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

Abstract

To provide compositions and methods for transient immunodepletion of specific subsets of a subject's immune cells.SOLUTION: The inventive methods comprise administering to the subject an effective amount of a radiolabeled antibody against CD19, CD20, CD33, CD38, CD45RA, CD52, or a combination thereof. The effective amount of the radiolabeled antibody depletes at least 50% of the targeted immune cells and less than 20% of the subject's stem cells. When used alone, these methods may target lymphomas, leukemias, and myelomas, and / or may additionally enable repopulation of non-autoreactive immune cells in patients with an autoimmune disease. When these methods precede certain cell-based therapies, such as adoptive cell therapy and / or hematopoietic stem cell therapy, the method can enhance the outcome of the cell-based therapies while minimizing adverse effects.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 838,646, filed April 25, 2019, which is incorporated herein in its entirety.

[0002] The present invention relates to compositions that selectively target and deplete cells of specific lineages without targeting stem cells, and methods using these compositions for transient immunodepletion, for example, prior to adoptive cell therapy. [Background technology]

[0003] Immunodepletion is a process that transiently reduces immune system cells while leaving the stem cell compartment intact. Immunodepletion can be useful and necessary in the context of adoptive cell therapy, where endogenous host cells must first be depleted before transplantation of autologous or allogeneic cells. This phenomenon is well established in the field of bone marrow transplantation and is also a widespread requirement for the efficacy of other adoptive cell therapies, such as CAR-T. These adoptively transferred cells can be either unmanipulated (bone marrow transplantation for the treatment of hematologic cancers) or engineered to ameliorate the pathogenic condition of the recipient (correction of autoimmune disease by removing autoreactive cells before autologous stem cell transplantation, or engineered CAR-T cells for cancer treatment). Because stem cells continuously generate new hematopoietic progenitors for both the myeloid and lymphoid compartments of the immune system, the removal of more differentiated cells is temporary; these depleted cells are eventually replaced by nascent mature cells derived from the stem cells. Immune depletion could also potentially be used without subsequent adoptive cell therapy, for example in the case of autoimmune diseases where differentiated autoreactive cells are depleted and then functional non-autoreactive immune cells are repopulated from healthy stem cells.

[0004] Chemotherapy-based immune depletion Prior to adoptive cell therapy methods such as CAR-T, it is common to use highly cytotoxic chemotherapy agents, particularly the combination of cyclophosphamide and fludarabine, to immunodeplete (i.e., lymphocyte depletion) patients. These agents reduce lymphoid cell counts. However, they are highly toxic. They not only deplete the immune system in a non-targeted manner, but can also damage other normal cells and tissues. Not all patients can tolerate them. Furthermore, sustained response rates, especially with CAR T-cell therapy, are typically less than 50%. Many patients eventually relapse after receiving CAR T-cell therapy and require further therapeutic intervention or stem cell transplantation (e.g., bone marrow transplantation).

[0005] Antibody-based immune depletion Antibodies have greater cell targeting specificity than chemotherapeutic agents.Therefore, antibodies against immune cell-specific antigens have been attracting attention as a potential substitute for chemotherapeutic agents as immune depletion agents.Although naked antibodies can sometimes achieve a certain level of cell killing, a more effective way to deplete immune cells would be to utilize the strong cytotoxic activity of radioactivity in the form of radionuclides conjugated to antibodies or antibody fragments.This can effectively and specifically target the cell killing ability of radionuclides to cells that express target proteins on the cell surface.

[0006] Thus, there is a need for alternative methods and therapy protocols that may improve the efficacy and reduce the toxicity of known immunodepletion regimens and adoptive cell therapies such as CAR-T, and / or reduce certain side effects of current adoptive cell therapy protocols. Summary of the Invention

[0007] The present invention provides a solution to the aforementioned problems by providing compositions useful for targeted and transient immunodepletion and methods of using these compositions in the treatment of malignant and non-malignant hematological diseases or prior to the administration of adoptive cell therapy.

[0008] The composition comprises an antibody against an antigen (e.g., cell surface marker) that is specific to a certain hematological lineage of cells but not to other lineages.This differential expression of surface markers can be used to selectively deplete cells of a specific lineage without affecting other immune cells such as stem cells.Exemplary surface markers that can be used to deplete distinct subsets of immune cells without targeting stem cells include CD2, CD3, CD5, CD11a, CD11b, CD11c, CD14, CD18, CD19, CD20, CD28, CD33, CD38, CD45RA, CD49a, CD52, CD96, CD116, CD122, CD132, CD152, CD154, CD244, CD272, CD305, CLA, IL-21, B7-HA, or any combination thereof.

[0009] Thus, the present invention generally relates to compositions comprising antibodies to any of CD2, CD3, CD5, CD11a, CD11b, CD11c, CD14, CD18, CD19, CD20, CD28, CD33, CD38, CD45RA, CD49a, CD52, CD96, CD116, CD122, CD132, CD152, CD154, CD244, CD272, CD305, CLA, IL-21, B7-HA, or combinations thereof. 131 I, 125 I, 123 I, 90 Y, 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 32 P, 225 Ac, 213 Bi, 213 Po, 211 At, 212 Bi, 213 Bi, 223 Ra, 227 Th, 149 Tb, 137 Cs, 212 Pb, and 103Pd.

[0010] The present invention further relates to a method for transient immunodepletion of a subset of blood cells, comprising administering an effective amount of any of the aforementioned compositions to a patient. Immunodepletion can be provided before, after, or both before and after adoptive cell therapy, such as administering a cell population expressing a CAR / TCR or TIL. The cell population expressing a CAR / TCR or TIL can be autologous cells, allogeneic cells derived from another human patient, or xenogeneic cells derived from an animal of a different species.

[0011] According to certain embodiments of the present invention, a cell population expressing a CAR / TCR or TIL can be isolated by leukapheresis, as in the case of autologous cells, and transduced and selected approximately four weeks before administration, or isolated from a healthy donor and prepared in advance for one or more patients, as in the case of so-called "off-the-shelf" allogeneic CAR-T cell therapy, and then stored, such as as a frozen preparation. The cell population expressing a CAR / TCR can include a population of activated T cells, natural killer (NK) cells, or dendritic cells that express an antigen-recognizing CAR / TCR. Dendritic cells have the ability to present antigens as well as the ability to directly kill tumors. Furthermore, the cell population can be a pluripotent stem cell population that can differentiate into a variety of different blood cell types.

[0012] According to one particular embodiment, the radiolabel is 131 I or 225 Ac, 131 An effective amount of an antibody labeled with I can be 25 mCi to 200 mCi (e.g., 50 mCi, 100 mCi, or 150 mCi), 225 An effective amount of an antibody labeled with Ac can be 0.1 μCi / kg (subject body weight) to 5.0 μCi / kg (subject body weight).

[0013] These methods may improve the therapeutic outcomes of malignant and non-malignant hematological diseases and / or reduce side effects associated with adoptive cell therapy, such as neurotoxicity, cytokine release syndrome (CRS), hypogammaglobulinemia, cytopenia, capillary leak syndrome (CLS), macrophage activation syndrome (MAS), tumor lysis syndrome (TLS), and combinations thereof.

[0014] The various aspects of the invention will be realized and attained by means of the combinations particularly outlined in the appended claims. The foregoing general description and the following detailed description and examples of the invention are provided to illustrate various aspects of the invention and should not be construed as limiting any of the described embodiments. [Brief explanation of the drawings]

[0015] [Figure 1A] We present a prior art adoptive cell therapy protocol including leukapheresis, cytoablation using standard chemotherapy agents, CAR / TCR T cell infusion, and the possibility of bone marrow conditioning in preparation for optional hematopoietic stem cell transplantation (HSCT). [Figure 1B] The present invention demonstrates an autologous or allogeneic adoptive cell therapy method involving the administration of a radiolabeled antibody instead of, or in addition to, standard cytoablative agents. [Figure 1C] 1 illustrates an autologous or allogeneic adoptive cell therapy method of the present invention that involves the administration of a radiolabeled antibody as a bone marrow conditioning agent prior to HSCT. [Figure 1D] The present invention demonstrates autologous or allogeneic adoptive cell therapy, which involves the administration of radiolabeled antibodies in place of or in addition to standard cytoablative agents, and the administration of radiolabeled antibodies as a bone marrow conditioning agent prior to HSCT. [Figure 1E] 1 shows pharmacokinetic data demonstrating exemplary clearance and administration times of lymphodepletion protocols according to the disclosed invention. [Figure 2] 1 shows the median change in absolute neutrophil counts after treatment with 131I-anti-CD45 (ie, 131I-BC8). [Figure 3A] 1 shows the results of immune cell analysis after 131I-anti-CD45 antibody targeted immune depletion in a mouse model using the surrogate anti-CD45 antibody 30F11. [Figure 3B] 1 shows the results of immune cell analysis after 131I-anti-CD45 antibody targeted immune depletion in a mouse model using the surrogate anti-CD45 antibody 30F11. [Figure 3C] 1 shows the results of immune cell analysis after 131I-anti-CD45 antibody targeted immune depletion in a mouse model using the surrogate anti-CD45 antibody 30F11. [Figure 3D] 1 shows the results of immune cell analysis after 131I-anti-CD45 antibody targeted immune depletion in a mouse model using the surrogate anti-CD45 antibody 30F11. [Figure 3E] 1 shows the results of immune cell analysis after 131I-anti-CD45 antibody targeted immune depletion in a mouse model using the surrogate anti-CD45 antibody 30F11. [Figure 3F] 1 shows the results of immune cell analysis after 131I-anti-CD45 antibody targeted immune depletion in a mouse model using the surrogate anti-CD45 antibody 30F11. [Figure 3G] 1 shows the results of immune cell analysis after 131I-anti-CD45 antibody targeted immune depletion in a mouse model using the surrogate anti-CD45 antibody 30F11. [Figure 3H] 1 shows the results of immune cell analysis after 131I-anti-CD45 antibody targeted immune depletion in a mouse model using the surrogate anti-CD45 antibody 30F11. [Figure 4A] 1 shows results from immunophenotyping of immune cell populations after 131I-anti-CD45 antibody targeted immunodepletion in mice. [Figure 4B] 1 shows results from immunophenotyping of immune cell populations after 131I-anti-CD45 antibody targeted immunodepletion in mice. [Figure 4C] 1 shows results from immunophenotyping of immune cell populations after 131I-anti-CD45 antibody targeted immunodepletion in mice. [Figure 4D]1 shows results from immunophenotyping of immune cell populations after 131I-anti-CD45 antibody targeted immunodepletion in mice. [Figure 4E] 1 shows results from immunophenotyping of immune cell populations after 131I-anti-CD45 antibody targeted immunodepletion in mice. [Figure 5A] Depletion of splenic T-reg cells is shown. [Figure 5B] Depletion of myeloid-derived suppressor cells (MDSCs) is shown. [Figure 5C] 1 shows preservation of bone marrow HSCs after targeted immunodepletion with 131I-anti-CD45 antibody in mice. [Figure 6] Selected open trials of autologous anti-CD19 CAR T-cell therapy for patients with B-cell non-Hodgkin's lymphoma (NHL) are shown. [Figure 7] Figure 1 shows a schematic of a preclinical study in mice of the effects of low-dose I-anti-CD45 radioimmunotherapy (surrogate 30F11) to investigate immunodepletion responses in specific immune cell types. Controls include lymphodepletion treatment with chemotherapy, cyclophosphamide (Cy) or cyclophosphamide / fludarabine (Flu / Cy), and no lymphodepletion treatment. [Figure 8] We present a preclinical model of adoptive T cell transfer after anti-CD45 radioimmunotherapy-mediated conditioning / immunodepletion in mice. In this model, E.G7 lymphoma tumor-bearing mice are conditioned with a single selective dose of 131I-anti-CD45 radioimmunotherapy prior to adoptive cell transfer of OVA-specific CD8+ T cells and monitored for engraftment of the transferred cells and the resulting anti-tumor response. [Figure 9] 1 shows clinical data from a low-dose 131I-BC8 study demonstrating lymphocyte depletion in human patients. [Figure 10] Pharmacokinetic data demonstrating a clearance rate of less than 25 cGy for a 100 mCi infusion of 131I-BC8 in human patients are shown. [Figure 11]1 shows pharmacokinetic data demonstrating the cumulative dose of 131I-BC8 to the spleen of a human patient following administration of a 100 mCi infusion of 131I-BC8. [Figure 12] 1 shows the blood clearance of 131I-BC8 from a human patient after administration of a 100 mCi infusion of 131I-BC8. [Figure 13A] 1 shows results from 131I-anti-CD45 targeted immunodepletion prior to adoptive cell therapy with OT I OVA-reactive T cells on day 4 in mice engrafted with E.G7-OVA tumors. [Figure 13B] 1 shows results from 131I-anti-CD45 targeted immunodepletion prior to adoptive cell therapy with OT I OVA-reactive T cells on day 4 in mice engrafted with E.G7-OVA tumors. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides radiolabeled antibody-based methods for immunodepleting a subject, as well as related compositions and articles of manufacture. Where these methods precede certain cell-based therapies, the methods can enhance the outcomes of the cell-based therapies while minimizing adverse effects.

[0017] definition In this application, certain terms are used which have the meanings set forth below.

[0018] The singular forms "a," "an," "the," etc. include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an" antibody includes both a single antibody and a plurality of different antibodies.

[0019] The term "about" when used before numerical designations, such as temperatures, times, amounts, and concentrations, indicates approximations that may vary by ±10%, ±5%, or ±1%.

[0020] As used herein, "administering" with respect to an antibody means delivering the antibody to the subject's body via any known method suitable for antibody delivery. Specific administration modes include, but are not limited to, intravenous, transdermal, subcutaneous, intraperitoneal, intrathecal, and intratumoral administration. Exemplary antibody administration methods may be substantially as described in International Publication No. 2016 / 187514, which is incorporated herein by reference.

