Radioactive labeling of anti-CD45 immunoglobulin and method of use

Abstract

This specification provides compositions and methods useful for the treatment of abnormal hemoglobin disorders and blood disorders. [Solution] A composition is provided comprising actinium-225-labeled anti-CD45 antibody (BC8), which is formulated as a single-victim dose that can be completely delivered to the affected body with a single dose. Actinium-225-labeled anti-CD45 may be administered alone or in combination with additional therapeutic agents such as other immunotherapies or radiosensitizers, or with additional therapeutic interventions such as bone marrow transplantation or adoptive cell therapy.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 901,290, filed on 17 September 2019, which is incorporated herein by reference in its entirety.

[0002] Sequence list reference This application includes a sequence listing showing the same sequences as those found herein, which is incorporated herein by reference as a supplemental file filed via EFS and shown in compliance with United States Patent Rules: 37 § 1.52(e)(5) and PCT Rule 13 1(a). This disclosure relates to a method for radioactively labeling a monoclonal antibody against CD45, a composition comprising a radioactively labeled monoclonal antibody against CD45, and a method for the use of a radioactively labeled anti-CD45 antibody for the treatment of malignant and non-malignant hematological disorders. [Background technology]

[0003] CD45 is a member of the protein tyrosine phosphatase (PTP) family and is a type I transmembrane glycoprotein that plays a major role in T cell receptor signaling and B cell receptor signaling. CD45 regulates the activation of Src family protein-tyrosine kinases Lck and Fyn. CD45 deficiency results in T lymphocyte and B lymphocyte dysfunction in the form of severe combined immunodeficiency. It has also been reported to play a significant 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 the common leukocyte 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 endogenous 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 hematopoietic cells except mature red blood cells and mature platelets express at least one isoform of CD45. High expression of CD45 is observed in most acute lymphoblastic leukemias and acute myeloid leukemias. Because CD45 is not found in non-hematopoietic tissues, its specific expression in leukemia has made it a good target for the development of therapeutics, including immunotherapies. For example, CD45 is expressed in circulating leukocytes and malignant B cells at a density of approximately 200,000 to 300,000 sites per cell. Certain anti-CD45 antibodies (BC8) have been investigated as candidate immunotherapies, either alone or in combination with chemotherapy or total body irradiation, for the treatment of leukemia. Anti-CD45 antibody-based lymphoexhaustion is also known (see, e.g., Louis, et al., 2009, Blood, 113:2442-2450). However, this approach has had drawbacks. For example, in Louis et al.'s study, eight patients experienced lymphoexhaustion with anti-CD45 antibodies and showed an increase in the desired frequency of T cells in peripheral blood after infusion. However, only three patients showed clinical benefit, and only one achieved a complete response.

[0005] CD45 exists as multiple isoforms due to alternative splicing of three of the 34 exons in the extracellular domain (exons 4, 5, and 6, indicated by 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 of O-linked glycosylation and are variably modified by sialic acid. As a result, the various isoforms differ substantially in size (391–552 amino acids, molecular weight ranging from 180–240 kDa), shape, and negative charge. The remaining membrane-proximal extracellular domain is highly N-glycosylated and contains three repeats of fibronectin type III following a high-cysteine ​​spacer region.

[0006] While eight isoforms of CD45 are possible, only six—RO (absent from 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)—have been identified in humans. These different isoforms are differentially expressed in subpopulations of B-cell and T-cell lymphocytes and are specific to the cellular activation and maturation states. For example, CD45-RA and CD45-RB are expressed in naive T cells, 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 various isoforms of CD45 have been identified. Furthermore, monoclonal antibodies (mAbs) that bind to an epitope common to all 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 for the treatment of patients requiring bone marrow transplantation. 131 Although the use of BC8 labeled in (I) has been considered (see International Publication No. 2017 / 155937, which is 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 hematological disorders. Specifically, there is a need for therapeutic compositions and methods that (i) use agents more specific than chemotherapeutic agents, (ii) have sufficient potency to be effective at low doses, and (iii) do not severely deplete at least some types of hematopoietic stem cells. [Overview of the project]

[0008] This disclosure utilizes the panspecific properties of BC8 monoclonal antibodies to provide compositions and methods useful for depletion, reversible immunosuppression, and / or ablation of specific cell populations, and further, methods for treating certain malignant and non-malignant hematological disorders using these compositions and methods. This disclosure provides compositions and methods of use thereof for the treatment of various disorders of the hematopoietic system, and in particular metabolic disorders, cancer, and autoimmune diseases. This disclosure further features a method for pre-conditioning a patient before hematopoietic stem cell transplantation therapy to facilitate the transplantation of hematopoietic stem cell grafts. The patient may suffer from one or more blood disorders, such as abnormal hemoglobinopathy or other hematopoietic conditions. The patient may require hematopoietic stem cell transplantation. As described herein, hematopoietic stem cells are capable of differentiating into many cell types in the hematopoietic lineage and can be administered to affected individuals to implant or re-implant cell types that are deficient in the affected individual. This disclosure provides for radiolabeled antibodies capable of targeting hematopoietic cells, specifically actinium-225, to (i) directly treat diseases described herein, particularly hematological disorders, metabolic disorders, cancer, or autoimmune diseases, by selectively depleting, reversibly suppressing, or ablating populations of CD45-expressing cells, such as ectopic blood cells, cancer cells, or autoimmune cells, and / or (ii) depleting, reversibly suppressing, or ablating populations of endogenous hematopoietic stem cells in affected individuals. 225 Ac) or Lutetium-177( 177 This method is characterized by treating the affected body with anti-CD45-immunoglobulin labeled with Lu.

[0009] The former activity allows for the direct treatment of a wide range of disorders associated with autoimmune lymphocytes, such as hematopoietic cells, including leukemia or lymphoma cells of the B cell or T cell lineage, and among several cell types, T cells expressing T cell receptors that cross-react with autoantigens. The latter activity, namely selective depletion, reversible suppression, or ablation of hematopoietic stem cells, creates vacancies that can subsequently be filled by transplantation of exogenous (e.g., autologous, allogeneic, or syngeneic) hematopoietic stem cell grafts. Accordingly, this disclosure provides methods for treating various non-cancerous hematopoietic conditions such as abnormal hemoglobin disorders (e.g., sickle cell disease or SCD, and β-thalassemia), congenital immunodeficiency (e.g., severe combined immunodeficiency or SCID, Fanconi anemia, Wiscott-Aldrich syndrome, Diamond-Blackfan anemia and Schwachmann-Diamond syndrome, adenosine deaminase deficiency), and viral infections (e.g., HIV infection and acquired immunodeficiency syndromes). This disclosure further provides methods for treating cancerous disorders such as hematological malignancies or solid tumors. Exemplary hematological malignancies include acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin lymphoma.

[0010] Therefore, this disclosure is not related to the actinium-225 ( 225 Ac) or Lutetium-177( 177 This invention relates to a stabilized composition comprising isolated anti-CD45 immunoglobulin (e.g., BC8 mAb clone) in a radioactively labeled form with Lu, and its therapeutic use for the treatment of malignant and non-malignant hematological diseases, as well as malignant and non-malignant hematological disorders. Due to its ability to bind to all isoforms of the CD45 antigen in humans, the BC8 antibody is expected to accumulate therapeutically high radioactive doses specifically and preferentially in high-density CD45 antigen-retaining cells. Therefore, this disclosure is, 225 Ac or 177Also relates to a method for radiolabeling an anti-CD45 immunoglobulin such as a BC8 antibody with a radionuclide such as Lu. According to a particular embodiment, the BC8 antibody is conjugated to a chelating agent such as S-2-(4-isothiocyanatobenzyl)-1,4,7,10 tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA; referred to as DOTA) to form DOTA-BC8, 225 radiolabeled with a radionuclide such as Ac, 225 Ac-DOTA-BC8 (i.e., 225 Ac-BC8) or 177 forms Lu to 177 provide Lu-DOTA-BC8 (i.e., 177 Lu-BC8).

[0011] 225 Ac-BC8 or 177 Lu-BC8 may be provided as a stabilized formulation comprising one or more pharmaceutically acceptable carriers, salts or excipients. As certain exemplary carriers or excipients, there may be mentioned saline, phosphate buffered saline (e.g., 50 mM PBS buffer, pH 7) and / or one or more of 0.5% to 5.0% (w / v) 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 an amino acid sequence as shown in SEQ ID NO: 1 and a heavy chain variable domain having an N-terminal amino acid sequence as shown in SEQ ID NO: 9. The BC8 antibody may comprise a light chain variable domain having at least one complementarity determining region (CDR) having an amino acid sequence as shown 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 shown in SEQ ID NO: 12 or SEQ ID NO: 13 (set for the). The BC8 antibody may contain a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 2, or a heavy chain variable domain having the N-terminal amino acid sequence shown in SEQ ID NO: 10. The BC8 antibody may contain a heavy chain variable domain having at least one complementarity-determining region (CDR) having the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. The BC8 antibody may contain a heavy chain having the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16.

[0013] In a particular embodiment, the BC8 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, where the amino acid at position 141 (relative to the N-terminal amino acid) is either ASP or ASN. The ASP:ASN ratio 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 a particular embodiment, the BC8 antibody comprises a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 2, or a heavy chain variable domain having the N-terminal amino acid sequence shown in SEQ ID NO: 10, where the amino acid at position 141 (relative to the N-terminal amino acid) of the heavy chain is ASP or ASN, and the ASP:ASN ratio within the BC8 protein population is in the range of 1:99 to 99:1, such as 10:90 to 90:10.

[0014] In a particular embodiment, any of the BC8 antibodies described above, i.e., one or more of SEQ ID NOs: 1 to 10, may be a chimeric antibody or a humanized antibody, i.e., BC8c. The BC8c antibody may include 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 shown in SEQ ID NOs: 17 to 19, a human IgG4 heavy chain constant region having the amino acid sequence shown in SEQ ID NOs: 20 (including mutant S228P), and / or a human kappa light chain constant region having the amino acid sequence shown in SEQ ID NOs: 21.

[0015] This disclosure relates to a method for directly treating subjects suffering from CD45-positive hematological malignancies, and an effective amount 225 Ac-BC8 or 177 The present invention provides a method comprising administering Lu-BC8 to the subject as a low-dose monotherapy agent, either alone or in combination with other therapies. This disclosure relates to a method for directly treating subjects suffering from CD45-positive hematological malignancies, and an effective amount 225 Ac-BC8 or 177 The present invention provides a method comprising administering Lu-BC8 to the subject as a low-dose monotherapy agent, either alone or in combination with other therapies using stem cell carriers.

[0016] This disclosure relates to a method for depleting, reversibly suppressing, or ablating target hematopoietic stem cells, and an effective amount 225 Ac-BC8 or 177 The present invention relates to a method comprising administering Lu-BC8 to the subject. This disclosure provides a dose that does not destroy bone marrow and therefore does not irreversibly deplete hematopoietic stem cells, and is effective in that dose. 225 Ac-BC8 or 177 The present invention provides a method for depleting or reversibly suppressing circulating tumor cells (as found in, for example, 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 that secrete IL-1 and / or IL-6, and any combination thereof. This disclosure relates to a method for depleting, reversibly suppressing, or ablating target lymphocytes, and an effective amount 225 Ac-BC8 or 177 The present invention further provides a method comprising administering Lu-BC8 to the subject.

[0017] This disclosure relates to a method for treating a subject suffering from a non-cancerous disorder, comprising an effective amount for depleting, reversibly suppressing, or ablating hematopoietic stem cells of the subject.225 Ac-BC8 or 177 The present invention also provides a method comprising administering Lu-BC8 to the subject. According to a particular embodiment, the disorder can be treated via gene-editing cell therapy, and the method is 225 Ac-BC8 or 177 The present invention further includes performing the treatment in the subject to treat the disorder in the subject after administration of Lu-BC8. In a particular embodiment, the disorder is SCD, and the treatment is gene-edited β-globin hematopoietic stem cell therapy. In a particular embodiment, the disorder is SCID, and the treatment is gene-edited hematopoietic stem cell therapy, where 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 may be, for example, allogeneic or autologous.

[0018] This disclosure relates to a method for treating a subject suffering from a cancerous disorder treatable via gene-editing cell therapy, wherein (i) an amount effective to deplete, reversibly suppress, or ablate the hematopoietic stem cells of the subject. 225 Ac-BC8 or 177 The present invention also provides a method comprising (ii) administering Lu-BC8 to the subject, and (ii) treating the subject's disorder by performing the treatment after an appropriate period of time. According to a particular embodiment, the treatment appropriate for treating the subject's disorder may be a bone marrow transplant or adoptive cell therapy. Finally, this disclosure (a) 225 Ac-BC8 or 177 The present invention provides a manufactured article comprising (b) a radioactively labeled anti-CD45 antibody such as Lu-BC8, and a label instructing the user to administer to a subject an amount of the antibody effective in depleting the subject's hematopoietic stem cells.

