before administering a dose of HST (e.g., bone marrow transplant) or engineered immune cells to them. The lymphodepletion process is important, and in fact considered essential, for the success of BMT and adoptive cell therapy (ACT) methods. This process creates sufficient space in the immune microenvironment (e.g., bone marrow) to allow transplantation of the transferred cells. It also creates a favorable immune homeostatic environment for successful transplantation, proliferation, and survival of the transferred cells by inducing a favorable cytokine profile, particularly within the peripheral immune niche (e.g., bone marrow, spleen, and lymph nodes) for the establishment and proliferation of the engineered cells. (e.g., Maine, et al., 2002, J. Clin. Invest., 110:157-159; Muranski, et al., 2006, Nat. Clin. Pract. Oncol., 3(12):668-681; Klebanoff, et al., 2005, Trends Immunol., 26(2): 111-117).

[0064] As shown above, bone marrow- and lymphoid-derived cells express CD45. This is an effective method for reducing blast counts without irreversibly depleting hematopoietic stem cells. 225 The dose of Ac radiolabeled anti-CD45 antibody delivers radiation exposure to the bone marrow below a threshold level. A dose of 2 Gy is considered a nonmyeloablative dose of radiation. Doses of at least 2 Gy may provide transient but reversible myelosuppression. Myeloablative doses can vary, such as within the range of 8-18 Gy, but typically include doses delivering greater than 10-12 Gy to the bone marrow. Therefore, a therapeutic low-dose range that can provide reversible immunosuppression (with or without a stem cell carrier) is greater than 2 Gy but less than a myeloablative dose, such as 8 Gy or less. For higher doses, a stem cell carrier may be required.

[0065] As used herein, an amount of radiolabeled anti-CD45 antibody is "effective" to deplete a specific target cell type if, upon administration, it reduces the cell population by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. For example, an amount of radiolabeled anti-CD45 antibody is "effective" to deplete a subject's peripheral blood lymphocytes if, upon administration, it depletes the subject's neutrophils without depleting the subject's neutrophils, or depletes the subject's peripheral blood lymphocytes with less than a 10% or less than a 20% reduction in the subject's neutrophils. An "effective" amount of a radiolabeled anti-CD45 antibody can also refer to, for example, an amount that depletes at least 20%, or 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of a subject's regulatory T cells, myeloid-derived suppressor cells, tumor-primed macrophages, activated macrophages that secrete IL-1 and / or IL-6, and combinations thereof.

[0066] As used herein, an amount of radiolabeled anti-CD45 antibody is "effective" to reversibly suppress a target cell type if, upon administration, the cell population, such as the HSC or lymphocyte levels of a subject, is reduced by more than 95%, for example, at least 96%, or 97%, or 98%, or even 99%. "Reversible immunosuppression" generally includes the use of low-dose therapeutic agents or combinations thereof, such as actinium-225-labeled BC8 disclosed herein, to deplete target cells to a greater extent than standard lymphodepletion without ablating the target cells, i.e., at a dose less than the myeloablative dose (non-myeloablative dose). Moreover, reversible immunosuppression may indicate that the targeted immune population (immune privileged cell population or immune privileged cell tissue) is only transiently depleted, while other non-target populations are unaffected.

[0067] As disclosed below, reversible immunosuppression of the present disclosure may generally involve administration of actinium-225 labeled BC8 and another agent, such as another immunotherapeutic agent or a radiosensitizer. As used herein, an amount of radiolabeled anti-CD45 antibody is "effective" to ablate a target cell type if, upon administration, it results in a 100% reduction in a cell population, such as HSC or lymphocyte levels in a subject (also known as myeloablation). According to certain aspects of the present disclosure, the radiolabeled anti-CD45 antibody is actinium-225 labeled BC8 ( 225 Ac-labeled BC8) 225 An effective amount of Ac-labeled BC8 is, for example, less than 5.0 μCi / kg (i.e., less than 5.0 μCi / kg administered to a subject). 225 (if the amount of Ac-BC8 delivers a radiation dose below 5.0 μCi per kilogram of subject body weight).

[0068] According to an aspect of the present disclosure, 225 The effective amount of Ac-labeled BC8 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, 0.05 μCi / kg, or 0.01 μCi / kg. 225 An effective amount of Ac-labeled BC8 is at least 0.01 μCi / kg, or 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 μCi / kg, 1.5 μCi / kg, 2 μCi / kg, 2.5 μCi / kg, 3 μCi / kg, 3.5 μCi / kg, 4 μCi / kg, or 4.5 μCi / kg. 225 Ac-labeled BC8 may be administered at a dose including any combination of upper and lower limits as described herein, for example, at least 0.1 μCi / kg to less than 5 μCi / kg, or at least 0.5 μCi / kg to less than 3 μCi / kg.

[0069] According to one particular embodiment, 225 An effective amount of Ac-labeled BC8 is less than 1.0 mCi, for example, less than 0.5 mCi (i.e., 225 Ac is administered to the subject in a non-mass-based dose). 225 The effective dose of Ac-labeled BC8 may be less than 1.0 mCi, for example, less than 0.9 mCi, 0.8 mCi, 0.7 mCi, 0.6 mCi, 0.5 mCi, 0.45 mCi, 0.4 mCi, 0.35 mCi, 0.3 mCi, 0.25 mCi, 0.2 mCi, 0.1 mCi, 90 μCi, 80 μCi, 70 μCi, 60 μCi, 50 μCi, 40 μCi, 30 μCi, 20 μCi, 10 μCi or 5 μCi. 225 An effective amount of Ac-labeled BC8 may be at least 2 μCi, e.g., at least 5 μCi, 10 μCi, 20 μCi, 30 μCi, 40 μCi, 50 μCi, 60 μCi, 70 μCi, 80 μCi, 90 μCi, 100 μCi, 120 μCi, 140 μCi, 160 μCi, 180 μCi, 200 μCi, 300 μCi, 400 μCi, 500 μCi, 600 μCi, 700 μCi, 800 μCi, or 900 μCi. 225 Ac-labeled BC8 may be administered at a dose including any combination of upper and lower limits as described herein, for example, at least 15 μCi to less than 120 μCi, or at least 20 μCi to less than 100 μCi, or at least 80 μCi to less than 500 μCi. 225 Ac-labeled BC8 may be administered in a low dose. An exemplary low dose is less than 150 μCi, for example, 10 μCi to 100 μCi. 225 The dose of Ac-BC8 may be less than 2 μCi / kg, for example, 0.01 μCi / kg to 1.5 μCi / kg, or 0.1 μCi / kg to 1.0 μCi / kg. According to certain aspects of the present disclosure, the radiolabeled anti-CD45 antibody is lutetium-177 labeled BC8 ( 177 Lu-labeled BC8) 177 An effective amount of Lu-labeled BC8 is, for example, less than 500 μCi / kg (i.e., less than 500 μCi / kg administered to a subject). 177(if the amount of Lu-BC8 delivers a radiation dose below 500 μCi per kilogram of subject body weight).

[0070] According to an aspect of the present disclosure, 177 An effective amount of Lu-labeled BC8 is less than 450 μCi / kg, 400 μCi / kg, 350 μCi / kg, 300 μCi / kg, 250 μCi / kg, 200 μCi / kg, 150 μCi / kg, 100 μCi / kg, 90 μCi / kg, 80 μCi / kg, 70 μCi / kg, 60 μCi / kg, 50 μCi / kg, 40 μCi / kg, 30 μCi / kg, 20 μCi / kg, 10 μCi / kg, 5 μCi / kg, or 1 μCi / kg. 177 An effective amount of Lu-labeled BC8 is at least 1 μCi / kg, 2.5 μCi / kg, 5 μCi / kg, 10 μCi / kg, 20 μCi / kg, 30 μCi / kg, 40 μCi / kg, 50 μCi / kg, 60 μCi / kg, 70 μCi / kg, 80 μCi / kg, 90 μCi / kg, 100 μCi / kg, 150 μCi / kg, 200 μCi / kg, 250 μCi / kg, 300 μCi / kg, 350 μCi / kg, 400 μCi / kg, or 450 μCi / kg. 177 Lu-labeled BC8 may be administered at a dose including any combination of upper and lower limits as described herein, for example, at least 5 μCi / kg to less than 50 μCi / kg, or at least 50 μCi / kg to less than 500 μCi / kg.

[0071] According to one particular embodiment, 177 An effective amount of Lu-labeled BC8 is less than 20 mCi, for example, less than 15 mCi, 10 mCi, 9 mCi, 8 mCi, 7 mCi, 6 mCi, 5 mCi, 3 mCi, 2 mCi, 1 mCi, 800 μCi, 600 μCi, 400 μCi, 200 μCi, 100 μCi or 50 μCi. 177An effective amount of Lu-labeled BC8 may be at least 10 μCi, e.g., at least 25 μCi, 50 μCi, 100 μCi, 200 μCi, 300 μCi, 400 μCi, 500 μCi, 600 μCi, 700 μCi, 800 μCi, 900 μCi, 1 mCi, 2 mCi, 3 mCi, 4 mCi, 5 mCi, 10 mCi, or 15 mCi. 177 Lu-labeled BC8 may be administered at a dose including any combination of upper and lower limits as described herein, for example, at least 10 μCi to less than 20 mCi, or at least 100 μCi to less than 3 mCi, or 3 mCi to less than 20 mCi.

[0072] As used herein, an "appropriate time period" after administering a radiolabeled anti-CD45 antibody to a subject and before administering an additional treatment to the subject is a time period sufficient to allow the administered antibody to deplete, reversibly suppress, or ablate the subject's target cells, such as the subject's HSCs and / or lymphocytes. According to certain embodiments, the appropriate time 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 time 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. According to certain embodiments, a suitable time period after administration of the radiolabeled anti-CD45 antibody during which the ACT procedure can be performed is 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, or 9 days, e.g., preferably 6 days, 7 days, or 8 days.

[0073] Throughout this application, various publications are cited.The disclosures of these publications are incorporated herein by reference in this application in order to more fully describe the state of the art to which this disclosure pertains.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.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.

[0074] Detailed Description of the Invention The present disclosure addresses an unmet need in the art by providing an unexpectedly superior method for depleting, reversibly immunosuppressing, or ablating specific cells in a subject, such as hematopoietic stem cells or lymphocytes. Reversible immunosuppression of these cells may be useful in the treatment of CD45-positive hematologic malignancies and can be achieved using low-dose therapeutic agents, such as administration of anti-CD45 antibodies at subsaturating radiation doses. Radiolabeled anti-CD45 antibodies have demonstrated the clinical potential of targeted myeloablative conditioning prior to bone marrow transplantation. CD45 is an attractive target for conditioning because it is highly expressed on all nucleated immune cells, including hematopoietic stem cells, lymphoid cells, and myeloid cells. The potent alpha-emitter 225 actinium ( 225 Ac) is a promising radionuclide for target preparation, with high linear energy transfer over short path lengths (80–100 keV / μm) and a long half-life of 9.9 days.

[0075] Moreover, high-dose therapy, i.e., depletion or ablation of these cells using higher radiation doses, may be a precursor model to cell-based therapies such as bone marrow transplantation and / or adoptive cell therapy (e.g., chimeric antigen receptor therapy, CAR T cell therapy, or TCR cell therapy) or gene-edited cell-based therapy (e.g., gene-edited β-globin hematopoietic stem cell therapy for sickle cell disease (SCD)). Thus, the present disclosure provides a method for depleting, reversibly immunosuppressing, or ablating specific cells in a subject, comprising: 225 Radiolabeled anti-CD45 antibodies such as Ac-BC8 are used. These antibodies can safely and effectively deplete or ablate the target hematopoietic stem cells or lymphocytes through targeted pretreatment. This approach avoids certain side effects caused by less specific agents such as chemotherapy drugs or external beam radiation.

[0076] The present disclosure provides methods for treating various disorders, such as, inter alia, cell-type diseases in the hematopoietic lineage, cancer, autoimmune diseases, metabolic disorders, and stem cell disorders. The compositions and methods described herein can (i) directly deplete or ablate populations of cells that cause pathology, such as populations of cancer cells (e.g., leukemia cells) and autoimmune cells (e.g., autoreactive T cells), and / or (ii) deplete or ablate populations of endogenous hematopoietic stem cells to promote engraftment of transplanted hematopoietic stem cells by providing a niche to which the transplanted cells can return. The above activity is 225 Ac-BC8 or 177 This can be achieved by administering a composition comprising Lu-BC8.In the case of direct treatment of disease, this administration can cause a reduction in the amount of cells that cause the desired pathology.In the case of preparing a patient for hematopoietic stem cell transplantation, this administration can cause selective depletion, reversible suppression or ablation of the endogenous hematopoietic stem cell population, thereby creating vacancies in hematopoietic tissues such as bone marrow or lymphocytes, which can then be filled by transplanted exogenous hematopoietic stem cells, i.e., bone marrow transplantation or adoptive cell transfer.