[0021] Furthermore, in the present invention, antibody can be formulated with one or more commonly used pharmaceutically acceptable carriers.Such carriers are well known to those skilled in the art.For example, injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymer injections, and can include excipients such as soluble modifiers (e.g., ethanol, propylene glycol, and sucrose) and polymers (e.g., polycaprylactone and PLGA).

[0022] As used herein, the term "antibody" includes, but is not limited to, (a) immunoglobulin molecules comprising two heavy chains and two light chains and recognizing an antigen, (b) polyclonal and monoclonal immunoglobulin molecules, (c) monovalent and divalent fragments thereof (e.g., Fab, di-Fab), and (d) bispecific forms thereof. Immunoglobulin molecules can be derived from any of the commonly known classes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. Antibodies can be of both natural and non-natural origin (e.g., IgG-Fc-silent). Furthermore, antibodies include chimeric antibodies, fully synthetic antibodies, single-chain antibodies (e.g., scFv), single- and dual-domain antibodies (e.g., VHH), and fragments thereof. Antibodies can be human, humanized, or non-human.

[0023] "Monoclonal antibody" refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope, or, in the case of multispecific monoclonal antibodies, binding specificities for two or more distinct epitopes. Thus, a "monoclonal antibody" refers to an antibody population having a single amino acid composition in each heavy and light chain, except for possible known variations such as removal of the C-terminal lysine from the antibody heavy chain. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, or monovalent, bivalent, or multivalent.

[0024] As used herein, an "anti-CDXX antibody" is an antibody that specifically binds to any available epitope of CDXX, where XX can be 2, 3, 5, 11a, 11b, 11c, 14, 18, 19, 20, 28, 33, 38, 45RA, 49a, 52, 96, 116, 122, 132, 152, 154, 244, 272, or 305 (i.e., CD2, CD3, CD5, CD11a, CD11b, CD11c, CD14, CD18, CD19, CD20, CD28, CD33, CD38, CD45RA, CD49a, CD52, CD96, CD116, CD122, CD132, CD152, CD154, CD244, CD272, or CD305). CDXX may also refer to CLA, IL-21, B7-HA, CLA, IL-21, or B7-HA. According to certain preferred embodiments, XX can be 19, 20, 33, 38, 45, or 52 (i.e., CD19, CD20, CD33, CD38, CD45RA, and CD52).

[0025] The anti-CDXX antibody may be a bispecific antibody that binds to two different epitopes, which may be at least one of CD19, CD20, CD33, CD38, CD45RA, and CD52 and another related epitope, or two different epitopes of a single cell surface target (two different epitopes of any one of CD19, CD20, CD33, CD38, CD45RA, or CD52). The bispecific antibody may be an antibody that targets lymphoid-derived cells but not stem cells, and / or an antibody that targets myeloid-derived cells but not stem cells. The lymphoid target may be CD3, CD2, CD28, CD96, CD122, CD152, or CD154, and / or the myeloid target may be CD11b, CD11c, CD14, CD33, or CD116. The bispecific antibody can be a recombinant antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or an antibody fragment.

[0026] The term "specific binding" means binding of at least 10 6 M -1 , usually about 10 6 M -1 ~about 10 8 M -1 It refers to the property of having high binding affinity to

[0027] As used herein, "cancer" includes, but is not limited to, solid cancers (e.g., tumors) and hematological malignancies.

[0028] As used herein with respect to a subject's immune cells, "depleting" or "targeted depletion" is intended to mean reducing the population of at least one type of immune cell in a subject (i.e., targeted immune cells) by administration of a particular antibody according to an embodiment of the invention. For example, targeted immune cells may include at least B cells, e.g., pro-B cells, pre-B cells, and plasma cells (e.g., cells comprising CD19, CD20 antigens); myeloid-derived cells, e.g., common myeloid progenitor cells, myeloid dendritic cells, mast cells, granulocyte-macrophage progenitor cells, monocytes, macrophages, myeloblasts, neutrophils, eosinophils, and basophils (e.g., CD33); natural killer cells, B cells, plasma cells, T cells, Tregs, dendritic cells, common myeloid progenitor cells, granulocyte-macrophage progenitor cells, myeloblasts, basophils, monocytes, megakaryocyte-erythroid progenitor cells, erythrocytes, megakaryocytes (e.g., CD38); lymphoid progenitor cells, double negative (CD4- These may include CD8- (T cells), B cells, NK cells, granulocyte / monocyte precursors, neutrophils, activated monocytes / macrophages (e.g., CD45RA); mature B and T lymphocytes, monocytes, NK cells, and dendritic cells (e.g., CD52). According to a preferred embodiment, depleting a subject's immune cells means reducing immune cell populations without also reducing stem cell populations, such as hematopoietic stem cells ("HSCs," i.e., pluripotent hematopoietic stem cells, also called hematoblasts).

[0029] According to certain preferred embodiments of the present disclosure, the reduction or depletion of a subject's immune cells is determined by measuring specific immune cell populations in the subject's peripheral blood. For example, if the population of at least one type of peripheral blood lymphocyte in the subject is reduced by 99% or less, the subject's peripheral blood lymphocyte population is depleted. For example, if the subject's peripheral blood T cell level is reduced by 50%, the subject's peripheral blood NK cell level is reduced by 40%, and / or the subject's peripheral blood B cell level is reduced by 30%, the subject's lymphocytes are depleted. In this example, the subject's lymphocytes are depleted even if the level of another immune cell type, such as neutrophils, is not reduced. According to certain embodiments, the subject's lymphocyte depletion is reflected by a reduction of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in the peripheral blood lymphocyte population. According to certain embodiments, the subject's HSC population is depleted by 20% or less, for example, 10% or less.

[0030] Methods for measuring peripheral blood immune cell populations are routine. These include, for example, flow cytometry in whole blood samples to determine the number of specific cell types based on labeling with fluorescent antibodies directed against specific cell surface markers. For example, lymphocyte counts can be determined using markers such as CD45, CD4, or CD8, and neutrophil counts can be determined using markers such as Ly6G. Methods for measuring HSC populations are routine and include, for example, flow cytometry and the use of fluorescent antibodies directed against cell surface markers such as Lin, CD34, CD38, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, and HLA DR (i.e., markers that recognize stem cells, such as CD34, and markers that specifically exclude stem cells, such as CD45RA).

[0031] As used herein, an amount of radiolabeled anti-CDXX antibody is "effective" if, when administered, the subject's targeted immune cells are depleted, for example, by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. An amount of radiolabeled anti-CDXX antibody is "effective" if, when administered, the subject's targeted immune cells are depleted without depletion of the subject's stem cells, or with a reduction of less than 20% or 10% of the subject's stem cells.

[0032] According to one particular embodiment, the radiolabeled anti-CDXX antibody 131 When labeled with I, the effective amount is, for example, less than 300 mCi (i.e., less than 100 mCi administered to a subject). 131 (The amount of I-anti-CDXX delivers a total body radiation dose of less than 300 mCi). 131 In the case of I-anti-CDXX, the effective amount is less than 250 mCi, less than 200 mCi, less than 150 mCi, less than 100 mCi, less than 50 mCi, less than 40 mCi, less than 30 mCi, less than 20 mCi, or less than 10 mCi. 131 For I-anti-CDXX, the effective amount is 1 mCi to 10 mCi, 1 mCi to 200 mCi, 10 mCi to 20 mCi, 10 mCi to 30 mCi, 10 mCi to 40 mCi, 10 mCi to 50 mCi, 10 mCi to 100 mCi, 10 mCi to 150 mCi, 10 mCi to 200 mCi, 20 mCi to 30 mCi, 30 mCi to 40 mCi , 40mCi to 50mCi, 50mCi to 100mCi, 50mCi to 150mCi, 50mCi to 200mCi, 60mCi to 140mCi, 70mCi to 130mCi, 80mCi to 120mCi, 90mCi to 110mCi, 100mCi to 150mCi, 150mCi to 200mCi, or 200mCi to 250mCi. 131 In the case of I-anti-CDXX, the effective amount is 10 mCi to 120 mCi, 20 mCi to 110 mCi, 25 mCi to 100 mCi, 30 mCi to 100 mCi, 40 mCi to 100 mCi, or 75 mCi to 100 mCi.131 For I-anti-CDXX, the effective amount is 1 mCi, 10 mCi, 20 mCi, 30 mCi, 40 mCi, 50 mCi, 60 mCi, 70 mCi, 80 mCi, 90 mCi, 100 mCi, 110 mCi, 120 mCi, 130 mCi, 140 mCi, 150 mCi, or 200 mCi.

[0033] According to one particular embodiment, the radiolabeled anti-CDXX antibody 225 When labeled with Ac, the effective amount is, for example, less than 5.0 μCi / kg (i.e., less than 5.0 μCi / kg administered to a subject). 225 (The amount of Ac-anti-CDXX delivers a radiation dose of less than 5.0 μCi per kilogram of subject body weight.) In certain embodiments, the antibody 225 In the case of Ac-anti-CDXX, the effective amount is less than 4.5 μCi / kg, 4.0 μCi / kg, 3.5 μCi / kg, 3.0 μCi / kg, 2.5 μCi / kg, 2.0 μCi / kg, 1.5 μCi / kg, 1.0 μCi / kg, 0.9 μCi / kg, 0.8 μCi / kg, 0.7 μCi / kg, 0.6 μCi / kg, 0.5 μCi / kg, 0.4 μCi / kg, 0.3 μCi / kg, 0.2 μCi / kg, 0.1 μCi / kg, or 0.05 μCi / kg. According to certain embodiments, the antibody 225 For Ac-anti-CDXX, the effective amount is 0.05μCi / kg~0.1μCi / kg, 0.1μCi / kg~0.2μCi / kg, 0.2μCi / kg~0.3μCi / kg, 0.3μCi / kg~0.4μCi / kg, 0.4μCi / kg~0.5μCi / kg, 0.5μCi / kg~0.6μCi / kg, 0.6μCi / kg~0.7μCi / kg, 0.7μCi / kg~0.8μCi / kg, 0.8μCi / kg~0.9 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.225 For Ac-anti-CDXX, the effective amounts are 0.05μCi / kg, 0.1μCi / kg, 0.2μCi / kg, 0.3μCi / kg, 0.4μCi / kg, 0.5μCi / kg, 0.6μCi / kg, 0.7μCi / kg, 0.8 μCi / kg, 0.9 μCi / kg, 1.0 μCi / kg, 1.5 μCi / kg, 2.0 μCi / kg, 2.5 μCi / kg, 3.0 μCi / kg, 3.5 μCi / kg, 4.0 μCi / kg, or 4.5 μCi / kg.

[0034] In radiolabeled antibodies, the majority of the composition administered to a subject typically consists of unlabeled antibody, with labeled antibody being a minority. The ratio of labeled to unlabeled antibody can be adjusted using known methods. Thus, according to certain aspects of the present invention, a radiolabeled antibody may contain both a labeled and an unlabeled fraction. The radiolabeled antibody may be provided as a single dose, and a single dose of radiolabeled antibody may contain labeled:unlabeled antibody in an amount of 0.1:10 to 1:1. The total protein amount may be up to 60 mg, e.g., 5 mg to 45 mg, or the total protein amount may be 0.2 mg / kg (patient body weight) to 0.6 mg / kg (patient body weight).

[0035] Adoptive cell therapy may involve the administration of cells expressing chimeric antigen receptors (CARs) or T cell receptors (TCRs), or may involve tumor-infiltrating lymphocytes (TILs). The cell population expressing CAR / TCRs may include activated T cells, natural killer (NK) cells, or dendritic cell populations that express CAR / TCRs that recognize antigens. Dendritic cells have the ability to present antigens and directly kill tumors. The cell population expressing CAR / TCRs may include gene-edited cell populations.

[0036] As used herein, the term "gene-edited" CAR T cells is synonymous with the terms "genetically engineered" CAR T cells and "engineered" CAR T cells. Gene-edited CAR T cells that "fail to properly express" a checkpoint receptor (e.g., PD1, Lag3, or TIM3) do not express a full-length, functional checkpoint receptor. For example, gene-edited CAR T cells that fail to properly express PD1 may do so because, but are not limited to, (i) the PD1 gene of the cells has been disrupted or (ii) the PD1 gene of the cells has been otherwise modified so that it does not produce a fully or partially functional PD1 product. In other words, according to certain embodiments, gene-edited CAR T cells that fail to properly express PD1 may do so because the PD1 gene of the cells has been modified to reduce PD1 expression. Similarly, gene-edited CAR T cells that "fail to properly express" a T cell receptor do not express a full-length, functional T cell receptor.

[0037] According to certain embodiments, functional endogenous T cell receptors are replaced by "knock-in" editing of an exogenously transduced CAR or recombinant TCR into the native TCR locus. Gene-edited CAR T cells include, but are not limited to: (i) allogeneic gene-edited CAR T cells that fail to properly express PD1 but properly express all other checkpoint receptors and T cell receptors, (ii) allogeneic gene-edited CAR T cells that fail to properly express a particular T cell receptor but properly express all checkpoint receptors and all other T cell receptors, and (iii) allogeneic gene-edited CAR T cells that fail to properly express PD1 and fail to properly express a particular T cell receptor but properly express all other checkpoint receptors and all other T cell receptors.

[0038] An example of T cell gene editing to generate allogeneic universal CAR T cells includes the work of Eyquem and colleagues (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. In this method, the recombinant CAR was placed under the control of the cell's natural TCR regulatory signals. By this same strategy, a CAR or recombinant TCR can be effectively inserted by knock-in into the T cell receptor beta constant locus (TRBC) or the beta-2 microglobulin (B2M) MHC-I-associated locus, which are known to be expressed in all T cells. Another example includes the work of Ren and colleagues (Ren, et al., 2017, Clin. Cancer Res 23:2255-2266). Recognizing that checkpoint receptors are immunosuppressive and can blunt the stimulation of exogenous autologous or allogeneic CAR T cells, this group utilized CRISPR / cas9 technology to disrupt the endogenous TCRα and β loci (TRAC and TRBC) and B2M genes, while silencing the endogenous PD1 gene. With this approach, the engineered cells did not induce graft-versus-host disease but resisted immune checkpoint receptor suppression.