[0019] This patent or application file includes at least one drawing performed in color. Copies of this patent or patent application publication containing (one or more) color drawings will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0020] [Figure 1] This diagram shows a schematic representation of how exons are used in various isoforms of CD45, which are generated by differential splicing of the human CD45 gene. [Figure 2] This document provides the protein sequences of the light chain (VL) and heavy chain (VH) complementarity-determining regions (CDRs), framework regions, and variable domain sequences of the anti-CD45 mAb BC8. The CDRs are shown in bold and underlined (SEQ ID NOs: 1 and 2). [Figure 3] This document provides the CDR and N-terminal protein sequences (SEQ ID NOs: 3 to 10) of the light and heavy chains of the anti-CD45 mAb BC8. [Figure 4A-1] This provides the complete nucleotide (SEQ ID NO: 11) and complete amino acid (SEQ ID NO: 12) sequences of the light chain of anti-CD45 mAb BC8. [Figure 4A-2] This provides the complete nucleotide (SEQ ID NO: 11) and complete amino acid (SEQ ID NO: 12) sequences of the light chain of anti-CD45 mAb BC8. [Figure 4B] This provides the amino acid sequence (SEQ ID NO: 13) of the light chain of anti-CD45 mAb BC8, which does not have a leader sequence. [Figure 5A-1] This document provides the complete nucleotide (SEQ ID NO: 14) and complete amino acid (SEQ ID NO: 15) sequences of the heavy chain of anti-CD45 mAb BC8, in which asparagine at position 141 (from the n-terminus of the protein sequence) has been found to be deaminated to aspartic acid in at least a portion of the protein population. [Figure 5A-2]This document provides the complete nucleotide (SEQ ID NO: 14) and complete amino acid (SEQ ID NO: 15) sequences of the heavy chain of anti-CD45 mAb BC8, in which asparagine at position 141 (from the n-terminus of the protein sequence) has been found to be deaminated to aspartic acid in at least a portion of the protein population. [Figure 5A-3] This document provides the complete nucleotide (SEQ ID NO: 14) and complete amino acid (SEQ ID NO: 15) sequences of the heavy chain of anti-CD45 mAb BC8, in which asparagine at position 141 (from the n-terminus of the protein sequence) has been found to be deaminated to aspartic acid in at least a portion of the protein population. [Figure 5B-1] This provides the amino acid sequence (SEQ ID NO: 16) of the heavy chain of anti-CD45 mAb BC8, which lacks a leader sequence, in which asparagine at position 141 (from the n-terminus of the protein sequence) has been found to be deaminated to aspartic acid in at least a portion of the protein population. [Figure 5B-2] This provides the amino acid sequence (SEQ ID NO: 16) of the heavy chain of anti-CD45 mAb BC8, which lacks a leader sequence, in which asparagine at position 141 (from the n-terminus of the protein sequence) has been found to be deaminated to aspartic acid in at least a portion of the protein population. [Figure 6A] Each of these provides the amino acid sequences of the human heavy chain constant region, SEQ ID NOs. 17-19. [Figure 6B] Each of these provides the amino acid sequences of the human heavy chain constant region, SEQ ID NOs. 17-19. [Figure 6C] Each of these provides the amino acid sequences of the human heavy chain constant region, SEQ ID NOs. 17-19. [Figure 6D] This provides the amino acid sequence (SEQ ID NO: 20) of the human heavy chain constant region containing the mutation S228P. [Figure 6E] This provides the amino acid sequence (SEQ ID NO: 21) of the constant region of the human kappa light chain. [Figure 7] This invention provides a method for depleting the lymphatic system of a subject before performing adoptive cell therapy according to certain aspects of this disclosure. [Figure 8] Pharmacokinetic data illustrating exemplary clearance and administration times for the lymph depletion protocol relating to this disclosure are provided. [Figure 9A] A schematic diagram of a method for radioactively labeling anti-CD45 mAb BC8 with actinium (225Ac) is provided, Figure 9A showing the attachment of the bifunctional chelating agent 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 Figure 9B showing the radioactive labeling of the DOTA-anti-CD45 conjugate with 225Ac to provide 225Ac-DOTA-anti-CD45. [Figure 9B] A schematic diagram of a method for radioactively labeling anti-CD45 mAb BC8 with actinium (225Ac) is provided, Figure 9A showing the attachment of the bifunctional chelating agent 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 Figure 9B showing the radioactive labeling of the DOTA-anti-CD45 conjugate with 225Ac to provide 225Ac-DOTA-anti-CD45. [Figure 10A] Elution profiles (size exclusion chromatography-high-performance liquid chromatography) for BC8 standard and 225Ac-DOTA-BC8 from SEC-HPLC are provided, with Figure 10A showing the elution of BC8 standard and Figure 10B showing the elution of 225Ac-DOTA-BC8 (the peak at 13 min is HSA added to stabilize the conjugate antibody). [Figure 10B] Elution profiles (size exclusion chromatography-high-performance liquid chromatography) for BC8 standard and 225Ac-DOTA-BC8 from SEC-HPLC are provided, with Figure 10A showing the elution of BC8 standard and Figure 10B showing the elution of 225Ac-DOTA-BC8 (the peak at 13 min is HSA added to stabilize the conjugate antibody). [Figure 11]This provides a graph showing the stability of 225Ac-DOTA-BC8 at various storage dilutions and temperatures as a function of time. [Figure 12] This provides graphs showing the 225Ac-DOTA-BC8 immunoreactivity to Ramos cells (CD45-positive cells) and EL4 cells (CD45-negative cells). [Figure 13A] The graphs provided show the binding of various antibody samples to cytotrol cells as measured by flow cytometry. Figure 13A compares the binding of naive BC8 antibody and naive 18B7 (non-specific control) antibody to cytotrol cells, and Figure 13B compares the binding of naive BC8 antibody and DOTA-BC8 antibody to cytotrol cells. [Figure 13B] The graphs provided show the binding of various antibody samples to cytotrol cells as measured by flow cytometry. Figure 13A compares the binding of naive BC8 antibody and naive 18B7 (non-specific control) antibody to cytotrol cells, and Figure 13B compares the binding of naive BC8 antibody and DOTA-BC8 antibody to cytotrol cells. [Figure 14A] This 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 immediately after labeling with 225Ac (i.e., 225Ac-DOTA-BC8), compared to the binding of 225Ac-18B7 (binding to Cytotrol cells) as measured by fractional radiation retained on cells after washing. [Figure 15A] The graph shows a comparison of DOTA-BC8 binding to different human multiple myeloma cell lines, namely H929 and U266, measured after labeling with 225Ac (i.e., 225Ac-DOTA-BC8; Figure 15B) by flow cytometry (Figure 15A) or by fractional radiation retained on the cells after washing. [Figure 15B]The graph shows a comparison of DOTA-BC8 binding to different human multiple myeloma cell lines, namely H929 and U266, measured after labeling with 225Ac (i.e., 225Ac-DOTA-BC8; Figure 15B) by flow cytometry (Figure 15A) or by fractional radiation retained on the cells after washing. [Figure 16A] The bar graphs show a comparison of the in vivo distribution of 225Ac-DOTA-BC8 (Figure 16A) and 225Ac-DOTA-18B7 (Figure 16B) antibodies in control mice at 1 hour, 4 hours, 24 hours, 48 ​​hours, and 96 hours. [Figure 16B] The bar graphs show a comparison of the in vivo distribution of 225Ac-DOTA-BC8 (Figure 16A) and 225Ac-DOTA-18B7 (Figure 16B) antibodies in control mice at 1 hour, 4 hours, 24 hours, 48 ​​hours, and 96 hours. [Figure 17A] The bar graphs show a comparison of the in vivo distribution of 225Ac-DOTA-18B7 (control; Figure 17A) and 225Ac-DOTA-BC8 (Figure 17B) antibodies in U266 SCID-NOD tumor-carrying mice and H929 SCID-NOD tumor-carrying mice at 1 hour, 4 hours, 24 hours, 48 ​​hours, and 96 hours. [Figure 17B] The bar graphs show a comparison of the in vivo distribution of 225Ac-DOTA-18B7 (control; Figure 17A) and 225Ac-DOTA-BC8 (Figure 17B) antibodies in U266 SCID-NOD tumor-carrying mice and H929 SCID-NOD tumor-carrying mice at 1 hour, 4 hours, 24 hours, 48 ​​hours, and 96 hours. [Figure 18A] This graph compares the tumor volume of H929 multiple myeloma xenograft-carrying SCID-NOD mice after radioimmunotherapy treatment with 225Ac-DOTA-BC8 or 225Ac-DOTA-18B7 (control). [Figure 18B] This graph compares the tumor volume of U266 multiple myeloma xenograft-carrying SCID-NOD mice after radioimmunotherapy treatment with 225Ac-DOTA-BC8 or 225Ac-DOTA-18B7 (control). [Figure 19] Histological analyses of tumors excised from U266 and H929 multiple myeloma xenograft-carrying SCID-NOD mice are shown. 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] MicroSPEC / CT scans of C57Bl / 6 mice injected (ip) with 111Ln-anti-CD45, obtained 1 hour, 24 hours, 48 ​​hours, 72 hours, 96 hours, and 6 days after injection, are shown. [Figure 21A] (A) Bar graphs showing the depletion of various immune cell subpopulations in tumor-free C57Bl / 6 mice after treatment with 177Lu-anti-CD45 or (B) 131I-anti-CD45. [Figure 21B] (A) Bar graphs showing the depletion of various immune cell subpopulations in tumor-free C57Bl / 6 mice after treatment with 177Lu-anti-CD45 or (B) 131I-anti-CD45. [Figure 22] (A) Bar graphs showing the depletion of various immune cell populations in the spleen of tumor-free C57Bl / 6 mice after treatment with 177Lu-anti-CD45 or (B) 131I-anti-CD45. [Figure 23A] The graph shows that 177Lu-anti-CD45 lymph depletion and 131I-anti-CD45 lymph depletion enable tumor control in an OT I adoptive cell therapy model, (A) showing that targeted pretreatment mediated by 177Lu-anti-CD45 and 131I-anti-CD45 prior to adoptive OT IT cells enabled control of EG.7 tumor growth, (B) showing tumor size for individual mice in each group, and (C) showing survival rates for control mice that were untreated or treated with OT IT cells, as well as mice pretreated with 177Lu-anti-CD45 and 131I-anti-CD45. [Figure 23B]The graph shows that 177Lu-anti-CD45 lymph depletion and 131I-anti-CD45 lymph depletion enable tumor control in an OT I adoptive cell therapy model, (A) showing that targeted pretreatment mediated by 177Lu-anti-CD45 and 131I-anti-CD45 prior to adoptive OT IT cells enabled control of EG.7 tumor growth, (B) showing tumor size for individual mice in each group, and (C) showing survival rates for control mice that were untreated or treated with OT IT cells, as well as mice pretreated with 177Lu-anti-CD45 and 131I-anti-CD45. [Figure 23C] The graph shows that 177Lu-anti-CD45 lymph depletion and 131I-anti-CD45 lymph depletion enable tumor control in an OT I adoptive cell therapy model, (A) showing that targeted pretreatment mediated by 177Lu-anti-CD45 and 131I-anti-CD45 prior to adoptive OT IT cells enabled control of EG.7 tumor growth, (B) showing tumor size for individual mice in each group, and (C) showing survival rates for control mice that were untreated or treated with OT IT cells, as well as mice pretreated with 177Lu-anti-CD45 and 131I-anti-CD45.

[0021] A brief explanation of arrays Sequence ID 1 is the amino acid sequence of the variable domain of the light chain of anti-CD45 mouse immunoglobulin BC8. Sequence ID 2 is the amino acid sequence of the variable domain of the heavy chain of anti-CD45 mouse immunoglobulin BC8. Sequence ID 3 is the amino acid sequence of CDR1 in the light chain of anti-CD45 mouse immunoglobulin BC8.

[0022] Sequence ID 4 is the amino acid sequence of CDR2 in the light chain of anti-CD45 mouse immunoglobulin BC8. Sequence ID 5 is the amino acid sequence of CDR3 in the light chain of anti-CD45 mouse immunoglobulin BC8. Sequence ID 6 is the amino acid sequence of CDR1 in the heavy chain of anti-CD45 mouse immunoglobulin BC8.

[0023] Sequence ID 7 is the amino acid sequence of CDR2 in the heavy chain of anti-CD45 mouse immunoglobulin BC8. Sequence ID 8 is the amino acid sequence of CDR3 in the heavy chain of anti-CD45 mouse immunoglobulin BC8. Sequence ID 9 is the N-terminal amino acid sequence of the light chain of anti-CD45 mouse immunoglobulin BC8.

[0024] Sequence ID 10 is the N-terminal amino acid sequence of the heavy chain of anti-CD45 mouse immunoglobulin BC8. Sequence ID 11 is the nucleotide sequence of the light chain of anti-CD45 mouse immunoglobulin BC8. Sequence ID 12 is the amino acid sequence of the light chain of anti-CD45 mouse immunoglobulin BC8, including the leader sequence. Sequence ID 13 is the amino acid sequence of the light chain of anti-CD45 mouse immunoglobulin BC8, which starts at the N-terminus of the protein (i.e., has no leader sequence). Sequence ID 14 is the nucleotide sequence of the heavy chain of anti-CD45 mouse immunoglobulin BC8. Sequence ID 15 is the amino acid sequence of the heavy chain of anti-CD45 mouse immunoglobulin BC8, including the leader sequence. Sequence ID 16 is the amino acid sequence of the heavy chain of anti-CD45 mouse immunoglobulin BC8, which starts at the N-terminus of the protein (i.e., has no leader sequence). Sequence ID No. 17 is the amino acid sequence of the constant region of the human IgG single-chain protein. Sequence ID No. 18 is the amino acid sequence of the constant region of the human IgG double chain. Sequence ID 19 is the amino acid sequence of the constant region of the human IgG quadruple chain. Sequence ID 20 is the amino acid sequence of the constant region of human IgG quadruple chain containing the mutation S228P. Sequence ID 21 is the amino acid sequence of the constant region of the human kappa light chain. [Modes for carrying out the invention]

[0025] Definitions and abbreviations Throughout this application, various publications are cited. The disclosures of these publications are incorporated herein by reference to provide a more complete description of the art relating to this disclosure. In this application, certain terms are used that have the meanings set forth below. The singular forms "a," "an," "the," and similar forms include multiple references unless explicitly specified by the context. Therefore, for example, a reference to "an" antibody includes both a single antibody and multiple different antibodies. The term "approximately" indicates an approximation that may vary by ±10%, ±5%, or ±1% when used before numerical representations such as temperature, time, quantity, and concentration (including ranges).

[0026] As used herein, “administration” of an antibody means the delivery of the antibody to the body of a subject via any known method appropriate for antibody delivery. Specific modes of administration include, but are not limited to, intravenous, transdermal, subcutaneous, intraperitoneal, and intrasacral administration. Exemplary methods of administration for antibodies may be substantially as described in International Publication No. 2016 / 187514, which is incorporated herein by reference in its entirety. Furthermore, according to aspects of this disclosure, antibodies may 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 solvents, suspensions, gels, microspheres, and polymer injections, and may 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) an immunoglobulin molecule comprising two heavy chains and two light chains that recognize an antigen, (b) polyclonal immunoglobulin molecules and monoclonal immunoglobulin molecules, (c) monovalent and bivalent fragments thereof (e.g., di-Fab), and (d) its bispecific forms. 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, including, but 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, almost all, or all of the amino acids outside the CDR domain of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of the humanized form of an antibody, some, almost all, or all of the amino acids outside the CDR domain are replaced with amino acids derived from human immunoglobulins, while some, almost all, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not impede the antibody's ability to bind to a particular antigen. "Humanized" antibodies retain similar antigen specificity to the original antibody. A "chimeric antibody" refers to an antibody in which the variable region originates from one species and the constant region originates from another species, such as an antibody in which the variable region originates from a mouse antibody and the constant region originates from a human antibody.

[0029] As used herein, “noncancerous disorders” or “nonmalignant disorders” include, but are not limited to, abnormal hemoglobin disorders (e.g., SCD), congenital immunodeficiency (e.g., SCID), autoimmune disorders (e.g., multiple sclerosis, rheumatoid arthritis, scleroderma, systemic lupus pallidum, type 1 diabetes mellitus, myasthenia gravis, Sjögren's disease, polymyositis, etc.), and viral infections (e.g., HIV infection). Noncancerous disorders do not include, for example, solid tumors (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 hematopoietic tissue, such as 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), pilocytic cell leukemia, and large granular lymphocytic leukemia), myelodysplastic syndromes (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocytosis, primary myelofibrosis, and chronic myeloid leukemia), lymphoma, multiple myeloma, MGUS and similar disorders, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, perisplenic zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies.

[0031] "Solid cancers" are not limited to, but include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, prostate cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, carcinoma of the Fallopian duct, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric tumors, bladder cancer, kidney or ureteral cancer, renal pelvis carcinoma, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem 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 “load” means quantity when used in relation to cancer cells. Thus, cancer cell “load” means quantity of cancer cells. Cancer cells have a load related to the origin of their tissue (i.e., the primary site of the disease), for example, a “myeloblast load” in the case of AML. Cancer cells also have a load related to one or more tissues other than that of origin, for example, a blast load in the blood, liver, and spleen in the case of AML. The term “peripheral load” refers to such cells. In the case of AML, the peripheral load of cancer cells, such as blasts, may be measured in different ways with different outcomes. For example, in the case of AML, the “peripheral blast load” may be measured as the total extramyeloblast population, or as the total blast population combined from the blood, spleen, and liver, or as the blood blast population measured simply by the number of cells per unit volume. When used herein in relation to AML and other cancers of bone marrow origin, unless otherwise specified, the term “peripheral cancer cell load” (e.g., peripheral blast load) refers to the population of cancerous cells in blood as measured per unit volume (e.g., cells / μL). This blood-based measurement is a useful substitute for more cumbersome measurements such as spleen load and hepatic load.

[0033] In this specification, when an agent targeting a hematological malignancy-associated antigen, such as the radiolabeled anti-CD45 antibody of this disclosure, is administered to a subject at the maximum safe dose, if the agent does not reach the primary site of the disease in an amount sufficient to bind to more than 90% of its target antigen at that site, the peripheral cancer cell load in the subject is "high". Conversely, when the agent is administered to a subject at the maximum safe dose, if the agent reaches the primary site of the disease in an amount sufficient to bind to more than 90% of its target antigen at that site, the peripheral cancer cell load in the subject is "low". In the case of AML, examples of low peripheral blast load include hematological blast loads of ≤1,000 / μL, ≤500 / μL, ≤400 / μL, ≤300 / μL, ≤200 / μL, ≤100 / μL, and ≤50 / μL.