[0077] Radiolabeled immunotherapeutic drugs According to an embodiment of the present disclosure, the anti-CD45 immunoglobulin BC8 comprises: 225 Ac or 177According to a particularly preferred embodiment, the anti-CD45 immunoglobulin BC8 is fused to the alpha-emitting radionuclide actinium-225 ( 225 It may be radiolabeled with Ac) conjugated to monoclonal antibody BC8. 225 The Ac load delivers high-energy alpha particles directly to the target cell(s), thereby producing lethal double-stranded DNA breaks. Because of its short path length, the high-energy alpha particle radiation extends only a small cell diameter, thereby limiting damage to nearby non-malignant or normal tissue. 225 Ac-BC8 is 177 It can provide a therapeutically effective dose with less heat loss than Lu-BC8.

[0078] moreover, 225 The Ac antibody conjugate offers a significant advantage over prior art antibody-drug conjugates because it has been found to be effective even in patients with tumors with low target antigen expression. 225 Due to the large cytotoxic effect of Ac, it stands in stark contrast to antibody-drug conjugates, which require hundreds of antibody molecules to bind to their respective antigens to exert an effect on a target cell or tissue. Other advantages of radioactive payload over drugs or toxins include: 1) the antibody delivering the radiation does not need to be internalized to kill cells, 2) the antibody does not need to target all cells within the target tissue or tumor, and 3) in contrast to antibody-drug conjugates, radioisotopes linked to antibodies are less likely to elicit significant immune responses that limit their subsequent use. Moreover, the work reported herein demonstrates that 225 This demonstrates the stability of Ac-labeled antibodies and their high target cytotoxicity.

[0079] According to certain aspects of the present disclosure, 225Ac may be attached or chelated by a chelator conjugated to the monoclonal antibody. As described in detail in Example 3 below, anti-CD45 immunoglobulins can be prepared by first forming a chelator-conjugated anti-CD45 ("conjugated anti-CD45") and then chelating a radionuclide with the conjugated anti-CD45 to form radiolabeled anti-CD45 (i.e., 225 Ac-BC8).

[0080] According to the method for forming radiolabeled anti-CD45 described herein, a monoclonal antibody against CD45 may be dissolved in a buffer solution containing a chelator. The pH may be selected to optimize the conditions for conjugation of the chelator to the antibody in the conjugation reaction mixture. The conjugation reaction mixture may contain a bicarbonate buffer or a phosphate buffer. The conjugation reaction mixture may have a pH of about 8.0 to about 9.2. For example, the conjugation reaction mixture may have a pH of about 8.0, about 8.1, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, or about 9.2. The temperature of the conjugation reaction mixture may be adjusted to facilitate conjugation of the chelator to the targeting moiety. For example, the conjugation reaction mixture may be incubated at about room temperature or about 37°C. The conjugation reaction mixture may be incubated for any time sufficient to provide for conjugation, such as, for example, about 1.5 hours.

[0081] The conjugated anti-CD45 may be dissolved in a buffer solution containing a radionuclide. The pH may be selected to optimize conditions for chelation of the radionuclide by the conjugated anti-CD45 in the chelation reaction mixture. The chelation reaction mixture may have a pH of about 5.5 to about 7.0. For example, the chelation reaction mixture may have a pH of about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

[0082] The temperature of the chelation reaction mixture may be adjusted to facilitate chelation of the radionuclide by the conjugated anti-CD45 immunoglobulin. For example, the chelation reaction mixture may be incubated at a temperature of about 37°C. The chelation reaction mixture may be incubated for about 1.5 hours. After a period of time, the solution may be quenched by the addition of a quencher chelating compound (e.g., diethylenetriaminepentaacetic acid (DTPA)), and the reaction mixture may be purified. After the addition of the quencher chelating compound, the chelation reaction mixture may be further incubated, for example, for about 30 minutes at about 37°C after the addition of the quencher chelating compound.

[0083] Chelators useful in the present disclosure are compounds that have the dual functionality of sequestering metal ions as well as the ability to covalently bind to biological carriers such as antibodies. Many chelators are known in the art.Exemplary chelating agents suitable for use in the present disclosure include S-2-(4-isothiocyanatobenzyl)-1,4,7,10 tetraazacyclododecanetetraacetic acid (p-SCN-Bn-DOTA), diethylenetriaminepentaacetic acid (DTPA); ethylenediaminetetraacetic acid (EDTA); 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA); p-isothiocyanato-benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA); 1,4,7,10-tetraazacyclododecane-N,N',N''-triacetic acid (DO3A); 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetrakis(2-propionic acid) (DOTMA); ,6,9-Triaza-12-oxa-3,6,9-tricarboxymethylene-10-carboxy-13-phenyl-tridecanoic acid ("B-19036"); 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA); 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA); triethylenetetraaminehexaacetic acid (TTHA); trans-1,2-diaminohexanetetraacetic acid (CYDTA); 1,4,7,10-tetraazacyclododecane-1-(2-hydroxypropyl)-4,7,10-triacetic acid (HP-DO3A); trans-cyclohexanediaminetetraacetic acid (CDTA); trans(1,2)-cyclohexanediethylenetriaminepentaacetic acid (trans(1,2)-cyclohexane Chelating agents include, but are not limited to, chelating agents such as dietylene triamine pentaacetic acid (CDTPA); 1-oxa-4,7,10-triazacyclododecane-N,N',N''-triacetic acid (OTTA); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis{3-(4-carboxyl)-butanoic acid}; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetic acid-methylamide); 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetrakis(methylenephosphonic acid); and derivatives thereof.

[0084] One or more steps may be used to separate the conjugated CD45 from other components of the conjugation reaction mixture, or the radiolabeled anti-CD45 from other components of the chelation reaction mixture. For example, the reaction mixture can be transferred to a filtration device (e.g., a Millipore centrifugal device) with a specific molecular weight cutoff so that the conjugated anti-CD45 or radiolabeled anti-CD45 can be separated from the other components of each reaction mixture by filtration of the reaction mixture through the filtration device. Filtration can be used to obtain conjugated anti-CD45 or radiolabeled anti-CD45 with a purity of at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5%.

[0085] According to certain embodiments of the present disclosure, the yield of conjugated anti-CD45 immunoglobulin or radiolabeled anti-CD45 immunoglobulin from separation (e.g., purification) is at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the final product. According to one embodiment of the present disclosure, a monoclonal antibody is first conjugated with a p-SCN-Bn-DOTA or DOTA chelator to form a conjugated anti-CD45 immunoglobulin, and then the p-SCN-Bn-DOTA or DOTA in the conjugated anti-CD45 immunoglobulin is conjugated to the monoclonal antibody. 225 Chelation of Ac may be performed to form radiolabeled anti-CD45 immunoglobulin. Thus, according to an embodiment of the present disclosure, to label anti-CD45 immunoglobulin, 225 Only a single step involving Ac is required.

[0086] According to certain aspects of the present disclosure, radiolabeled anti-CD45 immunoglobulin 225 Ac-BC8 is relatively stable. For example,225 More than 85% of Ac-BC8 can remain intact after 24 hours of storage at 4°C (see Figure 11). 225 Ac-BC8 exhibits specificity for CD45-expressing cells (see Figures 12, 15A, 15B) and CD45-expressing tissues (see Figure 16A). 225 The labeling efficiency, stability and immunoreactivity of Ac-BC8 combine to provide an effective therapeutic agent. 177 Lutetium-177 ( 177 Radiolabeling with Fluorescent Lu is also possible and within the scope of the present disclosure. 225 Ac-BC8 or 177 References to Lu-BC8 are, respectively, unless otherwise indicated. 225 Ac-DOTA-BC8 or 177 It may also contain a reference to either Lu-DOTA-BC8.

[0087] Methods for depleting or ablating target cells The present disclosure provides a method for depleting, reversibly suppressing, or ablating hematopoietic stem cells in a subject, comprising administering an effective amount of a radiolabeled anti-CD45 immunoglobulin, e.g., 225 The present disclosure provides a method for lymphodepleting a subject, the method comprising administering Ac-BC8 to the subject. 225 The present disclosure provides a method for depleting, reducing, or eliminating hematopoietic cancer blasts in a subject, comprising administering to the subject an effective amount of a radiolabeled anti-CD45 immunoglobulin, such as Ac-BC8. 225 Methods are provided that include administering to a subject an effective amount of a radiolabeled anti-CD45 immunoglobulin, such as Ac-BC8, either alone as a monotherapy or in combination with other treatments.

[0088] According to one particular aspect of this method, 225An effective dose of Ac-BC8 is 0.05 μCi / kg to 5.0 μCi / kg of the subject's body weight. Examples of effective doses include, but are not limited to, 0.05 μCi / kg to 5.0 μCi / kg, for example, 0.1 μCi / kg to 0.2 μCi / kg, 0.2 μCi / kg to 0.3 μCi / kg, 0.3 μCi / kg to 0.4 μCi / kg, 0.4 μCi / kg to 0.5 μCi / kg, 0.5 μCi / kg to 0.6 μCi / kg, 0.6 μCi / kg to 0.7 μCi / kg, 0.7 μCi / kg to 0.8 μCi / kg, and 0.8 μCi / kg to 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. According to one particular aspect of this method, 225 An effective amount of Ac-BC8 is less than 1 mCi, for example, less than 500 μCi. Examples of effective amounts include, but are not limited to, 1 μCi to 500 μCi, for example, 10 μCi to 400 μCi, or 10 μCi to 300 μCi, 10 μCi to 200 μCi, 10 μCi to 100 μCi, 15 μCi to 75 μCi, 20 μCi to 75 μCi, 10 μCi to 50 μCi, 50 μCi to 100 μCi, 100 μCi to 150 μCi, 150 μCi to 200 μCi, 200 μCi to 250 μCi, 250 μCi to 300 μCi, 300 μCi to 350 μCi, 350 μCi to 400 μCi, 400 μCi to 450 μCi, or 450 μCi to 500 μCi.

[0089] An exemplary low dose is less than 120 μCi, for example, 10 μCi to 100 μCi. 225 The dose of Ac-BC8 may be low, or less than 2 μCi / kg, for example, 0.01 μCi / kg to 1.5 μCi / kg, or 0.1 μCi / kg to 1.0 μCi / kg. 225An exemplary high dose of Ac-BC8 may be a medium to high dose of at least 120 μCi, e.g., 120 μCi to 500 μCi, or a dose of at least 2 μCi / kg, e.g., 2 μCi / kg to 5 μCi / kg, or 3 μCi / kg to 5 μCi / kg.

[0090] According to one particular aspect of this method, 225 An effective amount of Ac-BC8 is an amount effective to deplete hematopoietic stem cells or lymphocytes in a subject, e.g., an amount effective to deplete at least 25% of the subject's hematopoietic stem cells, or at least 50% of the subject's hematopoietic stem cells, or at least 70% of the subject's hematopoietic stem cells, or at least 80% of the subject's hematopoietic stem cells, or up to 90% of the subject's hematopoietic stem cells. According to one particular aspect of this method, 225 An effective amount of Ac-BC8 is an amount effective to reversibly immunosuppress a subject's hematopoietic stem cells or lymphocytes, e.g., at least 90% of the subject's hematopoietic stem cells, or at least 92%, or at least 94%, or at least 96%, or up to 98% of the subject's hematopoietic stem cells, without completely ablating the subject's hematopoietic stem cells or lymphocytes.

[0091] According to one particular aspect of this method, 225 An effective amount of Ac-BC8 is an amount effective to deplete a subject's circulating tumor cells, e.g., hematopoietic stem cells or lymphocytes, e.g., at least 25% of the subject's hematopoietic stem cells or lymphocytes, or at least 50% of the subject's hematopoietic stem cells or lymphocytes, or at least 70% of the subject's hematopoietic stem cells or lymphocytes, or at least 80% of the subject's hematopoietic stem cells or lymphocytes, or up to 90% of the subject's hematopoietic stem cells or lymphocytes, without myeloablative treatment. According to certain embodiments, this amount is 225The dose of Ac-BC8 may be low, e.g., less than 150 μCi or less than 120 μCi, e.g., between 10 μCi and 100 μCi, or less than 2 μCi / kg, e.g., between 0.01 μCi / kg and 1.5 μCi / kg, or between 0.1 μCi / kg and 1.0 μCi / kg, which may be administered alone or in combination with additional therapeutic agents as disclosed below.

[0092] According to one particular aspect of this method, 225 An effective amount of Ac-BC8 is an amount effective, either as a single agent or in combination with other treatments, to deplete, reduce, or eliminate 25%, 50%, or 100% of hematopoietic cancer blasts in a patient. Depending on the dose administered, a stem cell carrier may be required, depending on the dose effective to deplete, reduce, or eliminate cancer blasts in a patient. When such cells are depleted or reversibly suppressed, or after their complete ablation, a stem cell carrier can be provided to the patient.For example, the treatment of a patient with a high cancer cell burden may require a higher dose of radiolabeled anti-CD45 antibody, and thus may cause a significant proportion of the patient's hematopoietic stem cells to be depleted or suppressed.In such cases, a stem cell carrier may be required to cause the repopulation of these cells.In certain embodiments, a stem cell carrier may not be required, and there may be a sufficient amount of hematopoietic stem cells, which may be able to repopulate.