[0039] "Hematologic malignancies" or "malignant hematologic diseases," also known as blood cancers, are cancers that arise in blood-forming tissues, such as bone marrow or other cells of the immune system. Hematologic malignancies include leukemias (e.g., acute myeloid leukemia (AML), acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), acute mixed lineage leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia (CLL), hairy cell leukemia, and large granular lymphocytic leukemia), myelodysplastic syndromes (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocytosis, primary myelofibrosis, and leukemia), and leukemias (e.g., leukemias of various types, such as leukemias of various types, ... These include, but are not limited to, chronic myelogenous leukemia), lymphoma, multiple myeloma, MGUS and similar disorders, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies.

[0040] "Solid cancer" includes, but is not limited to, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, prostate cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, cancer of the fallopian tubes, endometrial cancer, cancer of the cervix, cancer of the vagina, cancer of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, cancer of the urethra, cancer of the penis, childhood tumors, cancer of the bladder, cancer of the kidney or ureter, cancer of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, and environmentally induced cancers including those induced by asbestos.

[0041] "Non-malignant hematological diseases" or "non-cancerous disorders" include, but are not limited to, hemoglobinopathies (e.g., SCD and β-thalassemia), congenital immunodeficiencies (e.g., SCID and Fanconi anemia), and viral infections (e.g., HIV infection). According to certain embodiments, the disorder is SCD and the therapy is gene-edited β-globin hematopoietic stem cell therapy. According to certain embodiments, the disorder is SCID and the therapy is gene-edited hematopoietic stem cell therapy, and the edited genes are the common gamma chain (γc) gene, the adenosine deaminase (ADA) gene, and / or the Janus kinase 3 (JAK3) gene. Stem cell therapy can be, for example, allogeneic or autologous.

[0042] As used herein, the term "subject" includes, but is not limited to, mammals such as humans, non-human primates, dogs, cats, horses, sheep, goats, cattle, rabbits, pigs, rats, and mice. When the subject is a human, the subject can be of any age. For example, the subject can be 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. Alternatively, the subject can be 50 years or younger, 45 years or younger, 40 years or younger, 35 years or younger, 30 years or younger, 25 years or younger, or 20 years or younger. In the case of a human subject with cancer, the subject can be newly diagnosed, or can be relapsed and / or refractory, or can be in remission.

[0043] As used herein, an "appropriate period of time" after administering a radiolabeled antibody to a subject and before administering adoptive cell therapy to the subject is a period of time during which the administered antibody is sufficient to lymphodeplete the subject's cells and / or the subject's cells remain lymphodepleted. According to certain embodiments, the appropriate period of time is less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, or less than 3 days. According to certain embodiments, the appropriate period of time 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.

[0044] As used herein, a "radioisotope" may be an alpha-, beta-, and / or gamma-emitting isotope. Examples of radioisotopes include: 131 I, 125 I, 123 I, 90 Y, 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 32 P, 225 Ac, 213 Bi, 213 Po, 211 At, 212 Bi, 213 Bi, 223 Ra, 227 Th, 149 Tb, 137 Cs, 212 Pb, and 103 Radioisotopes include Pd.Methods for attaching radioisotopes to antibodies (i.e., using radioisotopes to "label" antibodies) are well known.Some of these methods are described, for example, in International Patent Application Publication No. 2017 / 155937.

[0045] As used herein, "treating" a subject afflicted with a malignant or non-malignant hematological disease includes, but is not limited to, (i) slowing, halting, or reversing the progression of the disease, (ii) slowing, halting, or reversing the progression of symptoms of the disease, and / or (iii) reducing the likelihood of disease recurrence. According to certain embodiments, treating a subject afflicted with cancer means (i) reversing the progression of the cancer, ideally to the point where the cancer is eliminated, and / or (ii) reversing the progression of symptoms of the cancer, ideally to the point where the symptoms are eliminated, and / or (iii) reducing or eliminating the likelihood of recurrence (i.e., consolidation, which ideally results in the destruction of any remaining cancer cells).

[0046] Throughout this application, various publications are cited. The disclosures of these publications are incorporated herein by reference into this application in order to more fully describe the prior art to which this invention pertains. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing described herein, suitable methods and materials are described below.

[0047] Aspects of the present invention Targeted immune depletion The present invention solves an unmet need in the art by providing an unexpectedly superior method for transiently immunodepleting specific targeted subsets of immune cells. Cells of the hematopoietic system often express specific surface markers that are present on certain lineages of cells but not on others. This differential expression of surface markers can be exploited to selectively deplete cells of specific lineages while leaving other immune cells unaffected. Some potentially useful surface markers that can effectively deplete distinct subsets of immune cells without targeting stem cells include at least the following: CD19, CD20, CD33, CD38, CD45RA, and CD52.

[0048] CD19 is a membrane receptor that is expressed early in B cell differentiation and remains expressed until B cells are induced to terminally differentiate (i.e., first on pro-B cells and continuing throughout their development into plasma cells). CD19 is part of a protein complex found on the cell surface of B lymphocytes. The protein complex includes CD19, CD21 (complement receptor, type 2), CD81 (TAPA-1), and CD225 (Leu-13). CD19 is a key regulator of transmembrane signaling in B cells. Increased or decreased cell surface density of CD19 affects B cell development and function, leading to diseases such as autoimmunity or hypogammaglobulinemia. Exemplary commercially available antibodies against CD19 include blinatumumab.

[0049] CD20 is a transmembrane protein expressed on the surface of B lymphocytes. It is expressed during B lymphocyte development, from the early pre-B cell stage to terminal differentiation into plasma cells, at which stage CD20 expression disappears. CD20 is also expressed on malignant B cells. In particular, CD20 is expressed on more than 90% of B-cell non-Hodgkin's lymphoma (NHL) cells and more than 95% of B-type chronic lymphocytic leukemia (B-CLL) cells. Although its function is unknown, it is thought to be involved in B-cell activation, regulation of B-cell proliferation, and transmembrane calcium flux. Exemplary commercially available antibodies against CD20 include rituximab.

[0050] CD33 is a type I transmembrane receptor glycoprotein that can function as a sialic acid-dependent cell adhesion molecule. It is expressed on early myeloid progenitor cells, myeloid dendritic cells, mast cells, granulocyte-macrophage progenitor cells, monocytes, macrophages, myeloblasts, neutrophils, eosinophils, and basophils, but CD33 is not expressed on stem cells. Exemplary commercially available antibodies against CD33 include lintuzumab (HuM195), gemtuzumab, or vadastuximab.

[0051] CD38 is a type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain.+ is a bifunctional extracellular enzyme that can catalyze the conversion of NAD to cyclic ADP-ribose (cADPR) and cADPR to ADP-ribose, thus increasing the amount of extracellular NAD + Furthermore, cADPR regulates intracellular Ca 2+ CD38 has been shown to be a second messenger for mobilizing intracellular Ca. 2+ CD38 may be an essential component in regulating the flow of blood.CD38 has been described on epithelial / endothelial cells of different origins, including the glandular epithelium of the prostate, pancreatic islet cells, the ductal epithelium of glands including the parotid gland, bronchial epithelial cells, testicular and ovarian cells, and tumor epithelium of colorectal adenocarcinoma.Exemplary commercially available antibodies against CD38 include daratumumab, MOR202, or SAR650984.

[0052] CD45 is a protein expressed exclusively on cells of the hematopoietic system, including stem cells. 131 Pan-CD45 antibodies conjugated to I) have already been utilized in numerous clinical trials for the complete destruction of hematopoietic cells prior to bone marrow transplantation. Furthermore, pan-CD45 antibodies, when used at low doses, are contemplated as good candidates for immune depletion, resulting in the preferential depletion of more differentiated cells with higher levels of CD45 expression. Multiple isoforms of CD45 exist, including CD45RO (pan-specific), CD45RA, and CD45RB. Ideally, a CD45-targeting strategy would use a CD45 isoform, such as CD45RA, that is not expressed on stem cells. Thus, radiolabeled CD45RA antibodies deplete more differentiated immune cells but spare stem cells.

[0053] CD52 is a membrane glycoprotein found on mature lymphocytes, but not on the stem cells from which these lymphocytes are derived.The protein is expressed on more than 95% of peripheral blood lymphocytes, and is found at a higher density on T lymphocytes than on B lymphocytes.Exemplary commercially available antibodies against CD52 include alemtuzumab (Lemtrada, Campath).

[0054] These targets, and the cells they can deplete, are listed in Table 1 (i.e., CD19, CD20, CD33, CD38, CD45RA, and CD52). Additional targets that may be useful in various embodiments of the invention, and the cells they can deplete, are listed in Table 2 (i.e., CD2, CD5, CD11a, CD18, CD49a, CD122, CD132, CD244, CD272, CD305, CLA, IL-21 receptor, and B7-HA).

[0055] Furthermore, the use of combinations of targets may enable the rational depletion of multiple cell types in a therapeutically useful manner. For example, the generation of a bispecific antibody, with one arm binding to a lymphoid marker and the other to a myeloid marker, neither of which is present on stem cells, would be an ideal immunodepletion target. One such potential combination strategy could involve bispecific targeting of CD3 and CD11c. Targeting CD3 would eliminate T cells, while targeting CD11c would deplete a large number of myeloid cells, including monocytes and myeloid dendritic cells. Because neither of these markers is expressed on stem cells, this would result in the transient depletion of more differentiated immune cells and the eventual repopulation of the hematopoietic system with stem cells. Other potential markers for subsets or all lymphoid cells include CD3, CD2, CD28, CD96, CD122, CD152, and CD154. Potential markers for subsets or all myeloid cells include CD11b, CD11c, CD14, CD33, and CD116. Any combination of the listed lymphoid or myeloid markers can potentially be combined for rational depletion of desired immune cell subsets.

[0056] Treatment with radiolabeled antibodies against such cell surface markers provides an effective means to specifically target the cell-killing capacity of radionuclides to cells that express the protein of interest on their cell surface.

[0057] Thus, the present invention uses a radiolabeled anti-CDXX antibody, for example, a radiolabeled anti-CD19, or anti-CD20, or anti-CD33, or anti-CD38, or anti-CD45RA, or anti-CD52 antibody, or a combination thereof. [Table 1] [Table 2]

[0058] The present invention further provides radiolabeled antibodies such as those listed in Table 2, including radiolabeled antibodies such as radiolabeled anti-CD2, or anti-CD5, or anti-CD11a, or anti-CD18, or anti-CD49a, or anti-CD122, or anti-CD132, or anti-CD244, or anti-CD272, or anti-CD305, or anti-CLA, or anti-IL-21 receptor, or anti-B7-HA, or combinations thereof.

[0059] The present invention still further provides radiolabeled antibodies that specifically target lymphoid or myeloid cells, such as radiolabeled anti-CD2, or anti-CD3, or anti-CD11b, or anti-CD11c, or anti-CD14, or anti-CD28, or anti-CD96, or anti-CD116, or anti-CD122, or anti-CD152, or anti-CD154 antibodies, or combinations thereof.

[0060] This antibody can immunodeplete a subject with surprisingly low doses of protein and radiation.Therefore, this approach targets specific immune cell populations without affecting stem cells, thus avoiding certain adverse effects caused by less specific agents such as chemotherapeutic agents or beam radiation.Furthermore, the radioisotope of the present invention selected for labeling the antibody provides targeted killing of these specific immune cell populations while minimizing adverse effects on surrounding tissues.

[0061] Targeted immune depletion for autoimmune diseases Because stem cells continually generate new hematopoietic progenitors for both the myeloid and lymphoid compartments of the immune system, the removal of more differentiated cells is temporary, and these depleted cells are eventually replaced by nascent mature cells derived from stem cells. Thus, immune depletion can also be used in the case of autoimmune diseases, where differentiated autoreactive cells are depleted and functional non-autoreactive immune cells are repopulated from healthy stem cells.

[0062] Therefore, the present invention also relates to compositions and methods for immunodepleting a subject suffering from an autoimmune disease by administering a radiolabeled anti-CDXX antibody, for example, a radiolabeled anti-CD19, or anti-CD20, or anti-CD33, or anti-CD38, or anti-CD45RA, or anti-CD52 antibody, or a combination thereof, or a radiolabeled bispecific antibody that recognizes lymphoid and myeloid markers. Furthermore, the present invention relates to a method for reducing the amount of B cells or plasma cells that produce pathological antibodies in the body of a patient suffering from an autoimmune disease (i.e., reducing the number of B cells and / or plasma cells in the patient), which comprises treating the patient with a therapeutically effective amount of an antibody that has specific binding to any one or more of CD19, CD20, CD33, CD38, CD45RA, and CD52.

[0063] The term "pathological antibody" refers to an antibody that exhibits specific binding to an autoantigen (ie, the antibody is autoreactive) or that is capable of depleting differentiated autoreactive cells.

[0064] The method can further include treating the patient with an additional pharmaceutically active agent, therapeutically effective treatment, or other adjunctive therapy. The additional pharmaceutically active agent can be a chemotherapeutic agent, a complement activation inhibitor, an antimetabolite (e.g., methotrexate), a steroid, toleragen, an anti-B cell agent, or an anticoagulant (heparin, coumadin), an antiplatelet agent such as acetylsalicylic acid, TICLID® (ticlopidine HCl), PLAVIX® (clopidogrel bisulfate), or intravenous immunoglobulin. Therapeutically effective treatments include plasma exchange or leukapheresis.

