Combination of radioimmunotherapy and immune checkpoint therapy in treatment of cancer

Abstract

To provide improved methods to specifically target and kill cancer cells while simultaneously improving the immune response to neo-antigens released from the targeted cancer cells.SOLUTION: The present invention provides methods for treating a proliferative disease or disorder by administering a radioimmunotherapy to induce an immune response in combination with an immune checkpoint therapy to further enhance the immune response.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 783,510, filed December 21, 2018, which is incorporated herein in its entirety.

[0002] The present invention relates to methods for treating a subject with a proliferative disorder by administration of radioimmunotherapy and immune checkpoint therapy. [Background technology]

[0003] Cancer is a heterogeneous group of malignant diseases that causes millions of deaths worldwide each year. In 2018, the death toll from cancer in the United States exceeded 600,000. Despite decades of efforts, the majority of cancers remain incurable, mainly due to progression from localized to metastatic disease. Furthermore, cancer cells have developed means to evade standard checkpoints of the immune system. For example, cancer cells have been found to evade immune surveillance through reduced expression of tumor antigens, downregulation of MHC class I and II molecules resulting in reduced tumor antigen presentation, secretion of immunosuppressive cytokines such as TGFb, recruitment or induction of immunosuppressive cells such as regulatory T cells (Tregs) or myeloid-derived immunosuppressive cells (MDSCs), as well as overexpression of certain ligands [e.g., programmed cell death ligand-1 (PD-L1)] that inhibit the host's existing antitumor immunity.

[0004] Another major mechanism of immune suppression by cancer cells is a process known as "T cell exhaustion", which results from chronic exposure to tumor antigens and is characterized by the upregulation of inhibitory receptors. These inhibitory receptors act as immune checkpoints to prevent uncontrolled immune responses. Various immune checkpoints acting at different levels of T cell immunity have been described in the literature, including PD-1 (i.e., programmed cell death protein 1) and its ligands PD-L1 and PD-L2, CTLA-4 (i.e., cytotoxic T lymphocyte-associated protein-4), LAG3 (i.e., lymphocyte activation gene 3), B and T lymphocyte attenuator, T-cell immunoglobulin, TIM-3 (i.e., mucin domain-containing protein 3), and V-domain immunoglobulin suppressor of T cell activation.

[0005] Enhancement of the effectiveness of the immune system through therapeutic intervention is a particularly exciting development in cancer treatment. As shown, checkpoint inhibitors such as CTLA-4 and PD-1 prevent autoimmunity and generally protect tissues from immune collateral damage. In addition, stimulatory checkpoints such as OX40 (i.e., tumor necrosis factor receptor superfamily, member 4; TNFR-SF4), CD137 (i.e., TNFR-SF9), GITR (i.e., glucocorticoid-induced TNFR), CD27 (i.e., TNFR-SF7), and CD28 activate and / or promote T cell expansion. Modulation of the immune system by inhibition or overexpression of these proteins is a promising current area of ​​research. However, such modulation has not been very promising in the treatment of tumors with low mutational burden, i.e., immunologically cold tumors.

[0006] In recent years, it has been observed that localized radiation therapy can stimulate the immune system, thereby modulating the systemic regression of certain cancers, known as the radiation-induced abscopal effect (Grass, et al. Curr Probl Cancer 2016 40:10-24). That is, targeted radiation therapy was found to minimize or eradicate metastases at distant sites. Localized radiation therapy damages DNA in tumor cells, leading to apoptosis of the tumor cells. Tumor antigens, e.g., neoantigens, released from dying tumor cells may provide antigenic stimulation that induces antitumor-specific responses in these distant metastases. This hypothesis is supported by evidence from T cell-deficient mice, where a single tumor nodule was irradiated and distant antigenically associated nodules were found to regress (Demaria S, et al. Int J Radiat Oncol Biol Phys. 2004 58(3):862-70).

[0007] Targeted radiation therapy is not without significant drawbacks. Noncancerous tissues in the radiation path are damaged, and nonlocalized cancers such as metastatic and hematological cancers are not easily targeted. Furthermore, while radiation therapy may provide useful release of neoantigens for antigenic stimulation, cancer cells have developed mechanisms to evade the host immune system. Moreover, in patients who exhibit T cell exhaustion, newly released neoantigens may not prime the immune system to mount a response. Thus, what is needed are improved methods to specifically target and kill cancer cells while at the same time improving the immune response to neoantigens released from targeted cancer cells. Summary of the Invention

[0008] The present invention provides improved methods for the treatment of a wide range of cancers based on the use of radioimmunotherapy in combination with immune checkpoint therapy. The administration of radioimmunotherapy can induce an immune response that can be further enhanced by a subsequent administration of immune checkpoint therapy. Alternatively, suppression of the immune response, such as by T cell exhaustion, can be relieved by administration of immune checkpoint therapy followed by targeting of specific antigens with radioimmunotherapy. These and other combinations of radioimmunotherapy and immune checkpoint therapy are the subject of the present invention.

[0009] Thus, the present invention relates to a method for treating a subject with a proliferative disorder, comprising administering to the subject a therapeutically effective amount of radioimmunotherapy and a therapeutically effective amount of immune checkpoint therapy.Radioimmunotherapy and immune checkpoint therapy may be administered simultaneously or sequentially, for example, radioimmunotherapy may be administered before and / or after immune checkpoint therapy, or vice versa.Administration of radioimmunotherapy and / or immune checkpoint therapy may follow a dosing schedule, such as once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 21, or 28 days.

[0010] According to certain other embodiments, radioimmunotherapy may be administered 1, 2, 3, or 4 weeks prior to immune checkpoint therapy, after which administration of immune checkpoint therapy and / or radioimmunotherapy may follow any of the schemes described herein, i.e., immune checkpoint therapy and radioimmunotherapy, if continued, may be administered simultaneously or sequentially.

[0011] According to certain embodiments, immune checkpoint therapy may be administered 1, 2, 3, or 4 weeks prior to radioimmunotherapy, after which administration of radioimmunotherapy and / or immune checkpoint therapy may follow any of the schemes described herein, i.e., radioimmunotherapy and immune checkpoint therapy, if continued, may be administered simultaneously or sequentially.

[0012] The radioimmunotherapy may include antibodies against CD19, CD20, CD22, CD30, CD33, CD38, CD45, CD123, CD138, CS-1, B-cell maturation antigen (BCMA), MAGEA3, MAGEA3 / A6, KRAS, CLL1, MUC-1, HER2, HER3, DR5, IL13Rα2, and EphA2, EpCam, GD2, GPA7, PSCA, EGFR, EGFRvIII, ROR1, GPC3, CEA, mesothelin, PSMA, or combinations thereof. The radioimmunotherapy may include antibodies against protein products of genes mutated in acute myeloid leukemia, the genes being NPM1, Flt3, TP53, CEBPA, KIT, N-RAS, MLL, WT1, IDH1 / 2, TET2, DNMT3A, ASXL1, or combinations thereof. Radioimmunotherapy may include antibodies against CD33, CD38, CD45, HER3, DR5, or combinations thereof.

[0013] Radioimmunotherapy is 32 P, 211 At, 131 I, 137 Cs, 90 Y, 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 225 Ac, 213 Bi, 213 Po, 212 Bi, 223 Ra, 227 Th, 149 Tb, 64 Cu, 212 Pb, 89 Zr, 68 Ga, and 103 Pd, or combinations thereof.

[0014] According to a particular embodiment, the radioimmunotherapy comprises: 131 I or 225 Ac or 177The antibody includes an anti-CD33 antibody, an anti-CD38 antibody, an anti-CD45 antibody, an anti-HER3 antibody, an anti-DR5 antibody, or a combination thereof, labeled with Lu.

[0015] According to certain embodiments, two or more radioimmunotherapy may be administered to the patient, such as a radioimmunotherapy against any of the antibodies listed above and a radioimmunotherapy against a different one of the antibodies listed above. According to certain embodiments, the first radioimmunotherapy may be against CD33, CD38, CD45, HER3, or DR5, and the second radioimmunotherapy may be against a different one of CD33, CD38, CD45, HER3, or DR5. When two or more radioimmunotherapy are administered to the patient, they may be administered as a combination (i.e., administration of a single solution containing both radioimmunotherapy, or administered separately within a single administration session of both radioimmunotherapy). Alternatively, one of the two radioimmunotherapy may be administered before the immune checkpoint therapy, and the second radioimmunotherapy may be administered after the immune checkpoint therapy, for example, according to any of the administration schedules described above.

[0016] Immune checkpoint therapy may include antibodies against CTLA-4, PD-1, TIM-3, VISTA, BTLA, LAG-3, TIGIT, CD28, OX40, GITR, CD137, CD27, HVEM, PD-L1, PD-L2, PD-L3, PD-L4, CD80, CD86, CD137-L, GITR-L, CD226, B7-H3, B7-H4, BTLA, TIGIT, GALS, KIR, 2B4, CD160, CGEN-15049, or combinations thereof.

[0017] According to certain embodiments, immune checkpoint therapy may include antibodies against PD-1, PD-L1, PD-L2, CTLA-4, or combinations thereof.

[0018] According to a particular embodiment, the proliferative disorder is cancer or solid tumor. According to a particular embodiment, the proliferative disorder is a hematological disease or disorder selected from one or more of multiple myeloma, acute myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, and myeloproliferative neoplasm.

[0019] The present invention also relates to a pharmaceutical composition for treating a proliferative disease, or a hematological disease or disorder, the composition comprising a synergistic combination of radioimmunotherapy and immune checkpoint therapy, as described above.

[0020] According to a particular embodiment of the composition, the radioimmunotherapy may comprise an anti-CD33 antibody, such as lintuzumab, or an anti-CD38 antibody, such as daratumumab, or an anti-CD45 antibody, such as BC8, or an anti-HER3 antibody, or an anti-DR5 antibody, any of which may be 131 I or 225 Ac or 177 The antibody may be labeled with any of the radionuclides described herein, such as Lu, and the immune checkpoint therapy may include antibodies against PD-1, PD-L1, PD-L2, CTLA-4, or a combination thereof.

[0021] According to certain embodiments of the composition, the radioimmunotherapy may be included in a subject effective amount, including a total protein content of less than 16 mg / kg of subject's body weight, less than 10 mg / kg of subject's body weight, or less than 6 mg / kg of subject's body weight. 225 When Ac is included, the total radioactivity content can be 0.1-10 uCi / kg of subject's body weight, such as 0.2-6 uCi / kg of subject's body weight, or 0.4-5 uCi / kg of subject's body weight. 131When radioimmunotherapy includes a radionuclide, the total radioactivity content can be about 25 mCi, or 50 mCi, or 75 mCi, or 100 mCi, or 150 mCi, or 200 mCi, or 250 mCi, or 300 mCi, or 350 mCi, or 400 mCi, or 450 mCi, or 500 mCi, for example, 25 mCi to 500 mCi, or 50 mCi to 500 mCi, or 100 mCi to 500 mCi. 177 When Lu is included, the total radioactivity content can be less than 500 uCi / kg of subject's body weight, such as 5 uCi-450 uCi / kg of subject's body weight, or 20-250 uCi / kg of subject's body weight.

[0022] According to certain embodiments of the composition, the immune checkpoint therapy may be comprised in a subject effective amount, including doses of 0.1 mg to 50 mg / kg of patient body weight, for example, 0.1 to 5 mg / kg, or 5 to 30 mg / kg.

[0023] The objects of the invention are realized and attained by means of the combinations particularly outlined in the appended claims. The foregoing general description as well as the following detailed description and examples of the invention are provided to illustrate various aspects of the invention and should not be taken in any way as limiting any of the described embodiments. [Brief description of the drawings]

[0024] [Figure 1] 1 provides the amino acid sequence of human CD38 as set forth in GenBank accession number NP_001766. [Diagram 2] 1 provides the amino acid sequence of human CD33 as set forth in GenBank accession number NP_001763. [Diagram 3] 1 provides the amino acid sequence of the RABC isoform of the human CD45 protein. [Figure 4] 1 illustrates a combination therapy according to various embodiments of the present invention. [Diagram 5] 1 illustrates a combination therapy according to various embodiments of the present invention. [Figure 6]1 illustrates a combination therapy according to various embodiments of the present invention. [Figure 7] 1 illustrates a combination therapy according to various embodiments of the present invention. [Figure 8] The sequences of the complementarity determining regions (CDRs), framework regions, and variable domain sequences of the light chain (VL, SEQ ID NO: 4) and heavy chain (VH, SEQ ID NO: 5) of anti-CD45 mAb BC8 are provided, with the CDRs in bold and underlined. [Figure 9] The amino acid sequences including the CDRs and N-terminal portions of the light and heavy chains of anti-CD45 mAb BC8 are provided (SEQ ID NOs: 6-13). [Figure 10-1] The nucleotide sequence (SEQ ID NO:14) and amino acid sequence (SEQ ID NO:15) of the light chain of anti-CD45-immunoglobulin BC8 are provided. [Figure 10-2] The nucleotide sequence (SEQ ID NO:14) and amino acid sequence (SEQ ID NO:15) of the light chain of anti-CD45-immunoglobulin BC8 are provided. [Figure 11-1] The nucleotide sequence (SEQ ID NO:16) and amino acid sequence (SEQ ID NO:17) of the heavy chain of anti-CD45-immunoglobulin BC8 are provided. [Figure 11-2] The nucleotide sequence (SEQ ID NO:16) and amino acid sequence (SEQ ID NO:17) of the heavy chain of anti-CD45-immunoglobulin BC8 are provided. [Figure 11-3] The nucleotide sequence (SEQ ID NO:16) and amino acid sequence (SEQ ID NO:17) of the heavy chain of anti-CD45-immunoglobulin BC8 are provided. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] A brief description of the sequence SEQ ID NO:1 is the amino acid sequence of human CD38 as shown in GenBank Accession No. NP_001766.