[0093] According to one particular aspect of this method, 225 An effective amount of Ac-BC8 is an amount effective to ablate 100% of a subject's hematopoietic stem cells (also called myeloablation). Exemplary doses include those at least designated herein as high doses. The methods generally involve administering a single effective dose, such as a single patient-specific dose. 225 The method includes administering Ac-BC8 to the subject. The amount of lymphocytes or hematopoietic stem cells reduced may be determined by any of the methods disclosed herein above.225 The dose of Ac-BC8 may depend on the amount of depletion or immunosuppression desired. For example, hematopoietic stem cell depletion may be achieved by: 225 This may be achieved with low doses, such as less than 2 Gy of Ac-BC8. 225 Ablation may be achieved with a dose of Ac-BC8 of less than 8 Gy, for example, a dose of 2 Gy to 8 Gy. 225 This may be achieved with a high dose of Ac-BC8, such as 8 Gy or more, for example, about 10-12 Gy. The method involves administering an effective amount of the compound in fractionated doses, such as multiple administrations of portions of a single patient-specific dose or administration of multiple single patient-specific doses. 225 The method may further comprise administering Ac-BC8 to a subject. When administered together with a second agent, 225 The dose of Ac-BC8 may depend on the amount of depletion or immunosuppression desired.

[0094] Methods for treating non-malignant hematological disorders This depletion method (also referred to herein as a pretreatment method) can be useful, for example, to improve the outcome of a subsequent gene-edited cell-based therapy in which depletion of hematopoietic stem cells is desired. According to certain 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. The present disclosure also provides a method for treating a subject suffering from a non-cancer disorder treatable via gene-edited cell therapy, the method comprising: (i) administering to the subject a radiolabeled anti-CD45 antibody in an amount effective to deplete the subject's hematopoietic stem cells; and (ii) after an appropriate period of time, administering the treatment to the subject to treat the subject's disorder.

[0095] Examples of non-cancerous disorders include, but are not limited to, hemoglobinopathies (e.g., SCD and β-thalassemia), congenital immune deficiencies (e.g., SCID and Fanconi anemia), and viral infections (e.g., HIV infection). According to certain embodiments, the disorder is SCD, and the treatment is gene-edited β-globin hematopoietic stem cell therapy. Stem cell therapy can be, for example, allogeneic or autologous. According to certain embodiments, the disorder is SCID, and the treatment is gene-edited hematopoietic stem cell therapy, in which the edited gene is the common gamma chain (γc) gene, the adenosine deaminase (ADA) gene, and / or the Janus kinase 3 (JAK3) gene. Stem cell therapy can be, for example, allogeneic or autologous.

[0096] According to certain preferred embodiments of the subject methods, the radiolabeled anti-CD45 antibody comprises: 225 Radiolabeled BC8 such as Ac-BC8. 225 An effective amount of Ac-BC8 is an amount that is effective too deplete, reversibly suppress, or ablate hematopoietic stem cells in a subject, and can be, for example, 0.01 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 of the subject's mass. According to certain embodiments, the method includes: (i) administering 0.1 μCi / kg to 5.0 μCi / kg of Ac-BC8; 225 and (ii) administering Ac-BC8 to the subject 6, 7, or 8 days later, to treat the subject's disorder. In certain other embodiments, the method includes (i) administering 0.1 μCi / kg to 1.0 μCi / kg of Ac-BC8 to the subject. 225 In yet another embodiment, the method includes administering Ac-BC8 to a subject, and (ii) administering to the subject a treatment 6, 7, or 8 days later, to treat the disorder in the subject. 225In yet another embodiment, the method includes administering Ac-BC8 to a subject, and (ii) administering to the subject 6, 7, or 8 days later, a treatment to treat the disorder in the subject. 225 and (ii) 6, 7, or 8 days later, administering Ac-BC8 to the subject to treat the disorder.

[0097] Methods for treating malignant hematological disorders The present disclosure also provides a method for treating a subject suffering from a malignant disease or disorder, such as cancer. The method may generally comprise administering to the subject an amount of a radiolabeled anti-CD45 antibody effective to deplete, reversibly suppress, or ablate the subject's HSCs or lymphocytes or hematopoietic cancer blasts. According to at least one exemplary method, a low dose of a radiolabeled anti-CD45 antibody is administered to reduce or deplete the number of hematopoietic stem cells or lymphocytes and / or circulating tumor cells without bone marrow ablation. The dose may be a low dose as defined herein.

[0098] According to certain embodiments, the method may further comprise administering a stem cell carrier. According to certain embodiments, the method may further comprise, after an appropriate period of time, administering a conditioning treatment. The conditioning treatment may be a hematopoietic stem cell transplant, such as a bone marrow transplant, or adoptive cell therapy to the subject to treat the subject's cancer. According to a specific embodiment of this method, the subject is suffering from cancer and is about to undergo adoptive cell therapy to treat the cancer. Adoptive cell therapy is known, and includes, for example, CAR T cell therapy (e.g., autologous cell therapy and allogeneic cell therapy). Adoptive cell therapy provides a method for promoting cancer regression in a subject, and generally includes (i) collecting autologous T cells (leukapheresis), (ii) expanding (culturing) the T cells, (iii) administering non-myeloablative lymphodepleting chemotherapy to the subject, and (iv) administering the expanded T cells to the subject after administering non-myeloablative lymphodepleting chemotherapy. The method of the present disclosure includes using a radiolabeled anti-CD45 antibody instead of lymphodepleting chemotherapy and / or after administering expanded cells (e.g., T cells, NK cells, dendritic cells, etc.). This later administration of anti-CD45 antibody (i.e., after administration of the expanded cells) may be used in preparation for autologous stem cell (HSCT) transplantation or administration of a second effective amount or number of expanded cells.

[0099] Thus, the present disclosure provides a method for treating a proliferative disease, such as a hematological malignancy, comprising the administration of a radiolabeled anti-CD45 antibody and adoptive cell therapy. Adoptive cell therapy generally involves lymphodepletion with the radiolabeled anti-CD45 antibody followed by apheresis of autologous cells, which may be gene edited, prior to reinfusion (adoptive cell therapy, such as CAR T cell therapy). Alternatively, allogeneic cells may be reinfused after lymphodepletion to provide the adoptive cell therapy. According to the methods of the present disclosure, the radiolabeled anti-CD45 antibody may be administered as a single dose 3 to 9 days, e.g., 6 to 8 days, prior to the adoptive cell therapy. According to certain aspects of this method, the radiolabeled anti-CD45 antibody is radiolabeled BC8 as described above, provided in a dose as described above, where the dose generally corresponds to the specific radionuclide label (e.g., 225According to certain embodiments of this method, the appropriate time period after administration of the radiolabeled anti-CD45 antibody is 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, or 9 days, e.g., preferably 6 days, 7 days, or 8 days.

[0100] According to certain embodiments, the method for treating a subject afflicted with cancer comprises (i) administering to the subject a single dose of a radiolabeled anti-CD45 antibody effective to lymphodeplete the subject's cells, and (ii) after a suitable period of time (e.g., 6, 7, or 8 days), administering adoptive cell therapy to the subject to treat the cancer. According to certain embodiments, the method for treating a subject afflicted with cancer comprises (i) administering to the subject a single dose of a radiolabeled anti-CD45 antibody effective to lymphodeplete the subject's cells, and (ii) after a suitable period of time (e.g., 6, 7, or 8 days), administering adoptive cell therapy to the subject to treat the cancer. According to one particular aspect of this method, 225 The effective amount of Ac-BC8 is 0.01 μCi / kg to 5.0 μCi / kg of subject body weight.

[0101] According to certain embodiments, the method for treating a subject suffering from cancer comprises (i) administering to the subject a single dose of a radiolabeled anti-CD45 antibody effective to myelosuppress the subject, and (ii) after a suitable time period (e.g., 4, 5, 6, 7, or 8 days), performing a bone marrow transplant on the subject to treat the cancer in the subject. According to certain embodiments, the method for treating a subject suffering from cancer comprises (i) administering to the subject a single dose of a radiolabeled anti-CD45 antibody effective to deplete the subject's myelocytes, and (ii) after a suitable time period (e.g., 4, 5, 6, 7, or 8 days), performing a bone marrow transplant on the subject to treat the cancer in the subject. According to one particular aspect of this method, 225An effective amount of Ac-BC8 is a low dose, for example, a dose of less than 120 μCi, for example, a dose of 10 μCi to 100 μCi, or a dose of less than 2 μCi / kg, for example, a dose of 0.01 μCi / kg to 1.5 μCi / kg, or 0.1 μCi / kg to 1.0 μCi / kg.

[0102] Additional Therapeutic Agents The compositions and methods of the present disclosure may be used in combination with certain additional therapeutic agents. For example, additional immunotherapeutic agents may be administered in combination with the anti-CD45 antibody compositions disclosed herein. Exemplary additional immunotherapeutic agents include antibodies against at least CD33 and / or CD38 (see, e.g., International Publication No. WO 2019 / 094931, the entire contents of which are incorporated herein by reference), and / or antibodies against CD34, CD117, and / or CD135 (see, e.g., U.S. Provisional Patent Application Nos. 62 / 838,646 and 62 / 838,589, the entire contents of which are incorporated herein by reference).

[0103] The compositions and methods of the present disclosure may be used in conjunction with the disclosed radioimmunotherapy (e.g., 225 Ac-BC8) may be used in combination with radiosensitizers that may enhance the effectiveness of Bcl-2 inhibitors. 225 In combination with radiation, such as that provided by Ac-BC8, it may be active against many cancer cell lines. Additionally, small molecule inhibitors of Bcl-2 protein exhibit synergistic effects with other anti-cancer drugs, including, but not limited to, etoposide, doxorubicin, cisplatin, paclitaxel, and radiation.

[0104] Inhibiting apoptosis is widely accepted as a necessary step in the transition from normal cells to cancer cells, and several cancer treatments exert their effects by reversing this process. Apoptosis is triggered by permeabilization of the outer mitochondrial membrane, a process regulated by binding between different members of the Bcl-2 family. Furthermore, Bcl-2 family members bind to the endoplasmic reticulum, where they modulate processes such as the unfolded protein response and autophagy, which also trigger or modify different forms of cell death. Bcl-2 overexpression was first described in follicular lymphoma as a result of the t(14;18) translocation and as a poor prognostic marker in acute myeloid leukemia (AML) and non-Hodgkin's lymphoma. Bcl-2 overexpression was subsequently described in prostate, breast, and colon cancers, as well as glioblastoma. Overexpression of Mcl-1, another anti-apoptotic Bcl-2-related protein, was identified in relapsed AML and associated with poor prognosis. Other alterations in Bcl-2-related protein expression identified in cancer cells include different mutations in the Bax gene and altered ratios of pro-apoptotic to anti-apoptotic Bcl-2 proteins. Thus, the inability of cancer cells to execute the apoptotic program due to defects in the normal apoptotic machinery is often associated with increased resistance to radiation- and / or immunotherapy-induced apoptosis. Thus, the methods of the present disclosure may include the addition of a radiosensitizer, such as a Bcl-2 inhibitor, which may act synergistically with the radiolabeled anti-CD45 antibody to directly or indirectly induce apoptosis in cancer cells. Bcl-2 inhibitors include small molecule and antisense oligonucleotide drugs such as AT-101 ((-)gossypol), GENASENSE® (G3139 or oblimersen; a Bcl-2-targeted antisense oligonucleotide), IPI-194, IPI-565, ABT-737, ABT-263, GX-070 (obatoclax), and the like.

[0105] According to a preferred embodiment, the Bcl-2 inhibitor may be venetoclax, a drug approved for treating chronic lymphocytic leukemia ("CLL"). Venetoclax binds to the BH3-binding groove of BCL-2, thereby displacing pro-apoptotic proteins such as BIM, initiating mitochondrial outer membrane permeabilization ("MOMP"), cytochrome c release, and caspase activation, ultimately resulting in programmed cancer cell death (i.e., apoptosis). Ideally, by altering the balance between pro- and anti-apoptotic stimuli, venetoclax facilitates programmed cell death of cancer cells, thus improving patient outcomes.

[0106] According to certain aspects, radioimmunotherapy of the present disclosure (e.g., 225 Ac-BC8) is therapeutically effective for treating cancer when the amounts of (i) the BCL-2 inhibitor and (ii) the radiolabeled anti-CD45 antibody (e.g., 225 The present invention may also be used in combination with a BCL-2 inhibitor, such as venetoclax, to provide a method for treating a subject afflicted with cancer, comprising administering to the subject a combination of venetoclax and Ac-BC8.

[0107] The present disclosure provides venetoclax and 225 (i) a BCL-2 inhibitor, such as venetoclax; and (ii) amounts of Ac-BC8, when administered together, that are therapeutically effective for treating acute myeloid leukemia. 225 Also provided is a method for treating a human subject suffering from a blood disease or disorder, comprising administering to the subject Ac-BC8.