[0065] Targeted immune depletion combined with adoptive cell therapy Immunodepletion may be useful to improve the outcome of subsequent therapies, for example, where depletion of specific immune cells, such as lymphodepletion, is desirable prior to cell-based therapies such as CAR T cell therapy or TCR cell therapy.

[0066] According to certain embodiments of this method, the subject suffers from a malignant hematological disease and is undergoing adoptive cell therapy to treat the disease (e.g., a hematological disease such as multiple myeloma, or a malignant tumor such as a solid tumor). Adoptive cell therapy is known and includes, for example, CAR T cell therapy (e.g., autologous cell therapy and allogeneic cell therapy). Examples of approved CAR-T cell therapies include, but are not limited to, KYMRIAH® (tisagenlecleucel) for treating NHL and diffuse large B-cell lymphoma (DLBCL), and YESCARTA® (axicabtagene ciloleucel) for treating NHL.

[0067] These disclosed methods may improve treatment outcomes for hematological malignancies, including solid tumors, and / or reduce side effects associated with adoptive cell therapies, such as the CAR-T cell therapies KYMRIAH® and / or YESCARTA®. For example, side effects of adoptive cell therapy include neurotoxicity, cytokine release syndrome (CRS), hypogammaglobulinemia, cytopenia, capillary leak syndrome (CLS), macrophage activation syndrome (MAS), tumor lysis syndrome (TLS), and combinations thereof. Furthermore, the disclosed methods may prolong the persistence of cell populations expressing CAR / TCR or TIL when compared to methods that do not involve administration of a radiolabeled anti-CD45 antibody.

[0068] According to certain embodiments of the method, the subject is suffering from cancer (e.g., a hematological malignancy or solid 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 for promoting cancer regression in a subject and generally includes (i) collecting autologous T cells (leukapheresis), (ii) expanding (culturing) the T cells, (iii) administering non-myeloablative lymphodepleting chemotherapy to the subject, and (iv) administering the expanded T cells to the subject after administering non-myeloablative lymphodepleting chemotherapy (see 1A).

[0069] The disclosed inventive methods include using radiolabeled antibodies in place of lymphodepleting chemotherapy (FIG. 1B), after administration of expanded cells (e.g., T cells, NK cells, dendritic cells, etc.) (FIG. 1C), or both before and after administration of expanded cells for lymphodepletion (FIG. 1D). Late administration of radiolabeled antibodies (i.e., after administration of expanded cells) can be used in preparation for autologous stem cell transplantation (HSCT) or administration of a second effective amount of expanded cells.

[0070] Thus, the present invention provides methods for treating proliferative diseases, such as hematological malignancies or solid cancers, involving the administration of a radiolabeled antibody and adoptive cell therapy. Adoptive cell therapy generally involves apheresis of autologous cells, which can be gene-edited prior to reinfusion (adoptive cell therapy, such as CAR T cell therapy) after lymphodepletion with a radiolabeled anti-CD45 antibody. Alternatively, allogeneic cells can be reinfused after lymphodepletion to provide adoptive cell therapy. According to the methods of the present invention, the radiolabeled antibody can be provided as a single dose 3 to 9 days, e.g., 6 to 8 days, prior to adoptive cell therapy, as shown in Figure 1E.

[0071] Thus, the present invention provides a method for treating a subject afflicted with cancer, the method comprising (i) administering to the subject an amount of a radiolabeled antibody effective to lymphodeplete the subject's cells, and (ii) after a suitable period of time, administering adoptive cell therapy to the subject to treat the cancer. Preferably, the subject is human.

[0072] Targeted immunodepletion of immunosuppressive cells The present invention further provides a method for targeted immunodepletion of immunosuppressive cells, such as regulatory T (T-reg) cells and myeloid-derived suppressor cells (MDSCs). Both cell types (i.e., T-reg and MDSCs) can attenuate the activation and efficacy of CAR-T cell therapy. Furthermore, the present invention also provides a method for targeted lymphodepletion of immunosuppressive cells, such as monocytes and tumor-associated macrophages (TAMs), which are involved in cytokine release that contributes to toxicities such as cytokine release syndrome (CRS) and CAR T cell-associated neurotoxicity.

[0073] Both solid and liquid tumors have evolved methods to hijack and / or evade the immune system as a means to persist and grow. This has been termed a hostile tumor immune microenvironment (TME). A classic and pertinent example is the upregulated expression of the ligand PD-L1 on the surface of tumor cells, which binds to PD1 on the surface of T cells, resulting in downregulation of immune cell activation. Interestingly, while blockade of this mechanism has resulted in remarkable response rates and durable survival in several different cancer types, most patients do not respond to this form of therapy (i.e., anti-PD1 / PD-L1), suggesting that immune evasion in the tumor microenvironment is multifaceted and complex. In response, tumors may challenge the immune system, in part through oncogenic expression, signaling, and cytokine production, preventing the establishment of an effective antitumor response. This can result in an environment characterized by oxidative stress, nutrient depletion, acidic pH, and hypoxia. Furthermore, the presence of these suppressive immune cells (T-regs and MDSCs), as well as tumor-associated macrophages (TAMs), can effectively blunt immune cell activation through direct contact or release of suppressive soluble factors and cytokines.

[0074] While a patient's endogenous immune system may encounter such an environment and mount a compelling anti-tumor immune response, adoptive cell therapies such as CAR T cell therapy may also be susceptible to these immunosuppressive mechanisms, limiting the ability of these novel cell therapies to mount an effective response against the tumor.

[0075] CAR T therapy, and adoptive cell therapy in general, represents one of the most promising anti-cancer strategies to emerge from clinical studies. Response rates have been exceptional, ranging from 80% across these tumors, but durable responses have only ranged from approximately 40-50% (see, for example, Figure 6). Nevertheless, these results represent a significant improvement in the outcomes of these patients. It is unclear why some patients respond and others do not, but it is likely that the tumor immune microenvironment plays a role in modulating response to cell therapy.

[0076] In response, preclinical and clinical studies have shown that regulatory T cells (T-regs) influence the response to adoptive cell therapy in mice and patients with melanoma (Gattinoni, et al., 2005, JEM, 202:907; Yao, et al., 2012, Blood, 119:5688). In these studies, depletion of T-regs, whether by intentional depletion or conditioning with external beam radiation, favorably affected the antitumor response to adoptive cell therapy. Interestingly, these and other studies suggest that T-reg depletion is more persistent after radiation treatment than chemotherapy-induced conditioning, which was associated with a rapid rebound of T-regs and poor outcomes.

[0077] MDSCs and TAMs are other cell types involved in generating a hostile tumor immune microenvironment. Through upregulation of metabolic gene expression, such as indoleamine 2,3-dioxygenase (IDO), adenosine A2A receptor, and CD73, tumors can effectively create nutrient depletion in the tumor environment, which can blunt T cell activation. For example, tryptophan metabolism by IDO from tumors and MDSCs leads to T cell anergy and death, as well as T-reg accumulation at the tumor site. Furthermore, these immunosuppressive cells can secrete immunomodulatory cytokines, such as TGF-β, which can also negatively affect T cell activation.

[0078] The negative impact of a hostile tumor immune microenvironment can exist for both liquid and solid tumors, but may be even more pronounced in solid tumors. In response, early clinical results suggest that robust responses to CAR-T therapy in liquid tumors, such as lymphoma, have not been observed in solid tumors, suggesting the existence of factors or conditions in solid tumors that may present physical or metabolic barriers to the establishment of an effective CAR-T-mediated immune response (Newick, et al., 2016, Mol. Ther. - Oncolytics, 3:16006; D'Aloia, et al., 2018, Cell Death and Disease. 9:282-293).

[0079] The tumor immune microenvironment is also involved in two major adverse events associated with CAR-T therapy: cytokine release syndrome (CRS) and neurotoxicity. Recent preclinical studies have shown that cytokine release, which leads to CRS or neurotoxicity, is due to activated macrophages after recruitment of CAR-T and tumor cells to the tumor site. Mouse studies (Giavadris, et al., 2018, Nat. Med., 24:731) demonstrated that macrophages secrete IL-1 or IL-6 after recruitment and activation by CAR-T cells at the tumor site.

[0080] Conditioning has been shown to improve the immune homeostatic environment to enable successful CAR-T engraftment and expansion in vivo following adoptive cell therapy or infusion. However, the use of cytotoxic nonspecific chemotherapy can induce off-target toxicity and has been identified as a risk factor for CRS and neurotoxicity following CAR-T administration. Interestingly, most CAR-T programs utilize the combination of fludarabine and cyclophosphamide (flu / cy) as a pre-CAR-T conditioning regimen. These agents are often administered using a 2- to 5-day course of therapy, 2- to 7-days prior to adoptive cell therapy infusion.

[0081] The targeted conditioning therapy of the present invention provides a greatly improved strategy for enhancing CAR-T outcomes. The invention described herein not only targets lymphocytes for depletion, but also immune cell types involved in mediating the hostile tumor immune microenvironment and CAR-T adverse events such as CRS and neurotoxicity. The present invention targets normal immune cells, including T-regs, MDSCs, TAMs, and activated macrophages that secrete IL-1 and / or IL-6. By doing so, the protocols and methods of the present invention can dramatically improve CAR-T outcomes and safety.

[0082] Furthermore, the present invention targets a patient's cancer cells, primarily in hematopoietic tumors, to reduce tumor burden and increase the probability of a CAR-T anti-tumor response. More specifically, the present invention provides a therapeutic strategy targeting CD19, CD20, CD33, CD38, CD45RA, and / or CD52 antigens. For example, CD45 is also expressed on most lymphoid and leukemia tumor cells. The radiolabeled anti-CD45 antibodies of the present invention will affect significant and sustained suppression of immune cells involved in regulating CAR-T responses. In this way, radiation targets and affects the CD45 cell population while minimizing effects on normal tissues. More specifically, the radiolabeled anti-CD45 antibodies can be provided as a single dose at levels sufficiently effective to deplete circulating immune cells in the spleen, lymph nodes, and peripheral blood, while having limited effects on hematopoietic stem cells in the bone marrow. Importantly, in addition to lymphocyte depletion, macrophages, MDSCs, and T-regs are depleted to improve activation and response to CAR-T therapy and reduce adverse events such as CRS and neurotoxicity.

[0083] Radiolabeled antibodies The anti-CDXX antibodies of the present disclosure can be administered to patients intravenously, intramuscularly, or subcutaneously. Exemplary dosages and rates of administration of the compositions can be substantially as described in WO2016 / 187514, which is incorporated herein by reference.

[0084] A dose considered effective for safe depletion of circulating immune cells is one that delivers 2 Gy or less to the bone marrow, thereby reducing the negative impact on hematopoietic stem cells. Such a dose should deplete lymphocytes, immune cells involved in the hostile immune-tumor microenvironment, and tumor cells, all of which result in an enhanced response to ACT or CAR-T therapy. As shown in Table 1, 131 I-anti-CD45( 131 Calculations from dosimetry performed in patients receiving I-BC8) indicate that doses of less than 100 mCi result in delivery of targeted radiation doses in the range of 200 cGy (2 Gy) to the dose-limiting organ, the bone marrow. Such doses have also been found to deliver higher amounts of radiation to the spleen, sites of lymphoid and myeloid cells, for targeted lymphodepletion (see Table 2 and Figures 3A-3H and 4A-4E).

[0085] According to certain aspects of this method, radiolabeled anti-CDXX antibodies contemplated by the present invention include, but are not limited to: 131 I, 125 I, 123 I, 90 Y, 177 Lu, 186 Re, 188 Re, 89 It is labeled with Sr. 153 Sm, 32 P, 225 Ac, 213 Bi, 213 Po, 211 At, 212 Bi, 213 Bi, 223 Ra, 227 Th, 149 Tb, 131 I, 137 Cs, 212 Pb, and 103 Pd, or any combination thereof. Preferably, the radiolabel 131 I or 225 Antibodies can be labeled using the methods detailed in International Patent Application Publication No. WO 2017 / 155937.

[0086] According to one particular aspect of this method, 131 An effective amount of antibody I is 10 mCi to 200 mCi. Examples of effective amounts include, but are not limited to, 50 mCi to 100 mCi, 50 mCi to 150 mCi, 50 mCi to 200 mCi, 60 mCi to 140 mCi, 70 mCi to 130 mCi, 80 mCi to 120 mCi, 90 mCi to 110 mCi, 100 mCi to 150 mCi, 50 mCi, 60 mCi, 70 mCi, 80 mCi, 90 mCi, 100 mCi, 110 mCi, 120 mCi, 130 mCi, 140 mCi, 150 mCi, or 200 mCi. According to certain embodiments, the antibody 131 When labeled with I, the effective amount is 10 mCi to 120 mCi, 20 mCi to 110 mCi, 25 mCi to 100 mCi, 30 mCi to 100 mCi, 40 mCi to 100 mCi, 50 mCi to 100 mCi, or 75 mCi to 100 mCi. 131 Low lymphocyte-depleting doses of I include 300 mCi to 1,200 mCi. 131 This is surprising given the known myeloablative doses of I. For example, it was unexpected that these low doses would result in reduced lymphocyte levels. Furthermore, these low doses allowed patients to 131 It is possible for patients to go home immediately after receiving I, which is not possible for patients receiving a dose of 1,200 mCi, for example, because of the radiation risk posed to others in physical proximity to the patient.

[0087] According to one particular aspect of the method, 225An effective dose of Ac antibody is 0.05 μCi / kg to 5.0 μCi / kg (subject body weight). Examples of effective doses include 0.05 μCi / kg to 5.0 μCi / kg, for example, 0.1 μCi / kg to 0.2 μCi / kg, 0.2 μCi / kg to 0.3 μCi / kg, 0.3 μCi / kg to 0.4 μCi / kg, 0.4 μCi / kg to 0.5 μCi / kg, 0.5 μCi / kg to 0.6 μCi / kg, 0.6 μCi / kg to 0.7 μCi / kg, 0.7 μCi / kg to 0.8 μCi / kg, 0.8 μCi / kg to 0.9 μCi / kg, and 0.9 μCi / kg. These include, but are not limited to, 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.