[0026] SEQ ID NO:2 is the amino acid sequence of human CD33 as shown in GenBank Accession No. NP_001763.

[0027] SEQ ID NO:3 is the amino acid sequence of the RABC isoform of the human CD45 protein.

[0028] SEQ ID NO:4 is the amino acid sequence of the variable domain of the light chain of anti-CD45 mouse immunoglobulin BC8.

[0029] SEQ ID NO:5 is the amino acid sequence of the variable domain of the heavy chain of anti-CD45 mouse immunoglobulin BC8.

[0030] SEQ ID NO:6 is the amino acid sequence of CDR1 of the light chain of anti-CD45 mouse immunoglobulin BC8.

[0031] SEQ ID NO:7 is the amino acid sequence of the CDR2 of the light chain of anti-CD45 mouse immunoglobulin BC8.

[0032] SEQ ID NO:8 is the amino acid sequence of the CDR3 of the light chain of anti-CD45 mouse immunoglobulin BC8.

[0033] SEQ ID NO: 9 is the amino acid sequence of CDR1 of the heavy chain of anti-CD45 mouse immunoglobulin BC8.

[0034] SEQ ID NO: 10 is the amino acid sequence of CDR2 of the heavy chain of anti-CD45 mouse immunoglobulin BC8.

[0035] SEQ ID NO: 11 is the amino acid sequence of the CDR3 of the heavy chain of anti-CD45 mouse immunoglobulin BC8.

[0036] SEQ ID NO:12 is the amino acid sequence of a portion of the anti-CD45 mouse immunoglobulin BC8 containing the N-terminus of the light chain.

[0037] SEQ ID NO:13 is the amino acid sequence of a portion of the anti-CD45 mouse immunoglobulin BC8 containing the N-terminus of the heavy chain.

[0038] SEQ ID NO:14 is the nucleotide sequence of the light chain of anti-CD45 mouse immunoglobulin BC8.

[0039] SEQ ID NO: 15 is the amino acid sequence of the light chain of anti-CD45 mouse immunoglobulin BC8.

[0040] SEQ ID NO: 16 is the nucleotide sequence of the heavy chain of anti-CD45 mouse immunoglobulin BC8.

[0041] SEQ ID NO: 17 is the amino acid sequence of the heavy chain of anti-CD45 mouse immunoglobulin BC8.

[0042] The present invention uses radioimmunotherapy in combination with immune checkpoint therapy to provide a more durable cancer therapy that stimulates immune responses through potent tumor cell killing and the release of neo-antigens and the enhancement of immune responses to those neo-antigens.

[0043] Cancers with low mutational burden typically do not respond well to immune checkpoint therapies, such as antibodies against checkpoint inhibitors. Previous studies have shown that external radiation can result in an abscopal effect, where non-irradiated tumors, such as melanoma or colon cancer, respond to therapy, presumably due to stimulation of an immune response at the site of the irradiated tumor that can recognize and attack distant tumor lesions.

[0044] Radioimmunotherapy uses radiolabeled antibodies against tumor-specific antigens to deliver cytotoxic radiation to targeted tumor cells, which kills tumor cells primarily by causing single- or double-stranded breaks in DNA. In doing so, tumors may release numerous tumor-specific antigens that prime immune cells against these antigens. Thus, targeted radioimmunotherapy may reach local and distant tumor sites and promote antigen presentation by antigen-presenting cells (i.e., dendritic cells, macrophages). Therefore, targeted radioimmunotherapy may be able to induce an abscopal effect.

[0045] Because radioimmunotherapy does not require cell proliferation and is not prone to multidrug resistance mechanisms, many tumor types may be sensitive to this therapy, including cancers such as leukemias and lymphomas that exhibit a relatively low mutational burden and therefore may be less sensitive to immunomodulatory therapies such as antibodies against PD1 (i.e., programmed cell death protein-1) or CD137. For example, in acute myeloid leukemia, a relatively small number of genes are known to be mutated, including: NPM1, Flt3, TP53, CEBPA, KIT, N-RAS, MLL, WT1, IDH1 / 2, TET2, DNMT3A, and ASXL1. Facilitating the release and presentation of these mutated tumor antigens following targeted radioimmunotherapy may enable the establishment of a robust immune response against cancer cells that would otherwise be inadequate using conventional therapies.

[0046] Certain other genes are overexpressed and / or selectively expressed on cells of blood origin, such as CD33, CD38, and CD45. Targeting these cell types with radioimmunotherapy against CD33, CD38, and / or CD45 may provide therapy for malignant and non-malignant hematological diseases or disorders.

[0047] Other genes, such as HER3, are overexpressed in several types of cancer, such as breast, gastrointestinal, and pancreatic cancers. A correlation has been shown between HER2 / HER3 expression and the progression of these cancers from noninvasive to invasive stages. Agents that block HER3-mediated signaling, such as anti-HER3 antibodies, may allow the establishment of a robust immune response against cancer cells that would otherwise be inadequate using conventional therapies.

[0048] Apoptosis is essential in the physiological process of removing unnecessary or damaged cells and maintaining normal cell numbers in the body. Death receptor 5, DR5, is known to induce apoptosis in cells. The regulatory mechanisms of apoptosis are often impaired in cancer or immune diseases. Antibodies against DR5 can act in an agonistic manner on cells expressing the receptor (cancer cells or immune disease-related cells) to kill the cells.

[0049] Immune checkpoint therapies, such as antibodies against checkpoint inhibitors PD1 or PD-L1 (i.e., programmed death ligand-1), TIM3 (i.e., T cell immunoglobulin and mucin domain-containing-3), Lag3 (i.e., lymphocyte activation gene 3), or TIGIT (i.e., T cell immunoreceptor with Ig and ITIM domains), are known to release regulatory control over immune cells, particularly T cells, and stimulate suppressed or exhausted T cells. However, in the absence of an active immune response against the tumor, immune checkpoint therapies are relatively ineffective. Immune checkpoint therapies are typically effective in eliciting sustained responses in only about 20% of patients across a wide range of responsive tumors. In many patients, failure to respond is likely due to a weak or insufficient immune response against the tumor.

[0050] The present invention uses targeted radioimmunotherapy in synergistic combination with antibodies against immune checkpoint inhibitors and / or costimulatory therapy (GITR, OX40, and CD137) that can further activate T cells.This targeted radioimmunotherapy can be effective across all tumor types, especially in tumors with relatively low mutation load, and will be suitable for the treatment of both liquid and solid tumors.

[0051] Therefore, the present invention contemplates combination therapy, including combination of radioimmunotherapy and immune checkpoint therapy. Destructive therapy such as radioimmunotherapy may cause sufficient tumor cell death and allow antigen presentation through release or phagocytosis by phagocyte antigen presenting cells. Combination with immune checkpoint therapy (inhibitory and / or costimulatory) may result in sustained activation of antitumor immune response against newly released neoantigens (i.e., abscopal effect) and / or may be used to activate exhausted immune system so that immune response is possible (i.e., desuppression of immune suppression).

[0052] Definitions and Abbreviations Unless otherwise defined herein, all scientific and technical terms used in connection with this specification shall have the meanings commonly understood by those skilled in the art.Furthermore, in this specification and the appended claims, unless otherwise specified, the use of the singular includes the plural, and the plural includes the singular.For example, although this specification refers to "an" antibody, "a" radionuclide, and "the" immune checkpoint therapy, any one or more of these components and / or any other components described herein may be used.

[0053] As used herein and in the claims, the word "comprises" and forms of the word "comprises" do not limit the invention to exclude any modifications or additions. Moreover, although the invention is described in terms of "comprises," the processes, materials, and compositions detailed herein may be described as consisting essentially of or consisting of. For example, although certain embodiments of the invention are described with respect to a method comprising administering an effective amount of radioimmunotherapy and an effective amount of immune checkpoint therapy, methods "consisting essentially of" or "consisting of" administering an effective amount of radioimmunotherapy and an effective amount of immune checkpoint therapy are also within the scope. In this context, "consisting essentially of" means that any additional components do not substantially affect the effectiveness of the method.

[0054] Furthermore, other than in the examples, or where otherwise indicated, all numbers expressing, for example, amounts of ingredients used herein should be understood to be modified in all cases by the term "about". Thus, unless otherwise indicated, the numerical parameters described in the following specification are approximations that may vary depending on the desired properties obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be interpreted at least in light of the number of reported significant digits and by applying ordinary rounding techniques. Thus, the term "about" when used before numerical designations, such as temperature, time, amount, and concentration (including ranges), indicates approximations that may vary by ±10%, ±5%, or ±1%.

[0055] As used herein, the term "administer" with respect to a targeting agent such as an antibody, an antibody fragment, a Fab fragment, or an aptamer means a means for delivering the agent to the subject's body by any known method suitable for antibody delivery.Specific modes of administration include, but are not limited to, intravenous, transdermal, subcutaneous, intraperitoneal, intrathecal and intratumoral administration.Exemplary methods of administration of antibodies can be substantially as described in International Publication No. WO2016 / 187514, which is incorporated herein by reference.

[0056] Furthermore, in the present invention, antibody or antibody fragment can be formulated with one or more commonly used pharma-ceutically acceptable carriers.Such carriers are well known to those skilled in the art.For example, injectable drug delivery system includes solution, suspension, gel, microsphere and polymer injection, and may include excipients such as soluble modifier (e.g., ethanol, propylene glycol and sucrose) and polymer (e.g., polycaprylactone and PLGA).

[0057] As used herein, the term "antibody" includes, but is not limited to, (a) immunoglobulin molecules that contain two heavy chains and two light chains and recognize an antigen, (b) polyclonal and monoclonal immunoglobulin molecules, (c) monovalent and bivalent fragments thereof (e.g., di-Fab), and (d) bispecific forms thereof. Immunoglobulin molecules may be derived from any of the commonly known classes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. Antibodies may be of both natural and non-natural origin (e.g., IgG-Fc silent). Additionally, antibodies include chimeric antibodies, fully synthetic antibodies, single chain antibodies, and fragments thereof. Antibodies may be human, humanized, or non-human.

[0058] As used herein, "immunoreactivity" refers to a measure of the ability of an immunoglobulin to recognize and bind to a particular antigen. "Specific binding" or "specifically binds" or "binds" refers to an antibody that binds to an antigen or an epitope within an antigen with higher affinity than to other antigens. Typically, an antibody binds to an antigen with a greater affinity than to other antigens, ... -8 M or less, for example, about 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 M or less, or about 1 x 10 -12 The equilibrium dissociation constant (K D ), typically with its K for binding to a non-specific antigen (e.g., BSA, casein) D At least 100 times less than K D The dissociation constant is a constant that ...

[0059] As used herein, an "anti-CD33 antibody" is an antibody, antibody fragment, peptide, Fab fragment, or aptamer that binds to any available epitope of CD33. According to a particular embodiment, the anti-CD33 targeting agent is a humanized antibody against CD33, such as lintuzumab (HuM195), gemtuzumab, or vadastuximab. According to a particular embodiment, the anti-CD33 targeting agent binds to the epitope recognized by the monoclonal antibody "lintuzumab" or "HuM195". HuM195 is known, as is the method of making it.

[0060] An "anti-CD38 antibody" is an antibody that binds to any available epitope of CD38. According to a particular embodiment, the anti-CD38 antibody binds to the epitope recognized by the monoclonal antibody "daratumumab." Daratumumab is known, as are methods for producing it.

[0061] An "anti-CD45 antibody" is an antibody that binds to any available epitope of CD45. According to a particular embodiment, the anti-CD45 antibody binds to the epitope recognized by the monoclonal antibody "BC8". BC8 is known, as are methods for making it. The BC8 antibody may be a chimeric antibody (BC8c) that includes the constant regions of the heavy and / or light chains of a human IgG1-IgG4 molecule or a human Kappa molecule.

[0062] An "anti-HER3 antibody" is an antibody that binds to any available epitope of HER3. According to a particular embodiment, the anti-HER3 antibody binds to an epitope of HER3 recognized by patritumab, seribantumab, lumuletuzumab, elgemtumab, or GSK2849330. According to a particular embodiment, the anti-HER3 antibody is a bispecific antibody against any available epitope of HER3 / HER2, such as MM-111 and MM-141 / Istiratumab from Merrimack Pharmaceuticals, MCLA0-128 from Merus NV, and MEHD7945A / Durigotumab from Genetech.

[0063] An "anti-DR5 antibody" is an antibody that binds to any available epitope of DR5. According to a particular embodiment, the anti-CD5 antibody binds to the epitope of DR5 recognized by the antibodies tigatuzumab, conatumumab, or dorozizumab.

[0064] "Epitope" refers to a target molecule site (e.g., at least a portion of an antigen) that can be recognized and bound by a targeting agent such as an antibody, an antibody fragment, a Fab fragment, or an aptamer. In the case of a protein antigen, for example, it can refer to the region of the protein (i.e., the amino acids, and particularly their side chains) that are bound by the antibody. Overlapping epitopes contain at least 1-5 common amino acid residues. Methods for identifying antibody epitopes are known to those of skill in the art and include, for example, those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988).

[0065] As used herein, the terms "proliferative disorder" and "cancer" may be used interchangeably and can include, but are not limited to, solid cancers (e.g., tumors) and hematological malignancies.

[0066] A "hematological disease" or "hematological disorder" may be understood to refer to at least a blood cancer. Such cancers arise from blood-forming tissues, such as the bone marrow or other cells of the immune system. Hematological diseases or disorders include, but are not limited to, leukemia (e.g., acute myeloid leukemia (AML), acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), acute mixed lineage leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia (CLL), hairy cell leukemia, and large granular lymphocytic leukemia), myelodysplastic syndromes (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocytosis, primary myelofibrosis, and chronic myelogenous leukemia), lymphoma, multiple myeloma, MGUS, and similar disorders, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies.