[0034] As used herein, “low-dose” radiolabeled anti-CD45 antibodies of this disclosure are subsaturated and thus introduce fewer target antigen-binding sites (i.e., CD45 binding sites in the administered antibody) into the subject’s body than the target antigen (i.e., CD45 molecules). In certain embodiments, a low-dose radiolabeled anti-CD45 antibody has a CD45 binding site-to-CD45 molecule ratio of 9:10 or less, for example, 1:2 or less, or 1:5 or less, or 1:10 or less, or 1:20 or less, or 1:100 or less. Where used herein, the terms “subject” or “affected” are interchangeable and not limited to, but include humans, non-human primates, dogs, cats, horses, sheep, goats, cattle, rabbits, pigs, rats, and mice. If the subject is human, the subject may be of any age. In a particular embodiment, the subject is an infant. In a further embodiment, the subject is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years old. In yet another embodiment, the subject is 10 to 15 years old, or 15 to 20 years old. In yet another embodiment, the subject is 20 years or older, 25 years or older, 30 years or older, 35 years or older, 40 years or older, 45 years or older, 50 years or older, 55 years or older, 60 years or older, 65 years or older, 70 years or older, 75 years or older, 80 years or older, 85 years or older, or 90 years or older.

[0035] As used herein, “treatment” of a disabled subject includes, but is not limited to, (i) slowing, stopping, or improving the progression of the disability; (ii) slowing, stopping, or improving the progression of the symptoms of the disability; (iii) reducing, and ideally eliminating, the likelihood of recurrence of the disability; and / or (iv) reducing, and ideally eliminating, the likelihood of recurrence of the symptoms of the disability. In a particular preferred embodiment, treating a disabled subject means (i) improving the progression of the disability to the extent that it is ideally eliminated; and / or (ii) improving the progression of the symptoms of the disability to the extent that it is ideally eliminated; and / or (iii) reducing or eliminating the likelihood of recurrence. Ideally, treating a disabled subject means curing the disability by eliminating its genetic cause or, otherwise, rendering it incapacitated. As used herein, “depleting” a specific cell type of a subject means reducing its cell population within the subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. As used herein, “ablating” a specific cell type of a subject means reducing its cell population within the subject by more than 95%, for example, at least 96%, 97%, 98%, 99%, or 100%.

[0036] Specific cell types to be depleted using the compositions and methods of this disclosure include, at a minimum, hematopoietic stem cells (i.e., pluripotent hematopoietic stem cells, also known as hematoblasts) and lymphocytes, such as peripheral blood lymphocytes or myeloid lymphocytes. Hematopoietic stem cells ("HSCs") are pluripotent, self-replicating progenitor cells that arise from all differentiated blood cell types during the hematopoietic process. HSCs are thought to differentiate into two lineage-restricted lymphoid oligopotent progenitor cells and lineage-restricted myeloid oligopotent progenitor cells, but alternative "bone marrow-based" models of hematophylogeny describe novel intermediate myeloid lymphoid progenitor cells capable of producing offspring from both lineages.

[0037] As used herein, the term “hematopoietic stem cell” (“HSC”) refers to immature blood cells that self-replicate and have the ability to differentiate into mature blood cells, including, but are not limited to, a diverse range of lineages such as 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). Examples of such cells include CD34+ cytoplasm. CD34+ cells are immature cells that express the CD34 cell surface marker.

[0038] Routine methods exist for measuring the HSC population. 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). A decrease in immune cells in affected individuals may be detected in peripheral blood. Routine methods exist for measuring the peripheral blood lymphocyte population. These include, for example, flow cytometry in 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. Routine methods also exist for measuring the peripheral blood neutrophil population. These include, for example, flow cytometry in whole blood samples to determine neutrophil counts based on labeling with fluorescent antibodies against specific cell surface markers such as Ly6G.

[0039] According to certain aspects of this disclosure, a decrease in the lymphocytes of interest is determined by measuring the peripheral blood lymphocyte levels of the interest. As used herein, “peripheral blood lymphocytes” means mature lymphocytes circulating in the blood of the interest. 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 of the interest is reduced by 95% or less, the lymphocyte population of the interest is depleted. For example, if the level of peripheral blood T cells of the interest is reduced by 50%, the level of peripheral blood NK cells of the interest is reduced by 40%, and / or the level of peripheral blood B cells of the interest is reduced by 30%, the lymphocytes of the interest are depleted. In this example, the lymphocytes of the interest are depleted even if the levels of other immune cell types, such as neutrophils, are not reduced. In a particular embodiment, depletion of target lymphocytes is reflected in a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the peripheral blood lymphocyte population.

[0040] As used herein, patients “requiring” hematopoietic stem cell transplantation include patients exhibiting a deficiency or absence of one or more blood cell types, and patients having stem cell disorders, autoimmune diseases, cancer, or other medical conditions described herein. Hematopoietic stem cells generally exhibit 1) pluripotency (therefore capable of differentiating 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, and lymphocytes (e.g., NK cells, B cells, and T cells)), 2) self-renewal (therefore capable of producing daughter cells with equivalent potential as parent cells), and 3) the ability to be reintroduced into the transplant recipient, thereby returning to the hematopoietic stem cell niche and re-establishing proliferative and sustained hematopoiesis.

[0041] Additionally or alternatively, a patient “requiring” hematopoietic stem cell transplantation may be a patient who is diseased or not diseased but who nevertheless exhibits reduced levels (e.g., compared to levels in a otherwise healthy subject) of one or more endogenous cell types within the hematopoietic lineage, such as megakaryocytes, platelets, erythrocytes, mast cells, myeoblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes.

[0042] Anti-CD45 antibody As used herein, “anti-CD45 antibody” or “anti-CD45-immunoglobulin” is an antibody that binds to the CD45 epitope. In certain embodiments, an anti-CD45 antibody may bind to an epitope recognized by the monoclonal antibody “BC8,” known as a method for producing it. These methods are described, for example, in International Publication No. 2017 / 155937, which is incorporated in its entirety by reference herein, and in the examples provided herein.

[0043] A BC8 monoclonal antibody may include a light chain having the amino acid sequence shown in SEQ ID NO: 12, which includes a leader sequence (Figure 4A), or the amino acid sequence shown in SEQ ID NO: 13, which does not include a leader sequence (Figure 4B). A BC8 monoclonal antibody may include a light chain variable region having the amino acid sequence shown in SEQ ID NO: 1 (Figure 2). A BC8 monoclonal antibody may include a light chain having the N-terminal amino acid sequence shown in SEQ ID NO: 9 (Figure 3). According to a particular embodiment, the light chain includes at least one complementarity-determining region having the amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 (Figure 3). According to a particular embodiment, the light chain includes the N-terminal amino acid sequence shown in SEQ ID NO: 9 and at least one complementarity-determining region having the amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 (Figure 3). A BC8 monoclonal antibody may include a heavy chain having the amino acid sequence shown in SEQ ID NO: 15, which includes a leader sequence (Figure 5A), or the amino acid sequence shown in SEQ ID NO: 16, which does not include a leader sequence (Figure 5B). A BC8 monoclonal antibody may include a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 2 (Figure 2). A BC8 monoclonal antibody may include a heavy chain having the N-terminal amino acid sequence shown in SEQ ID NO: 10 (Figure 3). According to a particular embodiment, the heavy chain includes at least one complementarity-determining region having the amino acid sequence as shown in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 (Figure 3). According to a particular embodiment, the heavy chain includes the N-terminal amino acid sequence shown in SEQ ID NO: 10 and at least one complementarity-determining region having the amino acid sequence as shown in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 (Figure 3).

[0044] In a particular embodiment, the BC8 monoclonal antibody contains a heavy chain having the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, where the amino acid at position 141 (relative to the N-terminal amino acid) is either ASP or ASN. The ASP:ASN ratio at position 141 in the population of BC8 proteins may be in the range of 1:99 to 99:1, such as 10:90 to 90:10. In a particular embodiment, a BC8 monoclonal antibody includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 2, where the amino acid at position 141 of the constant region (relative to the N-terminal amino acid) is either ASP or ASN. The ASP:ASN ratio 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] In certain embodiments, the antibody against CD45 (anti-CD45 antibody) may be a chimeric antibody or a humanized antibody. For example, a 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 parental mouse variable (V) region grafted onto the human IgG1 constant region, human IgG2 constant region, or human IgG4 constant region for the heavy chain, or onto the human kappa region for the light chain. The IgG4 antibody is capable of Fab arm exchange by replacing the heavy chain and added light chain (half) with heavy-light chain pairs derived from other molecules, thereby producing a bispecific antibody. 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 the IgG4 antibody to undergo Fab arm exchange was attributed to an unstable core-hinge sequence in combination with a sequencing factor of the IgG4 CH3 domain. Substitution of the core-hinge residue Ser228 by Pro(S228P) results in partial stabilization of the IgG4 molecule in vitro and in vivo. Thus, in certain embodiments, IgG4 can contain S or P at position 228, where the mutation S228P may help stabilize 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 mouse heavy chain V region and BC8 mouse light chain V region, as well as the endogenous mouse signal sequence, in frame into a mammalian expression vector for heavy and light chains that already contains human heavy chain constant regions (IgG1, IgG2, or IgG4) or human C kappa. Therefore, according to certain embodiments, the BC8 monoclonal antibody may be a chimeric BC8, i.e., BC8c, and may include a human IgG1 heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 17, or a human IgG2 heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 18, or a human IgG4 heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 19, or a human IgG4 heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 20, or a human kappa light chain constant region having the amino acid sequence shown in SEQ ID NO: 21 (Figures 6A to 6E).

[0047] In 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 shown in any one of SEQ ID NOs: 17 to 20, and a human kappa light chain constant region having the amino acid sequence shown in SEQ ID NOs: 21 (Figures 6A to 6E).

[0048] In a particular embodiment, the chimeric BC8c monoclonal antibody may include a light chain variable region having the amino acid sequence shown in SEQ ID NO: 1 (Figure 2). The BC8c monoclonal antibody may include a light chain having the N-terminal amino acid sequence shown in SEQ ID NO: 9 (Figure 3). The BC8c monoclonal antibody may include a light chain having at least one complementarity-determining region having the amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 (Figure 3). In a particular embodiment, the chimeric BC8c monoclonal antibody may include a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 2 (Figure 2). The BC8c monoclonal antibody may include a heavy chain having the N-terminal amino acid sequence shown in SEQ ID NO: 10 (Figure 3). The BC8c monoclonal antibody may include a heavy chain having at least one complementarity-determining region having the amino acid sequence as shown in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 (Figure 3). In certain embodiments, the heavy chain of a BC8 or BC8c monoclonal antibody contains a C-terminal lysine, a C-terminal glycine (G) lacking a C-terminal lysine (K), or lacks both GK and K. When referring to an antibody containing a modified heavy chain constant region as described herein, the antibody may contain a provided sequence having a C-terminal GK or C-terminal K, or alternatively, lacking either GK or K.

[0049] Patient specific composition When used in this specification, 225 Compositions containing Ac-labeled BC8 include both actinium-225-labeled and unlabeled antibodies, with a small number being actinium-225-labeled antibodies. Similarly, 177 With respect to Lu-labeled BC8, the composition comprises both a population of labeled antibodies and a population of unlabeled antibodies. The ratio of labeled antibodies to unlabeled antibodies can be adjusted using known methods. Accordingly, according to certain embodiments of this disclosure, anti-CD45 antibodies may be provided in a total protein amount of up to 100 mg, for example, up to 60 mg, for example, 5 mg to 45 mg, or in a total protein amount of 0.001 mg / kg (affected body weight) to 3.0 mg / kg (affected body weight), for example, 0.005 mg / kg (affected body weight) to 2.0 mg / kg (affected body weight), or 0.01 mg / kg (affected body weight) to 1 mg / kg (affected body weight), or 0.1 mg / kg (affected body weight) to 0.6 mg / kg (affected body weight), or 0.3 mg / kg (affected body weight), or 0.4 mg / kg (affected body weight), or 0.5 mg / kg (affected body weight), or 0.6 mg / kg (affected body weight).

[0050] According to certain aspects of this disclosure, a radiolabeled anti-CD45 antibody (i.e., 225 Ac label BC8 or 177The Lu-labeled BC8) may comprise a labeled fraction and an unlabeled fraction, where the labeled:unlabeled ratio may be approximately 0.01:10 to 1:10, for example, 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 radioactively labeled anti-CD45 antibody may be provided as a single-dose composition tailored to a specific affected individual, where the amounts of labeled and unlabeled anti-CD45 antibody in the composition may depend at least on the mass, age, sex, and / or disease status or health condition of the affected individual. For example, see the method of administration disclosed in International Publication No. 2016 / 187514, which is incorporated herein by reference in whole. In a particular embodiment, the radioactively labeled anti-CD45 antibody may be provided in multiple doses, where each dose in the regimen may include a composition tailored to a specific affected individual, where the amounts of labeled and unlabeled anti-CD45 antibodies in the composition may depend at least on the mass, age, sex, and / or disease status or health condition of the affected individual.

[0051] The present invention's combination of labeled and unlabeled fractions of anti-CD45 antibody makes it possible to tailor the composition to a specific patient, where each of the monoclonal antibody's radiation dose and protein dose is individualized to that patient based on at least one patient-specific parameter. Thus, each vial of the composition may be prepared for a specific patient, and the entire contents of the vial are delivered to that 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, administered to the patient as a "single dose" (i.e., the entire contents in a vial administered in one dose). Subsequent doses may be formulated in a similar manner, so that each dose in the regimen provides a patient-specific dose in a single-dose container. One advantage of the compositions of this disclosure is that there is no residual radiation that healthcare professionals need to discard or handle, such as dilution or other operations to obtain a dose for a patient. When provided in single-dose containers, the containers are simply arranged in a single row within a set of infusion tubes for administration to the affected body. Furthermore, the quantities can be standardized to greatly reduce the likelihood of medical errors (i.e., delivery of an incorrect dose when the entire amount of the composition is to be administered in a single infusion).

[0052] Treatment of blood disorders Most malignancies of hematological origin express CD45 to varying degrees on the surface of tumor cells, whether they originate from bone marrow or lymphocytes. This includes 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), pilocytic cell leukemia, and large granular lymphocytic leukemia), myelodysplastic syndromes (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocytosis, primary myelofibrosis, and chronic myeloid leukemia), lymphoma, multiple myeloma, MGUS and similar disorders, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, perisplenic zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies. Therefore, when administered to an affected individual, a certain amount of radiolabeled anti-CD45 antibody is effective as a direct antitumor therapy to reduce the number of tumor blasts in the periphery as well as in immune cell compartments such as the bone marrow, spleen, and lymph nodes. Direct therapy with radiolabeled anti-CD45 antibodies may be used as a low-dose monotherapy necessary to reduce the number of tumor blasts, but reversibly as a reserve hematopoietic stem cell, or in combination with other therapeutic agents, such as chemotherapeutic agents or targeted therapies (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 cell count without irreversibly depleting hematopoietic stem cells. 225The dose of Ac radiolabeled anti-CD45 antibody delivers bone marrow radiation exposure below the threshold level. A dose of 2 Gy is considered a non-myeloablative dose. The ideal dose delivers at least 2 Gy, which is high enough to eliminate leukemia or lymphoma tumor cells and provide transient but reversible myelosuppression. Dose levels above 2 Gy but below the myeloablative dose are expected to be effective in controlling tumor burden in lymphoma and leukemia. Furthermore, when single low-dose radiolabeled anti-CD45 antibody treatment is used in combination with another targeted agent, potent antitumor activity can be achieved using lower-dose antibody radioconjugates, further avoiding hematopoietic stem cell depletion.

[0054] Typical low doses are less than 150 μCi, such as 10 μCi to 100 μCi. 225 The dose may be Ac-BC8, or a dose 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] Exhaustion of circulating tumor blasts and circulating myeloblasts Hematological malignancies, such as leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and multiple myeloma, present a unique set of problems for effective treatment. If killed too rapidly, the high burden of circulating tumor cells, often associated with leukemia, can be toxic to the affected body. Cytodeceptic therapy is a process that reduces the number of circulating blast cells. In certain embodiments, cytoreductive therapy may be used to treat hematological malignancies and generally involves the administration of low doses of radiolabeled anti-CD45, such as doses that deplete circulating tumor cells (e.g., leukemia, lymphoma, myeloma, MDS) but are not myeloablative and therefore do not irreversibly deplete HSCs. An example of a low dose is less than 150 μCi, such as 10 μCi to 100 μCi. 225The dose may be Ac-BC8, or a dose 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 includes the administration of hematopoietic stem cells, such as bone marrow transplantation (BMT). Hematopoietic stem cells may be administered in vivo to affected individuals who are deficient or lacking one or more cell types of hematopoietic lineages in order to reconstitute a cell deficit or deficiency population. For example, affected individuals may have cancer or abnormal hemoglobinopathy (e.g., non-malignant abnormal hemoglobinopathy), such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiscott-Aldrich syndrome. Subjects may also have adenosine deaminase severe combined immunodeficiency (ADA SCID), HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachmann-Diamond syndrome. The subjects may have or be suffering from a hereditary blood disorder (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 conditions.