[0108] In this specification, 225 Administering a BCL-2 inhibitor to a subject "in conjunction with" a radiolabeled anti-CD45 antibody such as Ac-BC8 225This refers to administering a BCL-2 inhibitor before, during, or after administration of Ac-BC8, including, but not limited to, the following scenarios: (i) first administering a BCL-2 inhibitor (e.g., once daily orally for 21 days, 28 days, 35 days, 42 days, 49 days, or longer as long as the cancer being treated does not progress and the BCL-2 inhibitor does not cause unacceptable toxicity), and second administering a BCL-2 inhibitor (e.g., once daily orally for 21 days, 28 days, 35 days, 42 days, 49 days, or longer as long as the cancer being treated does not progress and the BCL-2 inhibitor does not cause unacceptable toxicity). 225 administering Ac-BC8 (e.g., intravenously, in a single dose or in multiple doses over a multi-week period); (ii) administering a BCL-2 inhibitor; 225 Co-administered with Ac-BC8 (e.g., BCL-2 inhibitor, orally administered once daily for n days, 225 Ac-BC8 is administered intravenously as a single dose on one of days 2 to n-1 of the BCL-2 inhibitor regimen), (iii) a BCL-2 inhibitor 225 co-administered with Ac-BC8 (e.g., a BCL-2 inhibitor is administered orally for a period of more than one month (e.g., once daily orally for 35 days, 42 days, 49 days, or longer as long as the cancer being treated does not progress and the BCL-2 inhibitor does not cause unacceptable toxicity); 225 Ac-BC8 is administered intravenously as a single dose on day 1 of the first month of the BCL-2 inhibitor regimen), and (iv) initially 225 Ac-BC8 is administered (e.g., intravenously, in a single dose or in multiple doses over a period of several weeks) and a BCL-2 inhibitor is administered second (e.g., orally once daily for 21 days, 28 days, 35 days, 42 days, 49 days, or longer as long as the cancer being treated does not progress and the BCL-2 inhibitor does not cause unacceptable toxicity).

[0109] The amount of the administered radiolabeled anti-CD45 antibody can be sufficient to deplete, reversibly immunosuppress, or ablate hematologic stem cells in the patient. Generally, the dose of the radiolabeled anti-CD45 antibody is a subsaturating dose that can reversibly immunosuppress hematologic stem cells.

[0110] Additional radiosensitizers include, for example, histone deacetylase inhibitors (HDACi), such as vorinostat, belinstat, and romidepsin; metronidazole, misonidazole, intra-arterial Budr, intravenous iodinated deoxyuridine (IudR), nitroimidazole, 5-substituted-4-nitroimidazole, 2H-isoindoledione, [[(2-bromoethyl)-amino]methyl]-nitro-1H-imidazole-1-ethanol, nitroaniline derivatives, DNA-affinity hypoxia-selective cytotoxins, halogenated DNA ligands, 1,2,4 benzotriazine oxide, 2-nitroimidazole derivatives, fluorine-containing nitroazole derivatives, benzamides, nicotinamide, acridine-intercalators, 5-thiotretrazole derivatives, 3-nitro-1,2,4-triazole, 4,5-dinitroimidazole derivatives, hydroxylated texaphyrins, texaphrins, cisplatin, mitomycin, tiripazamine, nitrosoureas, mercaptopurine, methotrexate, fluorouracil, bleomycin, vincristine, carboplatin, epirubicin, doxorubicin, cyclophosphamide, vindesine, etoposide, paclitaxel, heat (hyperthermia), and the like.

[0111] The term "histone deacetylase inhibitor" or "HDACi" refers to histone deacetylase inhibitors that can be divided into four classes: hydroxamates (panobinostat (LBH-589), trichostatin A (TSA), vorinostat (SAHA), belinostat (PXD101), NVP-LAQ824, and gibinostat (ITF2357)), cyclic peptides (romidepsin (depsipeptide)), fatty acids (valproic acid (VPA) and sodium phenylbutyrate), and benzamides (MS-275, MGCD0103). HDACi are characterized as class I-specific HDAC inhibitors (MGCD0103, romidepsin, and MS-275) or as pan-HDAC inhibitors (TSA, panobinostat, vorinostat, and belinostat) that exhibit activity against both class I and class II HDACs. Histone deacetylase inhibitors are recognized to exert multiple cytotoxic effects in cancer cells, often through acetylation of nonhistone proteins. Some well-recognized mechanisms of HDACi lethality include interference with chaperone protein function, free radical generation, DNA damage induction, upregulation of endogenous inhibitors of cell cycle progression, and promotion of apoptosis, in addition to DNA relaxation and derepression of gene transcription. Interestingly, this class of agents is relatively selective for transformed cells, which has been shown to halt DNA repair after chemotherapy and promote chemotherapy efficacy.

[0112] According to certain aspects, radioimmunotherapy of the present disclosure (e.g., 225 Ac-BC8) is therapeutically effective for treating cancer when the amounts of (i) the HDACi and the radiolabeled anti-CD45 antibody are administered together with each other, 225 The compound may be used in combination with an HDACi, such as vorinostat, belinstat, or romidepsin, to provide a method for treating a subject afflicted with cancer, comprising administering to the subject a compound containing benzodiazepine, benzodiazepine, or benzocaine (Ac-BC8). The present disclosure provides HDACi and225 (i) an HDACi, such as vorinostat, belinostat, or romidepsin, and (ii) amounts of Ac-BC8, when administered together, are therapeutically effective for treating acute myeloid leukemia. 225 Also provided is a method for treating a human subject suffering from a blood disease or disorder, comprising administering to the subject Ac-BC8.

[0113] Similar to BCL-2 inhibitors, 225 Administering an HDACi to a subject "in conjunction with" a radiolabeled anti-CD45 antibody, such as Ac-BC8, 225 This refers to administering an HDACi before, during, or after the administration of Ac-BC8, including, but not limited to, the following scenarios: (i) administering an HDACi first (e.g., once daily orally or intravenously on days 1, 8, and 15 of a 28-day cycle for 21 days, 28 days, 35 days, 42 days, 49 days, or longer as long as the cancer being treated does not progress and the HDACi does not cause unacceptable toxicity), and secondly, 225 administering Ac-BC8 (e.g., intravenously, in a single dose or in multiple doses over a multi-week period); (ii) administering an HDACi 225 or co-administered with Ac-BC8 (e.g., HDACi administered orally once daily for n days or intravenously for n days, 225 Ac-BC8 is administered intravenously as a single dose on one of days 2 to n-1 of the HDACi regimen), (iii) HDACi 225 Ac-BC8 (e.g., an HDACi is administered orally for a period of more than one month as described herein, 225 Ac-BC8 is administered intravenously as a single dose on day 1 of the first month of the HDACi regimen), and (iv) initially 225 Ac-BC8 is administered (e.g., intravenously in a single dose or in multiple doses over a period of several weeks) and an HDACi is administered second (as described herein).

[0114] manufactured goods The present disclosure further provides an article of manufacture comprising (a) a radiolabeled anti-CD45 immunoglobulin and (b) a label instructing a user to administer to a subject an amount of the immunoglobulin effective to deplete hematopoietic stem cells in the subject. According to a specific embodiment of the subject article, the radiolabeled anti-CD45 immunoglobulin is administered in an effective amount of, for example, 0.01 μCi / kg to 5.0 μCi / kg or 0.01 μCi / kg to 1.0 μCi / kg. 225 Ac-BC8, or 1.0μCi / kg to 3.0μCi / kg 225 Ac-BC8, or 3.0μCi / kg to 5.0μCi / kg 225 Ac-BC8, or 10μCi~120μCi 225 Ac-BC8, or 100μCi~250μCi 225 Ac-BC8, or 200μCi~500μCi 225 Ac-BC8, or 5μCi to 80μCi 225 Can be Ac-BC8 225 It is Ac-BC8. According to a specific embodiment of the subject article, the radiolabeled anti-CD45 immunoglobulin is administered in an effective amount of, for example, 1 μCi / kg to 500 μCi / kg or 1 μCi / kg to 100 μCi / kg. 177 Lu-BC8, or 100μCi / kg to 300μCi / kg 177 Lu-BC8, or 300μCi / kg to 500μCi / kg 177 Can be Lu-BC8 177 It is Lu-BC8.

[0115] The present disclosure will be better understood by reference to the examples that follow; however, those skilled in the art will readily recognize that the specific examples described in detail are merely illustrative of the present disclosure as more fully described in the claims that follow.

[0116] Aspects of the present invention In the present application, the following aspects are disclosed. Aspect 1: A method for depleting hematopoietic stem cells in a subject, the method comprising administering to said subject an effective amount of a radiolabeled anti-CD45 immunoglobulin. Aspect 2. The method of aspect 1, wherein said effective amount of said radiolabeled anti-CD45 immunoglobulin depletes at least 25% of the subject's hematopoietic stem cells, or 50% of the subject's hematopoietic stem cells, or at least 70% of the subject's hematopoietic stem cells, or at least 80% of the subject's hematopoietic stem cells, or at least 90% of the subject's hematopoietic stem cells, or at least 95% of the subject's hematopoietic stem cells, or no more than 90% of the subject's hematopoietic stem cells, or no more than 95% of the subject's hematopoietic stem cells. Aspect 3. The method of aspect 1, wherein said effective amount of said radiolabeled anti-CD45 immunoglobulin depletes at least 90% of the subject's hematopoietic stem cells, or at least 95% of the subject's hematopoietic stem cells, or at least 98% of the subject's hematopoietic stem cells, or at least 99% of the subject's hematopoietic stem cells, or no more than 98% of the subject's hematopoietic stem cells, or no more than 99% of the subject's hematopoietic stem cells. Aspect 4 The method of any one of aspects 1-3, further comprising administering an effective amount of a second therapeutic agent comprising one or more of an immunotherapeutic agent, a radiosensitizer, or a chemotherapeutic agent. Embodiment 5 The method of embodiment 4, wherein said immunotherapeutic agent comprises one or more antibodies against CD33, CD34, CD38, CD119, and CD135.

[0117] Embodiment 6 The method of embodiment 4, wherein the radiosensitizer comprises a Bcl-2 inhibitor or an HDAC inhibitor (HDACi). Aspect 7 The method of aspect 1, wherein said effective amount of said radiolabeled anti-CD45 immunoglobulin depletes 100% of the subject's hematopoietic stem cells (i.e., ablates said hematopoietic stem cells). Embodiment 8. A method for lymphodepleting a subject, the method comprising administering to said subject an effective amount of a radiolabeled anti-CD45 immunoglobulin. Aspect 9. The method of aspect 8, wherein said effective amount of said radiolabeled anti-CD45 immunoglobulin depletes at least 25% of the subject's lymphocytes, or 50% of the subject's lymphocytes, or at least 70% of the subject's lymphocytes, or at least 80% of the subject's lymphocytes, or at least 90% of the subject's lymphocytes, or at least 95% of the subject's lymphocytes, or at least 98% of the subject's lymphocytes, or at least 99% of the subject's lymphocytes, or no more than 90% of the subject's lymphocytes, or no more than 95% of the subject's lymphocytes, or no more than 98% of the subject's lymphocytes, or no more than 99% of the subject's lymphocytes.

[0118] Aspect 10. The method of aspect 8, wherein said effective amount of said radiolabeled anti-CD45 immunoglobulin depletes at least 25% of the subject's hematopoietic cancer blasts, or 50% of the subject's hematopoietic cancer blasts, or at least 70% of the subject's hematopoietic cancer blasts, or at least 80% of the subject's hematopoietic cancer blasts, or at least 90% of the subject's hematopoietic cancer blasts, or at least 95% of the subject's hematopoietic cancer blasts, or at least 98% of the subject's hematopoietic cancer blasts, or at least 99% of the subject's hematopoietic cancer blasts, or no more than 90% of the subject's hematopoietic cancer blasts, or no more than 95% of the subject's hematopoietic cancer blasts, or no more than 98% of the subject's hematopoietic cancer blasts, or no more than 99% of the subject's hematopoietic cancer blasts. Embodiment 11. The method of embodiment 8, wherein said effective amount of said radiolabeled anti-CD45 immunoglobulin depletes 100% of said subject's lymphocytes (i.e., ablates said lymphocytes) or depletes 100% of said hematopoietic cancer blasts in said subject.

[0119] Aspect 12. The method of any one of Aspects 1 to 11, wherein the subject is suffering from a non-cancerous disorder treatable via gene editing cell therapy and is undergoing such therapy to treat the disorder, and wherein the effective amount of the radiolabeled anti-CD4 immunoglobulin is administered as a single dose. Embodiment 13. A method for treating a subject afflicted with a non-cancerous disorder treatable via gene editing cell therapy, comprising: (i) administering to the subject a radiolabeled anti-CD45 immunoglobulin in an amount effective to deplete hematopoietic stem cells in said subject; and (ii) after a suitable period of time, administering said therapy to said subject, thereby treating the disorder in said subject.

[0120] Embodiment 14 The method of embodiment 12 or 13, wherein said disorder is selected from the group consisting of a hemoglobinopathy, a congenital immune deficiency, and a viral infection. Embodiment 15: The method of embodiment 14, wherein said disorder is selected from the group consisting of sickle cell disease (SCD), severe combined immunodeficiency (SCID), β-thalassemia, and Fanconi anemia.

[0033] Aspect 16. The method of aspect 14, wherein said disorder is SCD and said treatment is gene-edited β-globin hematopoietic stem cell therapy.

[0033] Aspect 17. The method of aspect 14, wherein said disorder is SCID, said treatment is gene editing hematopoietic stem cell therapy, and said edited gene is selected from the group consisting of a common gamma chain (γc) gene, an adenosine deaminase (ADA) gene, and a Janus kinase 3 (JAK3) gene.