[0067] "Solid cancer" includes, but is not limited to, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, prostate cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers including those induced by asbestos.

[0068] According to certain embodiments, the radioimmunotherapy disclosed herein comprises a radiolabeled antibody against a hematologically expressed antigen, such as CD33, CD38, CD45, DR5, or HER3. The antibody may be labeled with a radioisotope. As used herein, a "radioisotope" may be an alpha-emitting isotope, a beta-emitting isotope, and / or a gamma-emitting isotope. Examples of radioisotopes include: 32 P, 211 At, 131 I,137 Cs, 90 Y, 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 225 Ac, 213 Bi, 213 Po, 212 Bi, 223 Ra, 227 Th, 149 Tb, 64 Cu, 212 Pb, 89 Zr, 68 Ga, and 103 Pd, or a combination thereof.

[0069] Methods for attaching radioisotopes to antibodies (i.e., "labeling" an antibody with a radioisotope) are known and are described, such as in International Publication No. WO2017 / 155937, which is incorporated herein in its entirety.

[0070] According to a particular embodiment, the radioimmunotherapy comprises: 131 The antibody may be radiolabeled with I (see " 131 I-labeled), an effective amount may be, for example, less than 1200 mCi (i.e., less than 1200 mCi administered to a subject). 131 (I delivers a total body radiation dose of less than 1200 mCi). 131 When labeled with I, the effective amount can be less than 1000 mCi, less than 750 mCi, less than 500 mCi, less than 250 mCi, less than 200 mCi, less than 150 mCi, less than 100 mCi, less than 50 mCi, less than 40 mCi, less than 30 mCi, less than 20 mCi, or less than 10 mCi. 131An effective amount of I-labeled antibody 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 a particular embodiment of this method, 131 An effective amount of I-labeled antibody is 200 mCi to 1200 mCi. Examples of effective amounts include, but are not limited to, 200 mCi to 300 mCi, 200 mCi to 400 mCi, 200 mCi to 500 mCi, 200 mCi to 600 mCi, 200 mCi to 700 mCi, 200 mCi to 800 mCi, 200 mCi to 900 mCi, 200 mCi to 1000 mCi, 200 mCi to 1100 mCi, 300 mCi to 1200 mCi, 400 mCi to 1200 mCi, 500 mCi to 1 Examples of the isomer include 200mCi, 600mCi to 1200mCi, 700mCi to 1200mCi, 800mCi to 1200mCi, 900mCi to 1200mCi, 1000mCi to 1200mCi, 50mCi, 100mCi, 150mCi, 200mCi, 300mCi, 400mCi, 500mCi, 600mCi, 700mCi, 800mCi, 900mCi, 1000mCi, and 1100mCi.

[0071] According to a particular embodiment, the radioimmunotherapy comprises: 225 The antibody may be radiolabeled with Ac (see 225 AC label), an effective amount may be, 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 administered delivers a radiation dose of less than 5.0 μCi per kilogram of subject body weight). According to certain embodiments, the antibody 225When labeled with AC, the effective amount is less than 4.5 μCi / kg, 4.0 μCi / kg, 3.5 μCi / kg, 3.0 μCi / kg, 2.5 μCi / kg, 2.0 μCi / kg, 1.5 μCi / kg, 1.0 μCi / kg, 0.9 μCi / kg, 0.8 μCi / kg, 0.7 μCi / kg, 0.6 μCi / kg, 0.5 μCi / kg, 0.4 μCi / kg, 0.3 μCi / kg, 0.2 μCi / kg, 0.1 μCi / kg, or 0.05 μCi / kg. According to a particular embodiment, the antibody 225 When labeled with AC, the effective amounts are 0.05μCi / kg~0.1μCi / kg, 0.1μCi / kg~0.2μCi / kg, 0.2μCi / kg~0.3μCi / kg, 0.3μCi / kg~0.4μCi / k g, 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~0.9μ Ci / kg, 0.9 μCi / kg to 1.0 μCi / kg, 1.0 μCi / kg to 1.5 μCi / kg, 1.5 μCi / kg to 2.0 μCi / kg, 2.0 μCi / kg to 2.5 μCi / kg, 2.5 μCi / kg to 3.0 μCi / kg, 3.0 μCi / kg to 3.5 μCi / kg, 3.5 μCi / kg to 4.0 μCi / kg, 4.0 μCi / kg to 4.5 μCi / kg, or 4.5 μCi / kg to 5.0 μCi / kg. According to certain embodiments, the antibody 225 When labeled with AC, 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, 0.8μ Ci / kg, 0.9μCi / kg, 1.0μCi / kg, 1.5μCi / kg, 2.0μCi / kg, 2.5μCi / kg, 3.0μCi / kg, 3.5μCi / kg, 4.0μCi / kg, or 4.5μCi / kg.

[0072] According to a particular embodiment, the radioimmunotherapy comprises: 177 The antibody may be radiolabeled with Lu (see " 177 Lu label”), 177An effective amount of Lu-labeled antibody is, for example, less than 12 mCi / kg (i.e., less than 10 mCi / kg administered to a subject). 177 The amount of Lu-labeled antibody delivers a radiation dose of less than 12 mCi per kilogram of subject body weight).

[0073] According to one particular embodiment, the antibody 177 When labeled with Lu, the effective amount is 12 mCi / kg, 11 mCi / kg, 10 mCi / kg, 9 mCi / kg, 8 mCi / kg, 7 mCi / kg, 6 mCi / kg, 5 mCi / kg, 4 mCi / kg, 3 mCi / kg, 2 mCi / kg, 1 mCi / kg, or 0.5 mCi / kg. 177 When labeled with Lu, the effective amount is at least 0.1 mCi / kg, 0.5 mCi / kg, 1 mCi / kg, 2 mCi / kg, 3 mCi / kg, 4 mCi / kg, 5 mCi / kg, 6 mCi / kg, 7 mCi / kg, 8 mCi / kg, or 9 mCi / kg. 177 The Lu-labeled antibody may be administered at a dose including any combination of the upper and lower limits described herein, such as at least 0.1 mCi / kg but less than 10 mCi / kg, or at least 5 mCi / kg but less than 8 mCi / kg.

[0074] According to an aspect of the present invention, 177 An effective amount of Lu-labeled antibody may be used for diagnostic or therapeutic purposes. 177 An effective diagnostic amount of Lu-labeled antibody may be 2.4mCi / kg, 2.2mCi / kg, 2mCi / kg, or 1.8mCi / kg, or 1.6mCi / kg, or 1.4mCi / kg, or 1.2mCi / kg, or 1.0mCi / kg, or 0.8mCi / kg, or 0.6mCi / kg, or 0.4mCi / kg, or 0.2mCi / kg, or 0.1mCi / kg. An effective therapeutic amount of 177Lu-labeled antibody may be less than 12mCi / kg, or 10mCi / kg, or 9mCi / kg, or 8mCi / kg, or 7mCi / kg, or 6mCi / kg, or 5mCi / kg, or 4mCi / kg, or 3mCi / kg.

[0075] According to an aspect of the present invention, 177 The effective diagnostic amount of the Lu-labeled antibody is 50 mCi to 200 mCi, for example, 50 mCi to 100 mCi, or 100 mCi to 150 mCi, or 150 mCi to 200 mCi. 177 An effective amount of Lu-labeled antibody is 200 mCi to 1000 mCi, for example, 200 mCi to 600 mCi, or 400 mCi to 600 mCi, or 200 mCi to 300 mCi, or 300 mCi to 400 mCi, or 400 mCi to 500 mCi, or 500 mCi to 600 mCi, or 600 mCi to 700 mCi, 700 mCi to 800 mCi, 800 mCi to 900 mCi, or 900 mCi to 1000 mCi.

[0076] Although specific radionuclide labels are disclosed herein, any of the list provided above are contemplated for labeling antibodies for radioimmunotherapy.

[0077] According to certain aspects of the invention, the majority of the targeting agents (antibodies, antibody fragments, etc.) administered to the subject typically consists of unlabeled targeting agents, with a minority of targeted agents labeled, such as with any of the labels described herein. The ratio of labeled to unlabeled targeting agents can be adjusted using known methods. Thus, according to certain aspects of the invention, radioimmunotherapy can be provided in a total protein amount of up to 100 mg, such as up to 60 mg, for example, 5 mg to 45 mg, or 0.01 mg / kg to 15.0 mg / kg of patient body weight, such as 0.01 mg / kg to 1.0 mg / kg, or 0.2 mg / kg to 0.6 mg / kg of patient body weight, or 0.3 mg / kg of patient body weight, or 0.4 mg / kg of patient body weight, or 0.5 mg / kg of patient body weight.

[0078] According to certain embodiments of the present invention, the radiolabeled antibody comprises a labeled fraction and an unlabeled fraction in a ratio of about 0.01:10 to 1:10 labeled:unlabeled, such as 0.01:5 to 0.1:5, or 0.01:3 to 0.1:3, or 0.01:1 to 0.1:1 labeled:unlabeled. Additionally, the radiolabeled antibody may be provided as a single dose composition tailored to a particular patient. See, for example, the administration methods disclosed in International Publication No. WO2016 / 187514, which is incorporated herein by reference in its entirety. According to certain embodiments, the radiolabeled antibody may be provided in multiple doses, with each dose in the regimen comprising a composition tailored to a particular patient.

[0079] The inventive combination of labeled and unlabeled fractions of an antibody or other biological delivery vehicle allows the composition to be tailored to a specific patient, with each of the antibody radiation dose and protein dose being personalized to that patient based on at least one patient-specific parameter. Thus, each vial of the composition may be made for a specific patient, and the entire contents of the vial are delivered to that patient in a single dose. If a treatment regimen requires multiple doses, each dose may be formulated as a patient-specific dose in a vial that is administered to the patient as a "single dose" (i.e., the entire contents of the vial are administered at one time). Subsequent doses may be formulated in a similar manner, so that each dose in the regimen provides a patient-specific dose in a single dose container.

[0080] As used herein, the term "subject" includes, but is not limited to, mammals such as humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs, rats, and mice. When the subject is 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 recurrent and / or refractory, or can be in remission.

[0081] As used herein, "treating" a subject suffering from cancer includes, but is not limited to, (i) slowing, stopping or reversing the progression of cancer, (ii) slowing, stopping or reversing the progression of symptoms of cancer, (iii) reducing the likelihood of cancer recurrence, and / or (iv) reducing the likelihood of symptoms of cancer recurrence. According to certain preferred embodiments, treating a subject suffering from cancer means (i) reversing the progression of cancer, ideally until the cancer is eliminated, and / or (ii) reversing the progression of symptoms of cancer, ideally until symptoms are eliminated, and / or (iii) reducing or eliminating the likelihood of recurrence (i.e., consolidation therapy, ideally destroying any remaining cancer cells).

[0082] "Chemotherapeutic drug" in the context of the present invention means a compound that inhibits or kills proliferating cells, and can be used or approved for use in the treatment of cancer.Exemplary chemotherapeutic agents include cytostatic agents that prevent, impede, disrupt, or delay cell division at the level of nuclear division or cell plasmosis.Such agents can stabilize microtubules, such as taxanes, particularly docetaxel or paclitaxel, and epothilones, particularly epothilones A, B, C, D, E, and F, or destabilize microtubules, such as vinca alkaloids, particularly vinblastine, vincristine, vindesine, vinflunine, and vinorelbine.

[0083] "Therapeutically effective amount" or "effective amount" refers to an amount effective at the dosage and for the period of time required to achieve the desired therapeutic result. The therapeutically effective amount may vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. Examples of indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved patient health, reduction in tumor burden, cessation or slowing of tumor growth, and / or absence of metastasis of cancer cells to other locations in the body. According to certain embodiments, "therapeutically effective amount" or "effective amount" refers to the amount of antibody that can deplete or reduce the overall number of cells expressing the antigen to which the antibody is targeted or reacts, or inhibit the growth of cells expressing the antigen.

[0084] As used herein, "deplete" with respect to cells expressing CD33, CD38, or CD45 shall mean to reduce the population of at least one type of cell (e.g., at least one type of peripheral blood lymphocytes of the subject or at least one type of bone marrow lymphocytes of the subject) that expresses or overexpresses CD33, CD38, or CD45. According to certain aspects of the present invention, lymphopenia in a subject is determined by measuring the peripheral blood lymphocyte levels of the subject. Thus, and by way of example, a subject's lymphocyte population is depleted if at least one type of population of peripheral blood lymphocytes of the subject is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99%, etc.

[0085] "Inhibiting growth" refers to a measurable reduction or delay in the growth of malignant cells or tissues (e.g., tumors) in vitro or in vivo when contacted with a therapeutic agent or combination of therapeutic agents, as compared to the reduction or delay in growth of the same cells or tissues in the absence of the therapeutic agent or combination of therapeutic agents. Inhibition of growth of malignant cells or tissues in vitro or in vivo can be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0086] The term "immune checkpoint therapy" refers to molecules that can regulate the function of immune checkpoint proteins in a positive or negative manner (particularly the interaction between antigen-presenting cells (APCs) such as cancer cells and immune T effector cells). The term "immune checkpoint" refers to proteins that, under normal physiological conditions, are directly or indirectly involved in immune pathways that are crucial for preventing uncontrolled immune responses and thus maintaining self-tolerance and / or tissue protection. One or more immune checkpoint therapies described herein can act independently at any step of T cell-mediated immunity, including clonal selection of antigen-specific cells, T cell activation, proliferation, trafficking to antigen and inflammatory sites, execution of direct effector functions, and signaling through cytokines and membrane ligands. Each of these steps is regulated by balancing stimulatory and inhibitory signals that fine-tune the response. In the context of the present invention, the term encompasses immune checkpoint therapies that can at least partially downregulate the function of inhibitory immune checkpoints (antagonists) and / or immune checkpoint therapies that can at least partially upregulate the function of stimulatory immune checkpoints (agonists).