[0088] An effective amount of radiolabeled antibody can be provided as a single dose. The majority of the antibody administered to a subject typically consists of unlabeled antibody, with a minority of labeled antibody. The ratio of labeled antibody to unlabeled antibody can be adjusted using known methods. Thus, according to certain aspects of the invention, the antibody may be provided in a total protein amount of up to 100 mg, for example, less than 60 mg, or 5 mg to 45 mg, or 0.1 ug / kg to 1 mg / kg of patient weight, for example, 1 ug / kg to 1 mg / kg of patient weight, or 10 ug / kg to 1 mg / kg of patient weight, or 100 ug / kg to 1 mg / kg of patient weight, or 0.1 ug / kg to 100 ug / kg of patient weight, or 0.1 ug / kg to 50 ug / kg of patient weight, or 0.1 ug / kg to 10 ug / kg of patient weight, or 0.1 ug / kg to 40 ug / kg of patient weight, or 1 ug / kg to 40 ug / kg of patient weight, or 0.1 mg / kg to 1.0 mg / kg of patient weight, for example, 0.2 mg / kg to 0.6 mg / kg of patient weight.

[0089] According to certain embodiments of the present invention, the radiolabeled antibody may comprise a labeled fraction and an unlabeled fraction, and the ratio of labeled fraction to unlabeled fraction may be about 0.01:10 to 1:1, e.g., 0.1:10 to 1:1 labeled fraction to unlabeled fraction.

[0090] Additionally, radiolabeled antibodies can be provided as single-dose compositions tailored to a particular patient, i.e., patient-specific therapeutic compositions, where the amount of labeled and unlabeled antibody in the composition can depend on at least the patient's weight, height, body surface area, age, sex, and / or medical or health condition. In this manner, the total volume of the patient-specific therapeutic composition can be provided in a vial configured to be administered entirely to a patient in one treatment session, such that little or no composition remains in the vial after administration.

[0091] Adoptive Cell Therapy Adoptive cell therapy is a powerful approach for treating cancer, as well as other diseases such as infectious diseases and graft-versus-host disease. Adoptive cell therapy is the passive transplantation of ex vivo expanded cells, most commonly immune-derived cells, into a host with the goal of transferring the immunological functions and characteristics of the graft.

[0092] Adoptive cell therapy can be autologous (e.g., isolated by leukapheresis, transduced and selected approximately four weeks immediately prior to administration), as is common in adoptive T cell therapy, or allogeneic, as is typical in the treatment of infectious diseases or graft-versus-host disease. Additionally, ACT can be xenogeneic.

[0093] Adoptive cell therapy may also involve the transfer of autologous tumor-infiltrating lymphocytes (TILs), which may be used to treat patients with advanced solid tumors, such as melanoma and hematological malignancies.

[0094] Adoptive cell therapy can also include the transplantation of "off-the-shelf" allogeneic lymphocytes that have been isolated, prepared, and stored (e.g., frozen) from healthy donors, which can be used to treat patients with advanced solid tumors, such as melanoma and hematologic malignancies.

[0095] Adoptive cell therapy can use cell types such as T cells, natural killer (NK) cells, delta-gamma T cells, regulatory T cells, dendritic cells, and peripheral blood mononuclear cells. Adoptive cell therapy can also use monocytes to induce differentiation into dendritic cells following contact with tumor antigens. Given that monocytes have a fixed mitotic index, permanent manipulation of the host may be reduced.

[0096] According to certain embodiments, the adoptive cell therapy can be CAR T cell therapy. CAR T cells can be engineered to target tumor antigens of interest by engineering a desired antigen-binding domain that specifically binds to the antigen on tumor cells. In the context of the present invention, "tumor antigen" or "proliferative disorder antigen" or "antigen associated with a proliferative disorder" refers to an antigen that is common to a particular proliferative disorder, such as cancer. The antigens discussed herein are included merely by way of example and are not intended to be exclusive, and further examples will be readily apparent to those skilled in the art.

[0097] According to certain embodiments, the CAR T cell therapy comprises one or more of the following: CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CS-1, B-cell maturation antigen (BCMA), MAGEA3, MAGEA3 / A6, KRAS, CLL1, MUC-1, HER2, EpCam, GD2, GPC3, GPA7, PSCA, EGFR, EGFRvIII, ROR1, mesothelin, CD33 / IL3Ra, c-Met, CD37, PSMA, glycolipid F77, GD-2, gp100, NY-ESO-1 CAR T cells are used that target TCR, FR alpha, CD24, CD44, CD133, CD166, CA-125, HE4, oval, estrogen receptor, progesterone receptor, uPA, PAI-1, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, or ULBP6, or a combination thereof (e.g., both CD33 and CD123). In certain embodiments, the present invention contemplates that the cancer-affected subject is a patient with a higher disease burden (greater than or equal to 5% myeloblasts), who has a higher incidence of adverse events such as cytokine release syndrome and shorter long-term survival after CAR T.

[0098] CAR T cells can contain antigen-binding domains capable of targeting two or more different antigens (i.e., bispecific or bivalent, trispecific or trivalent, tetraspecific, etc.). Thus, CAR T cells can contain a first antigen-binding domain that binds to a first antigen and a second antigen-binding domain that binds to a second antigen (e.g., tandem CAR). For example, CAR T cells can contain a CD19 binding domain and a CD22 binding domain, and thus can recognize and bind both CD19 and CD22. Alternatively, CAR T cells can contain a CD19 binding domain and a CD20 binding domain, and thus can recognize and bind both CD19 and CD20.

[0099] Alternatively, each cell of the cell population, or the entire cell population, may contain multiple distinct CAR T cells (e.g., constructs), and each CAR T cell construct may recognize a different antigen. For example, a CAR T cell population may target three antigens, such as HER2, IL13Rα2, and EphA2.

[0100] According to certain embodiments of the present invention, cell populations, whether autologous or allogeneic, can be manipulated using gene editing techniques such as CRISPR / cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9), zinc finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs). These techniques, recognized and practiced in the field of genetic engineering, allow for the selective editing, destruction, or insertion of targeted sequences to modify the genome of target cells. Thus, isolated autologous or allogeneic cells for adoptive transplantation practiced in the present invention can be edited to delete or replace known genes or sequences. For example, T cell receptors (TCRs) in allogeneic T cell populations can be deleted or replaced before or after CAR-T transduction as a means of eliminating graft-versus-host disease in recipient patients.

[0101] According to certain aspects of the invention, the cell population may comprise a population of T cells, NK cells, or dendritic cells expressing a CAR, wherein the CAR comprises an extracellular antibody or antibody fragment comprising a humanized anti-CD19 binding domain or a humanized anti-BCMA binding domain, a transmembrane domain, and one or more cytoplasmic costimulatory signaling domains. The cell population may comprise a population of cells expressing a CAR, wherein the CAR comprises an extracellular antibody or antibody fragment comprising two or more binding domains, such as a humanized anti-CD19 binding domain, a humanized anti-CD22 binding domain, and / or a humanized anti-BCMA binding domain, and a transmembrane domain, and one or more cytoplasmic costimulatory signaling domains.

[0102] Although CAR cell therapy has demonstrated unprecedented initial response rates in advanced B-cell malignancies, recurrence after CAR cell infusion and limited therapeutic success in solid tumors remain major hurdles in the success of CAR regimens. This latter limitation is primarily due to the hostile microenvironment of solid tumors. Anatomical barriers such as tumor stroma, as well as immunosuppressive cytokines and immune cells that are detrimental to the infiltration of infused CAR-modified cells into the tumor site, both limit the success of CAR cell therapy. Armored CARs can be used to circumvent some of these limitations. These CAR cells are further modified to express immunomodulatory proteins, such as cytokines (e.g., IL-2, IL-12, or IL-15), which can stimulate T cell activation and recruitment and thus help combat the tumor microenvironment. Thus, according to certain aspects of the present invention, a cell population can include a cell population that expresses a CAR and further expresses an immunomodulatory protein, such as IL-2, IL-12, or IL-15.

[0103] The adoptive cell therapy of the present invention involves a cell population expressing T cell receptors (TCRs). TCRs are antigen-specific molecules involved in recognizing antigenic peptides presented in the context of major histocompatibility complex (MHC) products on the surface of antigen-presenting cells or any nucleated cell (e.g., all human cells in the body except red blood cells). In contrast, antibodies typically recognize soluble or cell-surface antigens and do not require MHC presentation. This system confers upon T cells, via their TCRs, the potential to recognize the entire array of intracellular antigens expressed by cells (including viral proteins) that are processed intracellularly into short peptides, bound to intracellular MHC molecules, and delivered to the surface as peptide-MHC complexes. This system allows virtually any foreign protein (e.g., mutated cancer antigens or viral proteins) or aberrantly expressed proteins to serve as targets for T cells.

[0104] As noted above, target-specific and extracellular adverse events typical of cytokine release syndrome (CRS) have been observed with adoptive cell therapy. To maintain the benefits of these innovative therapies while minimizing risk, adjustable safety switches can be incorporated to control the activity level of CAR- or TCR-expressing cells. That is, inducible costimulatory chimeric polypeptides can be included, enabling sustained and regulated control of CAR or TCR activity. Ligand inducers can activate CAR- or TCR-expressing cells, for example, by multimerizing inducible chimeric signaling molecules that can induce intracellular signaling pathways, resulting in activation of target cells (T cells, NK cells, TILs, dendritic cells). In the absence of the ligand inducer, target cells are quiescent or have a basal level of activity.

[0105] Thus, according to certain embodiments of the present invention, a switch may be added to activate the CAR or TCR (i.e., a safety switch CAR or TCR, goCAR or goTCR). CAR- or TCR-expressing cells may be designed to be fully activated only when exposed to both a cancer cell (e.g., a target antigen) and a chemical agent (e.g., rimiduside, rapamycin), thus providing a means to control the degree of activation of the CAR or TCR cells by adjusting the administration schedule of the chemical agent. The CAR or TCR still acts in a targeted manner, but on a controllable schedule, which may reduce certain side effects of adoptive cell therapy. Thus, according to certain embodiments of the present invention, ACT may include goCAR or goTCR cell therapy.

[0106] According to certain embodiments of the present invention, engineered CAR cells can be allogeneic cells from a healthy donor and can be further engineered to disrupt or replace the endogenous TCR by gene editing techniques such as CRISPR / cas9, ZFN, or TALEN, where deletion of the endogenous TCR serves to eliminate CAR-driven graft-versus-host disease.

[0107] According to certain embodiments of the present invention, autologous cells (e.g., T cells, NK cells, or dendritic cells) can be collected from a subject. These cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spinal cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. According to certain embodiments of the present invention, allogeneic or xenogeneic cells can be used, typically isolated from a healthy donor. When the T cells, NK cells, dendritic cells, or pluripotent stem cells are allogeneic or xenogeneic cells, any number of cell lines available in the art can be used.

[0108] Cells can be obtained from a blood unit drawn from a subject using any of several techniques known to those skilled in the art, such as Ficoll™ separation. According to certain aspects of the present invention, cells from an individual's circulating blood can be obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.

[0109] Enrichment of a cell population by negative selection can be achieved using a combination of antibodies directed against surface markers specific to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. According to certain embodiments of the present invention, it may be desirable to enrich or positively select a cell population. For example, positive enrichment of regulatory T cells can use positive selection against CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+.

[0110] The harvested cells can be engineered to express CAR or TCR by any of several methods known in the art. Furthermore, the engineered cells can be propagated by any of several methods known in the art. As described above, the CAR or TCR can be bispecific, trispecific, or tetraspecific, the CAR or TCR can include a switch such as goCAR or goTCR, or a safety switch CAR or TCR, and the CAR or TCR can express an immunomodulatory protein such as armored CAR or TCR.

[0111] Additional agents known to suppress bone marrow, or can act on lymphocyte depletion, or can change tumor microenvironment, can be used in combination with the radiolabeled antibody described above.For example, gemcitabine can deplete myeloid-derived suppressor cells and / or bone marrow, while PD1, PD-L1, TIM3, or LAG-3 antagonist antibodies, GITR or OX40 costimulatory antibodies, IDO inhibitors, A2aR antagonists, or CD73 antagonists can change or shift tumor microenvironment.When the additional agent is an antibody, it can be provided as a separate agent or composition, or as one arm of a bispecific antibody combined with any of the antibody targets listed herein (i.e., CD19, CD20, CD33, CD38, CD45RA, CD52).

[0112] According to certain aspects of the present invention, collection of a blood sample or apheresis product from a subject can be any time prior to the time when expanded cells as described herein may be needed. Thus, the source of cells to be manipulated and expanded (or simply expanded, in the case of TILs) can be collected at any time needed, and the desired cells, such as T cells, NK cells, dendritic cells, or TILs, can be isolated and frozen for later use in adoptive cell therapy, such as the adoptive cell therapy described herein.

[0113] Therefore, according to certain aspects of the present invention, a blood sample or apheresis can be taken from a generally healthy subject, or a generally healthy subject who is at risk of developing a disease but has not yet developed the disease, and the cells of interest can be isolated and frozen for later use. The cells can be expanded, frozen, and later used. In certain cases, a cell sample can be taken from a subject immediately after diagnosis of a particular disease described herein, but before any treatment.

[0114] According to certain aspects of the invention, cells are isolated from a subject in conjunction with lymphodepletion (e.g., before, simultaneously with, or after lymphodepletion) using any of the radiolabeled antibodies of the invention and may be used fresh or frozen for later use.

