CLEC12axcd3 bispecific antibodies and methods for treatment of disease

Abstract

To provide means and methods for treating a subject for a CLEC12A positive cancer.SOLUTION: In some embodiments, the method comprises treating the subject in need thereof with two or more administrations of a bispecific antibody that binds CDS and CLEC12A, in which in a first administration a first amount of the bispecific antibody is administered, and in each of the subsequent administrations the amount of bispecific antibody is higher than the amount of bispecific antibody in the first administration. In some embodiments, CLEC12A positive cancer treatment methods are provided by dosing regimens at intervals that spare hemopoietic stem cell compartment, allowing for recovery of normal CLEC12A positive hemopoietic cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of antibodies, in particular to the field of therapeutic antibodies. The antibodies can be used for human therapy. More specifically, the present invention relates to antibodies, preferably bispecific antibodies, for the treatment of cancer. [Background technology]

[0002] Acute myeloid leukemia (AML) is the third most common leukemia worldwide, yet little progress has been made in disease outcome over the past 40 years (Arrighi, Soulas et al. 2003, Lowenberg, Ossenkoppele et al. 2009, Burnett, Wetzler et al. 2011). The annual incidence of AML in European adults is 5-8 per 100,000. In the United States, 4-5 new cases per 100,000 are diagnosed with AML each year, and 10,000 deaths occur annually from AML. The 5-year survival rate for young adults is approximately 40%, but this declines significantly with age, to less than 25% for older patients. The majority of patients who achieve a morphological complete remission after induction chemotherapy subsequently relapse within 3 years (Juliusson, Lazarevic et al. 2012, Oran and Weisdorf 2012). These patients are therefore in urgent need of the development of new agents with more effective mechanisms of action. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Arrighi, Soulas et al. 2003 [Non-Patent Document 2] Lowenberg,Ossenkoppele et al.2009 [Non-Patent Document 3] Burnett, Wetzler et al. 2011 [Non-Patent Document 4] Juliusson, Lazarevic et al.2012 [Non-Patent Document 5] Oran and Weisdorf 2012 Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention focuses on an antigen that is specifically expressed on AML blasts and leukemic stem cells (LSCs). This antigen is C-type lectin domain family 12 member A or CLEC12A (also called human myeloid inhibitory C-type lectin, hMICL, C-type lectin-like molecule-1, CLL-1, or CD371) (Bakker, van den Oudenrijn et al. 2004, van Rhenen, van Dongen et al. 2007). CLEC12A is a myeloid differentiation antigen expressed on leukemic cells in 90-95% of cases of either de novo or relapsed AML, regardless of subtype (Bakker, van den Oudenrijn et al. 2004, Zhao, Singh et al. 2010, Larsen, Roug et al. 2012). CLEC12A is selectively expressed in CD34POSCD38NEG LSCs but not in normal hematopoietic stem cells (HSCs), erythroid precursors, or megakaryocytes, based on in vitro and in vivo evidence (van Rhenen, van Dongen et al. 2007, Kikushige and Miyamoto 2013). [Means for solving the problem]

[0005] In the present invention, it is shown that the CLEC12AxCD3 bispecific antibody induces efficient T cell-mediated CLEC12A targeting and selectively eradicates leukemic cells (including leukemic stem cells) without affecting normal HSCs, thereby allowing rapid resumption of normal hematopoiesis after treatment, thereby limiting hematologic toxicity.

[0006] Many T cell engager formats face problems such as short serum half-life, immunogenicity, and manufacturing difficulties (Brinkmann and Kontermann 2017). The CLEC12AxCD3 bispecific antibody of the present invention has a good serum half-life. The means and methods of the present invention do not significantly induce immune responses against the therapeutic agent.

[0007] The invention provides a method of treating a subject with a CLEC12A-positive cancer, comprising treating a subject in need thereof with two or more administrations of a bispecific antibody that binds CD3 and CLEC12A, wherein in a first administration, a first amount of the bispecific antibody is administered, and in each subsequent administration, the amount of the bispecific antibody is greater than the amount of the bispecific antibody in the first administration.

[0008] The invention also provides a bispecific antibody that binds CD3 and CLEC12A for use in a method of treating a CLEC12A-positive cancer in a subject, the treatment comprising two or more administrations of a bispecific antibody that binds CD3 and CLEC12A, wherein in a first administration a first amount of the bispecific antibody is administered and in each subsequent administration the amount of the bispecific antibody is greater than the amount of the bispecific antibody in the first administration.