[0087] "Pharmaceutically acceptable salts" refers to acid addition salts of basic compounds, e.g., compounds containing a basic amino group, and basic salts of acidic compounds, e.g., compounds containing a carboxyl group, and amphoteric salts of compounds containing both acidic and basic moieties, such that these salts are suitable for administration in vivo, preferably to humans. A variety of organic and inorganic acids can be used to form acid addition salts. Pharmaceutically acceptable salts are derived from a variety of organic and inorganic counterions well known in the art. Pharmaceutically acceptable salts include, by way of example only, hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, etc., when the molecule contains a basic functional group, and by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, N-methylmorpholinium, etc., when the molecule contains an acidic functional group. In one embodiment, the pharmaceutically acceptable salt of ezatiostat is ezatiostat hydrochloride.

[0088] As used herein, a "synergistic combination" is a combination of monotherapies that can provide therapeutic efficacy comparable to the efficacy of the monotherapies while reducing adverse side effects, such as damage to non-target tissues, immune status, and other clinical indications. Alternatively, a synergistic combination can provide improved efficacy, which can be measured by total tumor cell count, length of time to recurrence, and other indications of patient health.

[0089] The synergistic combination of the present invention combines radioimmunotherapy with immune checkpoint therapy. The synergistic combination of the present invention combines two or more radioimmunotherapy with one or more immune checkpoint therapy.

[0090] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods described herein, suitable methods and materials are described below.

[0091] Radioimmunotherapy-targeted The radioimmunotherapy of the present invention involves antibodies labeled with a radionuclide, which may be recombinant, monoclonal, chimeric, humanized, human, or fragments thereof.

[0092] According to certain aspects of the invention, radioimmunotherapy involves antibodies against any known tumor-specific antigen or antigens that can target a particular cell type. Exemplary antigens include mesothelin, TSHR, CD19, CD123, CD22, CD33, CD30, CD45, CD171, CD138, CS-1, CLL-1, GD2, GD3, B-cell maturation antigen (BCMA), Tn Ag, prostate membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, interleukin-11 receptor a (IL-1R), IL-1R, ... lRa), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor-beta (PDGFR-beta), SSEA-4, CD20, folate receptor alpha (FRa), ERBB2 (Her2), ERBB3 (Her3), death receptor 5 (DR5), MUCl, epidermal growth factor receptor (EGFR), EGFRvIII, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6,E7, MAGE Al, MAGEA3, MAGEA3 / A6, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, prostein, survivin and telomerase, PCTA-l / galectin 8, KRAS, MelanA / MARTl, Ras mutants, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut These include hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, GPA7, and IGLL1.

[0093] According to certain embodiments, the radioimmunotherapy comprises antibodies against protein products of genes mutated in acute myeloid leukemia, including: NPM1, Flt3, TP53, CEBPA, KIT, N-RAS, MLL, WT1, IDH1 / 2, TET2, DNMT3A, and ASXL1.

[0094] According to a particular embodiment, antigens may be selected from tumors with normal or even high mutational burden, such as melanoma, renal cell carcinoma, and lung cancer.

[0095] According to certain aspects, the antigen may be selected from tumors known to be immunologically cold, i.e., the antigen may be selected from tumors with low mutational burden, such as antigens expressed by pancreatic tumors, neuroblastomas, and hematological diseases.

[0096] According to a particular embodiment, the antigen may be of hematopoietic origin, such as an antigen present on blood cells or tumor cells having hematopoietic origin.

[0097] According to particular embodiments, the antigen may be selected from the group including CD19, CD20, CD22, CD30, CD33, CD38, CD45, CD123, CD138, CS-1, B-cell maturation antigen (BCMA), MAGEA3, MAGEA3 / A6, KRAS, CLL1, MUC-1, HER2, HER3, DR5, IL13Rα2, and EphA2, EpCam, GD2, GPA7, PSCA, EGFR, EGFRvIII, ROR1, GPC3, CEA, mesothelin, and PSMA.

[0098] According to a particular embodiment, the antigen may be selected from antigens known to be expressed on cells involved in hematological disorders, such as, for example, CD33, CD38, or CD45.

[0099] According to particular embodiments, the antigen may be selected from CD38, CD33, CD45, DR5, or HER3.

[0100] Multiple myeloma cells uniformly overexpress CD38, a 45 kD transmembrane glycoprotein. Human CD38 has the amino acid sequence shown in GenBank accession number NP_001766 and SEQ ID NO: 1 (Figure 1). As shown in Figure 1, CD38 is a single transmembrane II membrane protein with amino acid residues 1-21 representing the cytoplasmic domain, amino acid residues 22-42 representing the transmembrane domain, and residues 43-300 representing the extracellular domain of CD38. The CD38 protein is a NAD + is a bifunctional extracellular enzyme that can catalyze the conversion of NAD to cyclic ADP-ribose (cADPR) and cADPR to ADP-ribose, and thus the conversion of extracellular NAD + CD38 expression is also upregulated in various hematological malignancies, including, but not limited to, B-cell chronic lymphocytic leukemia, B-cell acute lymphocytic leukemia, Waldenstrom's hypergammaglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, prolymphocytic / myeloid leukemia, acute myeloid leukemia, chronic myeloid leukemia, follicular lymphoma, NK cell leukemia, and plasma cell leukemia.

[0101] Furthermore, CD38 expression has been described on epithelial / endothelial cells of different origins, including the glandular epithelium of prostate, the islet cells of pancreas, the ductal epithelium of glands including parotid glands, bronchial epithelial cells, cells of testis and ovary, and the tumor epithelium of colorectal adenocarcinoma.Therefore, diseases in which CD38 expression may also be involved include, but are not limited to, bronchial epithelial carcinoma of the lung, breast cancer arising from malignant proliferation of the epithelial lining of breast ducts and lobules, pancreatic tumors arising from b-cells (e.g., insulinoma), tumors arising from intestinal epithelium (e.g., adenocarcinoma and squamous cell carcinoma).

[0102] According to certain aspects of the present invention, radioimmunotherapy may include monoclonal antibodies against CD38. Exemplary monoclonal antibodies include daratumumab, MOR202, or SAR650984, each of which has been found to bind to different parts of the extracellular domain of CD38 and shows different clinical responses (e.g., antitumor effects). Daratumumab, MOR202, or SAR650984 are available from Johnson&Johnson (Janssen Biotech) / Genmab as Darzalex®, from Celgene Corp. / Morphosys as Isatuximab, or from Sanofi / Immunogen, respectively.

[0103] Leukemic stem cells, which have been well characterized especially for acute myeloid leukemia (AML), express a characteristic set of cell surface antigens including, among others, CD33. The CD33 antigen is expressed on the blast cells of most cases of AML, with approximately 85-90% of AML cases expressing the CD33 antigen. In addition, the CD33 antigen is expressed in virtually all cases of chronic myeloid leukemia (CML). Patients over 60 years of age have a poor prognosis, with only 10%-15% of AML cases demonstrating a 4-year disease-free survival. This high relapse rate in AML patients and the poor prognosis in older patients may be due to the lack of CD33 expression. + This highlights the need for novel therapeutic agents that preferentially target cells.

[0104] Human CD33 has the amino acid sequence shown in GenBank accession number NP_001763 and SEQ ID NO: 2 (Figure 2). CD33 is a 67 Kd type I transmembrane receptor glycoprotein that can function as a sialic acid-dependent cell adhesion molecule. CD33 has a long N-terminal extracellular domain, a helical transmembrane domain, and a short C-terminal cytoplasmic domain. CD33 is expressed on early myeloid progenitor cells and myeloid leukemia (e.g., acute myeloid leukemia, AML) cells, but not on stem cells.

[0105] With reference to Figure 2, amino acid residues 1-259 represent the extracellular domain, amino acids 260-282 represent the helical transmembrane domain, and amino acids 283-364 represent the cytoplasmic domain (intracellular). There are at least three known single nucleotide polymorphisms ("SNPs") in the extracellular domain of CD33 (i.e., W22R, R69G, S128N). Thus, the extracellular domain of human (Homo sapiens) CD33 can have the amino acid sequence of SEQ ID NO:2 with any one or more of these SNPs.

[0106] Recent studies suggest a role for CD33 in regulating inflammatory and immune responses through its attenuating effect on tyrosine kinase-driven signaling pathways. For example, in vitro studies have demonstrated that CD33 constitutively suppresses the production of inflammatory cytokines such as IL-1β, TNF-α, and IL-8 by human monocytes in a sialic acid ligand-dependent and SOCS3-dependent manner. Conversely, reduction of cell surface CD33 or disruption of sialic acid binding can increase the activity of p38 mitogen-activated protein kinase (MAPK), enhancing cytokine secretion and cytokine-induced cell proliferation.

[0107] According to certain aspects of the present invention, radioimmunotherapy may include a monoclonal antibody against CD33. Exemplary monoclonal antibodies include lintuzumab (HuM195), gemtuzumab, and vadastuximab, each of which has been found to bind to a different portion of the extracellular domain of CD33. Furthermore, each of these antibodies shows different clinical responses (e.g., antitumor effects). Gemtuzumab is available from Pfizer as Mylotarg™, and vadastuximab is available from Seattle Genetics as vadastuximab-butarilin.

[0108] For example, the antibody lintuzumab (HuM195) has shown anti-leukemic effects in the treatment of AML. HuM195 is a recombinant humanized anti-CD33 monoclonal antibody originally produced by Protein Design Labs, Inc. (Fremont, California). M195 is a monoclonal IgG2a antibody that binds to CD33. M195 was derived from mice immunized with live human leukemic myeloblasts. HuM195 was constructed by grafting the complementarity determining regions of M195 into a human IgG1 framework and scaffold. HuM195 elicited antibody-dependent cell-mediated cytotoxicity using human peripheral blood mononuclear cells as effectors. Four clinical trials have investigated only native (i.e., unconjugated) HuM195 in patients with relapsed or refractory AML and CML. Fever, chills, and nausea were the most common toxicities. No human anti-human antibody responses were observed. Beneficial biological activity in terms of reduction of bone marrow blast cells was seen in some patients. Furthermore, each of these antibodies shows different clinical responses (eg, antitumor effects).

[0109] The majority of hematologic malignancies, whether of myeloid or lymphoid origin, express CD45 to varying degrees on the surface of tumor cells. This includes leukemias (e.g., acute myeloid leukemia (AML), acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), acute mixed lineage leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia (CLL), hairy cell leukemia and large granular lymphocytic leukemia), myelodysplastic syndromes (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocytosis, primary myelofibrosis and chronic myelogenous leukemia), lymphomas, multiple myeloma, MGUS and similar disorders, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies.

[0110] CD45 is not present on tissues of non-hematopoietic origin, making it a good target for the treatment of these malignancies. Among several clones of anti-CD45 mouse antibodies, BC8 recognizes all human isoforms of CD45 antigen (CD45 RABC isoform shown in FIG. 3), thus providing an excellent target for the development of therapeutic agents for human malignancies of hematopoietic origin, including leukemia and lymphoma. Therefore, the radioimmunotherapy of the present disclosure may include monoclonal antibodies against CD45.

[0111] According to certain embodiments, the anti-CD45 antibody may comprise the BC8 monoclonal antibody substantially as detailed in U.S. Patent No. 10,420,851, which is incorporated herein by reference. Exemplary compositions comprising the BC8 monoclonal antibody include those described in WO2017 / 155937.

[0112] The BC8 monoclonal antibody may have a light chain comprising the amino acid sequence set forth in SEQ ID NO: 15 (Figure 10). The BC8 monoclonal antibody may have a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4 (Figure 8). The BC8 monoclonal antibody may have a light chain comprising the N-terminal amino acid sequence set forth in SEQ ID NO: 12 (Figure 9). According to a particular embodiment, the light chain comprises at least one complementarity determining region comprising the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 (Figure 9). According to a particular embodiment, the light chain comprises the N-terminal amino acid sequence set forth in SEQ ID NO: 12 and at least one complementarity determining region comprising the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 (Figure 9).

[0113] The BC8 monoclonal antibody may have a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 17 (Figure 11). The BC8 monoclonal antibody may have a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5 (Figure 8). The BC8 monoclonal antibody may have a heavy chain comprising the N-terminal amino acid sequence set forth in SEQ ID NO: 13 (Figure 9). According to a particular embodiment, the heavy chain comprises at least one complementarity determining region comprising the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 (Figure 9). According to a particular embodiment, the heavy chain comprises the N-terminal amino acid sequence set forth in SEQ ID NO: 13 and at least one complementarity determining region comprising the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 (Figure 9).

[0114] As shown in Figure 11, the amino acid at position 141 (relative to the N-terminal amino acid) of the BC8 monoclonal antibody heavy chain may be either ASP or ASN. Thus, a population of BC8 antibody molecules may contain both ASP and ASN at position 141.

[0115] The light and heavy chains each contain a leader sequence and a constant region derived from a particular murine hybridoma, as shown in Figures 10 and 11. Any one or more of these regions may be replaced with the equivalent regions from a human antibody, i.e., human leader sequence, human IgG1-4 constant region, etc.

[0116] Proposed methods by which these antibodies eliminate antigen-positive cells, such as CD45, CD38, or CD33 positive cells, include antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and apoptosis.

[0117] According to certain embodiments, radioimmunotherapy may directly engage the apoptotic pathway, such as agonists of DR5 (death receptor 5; TRAIL-R2, also known as tumor necrosis factor-related apoptosis-inducing ligand receptor 2). DR5 is known to induce apoptosis when activated by its ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). DR5 has been found to be overexpressed on endothelial cells in solid tumors but not in normal tissues, yet tumor cells are often found to be resistant to TRAIL-induced apoptosis. Activation of DR5 by antibodies against the receptor is known to lead to tumor-biased cell death. Thus, DR5 presents an excellent target for radioimmunotherapy, both to specifically target tumor cells and to induce apoptosis of these cells. Exemplary antibodies against DR5 include at least tigatuzumab (CD-1008) from Daiichi Sankyo, conatumumab (AMG655) from Amgen, and doroditumab from Genentech. Initial studies in mouse models may use the surrogate mouse antibody TRA-8.