[0121] Aspect 18. The method of any one of Aspects 1 to 7, wherein the subject is suffering from a cancerous disorder and is undergoing a hematopoietic stem cell transplant, such as a bone marrow transplant, to treat the disorder, and wherein the effective amount of the radiolabeled anti-CD4 immunoglobulin is administered as a single dose. Aspect 19. The method of any one of Aspects 8 to 11, wherein the subject is suffering from a cancerous disorder treatable via gene editing cell therapy and is undergoing such therapy to treat the disorder, and wherein the effective amount of the radiolabeled anti-CD4 immunoglobulin is administered as a single dose. Embodiment 20: A method for treating a subject afflicted with a cancerous disorder treatable via gene editing cell therapy, comprising: (i) administering to the subject a radiolabeled anti-CD4 immunoglobulin in an amount effective to lymphodeplete the subject's cells; and (ii) after a suitable period of time, administering said therapy to the subject, thereby treating the disorder in the subject. Aspect 21: The method of any one of aspects 18 to 20, wherein said cancerous disorder is non-Hodgkin's lymphoma, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma.

[0122] Embodiment 22: The method of embodiment 19 or 20, wherein said gene editing cell therapy is adoptive cell therapy for treating said cancerous disorder. Embodiment 23: The method of embodiment 22, wherein said adoptive cell therapy is CAR T cell therapy, and wherein said CAR T cell therapy comprises administration of gene-edited CAR T cells, and wherein said gene-edited CAR T cells are incapable of appropriately expressing at least one checkpoint receptor and / or at least one T cell receptor. Embodiment 24: The method of embodiment 23, wherein said CAR T cell therapy is autologous cell therapy. Embodiment 25: The method of embodiment 23, wherein said CAR T cell therapy is an allogeneic cell therapy. Aspect 26: The radiolabeled anti-CD45 antibody 225 Ac-BC8 or 177 26. The method of any one of aspects 1 to 25, wherein the compound is Lu-BC8.

[0123] Aspect 27 225 the effective amount of Ac-BC8 is between 0.01 μCi / kg and 5.0 μCi / kg of the target mass, or between 0.01 μCi / kg and 1.0 μCi / kg of the target mass, or between 1.0 μCi / kg and 3.0 μCi / kg of the target mass, or between 3.0 μCi / kg and 5.0 μCi / kg of the target mass; or 225the effective amount of Ac-BC8 is between 2 μCi and less than 0.5 mCi, or at least between 2 μCi and less than 120 μCi, or between 10 μCi and less than 120 μCi, or between 50 μCi and less than 250 μCi; or 177 the effective amount of Lu-BC8 is between 1 μCi / kg and 500 μCi / kg of the target mass, or between 1 μCi / kg and 100 μCi / kg of the target mass, or between 100 μCi / kg and 300 μCi / kg of the target mass, or between 300 μCi / kg and 500 μCi / kg of the target mass; or 177 27. The method of claim 26, wherein the effective amount of Lu-BC8 is 10 μCi to 20 mCi, or 100 μCi to 3 mCi, or 3 mCi to 20 mCi.

[0124] Embodiment 28: The method of any one of embodiments 1 to 27, wherein said anti-CD45 immunoglobulin comprises BC8, and said BC8 comprises a light chain having an amino acid sequence as set forth in SEQ ID NO: 1, or a light chain N-terminal amino acid sequence as set forth in SEQ ID NO: 9. Embodiment 29: The method of any one of embodiments 1 to 28, wherein said anti-CD45 immunoglobulin comprises BC8, and wherein said light chain of said BC8 comprises at least one complementarity determining region having an amino acid sequence as set forth in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. Embodiment 30: The method of any one of embodiments 1 to 29, wherein said anti-CD45 immunoglobulin comprises BC8, and said BC8 comprises a light chain having an amino acid sequence as set forth in SEQ ID NO: 12 or SEQ ID NO: 13. Embodiment 31: The method of any one of embodiments 1 to 30, wherein said anti-CD45 immunoglobulin comprises BC8, and said BC8 comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 2, or a heaving chain N-terminal amino acid sequence as set forth in SEQ ID NO: 10.

[0125] Embodiment 32: The method of any one of embodiments 1 to 31, wherein said anti-CD45 immunoglobulin comprises BC8, and wherein said heavy chain of said BC8 comprises at least one complementarity determining region having an amino acid sequence as set forth in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. Embodiment 33: The method of any one of embodiments 1 to 32, wherein said anti-CD45 immunoglobulin comprises BC8, and said BC8 comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16. Embodiment 34: The method according to any one of embodiments 1 to 33, wherein said anti-CD45 immunoglobulin comprises BC8, and the heavy chain of said BC8 comprises the amino acid ASP or ASN at position 141 (relative to the N-terminal amino acid). Embodiment 35 The method of embodiment 34, wherein the ratio of ASP:ASN in the population of BC8 proteins is in the range of 1:99 to 99:1, such as 10:90 to 90:10.

[0126] Embodiment 36: The method according to any one of embodiments 1 to 35, wherein said anti-CD45 immunoglobulin comprises a heavy chain constant region derived from human IgG1, IgG2, or IgG4, i.e., BC8 modified to comprise an amino acid sequence as set forth in one of SEQ ID NOs: 17 to 19. Embodiment 37: The method according to any one of embodiments 1 to 36, wherein said anti-CD45 immunoglobulin comprises BC8 modified to comprise a heavy chain constant region derived from human IgG4 containing the mutation S228P, having an amino acid sequence as set forth in SEQ ID NO: 20. Embodiment 38: The method of any one of embodiments 1 to 37, wherein said anti-CD45 immunoglobulin comprises BC8 modified to include a light chain kappa constant region of human origin, having an amino acid sequence as set forth in SEQ ID NO: 21. Embodiment 39. An article of manufacture comprising: (a) a radiolabeled anti-CD45 antibody; and (b) a label instructing a user to administer to a subject an amount of the antibody effective to deplete or ablate hematopoietic stem cells in said subject or lymphocytes in said subject.

[0127] Aspect 40: wherein the radiolabeled BC8 225 Ac-BC8, 225 the effective amount of Ac-BC8 is 0.01 μCi / kg to 5.0 μCi / kg of the target mass, or 0.01 μCi / kg to 1.0 μCi / kg of the target mass, or 1.0 μCi / kg to 3.0 μCi / kg of the target mass, or 3.0 μCi / kg to 5.0 μCi / kg of the target mass, or 225 40. The article of claim 39, wherein the effective amount of Ac-BC8 is between 2 μCi and less than 0.5 mCi, or between 2 μCi and 250 μCi, or between 75 μCi and 400 μCi. [Example]

[0128] Example 1 - Generation of anti-CD45 immunoglobulin BC8 The mouse anti-CD45 mAb BC8 was prepared from a hybridoma (ATCC No. HB-10507) originally developed by fusing mouse myeloma NS1 cells with spleen cells from BALB / C mice hyperimmunized with human phytohemagglutinin (PHA)-stimulated mononuclear cells. After screening for microbial contamination, the original fused cells were cultured in JRH-Biosciences EXCell300 medium supplemented with 1–2% fetal bovine serum (FBS).

[0129] Hybridoma cell lines were adapted for culture in serum-free medium. Briefly, cells in culture were gradually and progressively depleted of serum albumin using combo medium supplemented with glutamine, cholesterol, insulin, and transferrin. Cells were then cultured at a concentration of 1 × 10 for up to 500 L scale. 6 The cells were grown to a density of >1000 cells / mL. The medium was collected and processed for purification of anti-CD45 antibody using a combination of cation exchange chromatography, protein A chromatography, and anion exchange membrane separation. The purified antibody was concentrated by nanofiltration (30 kD cutoff). The concentration of the purified product was measured at 5.2 mg / mL and stored at 2-8°C. The purified antibody was characterized by SDS-PAGE, IEF, and SEC-HPLC. A single product peak (99.4%) was recorded by SEC-HPLC with approximately 0.6% concentra- tion. Non-reducing SDS-PAGE showed a single band for the antibody. SDS-PAGE under reducing conditions confirmed the presence of both light and heavy chains (99.9%).

[0130] Example 2 - Sequencing of anti-CD45 immunoglobulin BC8 Total RNA was isolated from hybridoma cells according to the Trizol® Reagent technical manual. Total RNA was analyzed by agarose gel electrophoresis and reverse transcribed into cDNA using isotype-specific antisense or universal primers according to the PrimeScript™ First Strand cDNA Synthesis Kit technical manual. VH, VL, CH, and CL antibody fragments were amplified and cloned separately into standard cloning vectors using standard molecular cloning techniques. Colony PCR screening was performed to identify clones with inserts of the correct size. For each antibody fragment, more than five single colonies with inserts of the correct size were sequenced. The complete nucleotide sequences of the light and heavy chains are shown in Figures 4A, 4B, 5A, and 5B.

[0131] Anti-CD45 immunoglobulin (i.e., BC8 antibody) was sequenced using a mass spectrometry peptide mapping approach. BC8 antibody was deglycosylated, reduced, and digested with individual enzymes, namely, trypsin, Lys-C, and chymotrypsin. The peptide fragments were then analyzed by LC-coupled mass spectrometry using an MS / MS fragmentation analysis approach. Protein sequencing of the heavy and light chains of BC8 antibody showed that the actual amino acid sequence differed from that predicted by DNA sequencing by only a single amino acid in the heavy chain. As highlighted in Figures 5A and 5B, the codon encoding the amino acid at position 141 predicts ASN-141, but protein sequencing did not reveal actual ASP-141. Furthermore, sequencing of various batches of protein revealed different amounts of ASP and ASN at position 141, i.e., the protein was found to contain both ASN-141 and ASP-141 in ratios ranging from 1:99 to 99:1, e.g., 10:90 to 90:10 (ASN-141:ASP-141). See Table 1.

[0132] [Table 1]

[0133] This type of post-translational modification, i.e., deamination, may depend on the cellular environment and, in some cases, may be associated with protein age (e.g., may provide a signal for protein degradation). However, the fact that no other deaminated amino acids were identified may indicate an important and specific role for ASP-141. At a minimum, ASP-141 may be located within an exposed or accessible region of the folded protein. That is, ASP-141 may be solvent accessible and may reside within a conformationally flexible region of the antibody. The effect of deamination on the biological activity of the BC8 antibody may be determined from the results of human clinical trials.

[0134] Example 3-BC8: 225Labeling and purification to form Ac-BC8 Conjugation of BC8 and irrelevant control mAb 18B7 (mouse IgG1) with DOTA Antibodies against CD45 (i.e., BC8 antibody) and controls (i.e., mouse monoclonal antibodies reactive with the fungal polysaccharide glucuronoxylomannan; 2 mg each) were equilibrated with conjugation buffer (sodium carbonate buffer with 1 mM EDTA, pH = 8.5-9.0) by four ultrafiltration spins using Centricon filters with a MW cutoff of 50,000 or Vivaspin ultrafiltration tubes with a MW cutoff of 50,000. 1.5 milliliters (mL) of conjugation buffer was used per spin. For each spin, the antibodies were spun at 53,000 RPM for 5-20 minutes at 4 °C in a Thermo IEC Centra CL3R centrifuge with a fixed-angle rotor, resulting in a retentate volume of 100-200 microliters (µL). Spin times vary for different antibodies and different protein concentrations. To allow time for equilibration, the antibodies were incubated for 30 minutes at 4 °C after the second and third spins.

[0135] [Table 2]

[0136] For conjugation, a 3 mg / mL solution of DOTA-pSCN (MW = 678) in 0.15 M NHOAc was prepared by dissolving by vortexing. DOTA-pSCN and antibody (>5 mg / mL) were mixed together in Eppendorf tubes at molar ratios of 5, 7.5, and 15 and incubated at room temperature for 15 hours (see Figure 9A). For purification of the DOTA-antibody conjugate, unreacted DOTA-pSCN was removed by seven rounds of ultrafiltration as described above, followed by washing each time with 1.5 mL of 0.15 M NHOAc buffer (pH = 6.5 or less, approximately 100 μL). After the final wash, 0.15 M NHOAc buffer was added to bring each sample to a final concentration of approximately 1 mg / mL.

[0137] The final concentration of the DOTA-antibody conjugate was measured by a simplified Lowry method. The number of DOTA molecules conjugated to the antibody (DOTA-to-protein molar ratio) was determined as described in Dadachova et al., 1999, Spectrophotometric method for determination of bifunctional macrocyclic ligands in macrocyclic ligand-protein conjugates, Nuclear Medicine & Biology, 26:977-982. The results of the DOTA-to-protein molar ratio determination are shown in Table 2.

[0138] 225 Radiolabeling of DOTA-antibody conjugates with Ac A reaction containing 15 μL of 0.15 M NH4OAc buffer (pH = 6.5) and 2 μL (10 μg) of DOTA-BC8 (5 mg / mL) was mixed in an Eppendorf reaction tube, followed by 4 μL of DOTA-BC8 in 0.05 M HCl. 225 Ac (10 μCi) was added (see FIG. 9B). The contents of the tube were mixed with a pipette tip, and the reaction mixture was incubated at 37° C. for 90 minutes with shaking at 100 rpm. At the end of the incubation period, 3 μL of 1 mM DTPA solution was added to the reaction mixture and incubated at room temperature for 20 minutes to remove unreacted DTPA. 225 Ac 225 bound in the Ac-DTPA complex.