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

[0058] gene editing Gene editing technology has advanced substantially with the emergence of site-directed editing methods such as TALEN, CRISPR / Cas9, and zinc finger nuclease (ZFN) methods. These methods have therapeutic potential for individuals suffering from malignant and non-malignant genetic diseases. Gene editing precisely and permanently alters the sequence of genomic DNA that remains under endogenous gene regulation and control for the valid and appropriate expression of modified genetic elements. Currently, there are four major classes of nucleases for human genome gene editing: zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), meganucleases (MNs), and clustered, regularly arranged short palindromic sequence repeats (CRISPR / Cas9). Each of these can recognize and bind to a specific target sequence in DNA. Depending on the approach, the target DNA may be cleaved on one or both strands. To correct mutations, a corrective template is used for homology-directed repair of the cleavage introduced at the site of the target lesion. This technique can also be used to deexpress or ablate specific genes by incorporating the insertion or deletion of mutations. Furthermore, gene editing technology can be used to alter the specificity of T cells, for example, by functionally replacing a gene within the T-cell receptor alpha constant locus (TRAC) with another gene (Eyquem, et. al., 2017, Nature. 543:113-117).

[0059] Adoptive Cell Therapy (“ACT”) Adoptive cell therapy may include the administration of cells expressing a chimeric antigen receptor (CAR) or T cell receptor (TCR), or it may include tumor-infiltrating lymphocytes (TILs). The population of CAR / TCR-expressing cells may include a population of activated T cells or natural killer (NK) cells or dendritic cells that express an antigen-recognizing CAR / TCR. Dendritic cells are capable of antigen presentation and direct tumor death. The population of CAR / TCR-expressing cells may also include a population of gene-edited cells.

[0060] As used herein, the term “gene-edited” CAR T cell is synonymous with the terms “genetically modified” CAR T cell and “modified” CAR T cell. Gene-edited CAR T cells that are “unable to properly express” a checkpoint receptor (e.g., PD1, Lag3, or TIM3) will not express a fully functional checkpoint receptor. For example, but not limited to, a gene-edited CAR T cell that is unable to properly express PD1 may not be able to do so because (i) the cell’s PD1 gene has been ablated, or (ii) otherwise, the cell’s PD1 gene has been modified so that it does not produce a functional PD1 product, either completely or more partially. That is, according to certain embodiments, a gene-edited CAR T cell that is unable to properly express PD1 may not be able to do so because the cell’s PD1 gene has been modified to reduce PD1 expression. Similarly, a gene-edited CAR T cell that is “unable to properly express” a T cell receptor will not express a fully functional T cell receptor.

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

[0062] An example of T cell gene editing for generating allogeneic pluripotent 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. By this method, the recombinant CAR was effectively positioned under the control of the cell's innate TCR regulatory signaling. By this same strategy, the CAR or recombinant TCR may also be effectively inserted by knock-in into the T cell receptor beta constant locus (TRBC) or beta-2 microglobulin (B2M) MHC-I related 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 are immunosuppressive and may attenuate the stimulation of exogenous autologous CAR T cells or exogenous allogeneic CAR T cells, this group utilized CRISPR / cas9 technology to ablate the endogenous TCRα and TCRβ loci (TRAC and TRBC) as well as the B2M gene, while also deexpressing the endogenous PD1 gene. This approach resulted in manipulated cells that resisted immune checkpoint receptor suppression without inducing graft-versus-host disease.

[0063] Lymphatic depletion and bone marrow destruction It is common practice to perform lymphatic depletion in the affected body before administering a certain dose of HST (e.g., bone marrow transplant) or manipulated immune cells to the affected body. The lymphatic depletion process is considered important, and indeed essential, for the success of BMT and adoptive cell therapy (ACT) methods. This process creates sufficient space within the immune microenvironment (e.g., bone marrow) to enable transplantation of the transplanted cells. This also creates a favorable immunohomeostatic environment for the successful transplantation, proliferation, and survival of the transplanted cells by inducing a favorable cytokine profile. This cytokine profile is particularly induced within the peripheral immune niche (e.g., bone marrow, spleen, and lymph nodes) for the establishment and proliferation of manipulated 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-derived cells and lymphocyte-derived cells express CD45. This is effective in reducing blast cell counts without irreversibly depleting hematopoietic stem cells. 225 The dose of Ac radiolabeled anti-CD45 antibody delivers radiation exposure to the bone marrow below the threshold level. A dose of 2 Gy is considered a non-myeloablative dose of radiation. A dose of at least 2 Gy may provide transient but reversible myelosuppression. Myeloablative doses can vary, ranging from 8 to 18 Gy, but typically include doses that deliver more than 10 to 12 Gy to the bone marrow. Therefore, the therapeutic low-dose range that can provide reversible immunosuppression (with or without stem cell carriers) is greater than 2 Gy but below myeloablative doses such as 8 Gy or less. Stem cell carriers may be required for higher doses.

[0065] As used herein, a dose 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, a dose of radiolabeled anti-CD45 antibody is "effective" to deplete target peripheral blood lymphocytes if, upon administration, the target neutrophils are not depleted, or the target neutrophils are reduced by less than 10% or less than 20% and peripheral blood lymphocytes are depleted. The “effective” dose of radiolabeled anti-CD45 antibody may also relate to the amount that depletes, for example, at least 20%, or 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the target regulatory T cells, bone marrow-derived suppressor cells, tumor-sensitized macrophages, IL-1 and / or IL-6-secreting activated macrophages, and combinations thereof.

[0066] As used herein, a dose of radiolabeled anti-CD45 antibody is “effective” for reversibly suppressing a target cell type if, upon administration, the target cell population, such as HSC levels or lymphocyte levels, decreases by more than 95%, for example, at least 96%, or 97%, 98%, or 99%. “Reversible immunosuppression” generally includes the use of low-dose therapeutic agents or combinations thereof, such as actinium-225-labeled BC8 as disclosed herein, to deplete target cells to a greater extent than standard lymphoid depletion without ablation of the target cells, i.e., at a dose below the myelosolytic dose (non-myelosolytic dose). Furthermore, reversible immunosuppression may indicate that only the targeted immune population (immunely privileged cell population or immunely privileged cell tissue) is transiently depleted, while other non-target populations remain unaffected.

[0067] As disclosed below, the reversible immunosuppression of this disclosure may generally involve the administration of actinium-225-labeled BC8 with another agent, such as another immunotherapeutic agent or radiosensitizer. As used herein, the amount of radiolabeled anti-CD45 antibody is "effective" for ablation of the target cell type if, upon administration, the target cell population, such as the HSC level or lymphocyte level, is reduced by 100% (also known as myelostomy). According to certain aspects of this disclosure, the radioactively labeled anti-CD45 antibody is actinium-225 labeled BC8( 225 It is labeled with Ac (BC8). 225 The effective dose of Ac-labeled BC8 is, for example, less than 5.0 μCi / kg (i.e., administered to the subject). 225 (If the amount of Ac-BC8 delivers a radiation dose below 5.0 μCi per kilogram of the subject's body weight).

[0068] According to the manner of this 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. According to a particular embodiment, 225 The 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. According to a particular embodiment, 225 Ac-labeled BC8 may be administered in doses including any combination of the 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 a particular aspect, 225 The 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. According to a particular embodiment, 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 The effective amount of Ac-labeled BC8 may be at least 2 μCi, for example, 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. According to a particular embodiment, 225 Ac-labeled BC8 may be administered in doses including any combination of the 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 80 μCi to less than 500 μCi. According to a particular embodiment, 225 Ac-labeled BC8 may be administered at low doses. An exemplary low dose is less than 150 μCi, for example, 10 μCi to 100 μCi. 225 The dose may be that of Ac-BC8, 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. According to certain aspects of this disclosure, the radiolabeled anti-CD45 antibody is lutetium-177 labeled BC8( 177 Lu-labeled BC8) 177 The effective dose of Lu-labeled BC8 is, for example, less than 500 μCi / kg (i.e., administered to the subject). 177(If the amount of Lu-BC8 delivers a radiation dose of less than 500 μCi per kilogram of the subject's body weight).

[0070] According to the manner of this disclosure, 177 The 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. According to a particular embodiment, 177 The 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. According to a particular embodiment, 177 Lu-labeled BC8 may be administered in doses including any combination of the 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 a particular aspect, 177 The effective dose 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. 177The effective amount of Lu-labeled BC8 may be at least 10 μCi, for example, 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. According to a particular embodiment, 177 Lu-labeled BC8 may be administered in doses including any combination of the 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, the “appropriate time period” between administration of a radiolabeled anti-CD45 antibody to a subject and any further treatment of the subject is a sufficient time period to allow the administered antibody to deplete, reversibly suppress, or ablate the subject’s target cells, e.g., 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 a particular embodiment, the appropriate time period after administration of a radiolabeled anti-CD45 antibody, for which the ACT method can be performed, is 3, 4, 5, 6, 7, 8, or 9 days, for example, preferably 6, 7, or 8 days.

[0073] Throughout this application, various publications are referenced. The disclosures of these publications are incorporated herein by reference to provide a more complete description of the art relating to this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs. Methods and materials similar to or equivalent to those described herein may be used in the practices or tests described herein, but suitable methods and materials are described below.

[0074] Detailed description of the invention This 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 of the subject. Reversible immunosuppression of these cells may be useful in the treatment of CD45-positive hematological malignancies and can be achieved using low-dose therapeutic agents, such as the administration of anti-CD45 antibodies at subsaturated radiation doses. Radiolabeled anti-CD45 antibodies demonstrated clinical potential as targeted myeloablative conditioning before bone marrow transplantation. CD45 is an attractive target for conditioning because it is highly expressed in all nucleated immune cells, including hematopoietic stem cells, lymphoid cells, and bone marrow cells. The potent alpha-emitting agent 225 actinium ( 225 Ac) is a promising radionuclide for target pretreatment, exhibiting high linear energy transfer over short path lengths (80-100 keV / μm) and a long half-life of 9.9 days.

[0075] Furthermore, high-dose therapeutics, i.e., depletion or ablation of these cells using higher doses of radiation, may serve as a precursor model for 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 therapies (e.g., gene-edited β-globin hematopoietic stem cell therapy for sickle cell disease (SCD)). Therefore, this disclosure relates to methods for depleting, reversibly immunosuppressing, or ablating specific cells in a target. 225 Radiolabeled anti-CD45 antibodies such as Ac-BC8 are used. These antibodies can safely and effectively deplete or ablate target hematopoietic stem cells or lymphocytes via targeted pretreatment. This approach avoids certain side effects caused by less specific agents such as chemotherapy drugs or external beam radiation.

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

[0077] Radioactively labeled immunotherapy drugs According to aspects of this disclosure, anti-CD45 immunoglobulin BC8 is 225 Ac or 177It may also contain Lu. According to a particular preferred embodiment, anti-CD45 immunoglobulin BC8 is alpha-emitting radioactive nuclide actinium-225 ( 225 It may be radioactively labeled with Ac). It is conjugated with the monoclonal antibody BC8. 225 Depending on the Ac load, high-energy alpha particles are delivered directly to (one or more) target cells, thereby causing lethal double-strand DNA breaks. Due to its short pathway length, the range of its high-energy alpha particle emission is limited to the thickness of a small cell diameter, thereby limiting damage to nearby non-malignant or normal tissue. Therefore, 225 Ac-BC8 is, 177 It can deliver an effective therapeutic dose with less heat dissipation than Lu-BC8.

[0078] moreover, 225 Ac antibody conjugates have been found to be effective even in patients with tumors expressing low target antigens, thus offering a significant advantage over conventional antibody-drug conjugates. 225 This is due to Ac's strong cytotoxic effect, which is in stark contrast to antibody-drug conjugates, where hundreds of antibody molecules need to bind to their respective antigens in order to exert their effect on target cells or tissues. Other advantages of radioactive loadings over drugs or toxins include: 1) the radiation-delivering antibody does not need to be internalized to kill cells; 2) the antibody does not need to target all cells within the target tissue or target tumor; and 3) in contrast to antibody-drug conjugates, the radioisotopes linked to the antibody are less likely to induce significant immune responses that would limit subsequent use. Furthermore, the studies reported herein are: 225 This demonstrates the stability of Ac-labeled antibodies and their high target cell cytotoxicity.

[0079] According to certain aspects of this disclosure, 225Ac may be added to or chelated by a chelating agent conjugated to a monoclonal antibody. As described in detail in Example 3 below, anti-CD45 immunoglobulin first forms a chelating agent conjugated anti-CD45 ("conjugated anti-CD45"), and then chelates the radionuclide with the conjugated anti-CD45 to form radiolabeled anti-CD45 (i.e., 225 It may be prepared by forming 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 chelating agent. The pH may be selected to optimize the conditions for the conjugation of the chelating agent with the antibody in the conjugation reaction mixture. The conjugation reaction mixture may contain a bicarbonate buffer or a phosphate buffer. The pH of the conjugation reaction mixture may be about 8.0 to about 9.2. For example, the pH of the conjugation reaction mixture may be 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 promote the conjugation of the chelating agent with the target moiety. For example, the conjugation reaction mixture may be incubated at approximately room temperature or about 37°C. The conjugation reaction mixture may be incubated for any time sufficient to provide the conjugation, such as about 1.5 hours.

[0081] The conjugate anti-CD45 may be dissolved in a buffer solution containing the radionuclide. The pH may be selected to optimize the conditions for chelation of the radionuclide by the conjugate anti-CD45 in the chelation reaction mixture. The pH of the chelation reaction mixture may be about 5.5 to about 7.0. For example, the pH of the chelation reaction mixture may be 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 promote the chelation of radionuclides by the conjugate 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 some time, the solution may be inhibited by adding a reaction-inhibiting chelate compound (e.g., diethylenetriaminepentaacetic acid (DTPA)) and the reaction mixture may be purified. After the addition of the reaction-inhibiting chelate compound, for example, the chelation reaction mixture may be further incubated at about 37°C for about 30 minutes.

[0083] The chelating agents useful in this disclosure are compounds that have a dual functionality: the ability to sequester metal ions and to covalently bind to biological carriers such as antibodies. Many chelating agents are known in the art.Examples of chelating agents suitable for use in this 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-isothiocyanatobenzyl-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); 3 ,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); Transcyclohexanediaminetetraacetic acid (CDTA); Trans(1,2)-cyclohexanediethylenetriaminepentaacetic acid (trans(1,2)-cyclohexane Examples of chelating agents include, but are not limited to, 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 their derivatives.

[0084] One or more steps may be used to separate conjugate CD45 from other components of the conjugation reaction mixture, or radiolabeled anti-CD45 from other components of the chelation reaction mixture. For example, the reaction mixture can be transferred to a filter having a specific molecular weight cutoff (e.g., a Millipore centrifuge) so that the conjugate anti-CD45 or radiolabeled anti-CD45 can be separated from other components of each reaction mixture by filtration of the reaction mixture through the filter. Filtration may be used to obtain conjugate 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 aspects of the present disclosure, the yield of conjugate 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 aspect of this disclosure, first, a monoclonal antibody is conjugated with p-SCN-Bn-DOTA or a DOTA chelating agent to form a conjugated anti-CD45 immunoglobulin, and then p-SCN-Bn-DOTA or DOTA is used in the conjugated anti-CD45 immunoglobulin. 225 Ac may be chelated to form radioactively labeled anti-CD45 immunoglobulin. Therefore, according to an embodiment of this disclosure, in order to label anti-CD45 immunoglobulin 225 Only a single step, including Ac, is required.