[0118] According to certain embodiments, the radioimmunotherapy may include an antibody against human epidermal growth factor receptor 3 (HER3). HER3 is a type I transmembrane glycoprotein that is a member of the erythroblastic oncogene B (ErbB) family of tyrosine kinase receptors (EGFR, HER2, HER3, and HER4). Signaling through HER3 can be activated in a ligand-dependent or ligand-independent manner. In the absence of ligand, the HER3 receptor molecule is usually expressed on the cell surface as a monomer with a structure that prevents receptor dimerization, in which the dimerization loop of subdomain II makes intramolecular contact with a pocket on subdomain IV. Binding of HER3 ligands, such as neuregulins (NRGs), for example NRG1 (also known as heregulin, HRG) or NRG2, to subdomains I and III of the extracellular region causes a conformational change that results in the exposure of the dimerization loop of subdomain II, facilitating receptor dimerization and signaling.

[0119] Some cancer-associated mutations in HER3 may disrupt the interaction of subdomains II and IV required for the formation of an inactive "closed" structure, thereby causing constitutive presentation of the dimerization loop and activation of HER3-mediated signaling in the absence of ligand binding. Antibodies targeting HER3 may be useful for targeting certain cancer cells, particularly certain solid cancers. Exemplary antibodies against HER3 include monoclonal antibodies such as patritumab from Daiichi Sankyo, seribantumab (MM-121) from Merrimack Pharmaceuticals, lumletuzumab from Roche, elgemtumab from Novartis, and GSK2849330 from GlaxoSmithKline, as well as bispecific antibodies against HER3 / HER2, such as MM-111 and MM-141 / istiratumab from Merrimack Pharmaceuticals, MCLA0-128 from Merus NV, and MEHD7945A / durigotumab from Genetech.

[0120] "Antibody-dependent cellular cytotoxicity", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a mechanism for inducing cell death that depends on the interaction of antibody-coated target cells with effector cells with lytic activity, such as natural killer (NK) cells, monocytes, macrophages, and neutrophils, via Fc gamma receptors (FcγR) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcvRIIIa. Death of antibody-coated target cells, such as CD33-expressing cells, occurs as a result of effector cell activity by secretion of pore-forming proteins and proteases.

[0121] "Complement dependent cytotoxicity" or "CDC" refers to a mechanism for inducing cell death in which the Fc effector domain of a target-binding antibody binds and activates complement component C1q, which then activates the complement cascade, resulting in the death of the target cell. Activation of complement can also result in the deposition of complement components on the target cell surface that promote ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0122] "Apoptosis" refers to a mechanism of programmed cell death in which antibody binding to a target cell disrupts essential cell signaling pathways, leading to the self-destruction of the cell.

[0123] To evaluate the ADCC activity of the antibody that binds to specific antibody, the antibody may be added to antigen-expressing cells in combination with immune effector cells, which can be activated by antigen-antibody complexes to cause cell lysis of antigen-expressing cells. Cell lysis is usually detected by the release of label (e.g., radioactive substrate, fluorescent dye, or natural intracellular protein) from lysed cells. Exemplary effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells.

[0124] By way of example, in an exemplary assay of ADCC activity of an anti-CD33 antibody, CD33-expressing cells are 51Anti-CD33 antibodies may be added to the CD33-expressing cells at various concentrations, and the assay is initiated by the addition of effector cells (e.g., NK cells from peripheral blood mononuclear cells). After incubation at 37°C for various time intervals, the assay is stopped by centrifugation and the CD33-expressing cells are isolated from the lysed cells. 51 Cr release is measured in a scintillation counter. The percentage of cytotoxicity can be calculated as the % of maximum lysis that can be induced by the addition of 3% perchloric acid to CD33-expressing cells.

[0125] In an exemplary assay for cytotoxicity, tetrazolium salts may be added to CD33-expressing cells treated with various amounts of anti-CD33 antibodies. In live mitochondria, XTT is reduced by mitochondrial dehydrogenases to an orange product and transferred to the cell surface. The orange product can be optically quantified and reflects the number of live cells. Alternatively, esterases from live cells have been found to hydrolyze colorless calcein as a fluorescent molecule. Fluorescence can be measured and quantified and reflects the number of live cells in the sample. The total amount of dead cells may be measured using propidium iodide, which is excluded from live cells by intact membranes. Fluorescence from propidium iodide in dead cells can be quantified by flow cytometry.

[0126] To assess CDC, it may be necessary to include complement proteins in the cytotoxicity assay. Measurement of apoptosis induction does not require the addition of NK cells or complement proteins in the cytotoxicity assay.

[0127] Radioimmunotherapy may include antibodies that are multispecific. For example, radioimmunotherapy may include bispecific antibodies against any two different tumor-specific antigens, or two different epitopes of the same antigen. For example, radioimmunotherapy may include bispecific antibodies against a first epitope of CD33 and a second epitope of CD33, or against an epitope of CD33 and one or more additional different antigens, such as an epitope of an antigen selected from the list presented above (e.g., CD38, CD45, etc.). For another example, radioimmunotherapy may include bispecific antibodies against HER3 / HER2.

[0128] According to a particular embodiment of the present invention, the radioimmunotherapy comprises a multispecific antibody comprising at least a first target recognition component that specifically binds to an epitope of a first antigen and a second target recognition component that specifically binds to a different epitope of the first antigen or an epitope of a second antigen. The multispecific antibody can be a recombinant antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or an antibody fragment.

[0129] The first target recognition component may comprise one of a first full-length heavy chain and a first full-length light chain, a first Fab fragment, or a first single-chain variable fragment (scFv). For example, when the first target recognition component is directed to CD33, the first target recognition component may be derived from lintuzumab (HuM195), gemtuzumab, or vadastuximab. The second target recognition component may comprise one of a second full-length heavy chain and a second full-length light chain, a second Fab fragment, or a second single-chain variable fragment (scFv). In addition, the second target recognition component may be derived from any of the additional different antigens listed above, or from a different epitope of the first target recognition component (i.e., the same antigen, different epitope).

[0130] Alternatively, the present invention contemplates a method comprising the administration of two or more radioimmunotherapy.For example, the radioimmunotherapy may comprise a first antibody and at least a second antibody, where the first and second antibodies recognize different epitopes of the same antigen or different antigens.For example, the radioimmunotherapy may comprise a first antibody against at least one epitope of CD33 and a second antibody against an epitope of CD33 different from the first antibody, or against an epitope of a different antigen, such as an antigen selected from the list above.

[0131] Reference is made herein in an exemplary manner to CD33, but such reference should be understood to include reference to any of the radioimmunotherapy targets disclosed herein.

[0132] Radioimmunotherapy-labeling The radioimmunotherapy of the present invention includes antibodies labeled with radionuclides, so that in treating a patient with radioimmunotherapy, the radionuclides become localized to cells expressing the antigen, inducing damage to and potentially killing those cells. In addition to the many mechanisms by which antibodies can kill cells, the emission of ionizing radiation from radionuclides-labeled antibodies can kill cells in close proximity to the antibody bound to the antigen-expressing cell. Radionuclides emit radioactive particles that can damage the DNA of cells to the point that cell repair mechanisms are unable to keep the cells alive. Thus, when cells expressing the antigen are involved in a tumor, radionuclides can advantageously kill tumor cells.

[0133] Radionuclides that can be used to induce such damage to cells, such as cancer cells, are usually high-energy emitters. High-energy radionuclides preferably act over a short range so as to localize the cytotoxic effect to the target cells. In this way, radiation therapy is delivered more locally to reduce damage to non-target or non-cancerous cells.

[0134] Radionuclides useful for labeling antibodies for use in the radioimmunotherapy of the present invention include:32 P, 211 At, 131 I, 137 Cs, 90 Y, 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 225 Ac, 213 Bi, 213 Po, 212 Bi, 223 Ra, 227 Th, 149 Tb, 64 Cu, 212 Pb, 89 Zr, 68 Ga, and 103 Pd, or a combination thereof.

[0135] The antibody of the present invention may be labeled with a radionuclide by any means known in the art. According to one aspect of the present invention, the radionuclide may be bound or chelated by a chelator conjugated to the antibody substantially as described in WO 2019 / 027973 (herein incorporated by reference in its entirety). That is, the radionuclide-labeled antibody may be prepared by first forming a chelator-conjugated antibody (hereinafter, conjugated antibody), and then chelating the radionuclide with the conjugated antibody to form a radiolabeled antibody. The radionuclide may be chelated by the conjugated antibody at any time after conjugation.

[0136] Chelators useful in the present invention are compounds that have dual functionality, the ability to sequester metal ions as well as to covalently bind to biological carriers such as antibodies. Numerous chelators are known in the art. Exemplary chelators suitable for use in the present invention include, but are not limited to, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid (p-SCN-Bn-DOTA), diethylenetriaminepentaacetic acid (DTPA); ethylenediaminetetraacetic acid (EDTA); 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA); p-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,1 0-Tetraacetic acid (p-SCN-Bz-DOTA); 1,4,7,10-tetraazacyclododecane-N,N',N"-triacetic acid (DO3A); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(2-propionic acid) (DOTMA); 3,6,9-triaza-12-oxa-3,6,9-tricarboxymethylene-10-carboxy-13-phenyl-tridecanoic acid ("B-19036"); 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA). ;1,4,8,11-Tetraazacyclotetradecane-N,N',N'',N'"-tetraacetic acid (TETA);Triethylenetetraaminehexaacetic acid (TTHA);Trans-1,2-Diaminohexanetetraacetic acid (CYDTA);1,4,7,10-Tetraazacyclododecane-1-(2-hydroxypropyl)-4,7,10-triacetic acid (HP-DO3A);Trans-Cyclohexane-diaminetetraacetic acid (CDTA);Trans(1,2)-Cyclohexanediethylenetriaminepentaacetic acid (CDTPA); Examples of chelators include 1-oxa-4,7,10-triazacyclododecane-N,N',N"-triacetic acid (OTTA); 1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetrakis{3-(4-carboxyl)-butanoic acid}; 1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetrakis(acetic acid-methylamide); 1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetrakis(methylenephosphonic acid); and derivatives thereof.

[0137] According to certain aspects of the invention, the radiolabeled antibody may be stable for a sufficient period of time (e.g., days or weeks) for production and administration to a patient, but the radionuclide may destroy the antibody after it reaches the target cell (the cell expressing the antigen) and before it can cause damage to normal cells. For example, 225 It has been found that over 75% of Ac-labeled monoclonal antibodies can remain intact after 24 hours of storage at 4° C. This provides sufficient time for radioimmunotherapy to be produced, transported, and administered, and for the radionuclides to damage target cells. 225 Ac-labeled anti-CD33 is destroyed before it can cause significant damage to cells that do not express the CD33 antigen.

[0138] According to certain aspects of the invention, the radiolabeled antibody may be prepared as a composition by the methods disclosed in International Patent Application Publication No. WO2016 / 187514. Additionally, the radiolabeled antibody may be administered by the methods disclosed in the same publication.

[0139] According to a particular aspect of the invention, the antibody comprises 225 Ac, 131 I, or 177 Lu and may be at least 5, 10, 20, 50, or even 100 times more effective than a control in causing cell death of lymphoblasts, myeloma cells, myeloblast cells, or malignant plasma cells, and the control antibody may be 225 Ac, 131 I, or 177 Include an unlabeled antibody against the same epitope or antigen as the Lu-labeled antibody.

[0140] Immune checkpoint therapy The immune checkpoint therapy of the present invention includes molecules that completely or partially reduce, inhibit, interfere, or regulate one or more checkpoint proteins. Checkpoint proteins regulate the activation or function of T cells. Immune checkpoint therapy can unblock existing immune response inhibition by binding to or otherwise disabling checkpoint inhibition. Immune checkpoint therapy can include monoclonal antibodies, humanized antibodies, fully human antibodies, antibody fragments, small molecule therapeutics, or combinations thereof.

[0141] Exemplary immune checkpoint therapies may specifically bind and inhibit checkpoint proteins such as the inhibitory receptors CTLA-4, PD-1, TIM-3, VISTA, BTLA, LAG-3, and TIGIT, and / or the activating receptors CD28, OX40, GITR, CD137, CD27, and HVEM. Additionally, immune checkpoint therapies may bind to ligands of any of the aforementioned checkpoint proteins, such as PD-L1, PD-L2, PD-L3, and PD-L4 (ligands for PD-1), CD80 and CD86 (ligands for CTLA-4), CD137-L (ligand for CD137), and GITR-L (ligand for GITR). Other exemplary immune checkpoint therapies may bind to checkpoint proteins such as CD226, B7-H3, B7-H4, BTLA, TIGIT, GALS, KIR, 2B4 (which belongs to the CD2 family of molecules and is expressed on all NK, gamma delta, and memory CD8+ (alpha beta) T cells), CD160 (also referred to as BY55), and CGEN-15049.

[0142] Central to the immune checkpoint process are the CTLA-4 and PD-1 immune checkpoint pathways. The CTLA-4 and PD-1 pathways are thought to operate at different stages of the immune response. CTLA-4 is considered the "leader" of immune checkpoint inhibitors because it potentially stops autoreactive T cells, typically at the early stage of naive T cell activation in lymph nodes. The PD-1 pathway regulates previously activated T cells at later stages of the immune response, mainly in peripheral tissues. Furthermore, patients with advanced cancer have been shown to lack upregulation of PD-L1 by either tumor cells or tumor-infiltrating immune cells. Thus, immune checkpoint therapy targeting the PD-1 pathway may be particularly effective in tumors where this immune suppressive axis is operational, and reversing the balance toward an immune-protective environment would reactivate and strengthen existing antitumor immune responses. PD-1 blockade can be achieved by a variety of mechanisms, including antibodies that bind to PD-1 or its ligand PD-L1.