[0139] Rapid thin layer chromatography (ITLC) with 10 cm silica gel strips and a 10 mM EDTA / saline mobile phase was used to separate the free 225 Ac( 225 Ac-DTPA) 225 Ac-labeled BC8 ( 225 Ac-DOTA-BC8) 225The radiochemical purity of Ac-DOTA-BC8 was determined. In this system, the radiolabeled antibody remained at the application site. 225 Ac-DTPA migrates with the solvent front. To cut the strip in half and exclude its daughter 225 Counting was performed using a gamma counter equipped with a multichannel analyzer, using channels 72 to 110 for Ac. The results of selected radiolabeling experiments are shown in Table 2, which indicates that the conjugate formed at an initial molar ratio of DOTA to BC8 of 7.5 provided the highest conjugation ratio (DOTA to BC8 protein) and was selected for all follow-up experiments (Batch A) described below.

[0140] 225 Purification and purification of Ac-DOTA-BC8 225 HPLC of Ac-DOTA-BC8 on a PD10 column pre-blocked with 1% HSA or on a Vivaspin centrifugal concentrator with a MW cutoff of 50 kDa for 3 minutes per rotation with 2 × 1.5 mL washes. 225 The Ac-DOTA-BC8 sample was purified using a Waters HPLC system equipped with a flow-through Waters UV and Bioscan Radiation detector. 225 HPLC analysis of Ac-DOTA-BC8 was performed. The injection sample size was 30 μL. Elution was performed on a TSK3000SW XL column at a flow rate of 1 mL / min using PBS pH=7.4 as the eluent. An exemplary chromatogram is shown below. 225 The SEC-HPLC (size exclusion chromatography-HPLC) of Ac-DOTA-BC8 is provided in Figures 10A and 10B, where Figure 10A shows the BC8 standard and Figure 10B shows the 225 Ac-DOTA-BC8 is shown (the peak at 13 min is HSA added to stabilize the final formulation).

[0141] Example 4 225 Ac-BC8: Stability 225 Determination of Ac-DOTA-BC8 stability DOTA-BC8 (batch A) was used in all immunoreactivity experiments, as described in the procedure above, with a specific activity of 1 μCi / μg. 225 Radiolabeled with Ac. For stability determination, 225 Ac-DOTA-BC8 was tested in the original volume of 20 μL or diluted to 40 μL or 60 μL with working buffer (0.15 M NHOAc), incubated at room temperature (rt) for 48 h or at 4°C for 96 h, and tested by ITLC. All samples were analyzed in duplicate, and experiments were performed in triplicate. The results shown in Figure 11 demonstrate that actinium-225 labeled BC8( 225 It is shown that Ac-DOTA-BC8) was stable for up to 96 hours at 4°C.

[0142] Example 5 225 Ac-BC8: immunoreactive Cell lines were used 225 Ac-DOTA-BC8 immunoreactivity (IR) determination DOTA-BC8 (batch A) was used in all immunoreactivity experiments, as described in the procedure above, with a specific activity of 1 μCi / μg. 225 Ramos CD45-positive cells and control CD45-negative EL4 cells were radiolabeled with Ac. Ramos CD45-positive cells and control CD45-negative EL4 cells were used in duplicate at 1,000,000 to 7,500,000 cells per sample. Experiments were performed twice. The results shown in Figure 12 indicate that 225 We demonstrate that Ac-DOTA-BC8 specifically binds to Ramos cells, with 50% radiolabeled antibody binding to these cells compared with only approximately 10% binding to control EL4 cells. However, it is not cost-effective to continuously grow two cell lines in the laboratory for QC, and a more convenient assay for IR determination was desired. The following conditions were used as controls: Ramos cells pre-blocked with 1% BSA, EL4 cells, and EL4 cells pre-blocked with 1% BSA.

[0143] Cytotrol cells were used 225 Ac-DOTA-BC8 immunoreactivity (IR) determination We first used Cytotrol cells (Beckman Coulter) to determine the binding rate of naive BC8 antibody to these cells by flow cytometry relative to the control 18B7 antibody (a nonspecific control antibody against the fungal polysaccharide glucuronoxylomannan) (Figure 13A). Cells were harvested in RPMI medium, and the secondary antibody was PE-labeled rat anti-mouse IgG1 from Biolegend. Cytotrol cells are lyophilized human lymphocytes isolated from peripheral blood that display the CD45 surface antigen and were selected based on their commercial availability (Beckman Coulter) and consistency. The binding rate of native BC8 to Cytotrol cells was compared with that of DOTA-BC8 (Figure 13B). Native BC8 showed strong binding to Cytotrol cells, whereas the control 18B7 mAb only bound at background levels (Figure 13B). Attachment of DOTA to BC8 reduced its IR to approximately 70% of that of native BC8 (Figure 13B).

[0144] [Table 3]

[0145] IR determination of 225Ac-DOTA-BC8 was then performed. To measure the binding rate of radiolabeled antibody to Cytotrol cells, three tubes of Cytotrol cells (Lot 729154) were used for each sample, and binding rates were measured for duplicate samples. The vials were washed with 0.5 mL of renaturing buffer, and the cells were pooled. The vials were washed with two additional 0.5 mL aliquots, and the washes were pooled. The cells were collected by centrifugation at 4000 rpm for 4 minutes, blocked with 1 mL of RPMI containing 1% bovine serum albumin (BSA), spun again, and resuspended in 0.5 mL of RPMI / BSA. Approximately 25,000 CPM of labeled antibody was added per vial. The vials were incubated at 37°C for 1 hour, shaken at 150 RPM, and spun at 4000 RPM for 4 minutes. Three washes were collected, and the washes and cells were counted. Table 3 shows the IR determination for six samples of 225Ac-DOTA-BC8. The average IR was 64.8±2.14%.

[0146] Finally, after a side-by-side comparison of the binding rates of DOTA-BC8 samples to Cytotrol cells by flow cytometry (Figure 14B), 225 The same samples were immediately radiolabeled with Ac, and Cytotrol cell binding to the radiolabeled samples was measured (Figure 14A). The binding rate of DOTA-BC8 to cells (approximately 60% of the binding rate of native BC8) by flow cytometry was approximately 100% of the binding rate of Cytotrol cell radiolabeling. 225 The binding rate of Ac-DOTA-BC8 was matched accordingly with the Cytotrol assay, which routinely binds to cells at 64.8 ± 2.14%. 225 It proved to be a convenient and cost-effective method for assessing the IR of Ac-DOTA-BC8.

[0147] Example 6 225 Ac-BC8: Radioimmunotherapy for multiple myeloma 225Evaluation of the suitability of human H929 and U266 multiple myeloma cells as model cell lines for radioimmunotherapy (RIT) of multiple myeloma with Ac-DOTA-BC8 Multiple myeloma (MM) cell lines H929 and U266 were purchased from the American Type Tissue Collection (ATCC) and grown according to the ATCC instructions. The binding rate of unlabeled DOTA-BC8 to both cell lines was measured by flow cytometry (Figure 15A). 225 Ac-DOTA-BC8 was conjugated (Figure 15B). Next, 225 H929 and U266 cells were used for the in vitro killing assay with Ac-DOTA-BC8. 225 Ac-DOTA-BC8 (20 pCi / mL and 250 pCi / mL) was used. Cell incubation with radiolabeled antibody was performed in a 96-well plate in a total volume of 200 μL. The same two doses of control antibody were used. 225 Ac-DOTA-18B7 was used. Cells were washed from unbound radioactivity at 4 and 12 hours, and their viability was assessed after 3 days by Trypan Blue assay (Table 4). Killing of both cell lines was antibody-specific and dose-dependent. 225 For specific targeting by Ac-DOTA-BC8, they expressed sufficient amounts of CD45 on their surface and could be used for subsequent in vivo experiments.

[0148] [Table 4]

[0149] Example 7 225 Ac-BC8: Distribution in the body In naive mouse models 225 Biodistribution of Ac-DOTA-BC8 The purpose of this study was to determine baseline biodistribution and clearance in a naive mouse model in the absence of disease. 225for Ac-DOTA-18B7 antibody 225 The objective of this study was to evaluate the pharmokinetic biodistribution of Ac-DOTA-BC8. As detailed above, the DOTA-conjugated BC8 antibody (batch A) and the 18B7 antibody (produced at the same molar ratio as batch A; 7.5 moles of DOTA per Ab) were used. 225 The antibody was radiolabeled with Ac at a specific activity of 0.4 μCi / μg. 225 Ac-DOTA-BC8 immunoreactivity was tested on Cytotrol cells and showed 55% binding, thus meeting the minimum binding requirement of 50%.

[0150] Fifty healthy female CD-1 mice were randomly assigned to two groups. 225 Ac-DOTA-BC8 or control 225 Ac-DOTA-18B7 was injected intraperitoneally. Each mouse received 5 μg (2 μCi) of radiolabeled antibody in 100 μL of 0.15 M NHOAc buffer with ascorbic acid. The intraperitoneal route was chosen to avoid personnel, animal, and facility contamination (i.e., potential back pressure splash from the tail). 225 For long-lived radionuclides such as Ac, this is preferable to tail vein injection. According to our own data and that of other groups, intraperitoneally injected antibodies are completely cleared from the peritoneum within 1 hour after injection. Mice were euthanized at 1 hour, 4 hours, 24 hours, 48 ​​hours, and 96 hours (n = 5 mice per construct per time point). Tissue samples (brain, muscle, bone (femur with bone marrow), heart, lung, liver, spleen, kidney, stomach, intestine, and blood) were collected from each mouse and weighed. 225 The cumulative activity per tissue was counted in a gamma counter using the Ac energy window.

[0151] The percentage of injected dose per gram (ID / g (%)) is shown in Figures 16A and 16B. The results show that the biodistribution and pharmacokinetic clearance patterns of the two antibodies were very similar to each other, demonstrating the overall stability of the one-step labeled antibodies in vivo. Clearance from blood and blood-rich organs and control 225 Uptake of Ac-DOTA-18B7 was somewhat lower, which is explained by the lack of homology between the mouse protein and the 18B7 antigen (a fungal polysaccharide glucuronoxylomannan). Using the data in Figures 16A and 16B and Prizm 5.0 software (GraphPad, San Diego, CA, USA), the blood and blood-rich organs (lungs and heart) were analyzed. 225 Ac-DOTA-BC8 antibody half-life and 225 Calculations were also performed to determine the half-life of the Ac-DOTA-18B7 antibody. The results are shown in Table 5, which indicate: 225 The half-life of Ac-DOTA-BC8 is approximately 100 hours (4.2 days), which is typical for a full-sized mouse IgG1 against a mammalian antigen; 225 The half-life of Ac-DOTA-18B7 (also a murine IgG1) is shown to be only 30 hours (1.25 days), likely due to the exogenous nature of its respective antigen (fungal polysaccharide). Thus, the radiolabeled antibody appears to be stable in vivo, rapidly cleared from the blood and blood-rich organs, and suitable for use in subsequent pharmacokinetic studies in CD45-positive tumor-bearing mice.

[0152] [Table 5]

[0153] in myeloma-bearing SCID mice 225 Biodistribution of Ac-DOTA-BC8 mAb The purpose of this study was to compare the efficacy and safety of IFN-γ in a SCID mouse model of multiple myeloma. 225for Ac-DOTA-18B7 antibody 225 The purpose of this study was to understand the biodistribution of Ac-DOTA-BC8. 225 DOTA-conjugated BC8 (batch A) and 18B7 (as described above) were radiolabeled with Ac. Their immunoreactivity was tested on Cytotrol cells and showed 61% conjugation, which met the minimum conjugation requirement of 50%.

[0154] Fifty SCID-NOD (severe combined immunodeficiency non-obese diabetic) female mice aged 4-5 weeks (Charles River Laboratories) were inoculated with 10 mAb into the right flank. 7 Human multiple myeloma H929 cells (ATCC) were inoculated into the left flank of 7 Human multiple myeloma U266 cells (ATCC) were subcutaneously injected into the mice. After approximately 20 days, when tumors reached 3-4 mm in diameter, the mice were randomized into two groups of 25 mice each. 225 Ac-DOTA-BC8 or control 225 Ac-DOTA-18B7 mAb was injected intraorbitally. Each mouse then received 0.4 μCi (1 μg) of radiolabeled antibody in 50 μL of 0.15 M NHOAc buffer with ascorbic acid. As mentioned above, the intraorbital route is preferred over tail vein injection to avoid possible back pressure splash from the tail. Mice were euthanized at 1, 4, 24, 48, and 96 hours (n = 5 mice per construct per time point). Tumor and tissue samples (brain, muscle, femur, bone marrow, heart, lung, liver, spleen, kidney, stomach, intestine, and blood) were collected from each mouse and weighed. 225 The cumulative activity per tissue was counted in a gamma counter using the Ac energy window. The results are shown in Figures 17A and 17B as percent injected dose per gram (ID / g (%)). 225 Ac-DOTA-BC8 uptake was 225Both antibodies were rapidly cleared from the blood and blood-rich organs. Importantly, the increase in the bone marrow 225 There was no uptake of Ac-DOTA-BC8, demonstrating the absence of any homology to human CD45 in mouse bone marrow. 225 We show that Ac-DOTA-BC8 was specifically localized in H929 and U266 tumors and could therefore be used for further radioimmunotherapy (RIT) experiments.