[0086] According to certain aspects of this disclosure, radioactively labeled anti-CD45 immunoglobulin 225 Ac-BC8 is relatively stable. For example,225 More than 85% of Ac-BC8 may remain intact after being stored at 4 °C for 24 hours (see Figure 11). Moreover, 225 Ac-BC8 shows specificity for CD45-expressing cells (see Figures 12, 15A, and 15B) and CD45-expressing tissues (see Figure 16A). 225 The labeling efficiency, stability, and immunoreactivity of Ac-BC8 together provide an effective therapeutic agent. 177 Radioactive labeling with lutetium-177 ( 177 Lu) to provide Lu-DOTA-BC8 is also possible and within the scope of the present disclosure. Moreover, 225 References to Ac-BC8 or 177 Lu-BC8, unless otherwise indicated, each may include a reference to either 225 Ac-DOTA-BC8 or 177 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 of a subject, the method comprising administering to the subject an effective amount of a radioactively labeled anti-CD45 immunoglobulin, such as 225 Ac-BC8. The present disclosure provides a method for depleting lymphocytes of a subject, the method comprising 225 administering to the subject an effective amount of a radioactively labeled anti-CD45 immunoglobulin, such as Ac-BC8. The present disclosure provides a method for depleting, reducing, or removing hematopoietic cancer blasts of a subject, the method comprising 225 administering to the subject an effective amount of a radioactively labeled anti-CD45 immunoglobulin, such as Ac-BC8, alone as monotherapy or in combination with other therapies.

[0088] According to certain embodiments of this method, 225The effective amount of Ac-BC8 is 0.05 μCi / kg to 5.0 μCi / kg of the subject's body weight. Examples of the effective amount include, but are not limited to, 0.05 μCi / kg to 5.0 μCi / kg, such as 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, 0.8 μCi / kg to 0.9 μCi / kg, 0.9 μCi / kg to 1.0 μCi / kg, 1.0 μCi / kg to 1.5 μCi / kg, 1.5 μCi / kg to 2.0 μCi / kg, 2.0 μCi / kg to 2.5 μCi / kg, 2.5 μCi / kg to 3.0 μCi / kg, 3.0 μCi / kg to 3.5 μCi / kg, 3.5 μCi / kg to 4.0 μCi / kg, 4.0 μCi / kg to 4.5 μCi / kg, or 4.5 μCi / kg to 5.0 μCi / kg. According to certain embodiments of this method 225 The effective amount of Ac-BC8 is less than 1 mCi, such as less than 500 μCi. Examples of the effective amount include 1 μCi to 500 μCi, such as 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, but are not limited thereto.

[0089] Exemplary low doses are less than 120 μCi, such as 10 μCi to 100 μCi 225 A low dose of Ac-BC8, or less than 2 μCi / kg, such as 0.01 μCi / kg to 1.5 μCi / kg, or a dose of 0.1 μCi / kg to 1.0 μCi / kg may be used. 225An exemplary high dose of Ac-BC8 may be at least 120 μCi, for example, a medium to high dose of 120 μCi to 500 μCi, or at least 2 μCi / kg, for example, 2 μCi / kg to 5 μCi / kg, or 3 μCi / kg to 5 μCi / kg.

[0090] According to a particular aspect of this method, 225 An effective dose of Ac-BC8 is an amount effective in depleting the target hematopoietic stem cells or lymphocytes, for example, an affective amount in depleting at least 25%, or at least 50%, or at least 70%, or at least 80%, or up to 90% of the target hematopoietic stem cells. According to a particular aspect of this method, 225 An effective dose of Ac-BC8 is an amount effective for reversibly immunosuppressing target hematopoietic stem cells or lymphocytes, for example, at least 90%, or at least 92%, or at least 94%, or at least 96%, or up to 98%, of the target hematopoietic stem cells or lymphocytes without completely ablating them.

[0091] According to a particular aspect of this method, 225 An effective dose of Ac-BC8 is an amount sufficient to deplete target circulating tumor cells, e.g., hematopoietic stem cells or lymphocytes, e.g., at least 25% of target hematopoietic stem cells or lymphocytes, without bone marrow destruction, or an effective amount to deplete at least 50%, at least 70%, at least 80%, or up to 90% of target hematopoietic stem cells or lymphocytes. According to a particular embodiment, this amount is 225The dose of Ac-BC8 may be a low dose, for example, less than 150 μCi or less than 120 μCi, for example, 10 μCi to 100 μCi, 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. This dose may be administered alone or in combination with additional therapeutic agents as disclosed below.

[0092] According to a particular aspect of this method, 225 The effective dose of Ac-BC8, either as a monotherapy or in combination with other treatments, is the amount effective in depleting, reducing, or eliminating 25%, 50%, or 100% of hematopoietic carcinoma blasts in the affected body. Depending on the dose administered, a stem cell carrier may be required, depending on the dose that is effective in depleting, reducing, or eliminating carcinoma blasts in the affected body. When such cells are depleted or reversibly suppressed, or after their complete ablation, stem cell carriers may be provided to the affected body. For example, treatment of an affected body with a high cancer cell burden may require higher doses of radioactively labeled anti-CD45 antibody, which may result in a significant depletion or suppression of a large proportion of hematopoietic stem cells in the affected body. In such cases, stem cell carriers may be necessary to induce the regrowth of those cells. According to certain embodiments, stem cell carriers may not be necessary, and a sufficient number of hematopoietic stem cells may be present and capable of regrowth.

[0093] According to a particular aspect of this method, 225 The effective dose of Ac-BC8 is the amount effective in ablating (also known as myelolysis) 100% of the target hematopoietic stem cells. Exemplary doses include those indicated herein as at least high doses. The above method generally involves the effective amount of a single dose, such as a single-patient-specific dose. 225 This includes administering the drug to Ac-BC8. The amount of reduction in lymphocytes or hematopoietic stem cells may be determined by any of the methods disclosed herein above.225 The dose of Ac-BC8 may depend on the desired amount of depletion or immunosuppression. For example, hematopoietic stem cell depletion is 225 This may be achieved at low doses such as less than 2 Gy of Ac-BC8. Reversible immunosuppression of hematopoietic stem cells is 225 Ablation may be achieved with a dose of less than 8 Gy of Ac-BC8, for example, a dose of 2 Gy to 8 Gy. 225 High doses of Ac-BC8, such as 8 Gy or more, may be achieved at, for example, approximately 10-12 Gy. The above method uses a fractional dose, such as administering multiple portions of a single-symptom-specific dose or administering multiple single-symptom-specific doses, to obtain an effective amount 225 It may further include administering it to Ac-BC8. When administered together with the second agent, 225 The dose of Ac-BC8 may depend on the desired amount of depletion or immunosuppression.

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

[0095] Examples of non-cancerous disorders include, but are not limited to, abnormal hemoglobin disorders (e.g., SCD and β-thalassemia), congenital immunodeficiency (e.g., SCID and Fanconi anemia), and viral infections (e.g., HIV infection). In a particular embodiment, the disorder is SCD, and the treatment is gene-edited β-globin hematopoietic stem cell therapy. The stem cell therapy may be, for example, allogeneic or autologous. In a particular embodiment, 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. The stem cell therapy may be, for example, allogeneic or autologous.

[0096] According to a particular preferred embodiment of the method, the radioactively labeled anti-CD45 antibody is 225 This is radioactive BC8, such as Ac-BC8. 225 The effective dose of Ac-BC8 is an amount that is effective too to deplete, reversibly suppress, or ablate the target hematopoietic stem cells, and may 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 mass of the target. According to a particular embodiment, the method is (i) 0.1 μCi / kg to 5.0 μCi / kg 225 (ii) administering Ac-BC8 to the subject, and (ii) treating the subject 6, 7 or 8 days later to address the subject's impairment. According to certain other embodiments, the method includes (i) 0.1 μCi / kg to 1.0 μCi / kg 225 (ii) administering Ac-BC8 to the subject, and (ii) treating the subject after 6, 7, or 8 days to address the subject's impairment. In another embodiment, the method includes (i) 1.0 μCi / kg to 3.0 μCi / kg 225(ii) administering Ac-BC8 to the subject, and (ii) treating the subject after 6, 7, or 8 days to address the subject's impairment. In another embodiment, the method includes (i) 3.0 μCi / kg to 5.0 μCi / kg 225 (ii) administering Ac-BC8 to the subject, and (ii) treating the subject 6, 7 or 8 days later to address the subject's impairment.

[0097] Methods for treating malignant blood disorders This disclosure also provides methods for treating subjects suffering from malignant diseases or disorders, such as cancer. The methods may generally involve administering to a subject an effective amount of radiolabeled anti-CD45 antibody to deplete, reversibly suppress, or ablate the subject's hematopoietic stem cells (HSCs) or lymphocytes or hematopoietic carcinomas. According to at least one exemplary method, a low dose of radiolabeled anti-CD45 antibody is administered to reduce or deplete the number of hematopoietic stem cells or lymphocytes and / or circulating tumor cells without myelolysis. The dose may be a low dose as defined herein.

[0098] In a particular embodiment, the method may further include administering a stem cell carrier. In a particular embodiment, the method may further include performing a pre-conditioning treatment after an appropriate period of time. The pre-conditioning treatment may be a hematopoietic stem cell transplant, such as a bone marrow transplant, or adoptive cell therapy to treat the target cancer. According to a particular aspect of this method, the subject is suffering from cancer and is undergoing 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 to promote cancer regression in a subject and generally includes (i) harvesting autologous T cells (leukocyte extrusion), (ii) expanding the T cells (culture), (iii) administering non-myeloablative lymphocyte depletion chemotherapy to the subject, and (iv) administering the expanded T cells to the subject after the administration of non-myeloablative lymphocyte depletion chemotherapy. The method of this disclosure includes using a radiolabeled anti-CD45 antibody instead of and / or after the administration of expanded cells (e.g., T cells, NK cells, dendritic cells, etc.) in lieu of lymphocyte depletion chemotherapy. This later administration of anti-CD45 antibody (i.e., after administration of growing cells) may be used in preparation for transplantation of autologous stem cells (HSCTs) or for administration of a second effective dose or number of growing cells.

[0099] Accordingly, this disclosure provides a method for treating proliferative disorders such as hematological malignancies, comprising the administration of a radiolabeled anti-CD45 antibody and adoptive cell therapy. Adoptive cell therapy generally involves apheresis of autologous cells, which may be genes edited before reinfusion after lymphatic depletion with the radiolabeled anti-CD45 antibody (adoptive cell therapy such as CAR T-cell therapy). Alternatively, allogeneic cells may be reinfused after lymphatic depletion to provide adoptive cell therapy. According to the method of this disclosure, the radiolabeled anti-CD45 antibody may be provided as a single dose 3 to 9 days prior to adoptive cell therapy, for example, 6 to 8 days prior. According to a particular aspect of this method, the radioactively labeled anti-CD45 antibody is the radioactively labeled BC8 as described above, provided in the dose as described above, where the dose is generally specific radionuclide labeling (e.g., 225This depends on Ac-BC8). According to a particular aspect of this method, the appropriate time period after administration of the radiolabeled anti-CD45 antibody is 3, 4, 5, 6, 7, 8, or 9 days, for example, preferably 6, 7, or 8 days.

[0100] According to a particular embodiment, the method for treating a subject with cancer comprises (i) administering a single dose of a radiolabeled anti-CD45 antibody effective in depleting the subject's lymphocytes, and (ii) treating the subject's cancer by performing adoptive cell therapy on the subject after an appropriate time period (e.g., 6, 7, or 8 days). According to a particular embodiment, the method for treating a subject with cancer comprises (i) administering a single dose of a radiolabeled anti-CD45 antibody effective in depleting the subject's lymphocytes, and (ii) treating the subject's cancer by performing adoptive cell therapy on the subject after an appropriate time period (e.g., 6, 7, or 8 days). According to a particular aspect of this method, 225 The effective dose of Ac-BC8 is 0.01 μCi / kg to 5.0 μCi / kg of the subject's body weight.

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

[0102] Additional therapeutic agents The compositions and methods of this disclosure may be used in combination with certain additional therapeutic agents. For example, an additional immunotherapeutic agent 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, for example, International Publication 2019 / 094931, which is incorporated herein by reference in whole), and / or antibodies against CD34, CD117 and / or CD135 (see, for example, U.S. Provisional Patent Applications 62 / 838,646 and 62 / 838,589, which are incorporated herein by reference in whole).

[0103] The compositions and methods disclosed herein are disclosed radioimmunotherapy (e.g., 225 Ac-BC8) may be used in combination with a radiosensitizer that can enhance its effectiveness. For example, a Bcl-2 inhibitor is 225 It may be active against many cancer cell lines when used in combination with radiation, such as that provided by Ac-BC8. Additionally, small molecule inhibitors of the Bcl-2 protein show synergistic effects with other anticancer 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 some cancer therapies exert their effects by reversing this process. Involvement in apoptosis is triggered by the permeabilization of the mitochondrial outer membrane, a process regulated by the binding of different members of the Bcl-2 family. Furthermore, Bcl-2 family members also bind to the endoplasmic reticulum, modifying processes such as denaturing protein responses and autophagy, which also induce or modify different types of cell death. Bcl-2 overexpression was first described in follicular lymphoma as a result of t(14;18) transposition and as a poor prognostic marker in acute myeloid leukemia (AML) and non-Hodgkin lymphoma. Bcl-2 overexpression has subsequently been described in prostate cancer, breast cancer, and colon cancer, as well as glioblastoma. Overexpression of Mcl-1, another anti-apoptotic Bcl-2 related protein, has been 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 alterations in the ratio of pro-apoptotic Bcl-2 protein to anti-apoptotic Bcl-2 protein. Thus, in many cases, the inability of cancer cells to carry out the apoptotic program due to deficiencies in the normal apoptotic mechanism is associated with increased resistance to radiation and / or immunotherapy-induced apoptosis. Accordingly, the methods of the present disclosure may include the addition of a radiosensitizer, such as a Bcl-2 inhibitor, which may act synergistically with a radiolabeled anti-CD45 antibody to directly or indirectly induce apoptosis in cancer cells. Examples of Bcl-2 inhibitors include small molecule and antisense oligonucleotide drugs such as AT-101(-)gossypol, GENASENSE® (G3139 or oblimersen; Bcl-2 targeted antisense oligonucleotide), IPI-194, IPI-565, ABT-737, ABT-263, GX-070 (ovatocrax), and similar products.

[0105] In a preferred embodiment, the Bcl-2 inhibitor may be venetoclax, a drug approved for the treatment of 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, thereby ultimately leading to programmed cancer cell death (i.e., apoptosis). Ideally, by altering the balance between pro-apoptotic and anti-apoptotic stimuli, venetoclax facilitates programmed cell death in cancer cells and thus improves outcomes in affected individuals.

[0106] According to certain aspects, the radioimmunotherapy of this disclosure (for example, 225 Ac-BC8) is therapeutically effective in treating cancer when the amounts of a BCL-2 inhibitor and a radioactively labeled anti-CD45 antibody are administered together, (i) a BCL-2 inhibitor, (ii) a radioactively labeled anti-CD45 antibody (e.g., 225 It may be used in combination with a BCL-2 inhibitor such as venetoclax to provide a method for treating cancer-affected subjects, including administration to the subject together with Ac-BC8).

[0107] This disclosure is made by Venetocrax and 225 When the amounts of Ac-BC8 are administered together, they are therapeutically effective in treating acute myeloid leukemia, (i) BCL-2 inhibitors such as venetoclax, (ii) 225 The invention also provides a method for treating human subjects suffering from blood disorders or blood disorders, including administering Ac-BC8 to the subject together with the subject.