[0143] For example, immune checkpoint therapy may include an inhibitor of the PD-1 checkpoint, which may reduce, block, inhibit, suppress, or interfere with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and PD-L2. Additionally, the inhibitor of the PD-1 checkpoint may be an anti-PD-1 antibody, an antigen-binding fragment, a fusion protein, an oligopeptide, and other molecules that reduce, block, inhibit, suppress, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In some embodiments, the PD-1 checkpoint inhibitor reduces negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., enhanced effector responses to antigen recognition). In some embodiments, the PD-1 checkpoint inhibitor is an anti-PD-1 antibody.

[0144] Thus, according to certain aspects of the invention, immune checkpoint therapy may include monoclonal antibodies against CTLA-4, PD-1, or PD-L1.

[0145] For example, the immune checkpoint inhibitor may be an inhibitor of PD-1. The immune checkpoint inhibitor may be an anti-PD-1 antibody, such as nivolumab. For example, inhibitors of PD-1 biological activity (or ligands thereof) disclosed in U.S. Patent No. 7,029,674. Exemplary antibodies against PD-1 include: anti-mouse PD-1 antibody clone J43 from BioXcell (catalog no. BE0033-2), anti-mouse PD-1 antibody clone RMP1-14 from BioXcell (catalog no. BE0146), mouse anti-PD-1 antibody clone EH12, Merck's MK-3475 anti-mouse PD-1 antibody (Keytruda®, pembrolizumab, lambrolizumab), and AnaptysBio's anti-PD-1 antibody known as ANB011, antibody MDX-1 106 (ONO-4538), Bristol-Myers Squibb's human IgG4 monoclonal antibody nivolumab (Opdivo®, BMS-936558, MDX1106), AstraZeneca's AMP-514 and AMP-224, and pidilizumab (CT-011), CureTech Ltd.

[0146] According to a particular embodiment, the immune checkpoint inhibitor is an inhibitor of PD-L1. Exemplary immune checkpoint inhibitors include antibodies (e.g., anti-PD-L1 antibodies), RNAi molecules (e.g., anti-PD-L1 RNAi), antisense molecules (e.g., anti-PD-L1 antisense RNA), dominant negative proteins (e.g., dominant negative PD-L1 proteins), and small molecule inhibitors. Exemplary anti-PD-L1 antibodies include clone EH12. Exemplary antibodies against PD-L1 include: MPDL3280A (RG7446) from Genentech, anti-mouse PD-L1 antibody clone 10F.9G2 from BioXcell (catalog no. BE0101), anti-PD-L1 monoclonal antibodies MDX-1105 (BMS-936559) and BMS-935559, MSB0010718C, mouse anti-PD-L1 clone 29E.2A3 from Bristol-Meyer's Squibb, and MEDI4736 (durvalumab) from AstraZeneca.

[0147] According to certain embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L2 or reduces the interaction between PD-1 and PD-L2. Exemplary immune checkpoint inhibitors include antibodies (e.g., anti-PD-L2 antibodies), RNAi molecules (e.g., anti-PD-L2 RNAi), antisense molecules (e.g., anti-PD-L2 antisense RNA), dominant negative proteins (e.g., dominant negative PD-L2 proteins), and small molecule inhibitors. Antibodies include monoclonal antibodies, humanized antibodies, deimmunized antibodies, and Ig fusion proteins.

[0148] According to certain embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, such as an anti-CTLA-4 antibody. According to one embodiment, the immune checkpoint inhibitor may be ipilimumab. The anti-CTLA-4 antibody may block the binding of CTLA-4 to CD80 (B7-1) and / or CD86 (B7-2) expressed on antigen-presenting cells. Exemplary antibodies against CTLA-4 include: Bristol Meyers Squibb's anti-CTLA-4 antibody ipilimumab (also known as Yervoy®, MDX-010, BMS-734016, and MDX-101), Millipore's anti-CTLA4 antibody, clone 9H10, Pfizer's tremelimumab (CP-675, 206, ticilimumab), and Abcam's anti-CTLA-4 antibody clone BNI3. According to certain embodiments, the immune checkpoint inhibitor may be a nucleic acid inhibitor of CTLA-4 expression.

[0149] According to certain embodiments, immune checkpoint therapies include inhibitors of lymphocyte activation gene-3 (LAG-3), such as IMP321, a soluble Ig fusion protein that activates dendritic cells, inhibitors of B7, such as antibodies against B7-H3 (e.g., MGA271) and B7-H4, and inhibitors against TIM3 (i.e., T cell immunoglobulin domain and mucin domain 3).

[0150] Any suitable immune checkpoint inhibitor is contemplated for use with the compositions, dosage forms, and methods disclosed herein. The choice of immune checkpoint inhibitor depends on multiple factors. For example, factors to be considered include any additional drug interactions of the immune checkpoint inhibitor, and the length of time that the immune checkpoint inhibitor can be taken. In certain cases, the immune checkpoint inhibitor is an immune checkpoint inhibitor that can be taken long-term, for example, chronically. The immune checkpoint therapy of the present invention may include immune stimulants, T cell growth factors, interleukins such as IL-7 or IL-15, antibodies, vaccines such as dendritic cell (DC) vaccines, or any combination thereof.

[0151] According to certain aspects of the present invention, immune checkpoint therapy may include two or more modulators of immune checkpoint proteins. Thus, immune checkpoint therapy may include a first antibody or inhibitor against a first immune checkpoint protein and a second antibody or inhibitor against a second immune checkpoint protein. For example, according to certain aspects of the present invention, the first inhibitor may be an antibody against PD-1, and the second inhibitor may be an antibody against CTLA-4, PD-L1, or PD-L2.

[0152] Proliferative disorders The compositions and methods of the invention may be used to treat a proliferative disease or disorder. According to certain aspects of the invention, the proliferative disease or disorder may be cancer, including but not limited to hematological malignancies, solid tumors, primary tumors or metastatic tumors.

[0153] For example, the proliferative disorder may be one or more hematological cancers. Exemplary hematological cancers include at least B cell acute lymphoblastic leukemia, T cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma. , myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström's macroglobulinemia, asymptomatic myeloma, smoldering multiple myeloma, indolent myeloma, monoclonal gammopathy of undetermined significance, plasma cell dyscrasias, isolated myeloma, isolated plasmacytoma, extramedullary plasmacytoma, multiple plasmacytoma, systemic amyloid light chain amyloidosis, POEMS syndrome, and combinations thereof.

[0154] The proliferative disorder may be one or more solid cancers. Exemplary solid cancers include at least bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, prostate cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, splenic marginal zone lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, and the like. cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and any combination thereof.

[0155] According to certain aspects of the invention, the hematological cancer or hematological malignancy may be multiple myeloma, acute myeloid leukemia, myelodysplastic syndrome, and myeloproliferative neoplasms.

[0156] Methods of the Invention The present invention includes a method for treating, ameliorating, or reducing the severity of at least one symptom or sign, or inhibiting the growth of cancer in a subject by administering a therapeutically effective amount of radioimmunotherapy and a therapeutically effective amount of immune checkpoint therapy.The present invention includes a method for initiating, enhancing, or prolonging an anti-tumor response in a subject by administering a therapeutically effective amount of radioimmunotherapy and a therapeutically effective amount of immune checkpoint therapy.The present invention includes a method for treating a proliferative disease or disorder in a subject by administering a therapeutically effective amount of radioimmunotherapy and a therapeutically effective amount of immune checkpoint therapy.

[0157] According to certain embodiments of the invention, the methods may treat patients with tumors with standard or even high mutational burden, such as melanoma, renal cell carcinoma, and lung cancer, who are poor responders or non-responders to standard immunotherapy (e.g., patients with T cell exhaustion).

[0158] According to certain aspects, the method may treat patients with tumors known to be immunologically cold, i.e., the radioimmunotherapy administered in the method may target antigens from tumors with low mutational burden, such as antigens expressed by pancreatic tumors, neuroblastomas, and hematological diseases.

[0159] According to certain aspects, the method may treat patients with tumors or disorders that are of hematopoietic origin, i.e., the radioimmunotherapy administered in the method may target antigens from blood cells or tumor cells that have hematopoietic origin.

[0160] According to certain embodiments, radioimmunotherapy and immune checkpoint therapy may be administered simultaneously.Therefore, they may be provided as a single composition or as separate compositions that are administered simultaneously.The combination may be administered in a single dose.Alternatively, the combination may be administered according to a dosing schedule selected from the group consisting of once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 20, 24, or 28 days throughout the treatment period, and the treatment period includes at least two doses.

[0161] According to certain aspects, the radioimmunotherapy and immune checkpoint therapy may be administered sequentially. Furthermore, each therapeutic regimen, i.e., the radioimmunotherapy and the immune checkpoint therapy, may be administered according to a particular dosing schedule, and the method provides for the administration of each therapy sequentially according to the dosing schedule, i.e., the dosing schedule of the radioimmunotherapy is completed before the dosing schedule of the immune checkpoint therapy begins, or vice versa.

[0162] For example, radioimmunotherapy may be administered in one or more doses prior to administration of immune checkpoint therapy, as shown in FIG. 4, or immune checkpoint therapy may be administered in one or more doses prior to administration of radioimmunotherapy, as shown in FIG. 5.

[0163] According to certain embodiments, two or more radioimmunotherapy may be administered to the patient, and the first and second radioimmunotherapy may be administered simultaneously or sequentially. The immune checkpoint therapy may be administered before or after the first and second radioimmunotherapy, or after the first radioimmunotherapy and before the second radioimmunotherapy.

[0164] According to certain aspects of the present invention, radioimmunotherapy and immune checkpoint therapy may be administered according to a specific dosing schedule that is performed simultaneously. That is, the dose of radioimmunotherapy may be administered during the dosing schedule of immune checkpoint therapy, or vice versa. For example, the doses of radioimmunotherapy and checkpoint immunotherapy may be given as shown in Figures 6 and 7, with individual doses of each therapeutic agent being administered in overlapping dosing schedules.

[0165] According to certain aspects of the method of the present invention, radioimmunotherapy may be administered in a single dose. Alternatively, radioimmunotherapy may be administered according to a dosing schedule selected from the group consisting of once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 20, 24, or 28 days throughout the treatment period, the treatment period including at least two doses. Radioimmunotherapy may be administered during a weekly schedule, such as once on a weekday but not on a weekend (Saturday or Sunday). Moreover, each dose may be the same or different. For example, the first dose or dose set of radioimmunotherapy agent may be larger (induction dose(s)) than the additional doses or dose sets (continuation doses).

[0166] According to certain aspects of the present invention, the immune checkpoint therapy may be administered in a single dose. Alternatively, the immune checkpoint therapy may be administered according to a dosing schedule selected from the group consisting of once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 20, 24, or 28 days throughout the treatment period, the treatment period including at least two doses. The immune checkpoint therapy may be administered during a weekly schedule, such as once on a weekday but not on a weekend (Saturday or Sunday). Moreover, each dose may be the same or different. For example, the first dose or dose set of immune checkpoint therapy may be larger (induction dose(s)) than the additional doses or dose sets (continuation doses).

[0167] According to certain aspects of the invention, a therapeutically effective amount of radioimmunotherapy may include a radiation dose that depends on the selected radionuclide used for labeling. For example, 225 When a radionuclide such as Ac is selected for radioimmunotherapy, the radiation dose may be about 0.1-20 uCi / kg of patient weight, for example, 0.2-10 uCi / kg of patient weight, or 0.2-5 uCi / kg of patient weight, or 0.4-4 uCi / kg of patient weight, or 0.4-3 uCi / kg of patient weight, or even 0.4-2 uCi / kg of patient weight. Alternatively, 131 The radiation dose may be up to 1000 times higher when a radionuclide such as I is selected. Preferred radiation doses for select radionuclides are described herein above in the definitions section of this disclosure.

[0168] Effective doses for radioimmunotherapy generally include protein doses of less than 16 mg / kg of patient weight, for example, less than 10 mg / kg of patient weight, or less than 6 mg / kg of patient weight, or less than 5 mg / kg of patient weight, or less than 4 mg / kg of patient weight, or less than 3 mg / kg of patient weight, or even less than 2 mg / kg of patient weight. According to certain embodiments, the protein dose can be between 0.1 mg and 16 mg / kg of subject weight, for example, between 0.1 mg / kg and 10 mg / kg, or between 0.1 mg / kg and 6 mg / kg, or between 0.1 mg / kg and 4 mg / kg, or between 0.1 mg / kg and 2 mg / kg, or between 0.5 mg / kg and 16 mg / kg, or between 2 mg / kg and 16 mg / kg, or between 4 mg / kg and 16 mg / kg, or between 6 mg / kg and 16 mg / kg.

[0169] According to a particular embodiment of the invention, an effective dose of radioimmunotherapy is 10 mg / m 2 , or 8 mg / m 2 , or 6 mg / m 2 , or 5 mg / m 2 , or 4 mg / m 2 , or 3 mg / m 2 , or even 2 mg / m 2 The protein dosage may include a protein dosage based on the patient's body surface area, such as a dosage of less than one dose.

[0170] According to certain embodiments of the present invention, an effective amount of radioimmunotherapy may be the maximum tolerated dose (MTD) of the radioimmunotherapy, based on either or both of protein dose and radiation dose.