[0155] Example 8 225 Ac-BC8: Radioimmunotherapy of tumors in mice Radioimmunotherapy (RIT) of H929 and U266 tumors in SCID-NOD mice with 225Ac-DOTA-BC8 Forty SCID-NOD female 4-5 week old mice were used for the treatment of multiple myeloma xenografts in a mouse model. 225 The therapeutic potential of Ac-DOTA-BC8 was evaluated. 7 Mice were subcutaneously injected with 100 μg of H929 (right flank) and 100 μg of U266 (left flank) human multiple myeloma cells. Approximately 19 days later, when tumors reached 3–4 mm in diameter, mice were randomized into eight groups and administered 0.3 μCi of 100 μg ... 225 Ac-DOTA-BC8, 0.3 µCi 225 Mice were treated intraorbitally with Ac-DOTA-18B7 control mAb, a matching amount of unlabeled BC8, or left untreated. Tumor size was measured with an electronic caliper on the day of treatment and every three days thereafter. Mice were monitored for tumor size and health for 30 days. Figures 18A and 18B show the results of the RIT study. 225 Ac-DOTA-BC8 had a significant therapeutic effect. 225 The adaptive activity of Ac-DOTA-18B7 had some effect on tumor size, 225The activity was significantly (P = 0.02) lower than that of Ac-DOTA-BC8. Thus, it is clear that RIT in mice bearing multiple myeloma xenografts was effective in almost completely suppressing tumor growth without any undesirable side effects.

[0156] Histological analysis of tumors after RIT Upon completion of the RIT experiments, mice were sacrificed and their tumors were excised, placed in ethanol, then buffered formalin, paraffinized, cut into 5 μm sections, and stained with H&E. Figures 19A-19D show tumors from untreated and 225 1 shows H929 and U266 tumors obtained from Ac-DOTA-BC8-treated mice. Untreated tumors are much more coherent than RIT-treated tumors, demonstrating a lack of coherence and necrosis.

[0157] Example 9 225 Ac-30F11: Myeloablative effects of an anti-CD45 surrogate In this study, mice were treated with 100 mg / kg of IFN-γ-γ (100 mg / kg / day) for targeted conditioning prior to BMT. 225 We evaluated the tolerability and myeloablative effects of Ac-labeled anti-mouse pan-CD45 antibody clone 30F11. a For mice 225 The dose-dependent myeloablative effect of Ac anti-CD45 antibody (30F11) was evaluated. b We assessed the degree of engraftment and donor chimerism after congenic bone marrow transplantation (CD45 allotype differences to monitor chimerism).

[0158] Experimental Method: (1) Conjugation and labeling of 30F11. The anti-CD45 antibody 30F11 was conjugated with the chelator DOTA as described above. To test whether the DOTA-conjugated 30F11 retained immunoreactivity, cells demonstrating CD45 positivity were incubated with naked 30F11 and DOTA-30F11, and anti-rat IgG2b was used to detect bound antibodies.PE The amount of bound Ab was determined by flow cytometry using . As mentioned above, DOTA-30F11 111 In or 225 The antibodies were radiolabeled with Ac to a specific activity of 5 μCi / 1 μg (1:1) or 1 μCi / 1 μg (1:1), respectively, and to a radiochemical purity of 99±1.

[0159] (1) Biodistribution of anti-CD45 antibody 30F11 in C57B1 / 6 mice. C57Bl / 6 mice were dosed with 60 μg of a specific activity of 5 μCi / μg. 111 In-30F11 was intravenously injected. 1 to 240 hours after injection, the spleen 111 The highest uptake of In-labeled 30F11 was found, followed by bone marrow and liver. Kidney, ovary, lung, and blood showed the least uptake. The biodistribution for each organ was fitted to a time-activity curve to calculate the cumulative activity per organ. The dose to organ per administered activity was then reported in Table 6. 225 The equilibrium dose constant of Ac was applied.

[0160] [Table 6]

[0161] (2)B6-Ly 5a In mice 225 Dose-dependent tolerability of Ac-30F11. 225 To determine the tolerability of the Ac-CD45 antibody radioconjugate, C57Bl / 6 mice (5 per cohort) were treated with increasing doses on Day 0. 225Mice were treated with Ac-30F11. Three increasing dose levels (100 nCi, 250 nCi, and 500 nCi) of 30F11 antibody were administered in a total of 10 μg (approximately 0.5 mg / kg) injected intravenously into the tail vein (100–200 μL). Five untreated mice served as controls for this study. Immediately prior to pretreatment, pretreatment blood samples were obtained from control mice for baseline blood counts. RBC and WBC counts were measured at Weeks 1 and 2, and the mice were euthanized at Week 4. Blood was collected from euthanized mice and analyzed for liver and kidney toxicity. Blood urea nitrogen (BUN), creatine, alanine transaminase (ALT), and aspartate aminotransferase (AST) were measured. Kaplan-Meier graphs showed that the 100 nCi and 250 nCi doses were each well tolerated, but the 500 nCi dose showed a decreased probability of survival after 1 week.

[0162] (3)B6-Ly5 a In mice 225 Safety profile of Ac-30F11. To determine the safety profile of 225Ac-CD45 bone marrow transplantation, C57B1 / 6 mice were treated with 225Ac-CD45 bone marrow transplantation as follows: 225 Treatment with Ac-30F11 and reconstitution with donor bone marrow (CD45.1) was performed: 100 nCi or 250 nCi. 225 Ac-30F11 (as above) was injected on Day 0. Four days after pretreatment, half of the cohort received 10 7 C57Bl / 6-Ly5 mice at a target density of 100 nucleated cells in bone marrow b Mice received congenic bone marrow (BMT) harvested from the mice. Mice were monitored regularly for overt signs of changes in weight and health and behavior.

[0163] Engraftment and donor chimerism were assessed by blood collection, and mice were euthanized at week 12. Engraftment was assessed by total WBC, RBC, HSC, neutrophil, and platelet counts, as well as BUN, creatinine, ALT, and AST. Result: 500nCi 225 Ac-30F11 was found to be the maximum tolerated dose for this myeloablative regimen. 225 Mice treated with Ac-30F11 exhibited effective myeloablative conditioning and donor BM engraftment in a dose-dependent manner without any long-term hematological toxicity. Conclusion: 225 The pan-CD45-targeted antibody 30F11, armed with Ac, appears to be a safe and efficacious targeted conditioning approach for BMT. 225 This supports the development of CD45-targeted ablation prior to BMT using Ac-armed antibodies.

[0164] Example 10 177 Lu anti-CD45 and 131 Lymphodepletion with anti-CD45 It is common for patients to undergo a lymphodepletion step, often using high-dose chemotherapy, before receiving a dose of adoptive cell transfer, such as engineered autologous or allogeneic CAR-T cells. This process is thought to be important for creating sufficient space within the immune microenvironment, such as the bone marrow, to allow transplantation of the transferred cells. Furthermore, it is thought to induce a favorable cytokine profile for the establishment and expansion of donor lymphocytes. In this study, we used beta-emitters to mediate effective lymphodepletion in a mouse model. 177 The use of Lu (6.6 day half-life, 1.5 mm path length) will be tested to examine the response of targeted RIT lymphodepletion in specific immune cell types and the resulting changes in immune cytokine expression in a mouse model. 177 Lu-labeled and 131 Preclinical studies were performed using an I-labeled surrogate anti-mouse pan-CD45 antibody (30F11).

[0165] Nonmyeloablative dose 177After a single dose of Lu-CD45-RIT, peripheral blood, bone marrow, and spleen samples were collected from 8- to 12-week-old C57Bl / 6 mice at 96 h and 10 days after treatment for immunophenotyping to assess lymphocyte and myeloid subsets for lymphodepletion and serum for cytokine profiling. 177 We demonstrated that Lu-CD45-RIT effectively lymphodepletes both lymphocytes and myeloid cells, including immunosuppressive Tregs and MDSCs. We also present a study evaluating this targeted lymphodepletion regimen in E.G7 lymphoma-bearing mice prior to adoptive cell transfer with OVA-specific CD8+ T cells.

[0166] Methods and Materials Anti-mouse pan-CD45 antibody 30F11 was incubated with lutetium-177 ( 177 Lu-CD45) and iodine-131 ( 131 I-CD45) and used as a surrogate for radiolabeled pan-human BC8 to perform targeted lymphodepletion in mice. Immunoreactivity was confirmed to be >95% in a CD45+ cell-based binding assay. For lymphodepletion studies in mice, 20 μCi or 40 μCi of IgG was administered to determine its ability to selectively deplete immune cell subsets. 177 Lu or 50 μCi or 100 μCi 131 Female adolescent C57B1 / 6 mice were treated with 20 μg of 30F11 labeled with I. Quantitation of immune cell subsets was determined by flow cytometry. For lymphodepletion studies in the OT1 mouse model, female adolescent C57B1 / 6 CD45.1 mice were subcutaneously injected with OVA-expressing CD45+E.G7-OVA lymphoma cells until a tumor volume of 100 mm3 was reached. Approximately 7 days after tumor cell injection, mice were 177 Lu-CD45 (40 μCi), 131Mice were treated with ICD45 (100 μCi) or no lymphodepletion treatment. Four days after lymphodepletion, mice were administered isolated CD8+ T cells isolated from CD45.2OT I mice. Tumor volume and body weight were monitored, and tumors were measured when tumor volume reached 4000 mm. 3 Mice were sacrificed when the tumor size exceeded 100 μg / ml or became necrotic.

[0167] result Anti-CD45 antibody was conjugated to DOTA at a ratio of 20:1, followed by 5:1 111 60 μg of 10 ... 111 In-labeled anti-CD45 antibodies were injected intraperitoneally into C57B1 / 6 mice, and antibody distribution was monitored by microSPECT / CT at the indicated time points. CD45 antibodies were homed to immune system organs, namely, lymph nodes, spleen, and bone marrow (see Figure 20). Radiolabeled anti-CD45 antibody 177 Lu-CD45 and 131 We found that I-CD45 transiently depleted CD45+ immune cell subsets without affecting platelets, erythrocytes, or myeloid cells. As shown in Figure 21, (A) 20 μCi or 40 μCi of I-CD45 transiently depleted CD45+ immune cell subsets without affecting platelets, erythrocytes, or myeloid cells. 177 Lu-CD45 or (B) 50 μCi or 100 μCi 131 Treatment of non-tumor-bearing C57B / 6 mice with -I CD45 antibody was similarly effective in transiently lymphodepleting various immune cell populations without affecting myeloid cells, erythrocytes, or platelets.

[0168] On top of that, 177 Lu radiolabeled anti-CD45 antibody was found to transiently deplete CD45-expressing immune cell subsets in the spleen. As shown in Figure 22, 40 μCi of 177 Treatment of non-tumor-bearing C57B / 6 mice with Lu-CD45 antibodies was effective in transiently depleting various immune populations in the spleen, including regulatory T cells (T-regs), and this lymphodepletion enabled tumor control in an OT1 adoptive cell therapy model.

[0169] As shown in Figure 23, after E.G7 tumor implantation, mice received no pretreatment (untreated and OT I) or were pretreated with 40 μCi of 177Lu-CD45 or 100 μCi of 131I-CD45 on Day 0, and then administered 1 x 10 6 OT I CD8+CD45.2 OVA-reactive T cells were administered. Panel A shows the results of the previous study in which adoptively transferred OT I cells were able to control EG.7 tumor growth. 177 Lu-CD45 and 131 Panel B shows the results of targeted pretreatment mediated by I-CD45. Panel B shows the tumor size for individual mice in each group. At the time of sacrifice, persistence and expansion of OT1 T cells was confirmed in the mice. Panel C shows the results of targeted pretreatment mediated by I-CD45. Panel B shows the tumor size for individual mice in each group. At the time of sacrifice, persistence and expansion of OT1 T cells was confirmed in the mice. Panel C shows the results of targeted pretreatment mediated by I-CD45. 177 Lu-CD45 and 131 Overall survival of mice that also received targeted conditioning mediated by I-CD45 is shown.

[0170] conclusion These studies focused on the use of low-dose IFN-γ as a transient non-myeloablative targeted lymphodepletion regimen prior to adoptive cell therapy. 177 Lu-CD45 radioimmunotherapy or 131 demonstrate the feasibility of using I-CD45 radioimmunotherapy. 111 In-CD45 imaging demonstrated that CD45 targeting selectively delivered radiation to immune-privileged tissues. 177 Lu-CD45 or 100 μCi 131 We determined that I-CD45 was able to effectively deplete various immune cell subsets in mice, while sparing myeloid cells, erythrocytes, and platelets. In a model of adoptive cell therapy using CD45.1OT1 mice bearing EG.7-OVA tumors, mice that underwent RIT-mediated lymphodepletion showed enhanced tumor control compared with mice that did not undergo lymphodepletion. This data supports the use of a non-myeloablative dose of I-CD45. 131I-CD45RIT or 177 Support CD45-targeted lymphodepletion prior to adoptive cell therapy using Lu-CD45RIT.