[0115] According to certain aspects of the invention, the CAR / TCR-expressing cell population may be administered to a subject by dose fractionation, with a first fraction of the total dose being administered on the first day of treatment, a second fraction of the total dose being administered on the next day of treatment, and optionally a third fraction of the total dose being administered on yet a subsequent day of treatment.

[0116] An exemplary total dose is 10 3 ~10 11 cells / kg (subject weight), e.g., 10 3 ~10 10 cells / kg (subject weight), or 10 3 ~10 9 cells / kg (subject weight), or 10 3 ~10 8 cells / kg (subject weight), or 10 3 ~10 7 Cells / kg (subject weight), 10 3 ~10 6 cells / kg (subject weight), or 10 3 ~10 5 cells / kg of subject body weight. Further, an exemplary total dose is 10 4 ~10 11 cells / kg (subject weight), e.g., 10 5 ~10 11cells / kg (subject weight), or 10 6 ~10 11 cells / kg (subject weight), or 10 7 ~10 11 Contains cells / kg (subject weight).

[0117] An exemplary total dose may be administered based on the patient's body surface area rather than weight. 3 ~10 13 cells / m 2 may include:

[0118] Exemplary doses may be based on a fixed or fixed dosing schedule, rather than on body weight or body surface area. Fixed doses may avoid potential dose calculation errors. Furthermore, genotyping and phenotyping strategies, as well as therapeutic drug monitoring, may be used to calculate the appropriate dose. That is, dosing may be based on the patient's immune repertoire of immune suppressor cells (e.g., T-reg, MDSC) and / or disease burden. Thus, the total dose may be 10 3 ~10 13 The total number of cells may be 100.

[0119] According to certain aspects of the present invention, cells can be obtained from a subject immediately after treatment. In this regard, it has been observed that after treatment of certain cancers, particularly after treatment with drugs that damage the immune system, the quality of certain cells (e.g., T cells) obtained shortly after treatment during the period when a subject would normally recover from treatment can be optimal or improved in terms of their ability to expand ex vivo. Similarly, after ex vivo manipulation using the methods described herein, these cells can be in a favorable state for enhanced engraftment and in vivo expansion. Therefore, it is contemplated within the context of the present invention to collect blood cells, including T cells, NK cells, dendritic cells, or other cells of the hematopoietic system, during this recovery period.

[0120] According to a specific embodiment of the present invention, the radiolabeled antibody can be administered after the administration of an effective dose of a cell population expressing CAR / TCR or TIL. The effective amount of the radiolabeled antibody can be an amount sufficient to induce lymphocyte depletion in a subject. According to a specific embodiment, the effective amount of the radiolabeled antibody can be an amount sufficient to induce myeloablation in a subject.

[0121] According to certain aspects of the invention, the radiolabeled antibody may be administered 1 to 3 months after administration of the CAR / TCR- or TIL-expressing cell population, wherein the effective amount of the radiolabeled antibody is sufficient to induce lymphodepletion in the subject, such as in preparation for autologous stem cell transplantation or administration of a second effective amount of the CAR / TCR-expressing cell population.

[0122] According to certain aspects of the present invention, a radiolabeled antibody can be administered to a subject both before and after adoptive cell therapy. That is, the method can include an apheresis step to harvest a cell population engineered to express a CAR / TCR and expanded, followed by the administration of an effective amount of a first dose of a radiolabeled antibody. The cell population expressing a CAR / TCR can then be administered to the subject, followed by the administration of an effective amount of a second dose of a radiolabeled antibody. Then, a second dose of a cell population expressing a CAR / TCR can be administered. This second dose of a cell population expressing a CAR / TCR can be the same as or different from the first dose. That is, the first and second doses can be doses of the same cell population expressing the same CAR / TCR. Alternatively, the second dose can be a different effective amount of the same cell population expressing the same CAR / TCR.

[0123] According to certain embodiments of the present invention, the second dose can be the same or different effective amounts of different cell populations expressing the same or different CAR / TCRs. The difference in the CAR / TCRs can be in any aspect of the CAR / TCR, such as a different binding domain or antigen recognition domain, or costimulatory domain. The second dose can additionally or alternatively include secretory cells with IL-12, or even adjuvant immunotherapy using small molecule inhibitors such as BTK, P13K, or IDO inhibitors, either simultaneously or sequentially with the cell therapy infusion.

[0124] Thus, according to certain aspects of the present invention, the second dose of a cell population expressing a CAR / TCR may comprise a second cell population expressing a second CAR / TCR, where the second CAR / TCR is different from the CAR / TCR in the first dose. For example, the second dose may comprise a second population of activated T cells or NK cells or dendritic cells expressing a second CAR / TCR, where the second CAR / TCR may comprise a second CAR having one or more extracellular, transmembrane, or cytoplasmic costimulatory signaling domains different from those of the CAR in the first dose. For example, the second CAR may recognize a different antigen, be expressed on a different cell type, or comprise a different cytoplasmic costimulatory signaling domain, etc.

[0125] Administration of a second dose of radiolabeled antibody after administration of a CAR / TCR-expressing cell population can be used for subjects who do not experience a complete response (CR) after administration of a CAR / TCR-expressing cell population. Additionally, administration of a second dose of radiolabeled antibody after administration of a CAR / TCR-expressing cell population can be used when a subject has or is identified as having a recurrence of antigen-positive disease (e.g., CD-19+) or antigen-negative disease (CD-19-) after administration of a CAR / TCR-expressing cell population.

[0126] According to certain aspects of the invention, the methods may include the administration of one or more additional therapeutic agents. Exemplary therapeutic agents include chemotherapeutic agents, anti-inflammatory agents, immunosuppressants, immunomodulatory agents, or combinations thereof.

[0127] Therapeutic agent can be administered according to any standard dosage regimen known in the art.Exemplary chemotherapeutic agent includes mitotic inhibitors such as taxanes (e.g., docetaxine and paclitaxel) and vinca alkaloids (e.g., vindesine, vincristine, vinblastine and vinorelbine).Exemplary chemotherapeutic agent includes topoisomerase inhibitors such as topotecan.

[0128] Exemplary chemotherapeutic agents include growth factor inhibitors, tyrosine kinase inhibitors, histone deacetylase inhibitors, P38a MAP kinase inhibitors, angiogenesis, neovascularization, and / or other angiogenesis inhibitors, colony-stimulating factors, erythropoietic agents, anti-anergic agents, immunosuppressive and / or immunomodulatory agents, viruses, viral proteins, immune checkpoint inhibitors, BCR inhibitors (e.g., BTK, P13K, etc.), immunometabolic agents (e.g., IDO, arginase, glutaminase inhibitors, etc.). According to certain embodiments of the present invention, one or more therapeutic agents may include an antimyeloma agent. Exemplary antimyeloma agents include dexamethasone, melphalan, doxorubicin, bortezomib, lenalidomide, prednisone, carmustine, etoposide, cisplatin, vincristine, cyclophosphamide, and thalidomide, some of which are identified above as chemotherapeutic, anti-inflammatory, or immunosuppressive agents. [Example]

[0129] The following example provides the experimental design and specific results of the compositions and methods of the present invention using one of the radiolabeled antibodies according to the present invention: anti-CD45 (the monoclonal antibody is BC8).

[0130] Example 1 131 I-BC8 (Iomab-B) Iomab-B drug is a radioiodinated anti-CD45 mouse monoclonal antibody (mAb) ( 131 Iomab-B is a cytotoxic T cell-mediated ischemia-associated inflammatory cytokine (C1C)-mediated thrombus formation (THC) that is specific for the hematopoietic CD45 antigen. Iomab-B drug product is supplied as a sterile formulation contained in a container closure system consisting of a depyrogenated type 1 50 mL glass vial, a sterile gray chlorobutyl rubber stopper, and an open-top style aluminum seal. Each dose vial also contains a drug fill volume of 45 mL in the 50 mL vial. Similarly, it can be provided as a single-use dose for complete infusion during intravenous administration, ranging from 1 mCi to 200 mCi (e.g., 100 mCi or 150 mCi). 131 It contains 1 mg / kg of BC8 antibody and 6-60 mg (e.g., 6-45 mg) of patient-specific radioactivity. The BC8 antibody dose can be determined according to ideal body weight at a level of 0.5 mg / kg. The drug is co-administered to patients sequentially with 0.9% Sodium Chloride Injection, USP (normal saline) at a drug-to-saline ratio of 1:9. Because the infusion rate depends on the amount of BC8 antibody in the 45 mL drug fill volume, a total drug and saline infusion volume of approximately 430-450 mL is administered over a variable period of time.

[0131] WO 2017 / 155937 provides the complete structure of BC8, and 131 Methods for making I-BC8 are taught.

[0132] Example 2 225 Ac-BC8 Conjugation of anti-CD45 BC8 with DOTA and subsequent 225Labeling with Ac: Antibody BC8 (2 mg) was equilibrated with conjugation buffer (Na Carbonate buffer with 1 mM EDTA, pH = 8.5-9.0) by four ultrafiltration spins using a Centricon filter with a MW cutoff of 50,000 or a Vivaspin ultrafiltration tube with a MW cutoff of 50,000. 1.5 ml of conjugation buffer was used per spin. For each spin, the antibody was spun at 53,000 RPM and 4°C for 5-20 minutes until the residual retention volume was 100-200 μl. The antibody was incubated at 4°C for 30 minutes after the second and third spins to allow equilibration. For DOTA conjugation, a 3 mg / ml solution of S-2-(4-isothiocyanatobenzyl)-1,4,7,10 tetraazacyclododecanetetraacetic acid (p-SCN-Bz-DOTA, MW = 687) in 0.15 M NHOAc was prepared by dissolving and vortexing. DOTA-Bz-pSCN and BC8 antibody (>5 mg / ml) were mixed together in an Eppendorf tube at a 7.5 molar ratio (DOTA:antibody) and incubated at room temperature for 15 hours. For purification of the DOTA-antibody conjugate, unreacted DOTA-Bz-pSCN was removed by seven rounds of ultrafiltration using 1.5 ml of 0.15 M NHOAc buffer (pH = 6.5) to a volume of approximately 100 μl. After the final wash, 0.15 M NHOAc buffer was added to bring the material to a final concentration of approximately 1 mg / ml. The final concentration of the DOTA-BC8 conjugate was measured, and the number of DOTA molecules conjugated to the antibody was determined to be 1.2 to 1.5 DOTA per antibody.

[0133] 225 Radiolabeling of DOTA-antibody conjugates with Ac:DOTA-BC8 conjugates 225 For labeling with Ac, 15 μL of 0.15 M NH4OAC buffer, pH = 6.5, was mixed with 2 μL (10 μg) of DOTA-BC8 (5 mg / ml) in an Eppendorf reaction tube. 225Ac (10 μCi) was added sequentially, 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 a 1 mM DTPA solution was added to the reaction mixture and incubated at room temperature for 20 minutes to remove uncomplexed DTPA. 225 Instant thin layer chromatography (ITLC) was performed using 10 cm silica gel strips and a 10 mM EDTA / normal saline mobile phase. 225 Determine the radiochemical purity of Ac-anti-BC8 225 Ac-labeled BC8 225 After separation from Ac-DTPA, sections were counted in a gamma counter equipped with a multichannel analyzer. Radiolabeling efficiency over several runs was determined to be greater than 80%.

[0134] Example 3 - Changes in absolute neutrophil counts CD45 is a cell surface protein expressed on most immune cell types, including both lymphocytes and neutrophils. 131 I-BC8) radioimmunotherapy targets and delivers its beta-emitting payload to CD45-positive cells. All CD45-positive cell types are equally 131 It is predicted that I-BC8 is susceptible to depletion after administration. Data from a human clinical trial shown in Figure 2 demonstrate that a 10 mCi dose 131 Relative absolute neutrophil counts are shown for human subjects at various time points after I-BC8 administration, presented as fold increase or decrease. Absolute lymphocyte counts were significantly decreased, demonstrating persistent depletion over time, whereas median absolute neutrophil counts were 131 There was minimal decline and rapid recovery after I-BC8 administration, a surprising finding. 131 The limited effect of I-BC8 on neutrophils and its rapid rebound is expected to benefit patients by preventing infections that might otherwise occur.

[0135] Example 4 - Clinical Data Demonstrating Lymphocyte Depletion and Clearance Low dose levels 131 Clinical data from patients receiving I-BC8 show consistent peripheral lymphopenia. Pharmacokinetic data include: 131 Demonstrates rapid clearance of I-BC8, which limits its interaction with CAR T products. Increased disease control and sustained lymphocyte depletion 131 The time window between I-BC8 administration and CAR T infusion becomes flexible, which in turn prevents toxicity. For example, as shown in Figures 3A-3H, 131 Immune cell analysis after I-anti-CD45 antibody-targeted lymphodepletion demonstrates selective depletion of WBCs in peripheral blood (Figure 3A) and splenic immune cell populations (Figure 3B), with minimal impact on the myeloid compartment (Figure 3C) and suppressed effects on RBCs and platelets (Figures 3D and 3E, respectively). Figures 3F-3H provide bar graphs of similar trial results (days 2 and 4 only). Somewhat surprisingly, splenic T-reg levels were found to be persistently depressed (depleted) even after 21 days. Figure 12 shows the results of the splenic T-reg levels from the patient's circulating blood. 131 Figure 1 shows the rapid clearance of I-BC8. On average, 59 percent of the radiolabeled BC8 antibody was cleared from the blood with a biological half-life of 0.65 hours (39 minutes), and 41 percent was cleared with a biological half-life of 31 hours.

[0136] Example 5 - Various CAR-T cell therapies under development and their lymphodepletion regimens There are many CAR-T cell therapies in development, each with its own lymphodepletion regimen. The diagram in Figure 6 shows selected open clinical trials of anti-CD19 CAR T cell therapy for patients with B-cell NHL. Most of these trials use chemotherapy-based lymphodepletion regimens.