[0171] According to certain aspects of the invention, the radioimmunotherapy may comprise a mixture of a radiolabeled fraction of the antibody and an unlabeled (e.g., "naked") fraction of the antibody. The unlabeled fraction may comprise the same antibody against the same epitope as the labeled fraction. In this way, the total radioactivity of the radioimmunotherapy can be reduced or set, while the overall antibody concentration can be varied. For example, the total protein concentration and total radioactivity of the radioimmunotherapy may vary independently depending on the exact nature of the disease being treated, the age and weight of the patient, the identity of the antibody, and the label (e.g., radionuclide) selected for labeling the monoclonal antibody.

[0172] Exemplary doses for some of these immune checkpoint therapies include individual doses of 0.1 mg to 50 mg / kg of patient body weight, e.g., 0.1 to 40 mg / kg, or 0.1 to 30 mg / kg, or 0.1 to 20 mg / kg, or 0.1 to 10 mg / kg, or 0.1 to 5 mg / kg, or 0.1 to 4 mg / kg, or 0.1 to 3 mg / kg, or 0.1 to 2 mg / kg, or 1 to 50 mg / kg, or 2 to 40 mg / kg, or 5 to 30 mg / kg, or 5 to 20 mg / kg, or 10 to 20 mg / kg, or 1 to 5 mg / kg, or 1 to 10 mg / kg. For example, dosing of pembrolizumab (anti-PD-1, Keytruda®) and nivolumab (anti-PD-1, Opdivo®) may be 1-5 mg / kg, such as 2 mg or 3 mg / kg of patient body weight, dosing of durvalumab (anti-PD-L1, MEDI4736) may be 10 mg-20 mg / kg of patient body weight. Dosing of anti-CTLA-4 (Yervoy®) may be 1-15 mg / kg, such as 2 mg or 3 mg / kg of patient body weight, every three weeks for up to four doses.

[0173] Additional drugs The method of the present invention, comprising the administration of radioimmunotherapy and immune checkpoint therapy, may further comprise administering one or more additional therapeutic agents.The additional therapeutic agent may be related to the disease or condition being treated.Such administration may be simultaneous, sequential, or continuous with the administration of an effective amount of radioimmunotherapy and immune checkpoint therapy regimen as detailed herein.In the case of simultaneous administration, the agent may be administered as one composition or as separate compositions, as required.

[0174] Exemplary additional therapeutic agents include at least a chemotherapeutic agent, an anti-inflammatory agent, an immunosuppressant, an immunomodulatory agent, or a combination thereof. Exemplary chemotherapeutic and anti-inflammatory agents are well known in the art and are within the scope of the presently disclosed invention.

[0175] According to certain embodiments of the invention, the one or more therapeutic agents may include an anti-myeloma agent. Exemplary anti-myeloma agents include dexamethasone, melphalan, doxorubicin, bortezomib, lenalidomide, prednisone, carmustine, etoposide, cisplatin, vincristine, cyclophosphamide, and thalidomide, some of which are set forth above as chemotherapeutic, anti-inflammatory, or immunosuppressive agents.

[0176] The therapeutic agent may be administered according to any standard dosage regimen known in the art. For example, the therapeutic agent may be administered in the range of 1 to 500 mg / m 2 may be administered in concentrations ranging from 0.1 to 1.0 mg / kg, the amount being calculated as a function of the surface area of ​​the patient (m 2 For example, an exemplary dose of the chemotherapy agent paclitaxel is 15 mg / m 2 ~275mg / m 2 and an exemplary dose of docetaxel is 60 mg / m 2 ~100mg / m 2 An exemplary dose of epothilone may include 10 mg / m 2 ~20mg / m 2 An exemplary dose of calicheamicin is 1 mg / m 2 ~10mg / m 2Although exemplary doses are listed herein, they are provided for reference only and are not intended to limit the dose ranges of the inventive agents of the present disclosure.

[0177] Aspects of the invention The present application discloses the following aspects.

[0178] Embodiment 1: A method for treating a subject having a proliferative disorder, the method comprising administering a therapeutically effective amount of immune checkpoint therapy to the subject, and at least one week later, administering a therapeutically effective amount of radioimmunotherapy to the subject.

[0179] Embodiment 2: A method for treating a subject having a proliferative disorder, the method comprising administering a therapeutically effective amount of radioimmunotherapy to the subject, and at least one week later, administering a therapeutically effective amount of immune checkpoint therapy to the subject.

[0180] Embodiment 3: A method for treating a subject having a proliferative disorder, the method comprising administering a therapeutically effective amount of radioimmunotherapy to the subject; and administering a therapeutically effective amount of immune checkpoint therapy to the subject.

[0181] Aspect 4: The method of any one of aspects 1-3, wherein the administration of the radioimmunotherapy and / or immune checkpoint therapy is according to a dosing schedule selected from the group consisting of once every 1, 2, 3, 4, 5, 6, 7, 10, 12, 14, 21, or 28 days, and wherein the treatment period comprises at least two doses.

[0182] Aspect 5: Radioimmunotherapy comprises 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 5. The method of any one of aspects 1-4, comprising a radionuclide selected from the group consisting of: Pd, Pd, or a combination thereof.

[0183] Aspect 6: Radioimmunotherapy is 131 I, 177 Lu, or 225 The method according to any one of aspects 1 to 5, comprising a radionuclide selected from the group consisting of Ac.

[0184] Embodiment 7: The method of any one of embodiments 1 to 6, wherein the radioimmunotherapy comprises a radionuclide complexed by a chelating agent bound to an antibody of the radioimmunotherapy, and the chelating agent comprises 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or a derivative thereof.

[0185] Embodiment 8: The method of any one of embodiments 1 to 7, wherein the radioimmunotherapy comprises antibodies against CD19, CD20, CD22, CD30, CD33, CD38, CD45, HER3, DR5, CD123, CD138, CS-1, B-cell maturation antigen (BCMA), MAGEA3, MAGEA3 / A6, KRAS, CLL1, MUC-1, HER2, IL13Rα2, and EphA2, EpCam, GD2, GPA7, PSCA, EGFR, EGFRvIII, ROR1, GPC3, CEA, mesothelin, PSMA, or a combination thereof.

[0186] Embodiment 9: The method according to any one of embodiments 1 to 7, wherein the radioimmunotherapy comprises an antibody against a protein product of a gene mutated in acute myeloid leukemia, wherein the gene is NPM1, Flt3, TP53, CEBPA, KIT, N-RAS, MLL, WT1, IDH1 / 2, TET2, DNMT3A, ASXL1, or a combination thereof.

[0187] Embodiment 10: The method according to any one of embodiments 1 to 7, wherein the radioimmunotherapy comprises an antibody against CD33, CD38, CD45, HER3, DR5, or a combination thereof.

[0188] Embodiment 11: The method of embodiment 10, wherein the anti-CD33 antibody comprises lintuzumab, or the anti-CD38 antibody comprises daratumumab, or the anti-CD45 antibody comprises BC8.

[0189] Embodiment 12: The method of any one of embodiments 1 to 11, wherein the radioimmunotherapy comprises a first radioimmunotherapy directed against CD33, CD38, CD45, HER3, or DR5, and a second radioimmunotherapy directed against a different one of CD33, CD38, CD45, HER3, or DR5.

[0190] Embodiment 13: The method of embodiment 12, wherein the first radioimmunotherapy is an antibody against CD33, CD38, or CD45, and the second radioimmunotherapy is an antibody against HER3 or DR5.

[0191] Embodiment 14: The method of embodiment 12 or 13, wherein the first and second radioimmunotherapy are delivered simultaneously or sequentially.

[0192] Embodiment 15: The method of embodiment 12 or 13, wherein the first radioimmunotherapy is administered prior to the immune checkpoint therapy and the second radioimmunotherapy is administered after the immune checkpoint therapy, or the second radioimmunotherapy is administered prior to the immune checkpoint therapy and the first radioimmunotherapy is administered after the immune checkpoint therapy.

[0193] Embodiment 16: The method according to any one of embodiments 1 to 15, wherein the therapeutically effective amount of radioimmunotherapy comprises a protein dose of less than 16 mg / kg of the subject's body weight, or less than 10 mg / kg of the subject's body weight, or less than 6 mg / kg of the subject's body weight, or between 0.1 mg and 16 mg / kg of the subject's body weight.

[0194] Embodiment 17: The method according to any one of embodiments 1 to 16, wherein the therapeutically effective amount of radioimmunotherapy is the maximum tolerated dose (MTD).

[0195] Embodiment 18: The method of any one of embodiments 1-16, wherein the therapeutically effective amount of radioimmunotherapy comprises a labeling dose of 0.05-10 uCi / kg of subject's body weight, or 0.1-6 uCi / kg of subject's body weight, or 0.2-5 uCi / kg of subject's body weight.

[0196] Embodiment 19: The method of any one of embodiments 1 to 16, wherein the therapeutically effective amount of radioimmunotherapy comprises a labeling dose of 0.05 to 10 mCi / kg of subject's body weight, or 0.1 to 6 mCi / kg of subject's body weight, or 0.1 to 5 mCi / kg of subject's body weight, or 0.1 to 3 mCi / kg of subject's body weight.

[0197] Embodiment 20: The method of any one of embodiments 1 to 16, wherein the therapeutically effective amount of radioimmunotherapy comprises a single dose delivering to the subject a dose of less than 2 Gy, or less than 8 Gy, for example, between 2 Gy and 8 Gy.

[0198] Embodiment 21: The method of any one of embodiments 1 to 20, wherein the immune checkpoint therapy comprises an antibody against CTLA-4, PD-1, TIM-3, VISTA, BTLA, LAG-3, TIGIT, CD28, OX40, GITR, CD137, CD27, HVEM, PD-L1, PD-L2, PD-L3, PD-L4, CD80, CD86, CD137-L, GITR-L, CD226, B7-H3, B7-H4, BTLA, TIGIT, GALS, KIR, 2B4, CD160, CGEN-15049, or a combination thereof.

[0199] Embodiment 22: The method of any one of embodiments 1 to 20, wherein the immune checkpoint therapy comprises an antibody against PD-1, PD-L1, PD-L2, CTLA-4, or a combination thereof.

[0200] Embodiment 23: The method according to any one of embodiments 1 to 22, wherein the proliferative disorder is a hematological cancer selected from one or more of multiple myeloma, acute myeloid leukemia, myelodysplastic syndrome, and myeloproliferative neoplasms.

[0201] Embodiment 24: The method according to any one of embodiments 1 to 23, wherein the radioimmunotherapy comprises BC8, and wherein BC8 comprises a light chain having the amino acid sequence set forth in SEQ ID NO: 1, or a light chain N-terminal amino acid sequence set forth in SEQ ID NO: 9.

[0202] Embodiment 25: The method according to any one of embodiments 1 to 24, wherein the radioimmunotherapy comprises BC8, and the light chain of BC8 comprises at least one complementarity determining region having the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0203] Embodiment 26: The method according to any one of embodiments 1 to 23, wherein the radioimmunotherapy comprises BC8, and wherein BC8 comprises a light chain having the amino acid sequence as set forth in SEQ ID NO: 12 or SEQ ID NO: 13.

[0204] Embodiment 27: The method according to any one of embodiments 1 to 26, wherein the radioimmunotherapy comprises BC8, and BC8 comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2, or the heavy chain N-terminal amino acid sequence set forth in SEQ ID NO: 10.

[0205] Embodiment 28: The method according to any one of embodiments 1 to 27, wherein the radioimmunotherapy comprises BC8, and the heavy chain of BC8 comprises at least one complementarity determining region having the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0206] Embodiment 29: The method according to any one of embodiments 1 to 27, wherein the radioimmunotherapy comprises BC8, and BC8 comprises a heavy chain having the amino acid sequence as set forth in SEQ ID NO: 15 or SEQ ID NO: 16.

[0207] Embodiment 30: A method according to any one of embodiments 1 to 28, wherein the radioimmunotherapy comprises BC8, the heavy chain of which comprises the amino acid ASP or ASN at position 141 (relative to the N-terminal amino acid). EXAMPLES

[0208] Example 1: Toxicity of Actinium-225 Labeled CD33 According to a particular embodiment of the invention, the radioimmunotherapy is performed using actinium-225 (Ac 225 ) labeled monoclonal antibodies. 225 Lintuzumab conjugated to lintuzumab-Ac was tested for cytotoxicity against certain cell types expressing CD33. For example, suspensions of HL60 (white blood cells) were conjugated to various doses of radiolabeled lintuzumab (lintuzumab-Ac). 225 ) at which 50% of the cells were killed (LD 50 ) was found to be 8 pCi per mL of cell suspension.

[0209] In a study to exploit the reactivity of radiolabeled lintuzumab with peripheral blood and bone marrow cells from non-human primates and human frozen tissues, radiolabeled lintuzumab was reactive only with mononuclear cells, demonstrating specificity. Additionally, in a study to determine the stability of the radiolabel on the antibody, 10 normal mice (8-week-old female Balb / c mice from Taconic, Germantown, New York) were injected in the tail with 300 nCi of radiolabeled lintuzumab (0.12 ml). Serum samples collected over a 5-day period showed that actinium-225 remained bound to lintuzumab, demonstrating the in vivo stability of the radiolabel to the antibody.

[0210] The maximum tolerated dose (MTD) of a single injection of radiolabeled lintuzumab was determined to be 3 Uci / kg of patient body weight. As a split dose (e.g., two equal doses administered 4-7 days apart), the MTD was determined to be 2 Uci / kg per dose, or 4 Uci / kg total. This data was determined by injection in patients with relapsed / refractory AML: 21 patients were injected with escalating doses of radiolabeled lintuzumab (0.5 Uci / kg to 4 Uci / kg). The determination of the MTD was based on the severity of adverse effects observed at each dose level. Anti-leukemic effects included elimination of peripheral blood blasts in 13 of 19 evaluable patients. Twelve of 18 patients who were evaluable 4 weeks after treatment had a reduction in bone marrow blasts (including 9 who had a ≥50% reduction). Three patients treated with 1 uCi / kg, 3 uCi / kg and 4 uCi / kg, respectively, had ≦5% blasts after treatment.