[0171] Example 11 225 Ac-BC8: Sickle cell disease (SCD) This example describes HSC ablation (i.e., 100% depletion) prior to transplantation with gene-edited HSCs in patients with SCD. SCD is the most common hemoglobinopathy worldwide. The incidence of SCD among African Americans is approximately 1 in 500. It is estimated that 100,000 individuals in the United States are affected. SCD is caused by a single nucleotide mutation in the beta-globin gene, which produces sickle hemoglobin. Patients with SCD may exhibit anemia, vaso-occlusive crisis (VOC), hemolysis, chronic organ dysfunction, and premature death. The mortality rate in children with SCD is 0.5 per 100,000. However, the mortality rate in adults 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. Currently, the only curative treatment for SCD is hematopoietic stem cell transplantation (HSCT). Unfortunately, HSCT for SCD is not without problems. According to the International Blood and Marrow Transplant Research Center, 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 (e.g., graft-versus-host disease).

[0172] With the advent of gene editing technology, there is now an opportunity to cure SCD patients using autologous stem cells that have corrected the mutation in the β-globin gene that causes SCD. ZFN, TALEN, CRISPR / cas9, and other nuclease-mediated editing approaches have been used to repair stem cells or to 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 β-globin gene mutation in affected pleuripotent HSCs using TALEN. In addition, Dever et al. (Nature, 2016, 539:384-389) demonstrated efficient repair of a Glu6Val mutation that causes SCD in affected HSCs using CRISPR / cas9. Clinical trials using this approach for SCD are currently underway.

[0173] Unfortunately, standard myeloablative conditioning regimens (i.e., 100% HSC depletion regimens) using high-dose chemotherapy or total body irradiation are currently used for transplantation, including for autologous gene-edited cell transplantation. There is a need for safer and more effective conditioning methods for these patients. Radiolabeled BC8 (e.g., 225 The radiolabeled BC8 approach spares more of the patient's normal tissue. In particular, older patients with SCD may already have organ damage as a result of their disease, and exposure to nonspecific radiation or chemotherapy as a myeloablative conditioning regimen may make stem cell transplantation even more risky. The radiolabeled BC8 approach represents a better option for these patients.

[0174] Furthermore, due to the genetic nature of the disease, it is preferable to correct the disease by transplanting gene-edited HSCs as early as possible, since 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 will be used to treat infants or young children with SCD.For this purpose, radiolabeled BC8, especially 225 BC8 labeled with an alpha-emitting radionuclide such as Ac is ideal because of its very short, high-energy radiation path length. 225 By using alpha-emitting radionuclides such as Ac, radiation is concentrated in CD45-positive cells (myeloablative doses). 131 Effective myeloablation is possible without the need to isolate treated patients (as is necessary for pretreatment with I-BC8).

[0175] Example 12 225 Ac-BC8: Severe combined immunodeficiency (SCID) This example describes HSC ablation prior to transplantation with gene-edited HSCs in patients with severe combined immunodeficiency (SCID). SCID is a germline genetic disorder characterized by a severe T-cell deficiency in affected individuals, with or without a concomitant B-cell deficiency. SCID involves a defect in the adaptive immune response that prevents the individual from mounting an effective antibody response against pathogens. SCID is the most severe form of primary immune deficiency, with at least nine known genes causing SCID mutations. Because SCID patients are unable to mount an adaptive immune response, they are susceptible to infection and have a high rate of early mortality. SCID is also known as "bubble boy" disease because patients must be kept in a sterile environment to avoid life-threatening infections. The most frequent gene defect in SCID is the common gamma chain (γc), a protein shared by 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. Similar to SCD, treatment with stem cell transplantation alone is potentially curative for SCID. However, delayed immune recovery and GVHD are significant risks for these patients. Also, similar to SCD patients, SCID patients are young and therefore require effective and safe methods of treatment, including better conditioning regimens before transplantation.

[0176] Gene editing technology can precisely repair defects in SCID patients' own HSCs. When these engineered HSCs are reintroduced into the body, they are capable of producing normal lymphocytes and establishing a functional, adaptive immune response to protect against infection. Recently, Chang et al. (Cell Reports, 2015, 12:1668-1677) reported the effective restoration of normal lymphocyte development in mice through CRISPR / cas9-mediated repair of a mutation in the JAK3 gene. Furthermore, Alzubi et al. (Nature, Scientific Reports, 2017, 7:12475) recently demonstrated the use of TALEN technology to precisely repair a gene defect in IL2RG (common gamma chain), the gene responsible for X-SCID, in mice. It is important that safer and more effective methods for pre-treating human SCID patients be developed. To safely pre-treat these primarily young patients, e.g. 225 Alpha-emitter CD45 radioimmunotherapy with Ac-BC8 is indicated.

[0177] Example 13 225 Ac-BC8: Overview of treatments for non-malignant disorders Table 7 shows the results of the actinium radiolabeled BC8 antibody (i.e., pretreatment agent; 225This figure summarizes selected treatment regimens using HSC depletion via administration of Ac-BC8 followed by gene-edited stem cell administration. Another aspect of the present invention may be as follows. [1] A method for treating hemoglobinopathy or a blood disease or disorder, comprising administering an effective amount of a composition comprising a radiolabeled anti-CD45 antibody, The effective amount is administered as a single dose, and the radiolabeled anti-CD45 antibody is an actinium-225 labeled antibody ( 225 Ac) or lutetium-177 labeled antibody ( 177 Lu). [2] The radiolabeled anti-CD45 antibody has a light chain having the amino acid sequence shown in SEQ ID NO: 1 or a light chain having the N-terminal amino acid sequence shown in SEQ ID NO: 9. 225 The method according to [1] above, comprising Ac-BC8. [3] The radiolabeled anti-CD45 antibody has a light chain having at least one complementarity-determining region having the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. 225 The method according to [1] above, comprising Ac-BC8. [4] The radiolabeled anti-CD45 antibody has a light chain having the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13. 225 The method according to [1], comprising Ac-BC8. [5] The radiolabeled anti-CD45 antibody has a heavy chain having the amino acid sequence shown in SEQ ID NO: 2 or a heavy chain having the N-terminal amino acid sequence shown in SEQ ID NO: 10. 225 The method according to [1], comprising Ac-BC8. [6] The radiolabeled anti-CD45 antibody has a heavy chain having at least one complementarity-determining region having the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. 225 The method according to [1], comprising Ac-BC8. [7] The radiolabeled anti-CD45 antibody has a heavy chain having the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16. 225 The method according to [1], comprising Ac-BC8. [8] The radiolabeled anti-CD45 antibody has the amino acid ASP or ASN at position 141 (relative to the N-terminal amino acid). 225 The method according to [1], comprising Ac-BC8. [9] The method described in [8] above, wherein the ratio of ASP:ASN in the BC8 protein population is 1:99 to 99:1, for example, 10:90 to 90:10.

[10] The method according to [1], further comprising administering an effective amount of a second agent.

[11] The method according to

[12] , wherein the second agent comprises a radiosensitizer.

[12] The method according to

[15] , wherein the radiosensitizer comprises a Bcl-2 inhibitor, an HDAC inhibitor, or a combination thereof.

[13] The composition comprises an actinium-225 labeled BC8 antibody ( 225 2. The method of claim 1, wherein the dose of each of the labeled BC8 and the unlabeled anti-CD45 antibody is selected based on a patient-specific characteristic selected from the patient's mass, the patient's age, the patient's sex, and / or the patient's health status.

[14] The method of [1], wherein the effective amount of the radiolabeled anti-CD45 antibody depletes at least 50% of the subject's lymphocytes but does not induce bone marrow destruction in the subject.

[15] The method according to

[14] , wherein the effective amount of the radiolabeled anti-CD45 antibody depletes circulating tumor cells of hematopoietic origin.

[16] The method according to

[14] , wherein the effective amount of the radiolabeled anti-CD45 provides a radiation dose of 2 Gy or less to the bone marrow, or at least 2 Gy to less than 8 Gy to the bone marrow.

[17] The radiolabeled anti-CD45 225 The method according to

[15] above, comprising Ac-BC8, wherein the effective amount comprises a dose of 0.1 uCi / kg to 1.0 uCi / kg, or a dose of 10 uCi to 150 uCi.

[18] The method of

[26] , wherein the effective amount of the actinium-225 anti-CD45 depletes, but does not deplete, CD45+ circulating tumor cells.

[19] The method according to

[14] , wherein the patient requires a bone marrow transplant.

[20] The method according to

[19] , further comprising performing the bone marrow transplantation 4 days, 5 days, 6 days, 7 days, 8 days, or 9 days after the administration of the radiolabeled anti-CD45 antibody.

[21] The method of [1], wherein the effective amount of the radiolabeled anti-CD45 depletes regulatory T cells, myeloid-derived suppressor cells, tumor-sensitized macrophages, activated macrophages secreting IL-1 and / or IL-6, and combinations thereof.

[22] The method according to

[21] , wherein the effective amount of the radiolabeled anti-CD45 provides a radiation dose of greater than 8 Gy to the bone marrow.

[23] The method further comprises administering to the subject an effective amount of a cell population expressing a chimeric antigen receptor or a T cell receptor (CAR / TCR) 6, 7, or 8 days after the administration of the radiolabeled anti-CD45 antibody. The method according to

[16] or

[22] .

[24] The method of [1], wherein the subject is suffering from a non-cancerous disorder treatable via gene editing cell therapy and is undergoing such therapy to treat the disorder, and the effective amount of the radiolabeled anti-CD45 antibody is administered as a single dose.

[25] The method according to

[24] , wherein the disorder is selected from the group consisting of abnormal hemoglobinopathy, congenital immunodeficiency, and viral infection.

[26] The method according to

[24] , wherein the disorder is selected from the group consisting of sickle cell disease (SCD), severe combined immunodeficiency (SCID), beta-thalassemia, and Fanconi anemia.

[27] The method described in

[26] , wherein the disorder is SCD and the treatment is gene-edited β-globin hematopoietic stem cell therapy.

[28] The method of

[27] , wherein the disorder is SCID, the treatment is gene-editing 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.

[29] The method according to [1], wherein the blood disease is myelodysplastic syndrome (MDS), multiple myeloma (MM), acute myeloid leukemia (AML), myeloproliferative neoplasm, or a combination thereof.

[0178] Table 7

Claims

1. (i) an effective amount of a population of T cells expressing a chimeric antigen receptor or T cell receptor (CAR / TCR) in the treatment of a hematological malignancy. in preparation for administration to a subject, actinium-225 labeled ( 225 Ac) a non-myeloablative amount of a radiolabeled anti-CD45 antibody, wherein the anti-CD45 antibody is a radiolabeled anti-CD45 antibody; The composition, wherein the malignant hematological disease is multiple myeloma (MM).

2. The composition of claim 1, wherein the radiolabeled anti-CD45 antibody comprises radiolabeled BC8 having a light chain having the amino acid sequence set forth in SEQ ID NO: 1 or a light chain having the N-terminal amino acid sequence set forth in SEQ ID NO: 9 and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2 or a heavy chain having the N-terminal amino acid sequence set forth in SEQ ID NO:

10.

3. the radiolabeled anti-CD45 antibody comprises BC8 having a light chain having a complementarity-determining region having the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and a heavy chain having a complementarity-determining region having the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; The composition of claim 1, wherein SEQ ID NO: 3 is the amino acid sequence of CDR1, SEQ ID NO: 4 is the amino acid sequence of CDR2, SEQ ID NO: 5 is the amino acid sequence of CDR3, SEQ ID NO: 6 is the amino acid sequence of CDR1, SEQ ID NO: 7 is the amino acid sequence of CDR2, and SEQ ID NO: 8 is the amino acid sequence of CDR3.

4. The composition of claim 1, wherein the radiolabeled anti-CD45 antibody comprises BC8 having a light chain having the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13 and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO:

16.

5. 2. The composition of claim 1, wherein the radiolabeled anti-CD45 antibody comprises BC8 having the amino acid ASP or ASN at position 141 relative to the N-terminal amino acid.

6. 6. The composition of claim 5, wherein the ratio of ASP:ASN in the population of BC8 proteins is 1:99 to 99:

1.

7. The composition of any one of claims 1 to 6, wherein the non-myeloablative amount of the radiolabeled anti-CD45 provides a radiation dose of 2 Gy or less to bone marrow.

8. The composition of any one of claims 1 to 7, wherein the non-cancerous disorder is selected from the group consisting of hemoglobinopathies, congenital immune deficiencies, and viral infections.

9. The radiolabeled anti-CD45 comprises BC8 having a light chain with a complementarity determining region having the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and a heavy chain with a complementarity determining region having the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8. 225 Ac-labeled anti-CD45 antibody, and the non-myeloablative amount consists of a dose of 10 μCi to 150 μCi; The composition of any one of claims 1 to 8, wherein SEQ ID NO: 3 is the amino acid sequence of CDR1, SEQ ID NO: 4 is the amino acid sequence of CDR2, SEQ ID NO: 5 is the amino acid sequence of CDR3, SEQ ID NO: 6 is the amino acid sequence of CDR1, SEQ ID NO: 7 is the amino acid sequence of CDR2, and SEQ ID NO: 8 is the amino acid sequence of CDR3.

10. The composition of any one of claims 1 to 9, comprising administering an effective amount of a radiosensitizer comprising a Bcl-2 inhibitor, an HDAC inhibitor, or a combination thereof.

11. 2. The method of claim 1, wherein the actinium-225-labeled ( 225 Ac) A composition comprising a non-myeloablative amount of an anti-CD45 antibody.

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