[0137] Example 6 - Pre-KYMRIAH® 131 I-BC8-based lymphocyte depletion In this example, the present invention is used to treat a human subject with NHL or DLBCL. In a first instance, the method includes: (i) administering 25 mCi to 200 mCi (e.g., 100 mCi or 150 mCi) of ribozyme; 131 In a second instance, the method includes (i) administering I-BC8 to an NHL patient; and (ii) 6, 7, or 8 days later, administering KYMRIAH® (tisagenlecleucel) therapy to the patient according to known protocols. In a second instance, the method includes (i) administering 25 mCi to 200 mCi (e.g., 100 mCi or 150 mCi) of KYMRIAH® (tisagenlecleucel) therapy to the patient according to known protocols. 131 The method includes administering I-BC8 to a DLBCL patient, and (ii) 6, 7, or 8 days later, administering KYMRIAH® therapy to the patient according to its known protocol.

[0138] Example 7 - Pre-YESCARTA® 131 I-BC8-based lymphocyte depletion In this example, the present invention is used to treat a human subject with NHL. The method includes: (i) administering 25 mCi to 200 mCi (e.g., 100 mCi or 150 mCi) of ribozyme; 131 The method includes administering I-BC8 to an NHL patient, and (ii) 6, 7, or 8 days later, administering YESCARTA® (axicabtagene ciloleucel) therapy to the patient according to known protocols.

[0139] Example 8 - Preclinical modeling of anti-CD45 radioimmunotherapy-mediated conditioning / lymphocyte depletion Summary: Before receiving a dose of adoptive cell transplantation, such as engineered autologous or allogeneic CAR T cells, it is common to perform a lymphodepletion step, often using high-dose chemotherapy. This process is thought to be important for creating sufficient space in the immune microenvironment, e.g., bone marrow, to allow engraftment of the transplanted cells. Furthermore, it is believed to induce a favorable cytokine profile for the establishment and proliferation of donor lymphocytes. Anti-CD45 radioimmunotherapy is being investigated in a phase III clinical trial as a myeloablative regimen before allogeneic hematopoietic cell transplantation in patients with AML. Results from this trial suggest that low-dose chemotherapy may be beneficial. 131 These findings suggest that I-anti-CD45 radioimmunotherapy may be sufficient for lymphodepletion but not for myeloablation.

[0140] For example, a single dose of 50 mCi to 200 mCi 131 Studies have been conducted to investigate the cumulative radiation dose absorbed by the spleen of human patients receiving I-anti-CD45 antibody. As shown in Table 3, the time during which the residual absorbed dose to the spleen would not exceed 25 cGy is: 131 11 shows that the total dose of 100 mCi of anti-CD45 antibody 131 1 shows the cumulative dose rate to the spleen in patients receiving I-anti-CD45 antibody by infusion, where the dose rate to the spleen is plotted against time after infusion. [Table 3]

[0141] Low dose 131 Preclinical studies of the effects of I-anti-CD45 radioimmunotherapy (surrogate 30F11) have been conducted. This study investigated the lymphodepletion response to specific immune cell types and did not investigate changes in cytokine expression in response to this form of conditioning. For example, Figure 9 shows that after 5–20 mCi 131We demonstrate transient lymphodepletion in human patients receiving I-anti-CD45 antibody (median dose of 8.4 mCi in 13 patients). The results of such studies will be supportive in the development and planning of human clinical studies utilizing anti-CD45 radioimmunotherapy as a non-myeloablative conditioning regimen prior to the administration of autologous or allogeneic adoptive cell transfer (ACT).

[0142] Current studies include the use of CD45 radiolabeled ( ) to deplete bone marrow and peripheral blood of CD45+ immune cells for modeling CD45 radioimmunotherapy as a conditioning regimen for receiving ACT. 131 I) Include immunocompetent mice (e.g., 8-12 week-old female C57Bl / 6 mice) using anti-mouse receptor antibodies. The comparator is treatment with a combination chemotherapy of cyclophosphamide (Cy) and fludarabine (Flu). At up to three time points after treatment, the animals are sacrificed, and peripheral blood, spleen, and bone marrow samples are collected for immunophenotyping to evaluate lymphoid and myeloid subsets for lymphocyte depletion, and blood (serum) is collected for cytokine profiling in response to various treatment regimens.

[0143] Materials: Mice (3 per time point group), e.g., female adolescent C57Bl / 6 mice (30 mice total). Mouse surrogate anti-CD45 antibody 30F11 (supplier: Millipore Sigma: MABF321, rat IgG2b). For labeling. 131 I. Fludarabine (Flu) + Cyclophosphamide (Cy) (Treatment: 250 mg / kg Cy + 50 mg / kg Flu)

[0144] Part I: Labeling and in vitro characterization of surrogate CD45 antibody 30F11 - Iodination of 30F11 antibody will be performed; immunoreactivity testing against mouse CD45+ cells will be performed (e.g., B6-Ly5a splenocytes at 5 ng / ml for 1 hour; target >70% immunoreactivity).

[0145] Part II: (a) (i) Prepare dose for in vivo studies (0.5 mCi in 100 μg administered in 200 μl, according to Matthews, et al., 2001, Blood, 2:737-745). (ii) Matthews dosimetry results: 0.5 mCi dose: 54 Gy to spleen, 17 Gy to bone marrow, 8 Gy to lung, and 5 Gy to liver. Other studies have administered 0.05, 0.1, and 0.2 mCi in 100 μg to determine dose response.

[0146] (b) Radioimmunotherapy regimens are administered IP. Nine mice per group receive CD45 radioimmunotherapy or combination chemotherapy (Cy / Flu). Twelve mice are untreated and there are no treatment controls.

[0147] (c) At three time points (i.e., 24 hours, 48 ​​hours, and 96 hours), mice are sacrificed and samples are extracted (bone marrow, peripheral blood, and spleen, as well as serum). See Figure 7.

[0148] (d) Immunophenotyping will be performed on bone marrow and peripheral blood to evaluate (i) Tregs (CD4, CD25, FoxP3), (ii) CD4 and CD8 T cells, B cells, NK cells: (CD3, CD4, CD8, CD19, CD335), (iii) HSCs (Lin-, c-KIT, SCA1), and (iv) MDSCs, DCs, MACs, granulocytes: (CD11b, CD11c, CD244, syglec F, Ly6G, Ly6C).

[0149] (e) Cytokine profiling is performed using a panel 31-plex (MD31) by Luminex, including IL6, IL7, IL10, IL15, MIP1a, VEGF, and IFNg.

[0150] Example 9 - Preclinical model of adoptive T cell transfer after anti-CD45 radioimmunotherapy-mediated conditioning / lymphodepletion in mice Summary: Lymphocyte depletion is considered a critical step for conditioning patients to receive autologous or allogeneic cell therapies, such as CAR T. Lymphocyte depletion is a promising strategy for selectively depleting immune cells and modulating cytokine responses in mouse models in preparation for adoptive cell transfer. 131 A study to evaluate the efficacy of I-anti-CD45 radioimmunotherapy has been proposed. In this study, the use of CD45 radioimmunotherapy will be evaluated as a non-myeloablative conditioning regimen prior to adoptive T cell transfer. E.G7 lymphoma tumor-bearing mice will receive a single selection dose of I-anti-CD45 radioimmunotherapy prior to adoptive cell transfer of OVA-specific CD8+ T cells. 131 The mice were conditioned with I-anti-CD45 radioimmunotherapy and monitored for engraftment of the transplanted cells and the resulting anti-tumor response. The comparator was conditioning with Cy and Flu combination chemotherapy or no prior conditioning. See Figure 8.

[0151] Materials: (i) Mice (5 per group): Female adolescent C57Bl / 6 CD45.1 mice (3 groups, 15 mice total). (ii) Donor CD45.2 OT-1 mice (approximately 5 mice for the donor T cell pool). (iii) E.G7 tumor cell line. (iv) Mouse surrogate anti-CD45 antibody 30F11 (supplier: Millipore Sigma: MABF321 200 μg, rat IgG2bκ). (v) For labeling. 111 In. (vi) Fludarabine + cyclophosphamide (treatment: 250 mg / kg Cy + 50 mg / kg Flu).

[0152] Methods: (1) CD45.1 C57BL / 6 mice were each treated with 2 × 10 6 E.G7 tumor cells (without Matrigel) were placed in approximately 100 mm 3 The tumor is injected subcutaneously until a tumor volume of 1000 μg / mL is reached.

[0153] (2) Approximately 7 days after tumor cell injection, the mice were 131 I-anti-CD45 radioimmunotherapy (0.5 mCi 131Lymphocyte depletion was performed with either a single intraperitoneal injection of I-anti-CD45 antibody (100 μg antibody in 200 μl) or Flu / Cy, or no conditioning.

[0154] (3) CD8+ T cells are isolated from CD45.2 OT-1 transgenic mice and cultured and activated in vitro.

[0155] (4) 4 days after lymphocyte depletion, 2 × 10 6 Administer CD8+ T cells to each cohort via tail vein injection.

[0156] (5) Tumor volume and body weight are measured daily along with behavioral and health assessments.

[0157] (6) Based on Hsu, et al., 2015, Oncotarget, 6:44134-44150, tumor responses are recorded within 10 days after T cell administration. After measurement on day 10, mice are sacrificed and blood and tumors are collected.

[0158] (7) Tumors will be sectioned and stained for H&E, CD8+ cells, and Tregs.

[0159] (8) Blood is subsequently evaluated for the presence of engrafted CD8 cells (CD45.2+) and populations of Tregs and MDSCs.

[0160] (9) Cytokine profiling will be performed to assess terminal levels of IL-10, IL-12, IL-15, and IFNg.

[0161] Example 10 - Gene-edited T cells This example relates to lymphodepletion in cancer patients prior to administration of one or more doses of adoptive cell therapy containing gene-edited T cells.

[0162] CAR T cells have shown considerable clinical promise, with response rates exceeding 80% in refractory lymphoma patient populations and durable responses lasting for more than 6 months in nearly 50% of treated patients.

[0163] However, these engineered T cells remain susceptible to immunoregulatory controls, such as upregulation of immune checkpoint receptors like PD1, Lag3, or TIM3. These receptors mediate a state of exhaustion and limit the activation potential of engineered cells. In the case of allogeneic CAR T cells, these exogenously administered cells carry the risk of inducing graft-versus-host disease (GVHD). This risk is caused by the recognition of incompatible major histocompatibility antigens by the native T cell receptors present on the engineered allogeneic T cells.

[0164] These adverse events can be effectively mitigated by gene editing techniques such as CRISPR / Cas9. For example, disruption of the gene encoding PD1 eliminates the potential for checkpoint regulation of CAR T antitumor responses. Furthermore, gene editing to disrupt the endogenous TCR locus in allogeneic CAR T cell preparations effectively prevents GVHD (Ren, et al.). Lymphodepletion is a critical step that enables successful engraftment, proliferation, and persistence of administered CAR T cells. However, safer and more effective methods of lymphodepletion (e.g., the subject method) are needed to replace the use of nonspecific chemotherapy and radiation. Known nonspecific regimens may contribute to the emergence of CAR T-related toxicities, such as cytokine release syndrome (CRS), and gene-edited CAR T cells are not exempt from this risk. The subject CD45-based lymphodepletion method is a safer, targeted, and more effective way to deplete lymphocytes prior to gene-edited CAR T, regardless of whether the CAR T cells have their checkpoint receptors or endogenous TCRs disrupted.

[0165] Example 11 - Red Bone Marrow Dosimetry and Time to Decline in a Multicenter Pivotal Phase 3 Study of Iomab-B Carry out the calculations and 131 The time after infusion for the activity level of I-anti-CD45 antibody (Iomab-B) to decrease to a reduced or assumed "safe" level required to minimize the effect of radiation dose on red blood cells and enable and facilitate subsequent cellular bone marrow recovery therapy was evaluated.

[0166] After administration of Iomab-B, radiolabeled antibody activity exponentially declines from each of the major organs with significant uptake. Without wishing to be bound by theory, one possible explanation is that the dose rate to the red bone marrow decreases exponentially over time due to the combined effects of biological clearance and radioactive decay, resulting in a point at which the total residual dose to the bone marrow over an infinite period of time does not exceed 25 cGy. This value of 25 cGy represents one estimate of a relatively "safe" absorbed dose that will not adversely affect red bone marrow cell regeneration and recovery after pretreatment with high-dose Iomab-B.

[0167] Data Overview: Clinical Trials 131 I-anti-CD45 antibody ( 131 In a subset of 25 patients receiving IFN-γ-I-BC8), 131 The mean initial uptake of I-BC8 was 17.4%, which was cleared with a mean effective half-life of 45.1 hours. Figure 10 shows the mean dose rate (cGy / hour) to red bone marrow in Iomab-B patients for an infusion of 100 mCi. The dose rate to bone marrow is plotted against time after infusion. The elimination curve represents a single exponential function with a residence half-life of 45.1 hours. At 154 hours (approximately 6.5 days) after infusion, the area under the curve indicates that a total absorbed dose of no more than 25 cGy would be imparted to the red bone marrow during the entire remaining time (representing approximately 9% of the total dose of 271 cGy).

[0168] Figure 10 also shows that the average initial dose rate to red bone marrow was 4.16 cGy / hr. The time to reach the 25 cGy point increases with increasing activity injected. Table 4 shows the time to reach the 25 cGy point for 50 mCi, 75 mCi, 100 mCi, 150 mCi, and 200 mCi doses. 131 The 25 cGy time point for the infusion of I-anti-CD45 antibody is shown. [Table 4]

[0169] Preliminary conclusion: These data suggest that the initiation of cellular restorative therapy (ACT) is 131 A waiting period of 6 to 8 days after infusion of I-anti-CD45 antibodies, depending on the administered dose, is likely sufficient. For added safety and to account for patients with longer than average pharmacokinetic uptake and clearance half-lives, 131 After infusion of I-anti-CD45 antibody, the safety period may be extended by 1–2 days (9–10 days with 200 mCi).