[0108] In this specification, 225 Administering BCL-2 inhibitors "together" with radiolabeled anti-CD45 antibodies such as Ac-BC8 is, 225This means administering a BCL-2 inhibitor before, during, or after administration of Ac-BC8. This administration is not limited to the following scenarios, namely, (i) first administering a BCL-2 inhibitor (e.g., orally once daily for 21, 28, 35, 42, 49 days, or longer, until the treated cancer does not progress and the BCL-2 inhibitor does not cause unacceptable toxicity), and secondly 225 (ii) Administer Ac-BC8 (for example, intravenously as a single dose or as multiple doses over a period of several weeks), (ii) administer a BCL-2 inhibitor 225 Administer Ac-BC8 simultaneously (for example, administer a BCL-2 inhibitor orally once daily for n days). 225 (iii) Administer Ac-BC8 as a single intravenous dose during one of the BCL-2 inhibitor regimens from Day 2 to n-1), (iii) BCL-2 inhibitor 225 Administer Ac-BC8 concurrently (for example, administer a BCL-2 inhibitor orally for a period exceeding one month (e.g., 35 days, 42 days, 49 days, or once daily orally for a longer period, provided the treated cancer does not progress and the BCL-2 inhibitor does not cause unacceptable toxicity)). 225 (iv) First 225 Treatment options include administering Ac-BC8 intravenously (e.g., as a single dose or in multiple doses over a period of several weeks), followed by administering a BCL-2 inhibitor orally once daily for 21, 28, 35, 42, or 49 days, or longer, until the treated cancer does not progress and the BCL-2 inhibitor does not cause unacceptable toxicity.

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

[0110] Additional radiosensitizers include, for example, histone deacetylase inhibitors (HDACi) such as vorinostat, belinostat, and romidepsin; metronidazole, misonidazole, intra-arterial Budr, intravenous deoxyuridine iodide (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, benzamide, nicotinamide, acridine intercalators, 5-thiotretrazole derivatives, 3-nitro-1,2,4-triazole, 4,5-dinitroimidazole derivatives, and hydroxylated texaphylline. Examples include texaphrins, cisplatin, mitomycin, tiripazamine, nitrosourea, mercaptopurine, methotrexate, fluorouracil, bleomycin, vincristine, carboplatin, epirubicin, doxorubicin, cyclophosphamide, vindesine, etoposide, paclitaxel, heat (hyperthermia), and similar therapies.

[0111] The term "histone deacetylase inhibitor" or "HDACi" refers to histone deacetylase inhibitors that can be classified into four classes: hydrooxamates (panobinostat (LBH-589), trichostatin-A (TSA), vorinostat (SAHA), belinostat (PXD101), NVP-LAQ824, and gibinostat (ITF2357)), cyclic peptides (romidepsin (depshipeptide)), 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 non-histone proteins. Some well-known mechanisms of HDACi lethality include DNA relaxation and derepression of gene transcription, as well as interference with chaperone protein function, free radical generation, induction of DNA damage, upregulation of endogenous inhibitors of cell cycle progression, and promotion of apoptosis. Interestingly, this class of agents is relatively selective for transformed cells, which have been shown to halt DNA repair after chemotherapy and enhance the efficacy of chemotherapy.

[0112] According to certain aspects, the radioimmunotherapy of this disclosure (for example, 225 Ac-BC8) is therapeutically effective in treating cancer when the amounts of HDACi and radioactively labeled anti-CD45 antibody are administered together, (i) HDACi, (ii) radioactively labeled anti-CD45 antibody (e.g., 225 It may be used in combination with HDACi such as vorinostat, belinostat, or romidepsin to provide a method for treating cancer-affected subjects, including administration to the subject together with Ac-BC8). This disclosure concerns HDA Ci and225 When the amount of Ac-BC8 is administered together with other HDACi such as vorinostat, bellinostat, or romidepsin, (ii) 225 The invention also provides a method for treating human subjects suffering from blood disorders or blood disorders, including administering Ac-BC8 to the subject together with the subject.

[0113] Similar to BCL-2 inhibitors, 225 Administering HDACi "together" with radiolabeled anti-CD45 antibodies such as Ac-BC8 is, 225 This means administering HDACi before, during, or after administration of Ac-BC8. This administration is not limited to the following scenarios, namely (i) first administering HDACi (e.g., for 21, 28, 35, 42, 49 days, or longer, once daily orally, or intravenously, on Day 1, Day 8, and Day 15 of a 28-day cycle, for a period of 21, 28, 35, 42, 49 days, or longer, until the treated cancer does not progress and the HDACi does not cause unacceptable toxicity), and secondly 225 (ii) Administer Ac-BC8 (for example, intravenously as a single dose or as multiple doses over a period of several weeks), (ii) HDACi 225 Administer simultaneously with Ac-BC8 (for example, administer HDA-BCi orally once daily for n days, or intravenously for n days). 225 (iii) Administer Ac-BC8 as a single intravenous dose during one of the HDACi regimens from Day 2 to n-1), (iii) HDACi 225 Administer simultaneously with Ac-BC8 (for example, administer HDACi orally for a period exceeding one month as described herein). 225 Ac-BC8 is administered intravenously as a single dose on the first day of the HDACi regimen, and (iv) first 225 One example is administering Ac-BC8 (for example, intravenously as a single dose or in multiple doses over a period of several weeks), followed by administering HDACi (as described herein).

[0114] manufactured goods The disclosure further provides a manufactured article comprising (a) radioactively labeled anti-CD45 immunoglobulin and (b) a label instructing the user to administer to a target an effective amount of immunoglobulin to deplete target hematopoietic stem cells. According to certain embodiments of the subject article, the effective amount of radioactively labeled anti-CD45 immunoglobulin is, 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 1.0 μCi / kg to 3.0 μCi / kg 225 Ac-BC8, or 3.0 μCi / kg to 5.0 μCi / kg 225 Ac-BC8, or 10μCi~120μCi 225 Ac-BC8, or 100μCi~250μCi 225 Ac-BC8, or 200μCi~500μCi 225 Ac-BC8, or 5μCi~80μCi 225 It could be AcBC8. 225 It is Ac-BC8. According to certain embodiments of the subject article, the effective amount of radioactively labeled anti-CD45 immunoglobulin is, for example, 1 μCi / kg to 500 μCi / kg or 1 μCi / kg to 100 μCi / kg 177 Lu-BC8, or 100 μCi / kg to 300 μCi / kg 177 Lu-BC8, or 300 μCi / kg to 500 μCi / kg 177 It could be Lu-BC8. 177 It is Lu-BC8.

[0115] While this disclosure will be better understood by referring to subsequent embodiments, those skilled in the art will readily recognize that the specific embodiments described in detail are merely illustrative of the disclosure as described more fully in the subsequent claims.

[0116] Embodiments of the present invention The following embodiments are disclosed in this application. Embodiment 1 A method for depleting a target hematopoietic stem cell, comprising administering an effective amount of radiolabeled anti-CD45 immunoglobulin to the target. Embodiment 2 The method according to Embodiment 1, wherein the effective amount of the radioactively labeled anti-CD45 immunoglobulin depletes at least 25%, or 50%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or 90% or less, or 95% or less of the target hematopoietic stem cells. Embodiment 3 The method according to Embodiment 1, wherein the effective amount of the radioactively labeled anti-CD45 immunoglobulin depletes at least 90% of the target hematopoietic stem cells, or at least 95% of the target hematopoietic stem cells, or at least 98% of the target hematopoietic stem cells, or at least 99% of the target hematopoietic stem cells, or 98% or less of the target hematopoietic stem cells, or 99% or less of the target hematopoietic stem cells. Embodiment 4 The method according to any one of Embodiments 1 to 3, further comprising administering an effective amount of a second therapeutic agent comprising one or more immunotherapeutic agents, radiosensitizers, or chemotherapeutic agents. Embodiment 5: The method according to Embodiment 4, wherein the immunotherapeutic agent comprises one or more antibodies against CD33, CD34, CD38, CD119, and CD135.

[0117] Embodiment 6: The method according to Embodiment 4, wherein the radiosensitizer comprises a Bcl-2 inhibitor or an HDAC inhibitor (HDACi). Embodiment 7 The method according to Embodiment 1, wherein the effective amount of the radioactively labeled anti-CD45 immunoglobulin depletes 100% of the target hematopoietic stem cells (i.e., ablates the hematopoietic stem cells). Embodiment 8 A method for depleting target lymphocytes, comprising administering an effective amount of radiolabeled anti-CD45 immunoglobulin to the target. Embodiment 9 The method according to Embodiment 8, wherein the effective amount of the radioactively labeled anti-CD45 immunoglobulin depletes at least 25%, or 50%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98%, or at least 99%, or 90% or less, or 95% or less, or 98% or less, or 99% or less of the target lymphocytes.

[0118] Embodiment 10 The method according to Embodiment 8, wherein the effective amount of the radioactively labeled anti-CD45 immunoglobulin depletes at least 25%, or 50%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98%, or at least 99%, or 90% or less, or 95% or less, or 98% or less, or 99% or less, of the target hematopoietic carcinoma cells. Embodiment 11 The method according to Embodiment 8, wherein the effective amount of the radioactively labeled anti-CD45 immunoglobulin depletes 100% of the target lymphocytes (i.e., ablates the lymphocytes) or depletes 100% of the target hematopoietic carcinoma blasts.

[0119] Embodiment 12 The method according to any one of Embodiments 1 to 11, wherein the subject is suffering from a non-cancerous disorder treatable via gene-editing cell therapy and is about to undergo such therapy to treat the disorder, and is administered an effective amount of the radiolabeled anti-CD4 immunoglobulin as a single dose. Embodiment 13 A method for treating a subject suffering from a non-cancerous disorder treatable via gene-edited cell therapy, comprising: (i) administering to the subject an effective amount of radiolabeled anti-CD45 immunoglobulin to deplete the subject's hematopoietic stem cells; and (ii) treating the subject's disorder by performing the treatment on the subject after an appropriate time period.

[0120] Embodiment 14 The method according to Embodiment 12 or 13, wherein the disorder is selected from the group consisting of abnormal hemoglobin disorders, congenital immunodeficiency, and viral infections. Embodiment 15 The method according to Embodiment 14, wherein the disorder is selected from the group consisting of sickle cell disease (SCD), severe combined immunodeficiency (SCID), β-thalassemia, and Fanconi anemia. Embodiment 16 The method according to Embodiment 14, wherein the disorder is SCD and the treatment is gene-edited β-globin hematopoietic stem cell therapy. Embodiment 17 The method according to Embodiment 14, wherein the disorder is SCID, the treatment is gene-edited hematopoietic stem cell therapy, and the editing 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] Embodiment 18 The method according to any one of Embodiments 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 the effective amount of the radioactively labeled anti-CD4 immunoglobulin is administered as a single dose. Embodiment 19 The method according to any one of Embodiments 8 to 11, wherein the subject has a cancerous disorder treatable via gene-edited cell therapy and is seeking such therapy to treat the disorder, and is administered an effective amount of the radiolabeled anti-CD4 immunoglobulin as a single dose. Embodiment 20: A method for treating a subject suffering from a cancerous disorder treatable via gene-edited cell therapy, comprising: (i) administering to the subject an effective amount of radiolabeled anti-CD4 immunoglobulin to deplete the lymphocytes of the subject; and (ii) treating the disorder of the subject by performing the treatment on the subject after an appropriate period of time. Embodiment 21: The method according to any one of Embodiments 18 to 20, wherein the cancerous disorder is a non-Hodgkin lymphoma, specifically acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, or diffuse large B-cell lymphoma.

[0122] Embodiment 22: The method according to Embodiment 19 or 20, wherein the gene-editing cell therapy is adoptive cell therapy for treating the cancerous disorder. Embodiment 23: The method according to Embodiment 22, wherein the adoptive cell therapy is CAR T cell therapy, the CAR T cell therapy comprises administration of gene-edited CAR T cells, and the gene-edited CAR T cells are unable to adequately express at least one checkpoint receptor and / or at least one T cell receptor. Embodiment 24: The method according to Embodiment 23, wherein the CAR T cell therapy is autologous cell therapy. Embodiment 25: The method according to Embodiment 23, wherein the CAR T cell therapy is allogeneic cell therapy. Embodiment 26: The radioactively labeled anti-CD45 antibody 225 Ac-BC8 or 177 The method according to any one of embodiments 1 to 25, wherein Lu-BC8 is used.

[0123] Appearance 27 225 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 225The effective amount of Ac-BC8 is either 2 μCi to less than 0.5 mCi, or at least 2 μCi to less than 120 μCi, or 10 μCi to less than 120 μCi, or 50 μCi to less than 250 μCi, or 177 The effective amount of Lu-BC8 is 1 μCi / kg to 500 μCi / kg of the target mass, or 1 μCi / kg to 100 μCi / kg of the target mass, or 100 μCi / kg to 300 μCi / kg of the target mass, or 300 μCi / kg to 500 μCi / kg of the target mass, or 177 The method according to embodiment 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 according to any one of Embodiments 1 to 27, wherein the anti-CD45 immunoglobulin comprises BC8, and the BC8 comprises a light chain having the amino acid sequence shown in SEQ ID NO: 1, or a light chain N-terminal amino acid sequence shown in SEQ ID NO: 9. Embodiment 29: The method according to any one of Embodiments 1 to 28, wherein the anti-CD45 immunoglobulin comprises BC8, and the light chain of BC8 comprises 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. Embodiment 30: The method according to any one of Embodiments 1 to 29, wherein the anti-CD45 immunoglobulin comprises BC8, and the BC8 comprises a light chain having the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13. Embodiment 31: The method according to any one of Embodiments 1 to 30, wherein the anti-CD45 immunoglobulin comprises BC8, and the BC8 comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 2, or a heaving chain N-terminal amino acid sequence shown in SEQ ID NO: 10.

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

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

[0127] Embodiment 40 The radioactive label BC8 225 It is 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, 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 The article according to embodiment 39, wherein the effective amount of Ac-BC8 is 2 μCi to less than 0.5 mCi, or 2 μCi to 250 μCi, or 75 μCi to 400 μCi. [Examples]

[0128] Example 1 - Production of anti-CD45 immunoglobulin BC8 Mouse anti-CD45 mAb BC8 was prepared from a hybridoma (ATCC number HB-10507) initially developed by fusing spleen cells from BALB / C mice hyperimmunized with human vegetative hemagglutinin (PHA)-stimulated mononuclear cells with mouse myeloma NS1 cells. After screening for microbial contamination, the original fusion cells were cultured in JRH-Biosciences EXCell300 medium supplemented with 1-2% fetal bovine serum (FBS).

[0129] The hybridoma cell line was adapted for culture in serum-free medium. In short, the cells were cultured using a combo medium supplemented with glutamine, cholesterol, insulin, and transferrin, and serum albumin was gradually and gradually removed from the culture medium. The cells were then cultured at a scale of up to 500 L, at a rate of 1 × 10⁶ cells. 6 The antibodies were grown to a density of over 1 / mL. The culture medium was collected and processed for the purification of anti-CD45 antibodies using a combination of cation exchange chromatography, protein A chromatography, and anion exchange membrane separation. The purified antibodies were 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 antibodies were characterized by SDS-PAGE, IEF, and SEC-HPLC methods. SEC-HPLC of approximately 0.6% of the aggregate recorded a single product peak (99.4%). 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% of both).

[0130] Example 2 - Sequencing of anti-CD45 immunoglobulin BC8 Total RNA was isolated from hybridoma cells according to the technical manual for Trizol® reagents. Total RNA was analyzed by agarose gel electrophoresis and reverse transcribed to cDNA using isotype-specific antisense primers or general-purpose primers according to the technical manual for the PrimeScript® 1st Strand cDNA Synthesis Kit. Antibody fragments of VH, VL, CH, and CL were amplified and cloned separately into standard cloning vectors using standard molecular cloning techniques. Colony PCR screening was performed to identify clones with accurately sized inserts. For each antibody fragment, more than 5 single colonies with accurately sized inserts 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. The BC8 antibody was deglycosylated, reduced, and digested with individual enzymes, namely trypsin, Lys-C, and chymotrypsin. 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 the BC8 antibody showed that the actual amino acid sequence differed from that predicted by the DNA sequence, based solely on a single amino acid in the heavy chain. As highlighted in Figures 5A and 5B, ASN-141 was predicted by the codon encoding the amino acid at position 141, but the actual ASP-141 was not found by protein sequencing. Furthermore, sequencing of various batches of the protein revealed different amounts of ASP and ASN at position 141, indicating that the protein contains both ASN-141 and ASP-141 in ratios of 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 be dependent on the cellular environment and, in some cases, may be related to protein age (e.g., potentially providing a signal for proteolysis). However, the fact that no other deaminated amino acids have been identified may indicate an important and specific role for ASP-141. At the very least, ASP-141 may be located in an exposed or accessible region on the folded protein. That is, ASP-141 may be an accessible solvent and may reside in a conformationally flexible region of the antibody. The effect of deamination on the biological activity of the BC8 antibody can be determined from the results of human clinical trials.