[0211] Example 2: CD33 Human Maximum Tolerated Dose and Efficacy Phase I study: Lintuzumab-Ac 225 The MTD of a fractionated dose of lintuzumab-Ac was used to determine the MTD of a fractionated dose of lintuzumab-Ac followed by supportive therapy with granulocyte colony-stimulating factor (GCSF). A typical cycle is approximately 42 days. A cycle consists of lintuzumab-Ac on day 1, followed by supportive therapy with granulocyte colony-stimulating factor (GCSF). 225 Treatment begins with lintuzumab Ac in divided doses, followed by GCSF on day 9 and continuing GCSF according to appropriate dosing instructions until absolute neutrophil count (ANC) is greater than 1,000 (expected to occur within 5-10 days). Peripheral blood is assessed for paraprotein burden on days 14, 21, 28, 35, and 42. On day 42, a bone marrow aspirate is performed to assess plasma cell infiltration. If the response is partial or better but not complete on day 42 and the patient is otherwise eligible, the patient is re-administered in a new cycle at the same dose level on days 1-60 of the first cycle. If no dose-limiting toxicity is observed, the patient is treated with lintuzumab Ac at 4 μCi / kg. 225 The cycle continues using the above algorithm until a cumulative dose of

[0212] Example 3: CD33 combination therapy with chemotherapeutic agents In the Phase I study, 18 patients with relapsed AML will receive split doses on days 1 and 8 in combination with low-dose Ara-C (LDAC) 225 Treatment was found to induce remission in elderly patients with untreated AML at doses exceeding 0.5 uCi / kg / fraction. In the phase 2 portion of the clinical trial, 13 patients with early symptoms of AML who were deemed unsuitable for cytotoxic chemotherapy were treated with Ac-lintuzumab, administered in divided doses on days 1-8 without LDAC. 225 Treatment with Ac-lintuzumab. Preliminary data are available for 9 for median age 75 years (range: 65-82) and median PS 2 (2 patients 0-1, 3 patients 2, and 2 patients 3). Six (67%) had been previously treated for AHD (5 with MDS and 1 with atypical CML). At baseline, 5 patients had ANC ≥ 500 / μL, only 2 had ANC ≥ 1000 / μL, and only 1 had platelets > 50,000 / μL.

[0213] Myelosuppression was present in all evaluable patients, including grade 4 thrombocytopenia with bone marrow hypoplasia for >6 weeks after treatment in 3 patients. The only non-hematologic toxicities >grade 3 reported in ≥1 patients were pneumonia and cellulitis. No venous occlusive events occurred. Thirty-day mortality was 33% (disease progression, acute or chronic respiratory failure, and traumatic intracranial hemorrhage after falls).

[0214] Objective responses were confirmed in 5 of 9 patients (56%): complete response with incomplete platelet recovery (CRp) in 2 and complete response with incomplete hematologic recovery (CRi) in 3. Two patients had resistant disease.

[0215] The mean time to neutrophil recovery (ANC ≥ 500 / μl) was: 225The first dose of Ac-lintuzumab was administered 36 days (range: 20-60). Two patients with CRp had neutrophil recovery on days 60 and 36. Two of the patients with CRi did not reach ANC ≥ 500 / μL when the infection expired on days 65 and 56, and the third expired > 66 days without ANC recovery. Patients with antecedent hematological disorders (AHDs) may not have the ability to recover to normal neutrophil production, so patients without AHDs may benefit more. Of the three patients without AHDs, one had ANC recovery on day 36, one died from infection on day 56 without ANC recovery, and one did not have ANC recovery on day 66. No patient achieved a platelet count > 20,000 / μL without transfusion.

[0216] 2μCi / kg / fraction 225 Preliminary data from this phase 2 study of Ac-lintuzumab monotherapy confirm a 56% remission rate in elderly patients, many of whom have AHD, who are unsuitable for intensive therapy. Although myelosuppression at this dose was deemed longer than tolerated in this population, additional studies will continue at 1.5 μCi / kg / fraction with the aim of shortening recovery time.

[0217] Example 4: Production of anti-CD45 immunoglobulin BC8 According to a particular embodiment of the invention, the radioimmunotherapy may include a monoclonal antibody against CD45, such as BC8 or a chimeric form of BC8 (BC8c). The mouse anti-CD45 mAb BC8 was prepared from a hybridoma (ATCC number HB-10507) originally developed by fusing mouse myeloma NS1 cells with spleen cells from BALB / C mice hyperimmunized with human phytohemagglutinin (PHA)-stimulated mononuclear cells. After screening for microbial contamination, the original fused cells were cultured using JRH-Biosciences EXCell 300 medium supplemented with 1-2% fetal bovine serum (FBS).

[0218] The hybridoma cell line was adapted for culture in serum-free culture medium. Briefly, the cells in culture were slowly and gradually weaned from serum albumin using a combination medium supplemented with glutamine, cholesterol, insulin, and transferrin. The cells were then cultured at a concentration of 1 × 10 6 The cells were grown to a density of over 1000 cells / mL. The medium was harvested and processed for purification of the anti-CD45 antibody using a combination of cation exchange chromatography, protein A chromatography, and anion exchange membrane separation. The purified antibody was concentrated by nanofiltration (30 kD cutoff). The concentration of the purified product was measured at 5.2 mg / mL and stored at 2-8 °C.

[0219] The purified antibody was characterized by SDS-PAGE, IEF, and SEC-HPLC techniques. A single product peak (99.4%) was recorded by SEC-HPLC with approximately 0.6% aggregates. Non-reducing SDS-PAGE showed a single band for the antibody. SDS-PAGE under reducing conditions confirmed the presence of light and heavy chains (99.9% combined).

[0220] Example 5: Sequencing of anti-CD45-immunoglobulin BC8 Total RNA was isolated from hybridoma cells according to the Trizol® reagent technical manual. Total RNA was analyzed by agarose gel electrophoresis and reverse transcribed into cDNA using isotype-specific antisense primers or universal primers according to the PrimeScript™ 1st Strand cDNA Synthesis Kit technical manual. VH, VL, CH, and CL antibody fragments were amplified and cloned separately into standard cloning vectors using standard molecular cloning procedures. Colony PCR screening was performed to identify clones with the correct size insert. Six or more single colonies with the correct size insert were sequenced for each antibody fragment. The complete nucleotide sequences of the light and heavy chains are shown in Figures 10 and 11.

[0221] Anti-CD45-immunoglobulin (i.e., BC8 antibody) was sequenced using a mass spectrometry peptide mapping approach. BC8 antibody was deglycosylated, reduced, and digested with the individual enzymes trypsin, Lys-C, and chymotrypsin. The peptide fragments were then analyzed by LC-coupled mass spectrometry using an MS / MS fragmentation analysis approach. Protein sequencing of the heavy and light chains of BC8 antibody showed that the actual amino acid sequence differs from that predicted by the DNA sequence by only a single amino acid in the heavy chain. As highlighted in Figure 11, the codon encoding the amino acid at position 141 predicts ASN-141 rather than the actual ASP-141 found by protein sequencing. Furthermore, sequencing of various batches of protein showed different amounts of ASP and ASN at position 141.

Claims

1. 1. A method for treating a subject having a proliferative disorder, comprising: administering a therapeutically effective amount of an immune checkpoint therapy to the subject; administering to said subject a therapeutically effective amount of radioimmunotherapy.

2. 2. The method of claim 1, wherein the radioimmunotherapy is administered at least one week prior to the immune checkpoint therapy, or the immune checkpoint therapy is administered at least one week prior to the radioimmunotherapy.

3. The radioimmunotherapy 131 I, 125 I, 123 I, 90 Y. 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 32 P. 225 A c, 213 Bi, 213 Po, 211 At, 212 Bi, 213 Bi, 223 Ra, 227 Th, 149 Tb, 137 Cs, 212 Pb or 103 3. The method of claim 1 or 2, comprising a radionuclide selected from the group comprising: Pd, Pd, or a combination thereof.

4. 3. The method of claim 1 or 2, wherein the radioimmunotherapy comprises antibodies against CD19, CD20, CD22, CD30, CD33, CD38, CD45, CD123, CD138, CS-1, B-cell maturation antigen (BCMA), MAGEA3, MAGEA3 / A6, KRAS, CLL1, MUC-1, HER2, HER3, DR5, IL13Rα2, and EphA2, EpCam, GD2, GPA7, PSCA, EGFR, EGFRvIII, ROR1, GPC3, CEA, mesothelin, PSMA, or a combination thereof.

5. 3. The method of claim 1 or 2, wherein the radioimmunotherapy comprises an antibody against a protein product of a gene mutated in acute myeloid leukemia, the gene being NPM1, Flt3, TP53, CEBPA, KIT, N-RAS, MLL, WT1, IDH1 / 2, TET2, DNMT3A, ASXL1, or a combination thereof.

6. 3. The method of claim 1 or 2, wherein the radioimmunotherapy comprises antibodies against CD33, CD38, CD45, HER3, DR5, or a combination thereof.

7. The radioimmunotherapy 131 I, 177 Lu, and 225 3. The method of claim 1 or 2, comprising an antibody against CD33, HER3, or DR5 labeled with a radionuclide selected from the group consisting of CD33, HER3, or DR5.

8. The radionuclide is 225 8. The method of claim 7, wherein the therapeutically effective amount of the radioimmunotherapy is 0.1-10 uCi / kg of the subject's body weight, or 0.2-6 uCi / kg of the subject's body weight, or 0.4-5 uCi / kg of the subject's body weight.

9. The radionuclide is 131 8. The method of claim 7, wherein the therapeutically effective amount of the radioimmunotherapy is I and comprises a radiation dose of 25 mCi to 500 mCi, or 50 mCi to 400 mCi.

10. 3. The method of claim 1 or 2, wherein the radioimmunotherapy comprises a radionuclide label conjugated with a chelating agent bound to an antibody, the chelating agent comprising 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or a derivative thereof.

11. 3. The method of claim 1 or 2, wherein the therapeutically effective amount of the radioimmunotherapy is a maximum tolerated dose (MTD).

12. 3. The method of claim 1 or 2, wherein the immune checkpoint therapy comprises an antibody against CTLA-4, PD-1, TIM-3, VISTA, BTLA, LAG-3, TIGIT, CD28, OX40, GITR, CD137, CD27, HVEM, PD-L1, PD-L2, PD-L3, PD-L4, CD80, CD86, CD137-L, GITR-L, CD226, B7-H3, B7-H4, BTLA, TIGIT, GALS, KIR, 2B4, CD160, CGEN-15049, or a combination thereof.

13. 3. The method of claim 1 or 2, wherein the immune checkpoint therapy comprises an antibody against PD-1, PD-L1, PD-L2, CTLA-4, or a combination thereof.

14. 2. The method of claim 1, wherein said radioimmunotherapy comprises a first and a second radioimmunotherapy, both of which are administered at least one week prior to said immune checkpoint therapy, or said radioimmunotherapy comprises a first radioimmunotherapy administered at least one week prior to said immune checkpoint therapy and a second radioimmunotherapy administered at least one week after said immune checkpoint therapy, or said radioimmunotherapy comprises a first radioimmunotherapy administered together with said immune checkpoint therapy and a second radioimmunotherapy administered at least one week after said immune checkpoint therapy, or said radioimmunotherapy comprises a first and a second radioimmunotherapy, both of which are administered at least one week after said immune checkpoint therapy.

15. 15. The method of claim 14, wherein the first radioimmunotherapy comprises an antibody to one of CD33, CD38, CD45, DR5, or HER3, and the second radioimmunotherapy comprises an antibody to another of CD33, CD38, CD45, DR5, or HER3.

16. 2. The method of claim 1, wherein the proliferative disorder is a hematological cancer selected from one or more of multiple myeloma, acute myeloid leukemia, myelodysplastic syndrome, and myeloproliferative neoplasms.

17. 1. A method for treating a subject having a proliferative disorder, comprising: administering to the subject a therapeutically effective amount of an immune checkpoint therapy selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, or a combination thereof; At least one week later, administering to the subject a therapeutically effective amount of a radioimmunotherapy selected from the group consisting of CD33, CD38, CD45, DR5, HER3, or a combination thereof, wherein the radioimmunotherapy comprises: 131 I, 177 Lu, and 225 and administering, to the subject a compound comprising the compound, a compound labeled with a radionuclide selected from the group consisting of: Ac, C1C, C2C, C3C, C4C, C5C, C6C, C7C, C8C, C9C, C10C, C11C, C12C, C13C, C14C, C15C, C16C, C17C, C18C, C29C, C30C, C4C, C

18. 1. A method for treating a subject having a proliferative disorder, comprising: administering to the subject a therapeutically effective amount of a radioimmunotherapy selected from the group consisting of CD33, CD38, CD45, DR5, HER3, or a combination thereof, wherein the radioimmunotherapy comprises: 131 I, 177 Lu, and 225 4. Administering a medicament for use in treating a patient with a disease comprising administering to said patient a therapeutically effective amount of a medicament for ... and at least one week later, administering to the subject a therapeutically effective amount of an immune checkpoint therapy selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, or a combination thereof.

19. The radionuclide is 225 20. The method of claim 17 or 18, wherein the therapeutically effective amount of the radioimmunotherapy is 0.1-10 uCi / kg body weight of the subject and a protein dose of 0.1 mg-16 mg / kg body weight of the subject.

20. The radionuclide is 131 19. The method of claim 17 or 18, wherein the therapeutically effective amount of the radioimmunotherapy is I and comprises a radiation dose of 0.1-12 mCi / kg body weight of the subject, and a protein dose of 0.1 mg-16 mg / kg body weight of the subject.

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