[0170] Individual patient variability: Patients differed in biodistribution and clearance kinetics of Iomab. The mean initial uptake in red bone marrow was 17.4% of the total injected dose, but the highest red bone marrow uptake observed after infusion was 36% in one patient. The mean clearance half-life from bone marrow was 45.1 hours, but the longest clearance half-life observed was 71 hours. The mean absorbed dose to red bone marrow was 2.71 cGy / mCi. 131 I, whereas the highest value observed with the current protocol was 4.24 cGy / mCi.

[0171] Example 12 - In mice 131 Immunophenotyping of T-reg cells after I-BC8-targeted immune depletion. Towards radioimmunotherapy 131Targeted immune depletion studies in mice using I-anti-CD45 antibodies demonstrate the ability to define doses that can target lymphocytes, including T-regs, for depletion while effectively suppressing effects on the bone marrow. Figures 4A-4E and 5A-5C show the efficacy of doses of 50-200 μCi. 131 We demonstrate that I-BC8 depletes lymphocytes and T-regs without significant effects on myeloid cells. Dose levels 1.5 to 4 times higher than those evaluated for μ-lymphocyte depletion have been demonstrated in mouse tumor models of B-cell lymphoma, demonstrating antitumor efficacy. The lower lymphocyte-depleting doses of the present invention also target CD45-positive tumor cells, contributing to antitumor efficacy, reducing tumor burden, including PD1-positive tumors, and improving ACT or CAR-T outcomes. In summary, when used in the preparation of ACT, the present invention targets the immunosuppressive tumor microenvironment, resulting in improved ACT engraftment, response, and antitumor outcomes.

[0172] Example 13 - CD45-targeted immunodepletion is safe for adoptive cell therapy. Towards radioimmunotherapy 131 Studies of targeted immune depletion in mice using anti-CD45 antibodies have shown that such forms of immune depletion are safe prior to adoptive cell therapy. Mice bearing E.G7-OVA tumor engraftments were treated with 100 uCi of IgG1. 131 Conditioned with I-CD45, 1 x 10 6 OT I OVA-reactive T cells were administered on day 4. As shown in Figures 13A and 13B, the adoptively transferred cells were able to persist in the spleen and control tumor growth.

[0173] The following aspects are disclosed in the present application:

[0174] Embodiment 1. A method for targeted immunodepletion of specific subsets of immune cells in a subject, comprising administering to the subject a therapeutically effective amount of ... or a combination thereof, or CD19, CD20, CD33, CD38, CD45RA, CD52, or a combination thereof, or CD2, CD5, CD11a, CD18, CD49a, CD122, CD132, CD244, CD272, CD305, CLA, IL-21 receptor, and B7-HA, or a combination thereof, to a subject.

[0175] Embodiment 2. The method of embodiment 1, wherein the radiolabeled antibody is a bispecific antibody comprising a lymphoid-derived target and a myeloid-derived target, wherein the lymphoid target is CD3, CD2, CD28, CD96, CD122, CD152, or CD154, and / or the myeloid target is CD11b, CD11c, CD14, CD33, or CD116.

[0176] Embodiment 3. The method of embodiment 1 or embodiment 2, wherein an effective amount of the radiolabeled antibody depletes at least 50% of the targeted immune cells, or at least 70% of the targeted immune cells, or at least 80% of the targeted immune cells, and wherein an effective amount of the radiolabeled antibody depletes less than 20% of the subject's stem cells, or less than 10% of the subject's stem cells.

[0177] Embodiment 4. The radiolabeled antibody is 131 I, 125 I, 123 I, 90 Y, 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 32 P, 225 Ac, 213Bi, 213 Po, 211 At, 212 Bi, 213 Bi, 223 Ra, 227 Th, 149 Tb, 137 Cs, 212 Pb, or 103 The method of any one of aspects 1 to 3, wherein the compound is labeled with Pd.

[0178] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the specific subset of immune cells of the subject comprises cells of lymphoid origin but which are not stem cells.

[0179] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the specific subset of immune cells of the subject comprises cells that are of myeloid origin but are not stem cells.

[0180] Embodiment 7. The radiolabeled antibody is 131 7. The method of any one of aspects 1-6, wherein the compound is labeled with I, and the effective amount is between 10 mCi and 200 mCi, or the effective amount is less than 200 mCi, or the effective amount is less than 100 mCi.

[0181] Embodiment 8. The radiolabeled antibody is 225 The method according to any one of aspects 1 to 6, wherein the compound is labeled with Ac, and the effective amount is 0.1 μCi / kg (body weight of the subject) to 5.0 μCi / kg (body weight of the subject).

[0182] Embodiment 9 The method of any one of embodiments 1 to 8, wherein the effective amount of the radiolabeled antibody does not induce myeloablation in the subject.

[0183] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the effective amount of the radiolabeled antibody is administered as a single dose.

[0184] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the effective amount of the radiolabeled antibody provides a radiation dose of 2 Gy or less to the bone marrow.

[0185] Embodiment 12. The method of any one of embodiments 1 to 11, wherein the subject is suffering from cancer and is undergoing adoptive cell therapy to treat the cancer.

[0186] Embodiment 13. The method of any one of embodiments 1-12, further comprising administering to the subject an adoptive cell therapy, wherein the adoptive cell therapy comprises administering to the subject an effective amount of a population of cells expressing a chimeric antigen receptor or T cell receptor (CAR / TCR).

[0187] Embodiment 14 The method of embodiment 11 or 13, wherein the adoptive cell therapy is performed 6, 7, or 8 days after administration of the radiolabeled antibody.

[0188] Embodiment 15. The method of any one of embodiments 11 to 14, wherein the adoptive cell therapy is an autologous cell therapy, or wherein the adoptive cell therapy is an allogeneic cell therapy.

[0189] Embodiment 16 The method of any one of embodiments 11-15, wherein the adoptive cell therapy comprises administration of gene-edited CAR T cells, wherein the gene-edited CAR T cells fail to properly express at least one checkpoint receptor and / or at least one T cell receptor.

[0190] Embodiment 17. The cell population expressing a CAR / TCR is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CS-1, B-cell maturation antigen (BCMA), MAGEA3, MAGEA3 / A6, KRAS, CLL1, MUC-1, HER2, EpCam, GD2, GPC3, GPA7, PSCA, EGFR, EGFRvIII, ROR1, mesothelin, CD33 / IL3Ra, c-Met, CD37, PSMA, glycolipid F77, GD-2, gp100, NY-ESO-1 17. The method of any one of aspects 11-16, wherein the method targets TCR, FR alpha, CD24, CD44, CD133, CD166, CA-125, HE4, oval, estrogen receptor, progesterone receptor, uPA, PAI-1, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, or ULBP6, or a combination thereof.

[0191] Embodiment 18. The method of any one of embodiments 11 to 17, wherein the cell population expressing a CAR / TCR targets CD19, CD20, CD22, or a combination thereof.

[0192] Embodiment 19. The method of any one of embodiments 1 to 11, wherein the subject is suffering from an autoimmune disease, and the particular subset of immune cells of the subject comprises B cells, plasma cells, or a combination of both.

[0193] Embodiment 20. An article of manufacture comprising: (a) a radiolabeled antibody to CD2, CD3, CD5, CDl la, CDl lb, CDl lc, CD14, CD18, CD19, CD20, CD28, CD33, CD38, CD45RA, CD49a, CD52, CD96, CDl 16, CD122, CD132, CD152, CD154, CD244, CD272, CD305, CLA, IL-21, B7-HA, or a combination thereof; and (b) a label instructing a user to administer to a subject an amount of the antibody effective to deplete at least 50% of the subject's targeted immune cells and less than 20% of the subject's stem cells, or wherein the amount of antibody is effective to deplete at least 90% of the subject's targeted immune cells and less than 10% of the subject's stem cells.

[0194] Embodiment 21 The article of manufacture of embodiment 20, wherein the radiolabeled antibody is directed against CD19, CD20, CD33, CD38, CD45RA, CD52, or a combination thereof.

[0195] Embodiment 22. The article of manufacture of embodiment 20, wherein the radiolabeled antibody is directed against CD2, CD5, CD11a, CD18, CD49a, CD122, CD132, CD244, CD272, CD305, CLA, IL-21 receptor, and B7-HA, or a combination thereof.

[0196] Embodiment 23 The article of manufacture of embodiment 20, wherein the radiolabeled antibody is a bispecific antibody comprising a lymphoid-derived target and a myeloid-derived target.

[0197] Embodiment 24. The article of manufacture of embodiment 23, wherein at least one of the targets comprises CD19, CD20, CD33, CD38, CD45RA, or CD52.

[0198] Embodiment 25. The article of manufacture of embodiment 23, wherein the lymphoid target is CD3, CD2, CD28, CD96, CD122, CD152, or CD154, and / or the myeloid target is CD11b, CD11c, CD14, CD33, or CD116.

[0199] 26. The antibody is 131 I and administered in an amount of 10 mCi to 200 mCi, or the antibody 225 26. The article of manufacture of any one of aspects 20 to 25, wherein the article is radiolabeled with Ac and administered in an amount of 0.1 μCi / kg to 5.0 μCi / kg of subject body weight.

Claims

1. 1. A composition for targeted immunodepletion of a specific subset of immune cells in a subject, said composition comprising: administering to the subject an effective amount of a radiolabeled antibody to CD2, CD3, CD5, CD11a, CD11b, CD11c, CD14, CD18, CD19, CD20, CD28, CD33, CD38, CD45RA, CD49a, CD52, CD96, CD116, CD122, CD132, CD152, CD154, CD244, CD272, CD305, CLA, IL-21, B7-HA, or a combination thereof; wherein the effective amount of the radiolabeled antibody depletes at least 50% of the targeted immune cells, and the effective amount of the radiolabeled antibody depletes less than 20% of the subject's stem cells; the radiolabeled antibody 131 I, 125 I, 123 I, 90 Y. 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 32 P. 225 Ac, 213 Bi, 213 Po, 211 At, 212 Bi, 213 Bi, 223 Ra, 227 Th, 149 Tb, 137 Cs, 212 Pb, or 103 A composition that is labeled with Pd.

2. The composition described in claim 1, wherein the radiolabeled antibody is directed against CD19, CD20, CD33, CD38, CD45RA, CD52, or a combination thereof.

3. The composition of claim 1, wherein the radiolabeled antibody is directed against CD2, CD5, CD11a, CD18, CD49a, CD122, CD132, CD244, CD272, CD305, CLA, IL-21 receptor, and B7-HA, or a combination thereof.

4. The composition described in claim 1, wherein the specific subset of immune cells of the subject includes cells of lymphoid origin but which are not stem cells, or cells of myeloid origin but which are not stem cells.

5. The composition of claim 1, wherein the radiolabeled antibody is a bispecific antibody comprising a lymphoid-derived target and a myeloid-derived target.

6. The composition described in claim 5, wherein at least one of the targets includes CD19, CD20, CD33, CD38, CD45RA, or CD52.

7. The composition described in claim 5, wherein the lymphoid target is CD3, CD2, CD28, CD96, CD122, CD152, or CD154, or the myeloid target is CD11b, CD11c, CD14, CD33, or CD116.

8. The composition of claim 1, wherein the radiolabeled antibody is labeled with 131 I and the effective amount is 10 mCi to 200 mCi.

9. The composition of claim 1, wherein the radiolabeled antibody is labeled with 225 Ac, and the effective amount is 0.1 μCi / kg (subject body weight) to 5.0 μCi / kg (subject body weight).

10. The composition described in claim 1, wherein the effective amount of the radiolabeled antibody does not induce bone marrow destruction in the subject.

11. The composition described in claim 1, wherein the effective amount of the radiolabeled antibody is administered as a single dose.

12. The composition described in claim 1, wherein the effective amount of the radiolabeled antibody provides a radiation dose of 2 Gy or less to bone marrow.

13. The composition described in claim 1, wherein the subject is suffering from an autoimmune disease and the specific subset of immune cells of the subject comprises B cells, plasma cells, or a combination of both.

14. The composition described in claim 1, wherein the subject is suffering from cancer and is undergoing adoptive cell therapy to treat the cancer.

15. The composition described in claim 1, wherein the subject is a subject undergoing adoptive cell therapy, and the adoptive cell therapy comprises administering to the subject an effective amount of a cell population expressing a chimeric antigen receptor or a T cell receptor (CAR / TCR).

16. The composition of claim 15, wherein the adoptive cell therapy is performed 6, 7, or 8 days after administration of the radiolabeled antibody.

17. The composition described in claim 15, wherein the adoptive cell therapy is autologous cell therapy or allogeneic cell therapy.

18. The composition of claim 15, wherein the adoptive cell therapy comprises administration of gene-edited CAR T cells, wherein the gene-edited CAR T cells fail to properly express at least one checkpoint receptor and / or at least one T cell receptor.

19. The cell population expressing the CAR / TCR comprises any one of CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CS-1, B-cell maturation antigen (BCMA), MAGEA3, MAGEA3 / A6, KRAS, CLL1, MUC-1, HER2, EpCam, GD2, GPC3, GPA7, PSCA, EGFR, EGFRvIII, ROR1, mesothelin, CD33 / IL3Ra, c-Met, CD37, PSMA, glycolipid F77, GD-2, gp100, and NY-ESO-1.

16. The composition of claim 15, which targets TCR, FR alpha, CD24, CD44, CD133, CD166, CA-125, HE4, oval, estrogen receptor, progesterone receptor, uPA, PAI-1, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, or ULBP6, or a combination thereof.

20. The composition described in claim 15, wherein the cell population expressing the CAR / TCR targets CD19, CD20, CD22, or a combination thereof.

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