[0134] Example 3-BC8: 225Labeling and purification for forming Ac-BC8 Conjugation of BC8 and unrelated control mAb18B7 (mouse IgG1) with DOTA Antibodies against CD45 (i.e., BC8 antibody) and controls (i.e., mouse monoclonal reactivity to 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 rotations using a Centricon filter with a MW cutoff of 50,000 or a Vivaspin ultrafiltration tube with a MW cutoff of 50,000, using 1.5 ml (mL) of conjugation buffer per rotation. For each rotation, the antibodies were rotated at 53,000 RPM for 5–20 minutes at 4°C in a Thermo IEC Centra CL3R centrifuge with a fixed-angle rotor to obtain a residue volume of 100–200 microliters (μL). Rotation times varied 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 rotations.

[0135] [Table 2]

[0136] For conjugation, a solution of 3 mg / mL DOTA-pSCN (MW=678) in 0.15 M NH4OAc was prepared by vortexing. DOTA-pSCN and antibody (>5 mg / mL) were mixed together in Eppendorf tubes in 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 ultrafiltration seven times as described above, and each time the tube was washed with 1.5 mL of 0.15 M NH4OAc buffer (pH ≤6.5, approximately 100 μL). After the final wash, 0.15 M NH4OAc 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 using a simplified Lowry method. The number of DOTA molecules conjugated to the antibody (molar ratio of DOTA to protein) 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 determining the molar ratio of DOTA to protein are shown in Table 2.

[0138] 225 Radioactive labeling of DOTA-antibody conjugates with Ac The reaction mixture 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 in 0.05 M HCl. 225 Ac(10μCi) was added (see Figure 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 any unreacted particles. 225 Ac 225 It bound to the Ac-DTPA complex.

[0139] Rapid thin-layer chromatography (ITLC) using a 10 cm silica gel strip and a 10 mM EDTA / saline mobile phase was used to extract free 225 Ac( 225 From Ac-DTPA) 225 Ac-labeled BC8 ( 225 By separating 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 migrated with the solvent front. The strip was cut in half and counted with a gamma counter equipped with a multi-channel analyzer using channels 72-110 for Ac to exclude its daughter. 225 The results of the selected radiolabeling are shown in Table 2, which shows that the conjugate formed with 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 the following tracer experiments (Batch A).

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

[0141] Example 4- 225 Ac-BC8: Stability 225 Determination of the stability of Ac-DOTA-BC8 DOTA-BC8 (Batch A) was used in all immunoreactivity experiments, and the specific activity was set to 1 μCi / μg as described in the method above. 225 It was labeled with Ac for radioactivity. For stability determination, 225 Ac-DOTA-BC8 was tested in its original volume of 20 μL, or diluted to 40 μL or 60 μL with working buffer (0.15 M NH4OAc), incubated at room temperature (rt) for 48 hours or at 4°C for 96 hours, and tested by ITLC. All samples were analyzed repeatedly, and the experiment was performed three times. The results shown in Figure 11 indicate that actinium-225 labeled BC8( 225 The results show that Ac-DOTA-BC8 remained stable for up to 96 hours at 4°C.

[0142] Example 5- 225 Ac-BC8: Immunoreactivity Using cell systems 225 Determination of Ac-DOTA-BC8 immunoreactivity (IR) DOTA-BC8 (Batch A) was used in all immunoreactivity experiments, and the specific activity was set to 1 μCi / μg as described in the method above. 225 The cells were radioactively labeled with Ac. Ramos CD45-positive cells and control CD45-negative EL4 cells were used in duplicate, in quantities ranging from 1,000,000 to 7,500,000 cells per sample. The experiment was performed twice. The results shown in Figure 12 indicate that 225 Ac-DOTA-BC8 specifically binds to Ramos cells, with a binding rate of 50% to these cells compared to only about 10% to control EL4 cells. However, sequential growth of the two cell lines in the laboratory for QC is not cost-effective, and a simpler assay was desired for IR determination. As a control, the following conditions were used: Ramos cells, EL4 cells, and EL4 cells pre-blocked with 1% BSA.

[0143] Cytotrol cells 225 Determination of Ac-DOTA-BC8 immunoreactivity (IR) First, using Cytotrol cells (Beckman Coulter), the binding rate of naive BC8 antibody to control 18B7 antibody (a nonspecific control antibody against the fungal polysaccharide glucuronoxylomannan) to these cells was determined by flow cytometry (Figure 13A). Cells were taken into RPMI medium, and the secondary antibody was Biolegend's PE-labeled rat anti-mouse IgG1. Cytotrol cells were lyophilized human lymphocytes isolated from peripheral blood exhibiting the CD45 surface antigen and were selected based on their commercial availability (Beckman Coulter) and consistency. The binding rate of naive BC8 to Cytotrol cells was compared with that of DOTA-BC8 (Figure 13B). Naive BC8 showed strong binding to Cytotrol cells, while the control 18B7 mAb only bound at a background level (Figure 13B). Attachment of DOTA to BC8 reduced its IR to approximately 70% of that of naive BC8 (Figure 13B).

[0144] [Table 3]

[0145] Next, IR determination was performed for 225Ac-DOTA-BC8. To measure the binding rate of the radiolabeled antibody to Cytotrol cells, three tubes of Cytotrol cells (lot 729154) were used for each sample, and the binding rate was measured for duplicate samples. The vials were washed with 0.5 mL of reconstitution buffer, and the cells were pooled. The vials were further washed with two 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), rotated again, and resuspended in 0.5 mL of RPMI / BSA. Approximately 25,000 CPM of labeled antibody was added to each vial. The vials were incubated at 37°C for 1 hour, shaken at 150 RPM, and rotated 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 mean IR was 64.8 ± 2.14%.

[0146] Finally, after comparing the binding rates of DOTA-BC8 samples to Cytotrol cells using flow cytometry (Figure 14B), 225 The same sample was rapidly radioactively labeled with Ac, and Cytotrol cells were bound to the radioactively labeled sample (Figure 14A). The binding rate of DOTA-BC8 to cells by flow cytometry (approximately 60% of the naive BC8 binding rate) was observed in the radioactively labeled Cytotrol cells. 225 The binding rate of Ac-DOTA-BC8 was matched. Therefore, the cytotrol assay routinely binds to cells at 64.8 ± 2.14%. 225 It proved to be a convenient and cost-effective method for evaluating the IR of Ac-DOTA-BC8.

[0147] Example 6- 225 Ac-BC8: Immunoradiotherapy for Multiple Myeloma 225Evaluation of the compatibility of human H929 multiple myeloma cells and human U266 multiple myeloma cells as model cell lines for radioimmunotherapy (RIT) of multiple myeloma using Ac-DOTA-BC8. Multiple myeloma (MM) cell lines H929 and U266 were purchased from the American Type Tissue Collection (ATTC) and grown according to ATCC instructions. After measuring the binding rate of unlabeled DOTA-BC8 to both cell lines by flow cytometry (Figure 15A), the cells were then subjected to... 225 Ac-DOTA-BC8 was coupled (Figure 15B). Next, 225 H929 and U266 cells were used for an in vitro cell death assay with Ac-DOTA-BC8. Two doses were used. 225 Ac-DOTA-BC8 (20 pCi / mL and 250 pCi / mL) was used. Cell incubation with radioactively labeled antibodies was performed in a 200 μL total volume 96-well plate. The same two doses of control antibodies were used. 225 Ac-DOTA-18B7 was used. Cells were washed from unbound radioactivity at 4 and 12 hours, and their viability was assessed by Trypan blue assay after 3 days (Table 4). Death of both cell lines was antibody-specific and dose-dependent. That is, both cell lines were antibody-specific and dose-dependent. 225 Regarding specific targeting with Ac-DOTA-BC8, it was possible to express a sufficient amount of CD45 on their surface and use them for subsequent in vivo experiments.

[0148] [Table 4]

[0149] Example 7- 225 Ac-BC8: Distribution in the body In the Naive Mouth Model 225 Distribution of Ac-DOTA-BC8 in the body The purpose of this study is to confirm the baseline distribution and clearance of the organism in a naive mouse model in the absence of disease. 225against Ac-DOTA-18B7 antibody 225 The objective was to evaluate the pharmacokinetic biodistribution of Ac-DOTA-BC8. As detailed above, DOTA-conjugated BC8 antibody (batch A) and 18B7 antibody (produced in the same molar ratio as batch A; 7.5 moles of DOTA relative to Ab) were used. 225 The antibodies were radioactively labeled with Ac. The antibodies were radioactively labeled with a specific activity of 0.4 μCi / μg. 225 The immunoreactivity of Ac-DOTA-BC8 was tested in Cytotrol cells and showed a binding rate of 55%, which met the minimum binding rate 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 administered intraperitoneally. Each mouse received 5 μg (2 μCi) of radiolabeled antibody in 100 μL of 0.15 M NH4OAc buffer along with ascorbic acid. The intraperitoneal route was chosen to avoid contamination of personnel, animals, and facilities (i.e., due to the possibility of back pressure splash from the tail). 225 For long-lived radionuclides such as Ac, intraperitoneal injection is preferable to tail vein injection. According to the inventors' own data and data from other groups, antibodies injected intraperitoneally disappear completely from the peritoneum within one 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, intestines, and blood) were collected from each mouse and weighed. 225 The cumulative activity per tissue was counted using a gamma counter with the Ac energy window.

[0151] The percentage of injection dose per gram (ID / g(%)) is shown in Figures 16A and 16B. The results show that the biodistribution (biodistrbution) and pharmacokinetic clearance patterns of the two antibodies were very similar, which demonstrates the overall stability of the one-step labeled antibody in vivo. Clearance from blood and blood-rich organs, as well as control 225 The uptake of Ac-DOTA-18B7 was somewhat lower, which can be explained by the lack of homology between the mouse protein and the 18B7 antigen (fungal polysaccharide glucuronoxylomannan). Using the data in Figures 16A and 16B, and Prizm 5.0 software (GraphPad, San Diego, California, USA), the following data were obtained regarding blood and blood-rich organs (lungs and heart). 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 indicates that 225 The half-life of Ac-DOTA-BC8 is approximately 100 hours (4.2 days), which is typical for full-size mouse IgG1 against mammalian antigens. 225 The half-life of Ac-DOTA-18B7 (and similarly mouse IgG1) is shown to be only 30 hours (1.25 days), likely due to the exogenous nature of their respective antigens (fungal polysaccharides). Therefore, the radiolabeled antibodies appear to be stable in vivo, rapidly disappearing from blood and blood-rich organs, and suitable for use in subsequent pharmacokinetic experiments in CD45-positive tumor-bearing mice.

[0152] [Table 5]

[0153] In myeloma-carrying SCID mice 225 Distribution of Ac-DOTA-BC8 mAb in the body The purpose of this study is to provide a control in the multiple myeloma (SCID) mouse model. 225against Ac-DOTA-18B7 antibody 225 The objective was to understand the in vivo distribution of Ac-DOTA-BC8. Its specific activity is 0.4 μCi / μg, as described above. 225 DOTA conjugates BC8 (Batch A) and 18B7 (as described above) were radiolabeled with Ac. Their immunoreactivity was tested in Cytotrol cells, showing a binding rate of 61%, which met the minimum binding rate requirement of 50%.

[0154] 50 female 4-5 week old mice with SCID-NOD (severe combined immunodeficiency non-obese diabetes) (Charles River Laboratories) had 10 samples placed on their right flank. 7 Ten human multiple myeloma H929 cells (ATCC) were placed in the left flank. 7 Human multiple myeloma U266 cells (ATCC) were subcutaneously injected. After approximately 20 days, when the tumors reached a diameter of 3-4 mm, 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 was then administered 0.4 μCi (1 μg) of radiolabeled antibody in 50 μL of 0.15 M NH4OAc buffer along with ascorbic acid. As described above, the intraorbital route is preferred over tail vein injection to avoid the possibility of back pressure splash from the tail. Mice were euthanized at 1 hour, 4 hours, 24 hours, 48 ​​hours, 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, intestines, and blood) were collected and weighed from each mouse. 225 The cumulative activity per tissue was counted using a gamma counter with the Ac energy window. The results are shown in Figures 17A and 17B as the percentage of injection dose per gram (ID / g(%)). This applies to H929 tumors and U266 tumors. 225 Acquisition of Ac-DOTA-BC8 is performed as follows: 225The levels were significantly higher (P=0.01) than those of Ac-DOTA-18B7. Both antibodies were rapidly eliminated from the blood and blood-rich organs. Importantly, in the bone marrow... 225 There was no uptake of Ac-DOTA-BC8, which demonstrates that there was no homology to human CD45 in mouse bone marrow. These results indicate that 225 This study demonstrates that Ac-DOTA-BC8 specifically localized in H929 and U266 tumors, and therefore could be used for further radioimmunotherapy (RIT) experiments.

[0155] Example 8- 225 Ac-BC8: Radioimmunotherapy for Tumors in Mice Radioimmunotherapy (RIT) of H929 and U266 tumors in SCID-NOD mice using 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. Similar to in vivo distribution experiments, 10 7 Human multiple myeloma cells from H929 (right flank) and U266 (left flank) were subcutaneously injected into mice. After approximately 19 days, when the tumors reached a diameter of 3-4 mm, the mice were randomized into eight groups and given 0.3 μCi of human multiple myeloma cells. 225 Ac-DOTA-BC8, 0.3μCi 225 Mice were either intraorbitally treated with an Ac-DOTA-18B7 control mAb or a suitable 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 30 days for the size of their tumors and their overall health. Figures 18A and 18B show the results of the RIT trial for H929 tumors and U266 tumors. 225 Ac-DOTA-BC8 showed a significant therapeutic effect. 225 The compatibility activity of Ac-DOTA-18B7 had some effect on tumor size, 225The compatibility activity was significantly lower (P=0.02) than that of Ac-DOTA-BC8. Therefore, it is clear that RIT in mice carrying 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 experiment, the mice were sacrificed, their tumors were excised, placed in ethanol, then in buffered formalin, treated with paraffin (paraffinized), cut into 5 μm sections, and stained with H&E. Figures 19A–19D show untreated mice and 225 The images show H929 and U266 tumors obtained from Ac-DOTA-BC8 treated mice. Untreated tumors are significantly more coherent than RIT-treated tumors, which exhibit a lack of coherence and necrosis.

[0157] Example 9- 225 Ac-30F11: Myeloablative effect of an anti-CD45 substitute. In this study, in mice used for targeted pretreatment before BMT 225 The tolerability and myelolytic effects of Ac-labeled anti-mouse pan-CD45 antibody clone 30F11 were evaluated. Therefore, B6-Ly5 a against mice 225 The dose-dependent myelolytic effect of Ac anti-CD45 antibody (30F11) was evaluated. Furthermore, the study evaluated B6-Ly5 b The degree of transplant and donor chimerism after genetically related bone marrow transplantation was evaluated (CD45 allotype difference to monitor chimerism).

[0158] Experimental method: (1) Conjugation and signage for 30F11. As described above, the anti-CD45 antibody 30F11 was conjugated with the chelating agent DOTA. To test whether DOTA-conjugated 30F11 retains immunoreactivity, CD45-positive cells were incubated with bare 30F11 and DOTA-30F11, and anti-rat IgG2b was detected to detect the binding antibody.PE The amount of bound Ab was determined by flow cytometry using [the specified method]. As mentioned above, DOTA-30F11 111 In or 225 The antibodies were radioactively labeled with Ac, and the specific activity was set to 5 μCi / 1 μg (1:1) or 1 μCi / 1 μg (1:1), respectively, with a radiochemical purity of 99 ± 1.

[0159] (1) In vivo distribution of the anti-CD45 antibody 30F11 in C57B1 / 6 mice. 60 μg of C57Bl / 6 mice with a specific activity of 5 μCi / μg 111 In-30F11 was administered intravenously. 1 to 240 hours after the injection, the spleen... 111 In-labeled 30F11 was found to have the highest uptake, followed by the bone marrow and liver. The kidneys, ovaries, lungs, and blood showed the lowest uptake. The in vivo distribution for each organ was fitted to a time-activity curve to calculate the cumulative activity for each organ. Then, to obtain the dose to each organ per dose of activity 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 Ac-CD45 antibody radioconjugates, C57Bl / 6 (5 animals per cohort) were subjected to increasing doses on Day 0. 225The mice were treated with Ac-30F11. A total of 10 ug (approximately 0.5 mg / kg) of 30F11 antibody (100-200 uL) was administered intravenously via tail vein injection at three increased dose levels (100 nCi, 250 nCi, and 500 nCi). Five untreated mice were provided as controls for this study. Immediately before pretreatment, pre-treatment blood samples were obtained from control mice for baseline blood cell count measurement. RBCs and WBCs were measured in Week 1 and Week 2, respectively, and the mice were euthanized in Week 4. Blood was collected from the euthanized mice and analyzed for hepatotoxicity and nephrotoxicity. Specifically, blood urea nitrogen (BUN), creatine, alanine transaminase (ALT), and aspartate aminotransferase (AST) were measured. Kaplan-Meier graphs showed that doses of 100 nCi and 250 nCi were well tolerated, but the 500 nCi dose was associated with a decrease in survival probability at one week.

[0162] (3) B6-Ly5 a In mice 225 Safety profile of the AC-30F11. To determine the safety profile of 225Ac-CD45 bone marrow transplantation, C57B1 / 6 mice were used as follows: 225 The cells were treated with Ac-30F11 and reconstituted with donor bone marrow (CD45.1). That is, 100 nCi or 250 nCi cells were used. 225 Ac-30F11 was injected on Day 0 (as described above). Four days after pretreatment, half of the cohort received 10 via tail vein injection. 7 Target density of individual nucleated cells in bone marrow: C57Bl / 6-Ly5 b The mice were administered biomedical bone marrow (BMT) extracted from mice. They were regularly monitored for any obvious changes in body weight, health, and behavior.

[0163] Transplantation and donor chimerism were evaluated by blood collection, and mice were euthanized at 12 weeks. Transplantation was evaluated by total WBC count, total RBC count, total HSC count, total neutrophil count, and total platelet count, as well as BUN, creatinine, ALT, and AST. Result: 500nCi 225 Ac-30F11 was found to be the maximum tolerated dose for this myelosophistication phase. 250 nCi and 100 nCi 225 Mice treated with Ac-30F11 showed effective myeloablative conditioning and donor BM transplantation in a dose-dependent manner, without any long-term hematological toxicity. Conclusion: 225 Ac-armed pan-CD45 targeted antibody 30F11 appears to be a safe and effective targeted pre-treatment approach for BMT. This data suggests that 225 This supports the development of CD45-targeted ablation prior to BMT using Ac-equipped antibodies.

[0164] Example 10- 177 Lu resistance CD45 and 131 Lymphatic depletion due to anti-CD45 It is common practice to perform a lymph depletion step, often using high-dose chemotherapy, before the affected patient receives a certain dose of adoptive cell transfer, such as engineered autologous CAR-T cells or engineered allogeneic CAR-T cells. This process is considered important to create sufficient space within the immune microenvironment, such as the bone marrow, to enable transplantation of the transferred cells. Furthermore, it is thought to induce a favorable cytokine profile for the establishment and proliferation of donor lymphocytes. In this study, beta-emitting cells were used to mediate effective lymph depletion in a mouse model. 177 The use of Lu (half-life of 6.6 days, pathway length of 1.5 mm) will be tested. In a mouse model, the response to targeted RIT lymphoid depletion in specific immune cell types and the resulting changes in immune cytokine expression will be investigated. 177 Lu indicator and 131 Preclinical trials were conducted using I-labeled surrogate anti-mouse pan-CD45 antibody (30F11).

[0165] Non-myeloablative dose 177Following a single dose of Lu-CD45-RIT, peripheral blood, bone marrow, and spleen samples were collected from 8-12 week old C57Bl / 6 mice 96 hours and 10 days after treatment for immunophenotyping to evaluate lymphocyte subsets and bone marrow subsets for lymph depletion, as well as serum for cytokine profiling. 177 Lu-CD45-RIT has been shown to effectively lymphodeplete both lymphocytes, including immunosuppressive T regs and MDSCs, and myeloid cells. We also present a study evaluating this targeted lymphodepletion regimen in E.G7 lymphoma-carrying mice prior to adoptive cell transfer with OVA-specific CD8+ T cells.

[0166] Methods and materials Anti-mouse pan-CD45 antibody 30F11 is used with lutetium-177 ( 177 Lu-CD45) and iodine-131 ( 131 Target lymphoid depletion was performed in mice using BC8 cells labeled with I-CD45 and used as a substitute for radiolabeled pan-human BC8 cells. Immunoreactivity was confirmed to be over 95% in CD45+ cell-based binding assays. For lymph depletion studies in mice, to determine the ability to selectively deplete a subset of immune cells, 20 μCi or 40 μCi of either were used. 177 Lu or 50 μCi or 100 μCi 131 Female pubescent C57Bl / 6 mice were treated with 20 ug of 30F11 labeled with 1I. Immune cell subsets were quantified by flow cytometry. For a lymphatic depletion study in the OT I mouse model, female pubescent C57Bl / 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 injection of tumor cells, the mice were... 177 Lu-CD45 (40μCi), 131The mice were either treated with ICD45 (100 μCi) or not subjected to lymphatic depletion. Four days after lymphatic depletion, isolated CD8+ T cells isolated from CD45.2OT I mice were administered to the mice. Tumor volume and body weight were monitored, and when the tumor volume reached 4000 mm³, 3 Mice were euthanized if the blood pressure exceeded a certain level or if necrosis occurred.

[0167] result The anti-CD45 antibody was conjugated to DOTA in a 20:1 ratio, and then in a 5:1 ratio. 111 Labeled with In. 60 μg of a specific activity of 5 μCi / μg. 111 In-labeled anti-CD45 antibody was intraperitoneally injected into C57Bl / 6 mice, and antibody distribution was monitored by microSPECT / CT at the indicated time points. The CD45 antibody returned to immune system organs, namely lymph nodes, spleen, and bone marrow (see Figure 20). Radiolabeled anti-CD45 antibody 177 Lu-CD45 and 131 I-CD45 was found to transiently deplete a subset of CD45+ immune cells without affecting platelets, red blood cells, or bone marrow cells. As shown in Figure 21, (A) 20 μCi or 40 μCi 177 Lu-CD45 or (B) 50μCi or 100μCi 131 Treatment of tumor-free C57B / 6 mice with -I CD45 antibody was equally effective in transiently depleting various immune cell populations of lymph without affecting bone marrow cells, red blood cells, or platelets.

[0168] On top of that, 177 Lu radioactively labeled anti-CD45 antibody was found to transiently deplete a subset of CD45-expressing immune cells in the spleen. As shown in Figure 22, 40 μCi 177 Treatment of tumor-free C57B / 6 mice with Lu-CD45 antibody was effective in transiently depleting various immune populations in the spleen, including regulatory T cells (T-regs). This lymphoid depletion enabled tumor control in the OT1 adoptive cell therapy model.

[0169] As shown in Figure 23, after E.G7 tumor transplantation, mice were either untreated (untreated and OT I) or treated on Day 0 with 40 μCi of 177Lu-CD45 or 100 μCi of 131I-CD45, and then on Day 4, 1 × 10 6 Each individual OT I CD8+CD45.2OVA-reactive T cell was administered. Panel A shows the results before adoptive OT IT cells enabled control of EG.7 tumor growth. 177 Lu-CD45 and 131 The results of targeted pretreatment mediated by I-CD45 are shown. Panel B shows tumor size for individual mice in each group. At sacrifice, residual and enlarged OT1 T cells were observed in the mice. Panel C shows control mice (i.e., those not treated or administered OT1 T cells), mice administered OT1 T cells, and 177 Lu-CD45 and 131 This shows the overall survival rate of mice that also received targeted pretreatment mediated by I-CD45.

[0170] conclusion These trials use low doses as a transient non-myeloablative targeted lymphocyte depletion regimen prior to adoptive cell therapy. 177 Lu-CD45 radioimmunotherapy or 131 This demonstrates the feasibility of using I-CD45 radioimmunotherapy. 111 In-CD45 imaging demonstrated that CD45 targeting selectively delivers radiation to immune-privileged tissues. The study used 40 μCi 177 Lu-CD45 or 100μCi 131 We determined that I-CD45 was capable of effectively depleting various immune cell subsets in mice while preserving myeloid cells, erythrocytes, and platelets. In a adoptive cell therapy model using CD45.1OT1 mice carrying EG.7-OVA tumors, mice subjected to RIT-mediated lymphoid depletion showed enhanced tumor control compared to mice that did not receive lymphoid depletion. This data supports non-myeloablative doses. 131I-CD45RIT or 177 Supports CD45-targeted lymphoid depletion 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 SCD-affected individuals. Sickle hemoglobin (SCD) is the most common abnormal hemoglobin disorder worldwide. The prevalence of SCD among African Americans is approximately 1 in 500. In the United States, an estimated 100,000 individuals are affected. SCD is caused by a single nucleotide mutation in the β-globin gene that produces sickle hemoglobin. Individuals with SCD may exhibit anemia, vasoconstrictive organ failure (VOC), hemolysis, chronic organ dysfunction, and premature death. The childhood mortality rate for SCD is 0.5 per 100,000 people. However, the mortality rate in adults is over 2.5 per 100,000 people, and the median life expectancy for both men and women with SCD is under 50 years. Currently, the only curative treatment for SCD is hematopoietic stem cell transplantation (HSCT). Unfortunately, HSCT for SCD is not without its problems. According to the International Blood and Bone Marrow Transplant Research Center, only 1,089 SCD patients 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] The advent of gene editing technology now presents the opportunity to cure SCD-affected individuals using autologous stem cells with corrected mutations in the β-globin gene that cause SCD. Editing approaches mediated by ZFNs, TALENs, CRISPR / Cas9, and other nucleases have been available to repair stem cells or to remove and replace stem cells from SCD-affected individuals. For example, Sun and Zhao (Biotech. And Bioeng., 2014, 111(5)) demonstrated successful repair of human β-globin gene mutations in affected pleuripotent HSCs using TALENs. In addition, Dever et al. (Nature, 2016, 539:384-389) demonstrated efficient repair of SCD-affected Glu6Val mutations in affected HSCs using CRISPR / Cas9. Clinical trials using this approach for SCD are now underway.

[0173] Unfortunately, standard myeloablative conditioning regimens using high-dose chemotherapy or total body irradiation (i.e., 100% HSC depletion regimens) 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 affected individuals. Radiolabeled BC8 (e.g., 225 Ac-BC8 preserves more normal tissue from affected individuals. In particular, older affected individuals with SCD may already have organ damage as a result of their disease, and exposure to nonspecific radiation or chemotherapy as myeloablative conditioning regimens could make stem cell transplantation even more risky. The radiolabeled BC8 approach presents a better option for these affected individuals.

[0174] Furthermore, due to the genetic nature of the disease, disease modification by transplanting gene-edited HSCs is preferable to be performed as early as possible, as disease complications may be irreversible and may have a negative impact on the long-term survival of affected individuals. Therefore, it is conceivable to treat infants or young children with SCD using gene-edited HSCs. For this purpose, radiolabeled BC8, in particular, 225 BC8 labeled with alpha-emitting radionuclides such as Ac is ideal. It has a very short, high-energy radiation path length. 225 The use of alpha-emitting radionuclides such as Ac concentrates radiation into CD45-positive cells, (myeloablative dose) 131 Effective myelostomy becomes possible without the need to isolate treated infected bodies (as is required for pre-treatment in 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 associated B-cell deficiency. SCID involves a failure of adaptive immune response that prevents affected individuals from initiating an effective antibody response against pathogens. SCID is the most severe form of primary immune deficiency, and there are at least nine different known genes through which mutations result in SCID. Because individuals with SCID are unable to initiate an adaptive immune response, they are susceptible to infections and have a high rate of early mortality. SCID is also known as "bubble boy" disease because affected individuals must be kept in a sterile environment to avoid life-threatening infections. The most frequent gene deficiency 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, only treatment with stem cell transplantation is potentially curative for SCID. However, delayed immune recovery and GVHD are significant risks for these affected individuals. Also, like SCD affected individuals, SCID affected individuals are young and therefore require effective and safe methods for treatment, including better pre-transplant conditioning regimens.

[0176] Gene editing technology makes it possible to precisely repair defects in HSCs (Hypercytocytes) in SCID-affected organisms themselves. When these modified HSCs are returned to the body, they become capable of producing normal lymphocytes and establishing an adaptive immune response that acts to protect against infection. Recently, Chang et al. (Cell Reports, 2015, 12:1668-1677) reported the effective restoration of normal lymphocyte development through CRISPR / cas9-mediated repair of mutations in the JAK3 gene in mice. Furthermore, Alzubi et al. (Nature, Scientific Reports, 2017, 7:12475) recently demonstrated the use of TALEN technology to precisely repair gene defects in IL2RG (common gamma strand), the gene responsible for X-SCID, in mice. It is important to develop safer and more effective methods for pre-treating human SCID patients. For example, to safely pre-treat these mainly young patients, 225 Ac-BC8-mediated alpha-emitting CD45 radioimmunotherapy is necessary.

[0177] Example 13- 225 Ac-BC8: Overview of treatments for non-malignant injuries Table 7 shows the actinium-labeled BC8 antibody (i.e., pretreatment agent; 225This summarizes selected treatment regimens using gene-edited stem cell administration after HSC depletion via Ac-BC8 administration.

[0178] [Table 7]

Claims

1. (i) A population of T cells expressing a chimeric antigen receptor or T cell receptor (CAR / TCR) in the treatment of malignant hematological diseases In preparation for administration to the subject, a non-myeloablative amount of radiolabeled anti-CD45 antibody, which is an actinium-225-labeled (225Ac) anti-CD45 antibody, is included for administration to the subject as a single dose. The aforementioned malignant blood disorder is lymphoma, in this composition.

2. The composition according to claim 1, wherein the radioactively labeled anti-CD45 antibody comprises a radioactively labeled BC8 having 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, and 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.

3. The radioactively labeled anti-CD45 antibody comprises BC8 having a light chain having a complementarity-determining region having the amino acid sequence 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 sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:

8. The composition according to 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 according to claim 1, wherein the radioactively labeled anti-CD45 antibody comprises BC8 having a light chain having the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13 and a heavy chain having the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO:

16.

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

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

1.

7. The composition according to 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 the bone marrow.

8. The composition according to any one of claims 1 to 7, wherein the non-cancerous disorder is selected from the group consisting of abnormal hemoglobin disorders, congenital immunodeficiency, and viral infections.

9. The radioactively labeled anti-CD45 is a BC8-containing 225Ac-labeled anti-CD45 antibody having a light chain having a complementarity-determining region having the amino acid sequence 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 sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, and the dose is the non-myeloablative amount consisting of a dose of 10 μCi to 150 μCi. The composition according to 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 according to 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. A composition comprising a non-myelogenically destructive amount of an actinium-225-labeled (225Ac) anti-CD45 antibody containing radiolabeled BC8 for administration as a single dose to a target in preparation for administering an effective amount to a target cell population expressing a chimeric antigen receptor or T cell receptor (CAR / TCR) in the treatment of lymphoma.

12. The composition according to claims 1 to 11, wherein the lymphoma is Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), mediastinal primary B-cell large cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, perisplenic zone lymphoma, lymphocytic lymphoma, or T-cell lymphoma.

13. A composition for treating diffuse large B-cell lymphoma, Contains an effective amount of radioactively labeled 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 A composition containing Lu.

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