[0009] In one aspect, the present invention provides a method for purging CLEC12A-positive hematopoietic cells, preferably CLEC12A-positive malignant cells, from a subject and repopulating the hematopoietic system of the subject with normal cells. The method comprises administering to a subject in need thereof, at intervals, a bispecific antibody that binds CD3 and CLEC12A, thereby killing CLEC12A-positive malignant cells, and stimulating hematopoietic stem cells and / or hematopoietic progenitor cells of the subject to repopulate the hematopoietic system with newly formed hematopoietic stem cell-derived cells, including CLEC12A-positive cells. Hematopoietic stem cells reside in the bone marrow. The presence of harmful CLEC12A-positive cells deprives healthy cells of nutrients and clogs the compartment, thereby restricting the normal growth and development of granulocyte, macrophage stem cells. Macrophage stem cells themselves give rise to basophils, neutrophils, eosinophils and monocytes. By removing harmful CLEC12A positive cells from the bone marrow compartment by the bispecific antibody administration methods described herein, the recruitment of healthy hematopoietic stem cells is stimulated by increasing the availability of nutrients and space in the bone marrow compartment, leading to their growth, survival and downstream differentiation into CLEC12A positive and CLEC12A negative healthy cells such as basophils, neutrophils, eosinophils and monocytes.

[0010] The subject is preferably one diagnosed with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myelofibrosis, or myeloproliferative neoplasms blast stage (MPN-BP).

[0011] Also provided is a method of preparing an autologous bone marrow cell graft for a subject undergoing treatment for a CLEC12A-positive cancer, the method comprising incubating a preparation of bone marrow cells from the subject with a bispecific antibody that binds CD3 and CLEC12A under conditions suitable to kill CLEC12A-positive cells, and subsequently harvesting the bone marrow cells from the incubation.

[0012] The invention further provides a method of treating a CLEC12A-positive cancer in a subject, the method comprising incubating a bone marrow cell preparation from the subject with a bispecific antibody that binds CD3 and CLEC12A under conditions suitable to kill CLEC12A-positive cells, treating the subject with hematopoietic system-ablative therapy, and providing to the subject a bone marrow cell graft comprising the incubated bone marrow cells.

[0013] Further provided is a method of providing a hematopoietic stem cell sparing cancer treatment to a subject, the method comprising administering a bispecific antibody that binds CD3 and CLEC12A to a subject in need thereof, the subject preferably having a CLEC12A positive cancer. [Brief description of the drawings]

[0014] [Figure 1A] The CLEC12AxCD3 bispecific antibody specifically binds to CLEC12APOS and CD3POS target cells. Binding of the CLEC12AxCD3 bispecific antibody to CLEC12A and CD3 was measured on a panel of tumor cell lines by flow cytometry: HL60: CLEC12APOS promyelocytic cell line; CLEC12A-CHO-K1: CHO-K1 cell line stably expressing human CLEC12A (validated with a reference anti-CLEC12A antibody, data not shown); CHO-K1: CLEC12ANEG parental CHO-K1 cell line; Jurkat E6.1: CD3POST cell line; J.RT-T3.5: CD3NEGT cell line. [Figure 1B] CLEC12AxCD3 bispecific antibody specifically binds to CLEC12APOS and CD3POS target cells. CLEC12AxCD3 bispecific antibody binding profile in normal peripheral blood by flow cytometry. The gating strategy for the analyzed subsets was: CD4T cells: CD4POS; CD8T cells: CD8POS; NK cells: CD3NEGCD56POS; NKT cells: CD3POSCD56POS; B cells: CD19POS; myeloid dendritic cells (DC): BDCA1POSCD19NEG; granulocytes: based on SSC-FSC; monocytes: CD14POSCD33POS. [Figure 1C] CLEC12AxCD3 bispecific antibody specifically binds to CLEC12APOS and CD3POS target cells. CLEC12AxCD3 bispecific antibody binds within the CD34POS progenitor compartment of normal bone marrow by flow cytometry. HSC: hematopoietic stem cell; MPP: multipotent progenitor; CMP: common myeloid progenitor; GMP: granulocyte-macrophage progenitor; MEP: megakaryocyte-erythroid progenitor; CLP: common lymphoid progenitor. Data shown for antibodies MF4327 and MF5196 and isotype control IgG formatted in final Fc format (Fc effector function silenced). [Figure 2A] Antibody MF4327xMF5196 mediates CLEC12A-specific activation of T cells and redirects these T cells to induce lysis of CLEC12APOSHL60 cells. Resting T cells from healthy donors were purified by negative selection and co-cultured with CFSE-labeled HL60 cells in the presence of antibody MF4327xMF5196, MockxCD3, control IgG or PBS. Cells were co-incubated at an IgG concentration of 1000ng / mL with the indicated IgGs (all with WTFc effector function) at an E:T ratio of 5:1. The ability of antibody MF4327xMF5196 prototypes with WTFc effector function to induce T cell activation was quantified after 24 / 48 / 72 hours by flow cytometry compared to PBS conditions. Data shown for 2 healthy donors representative of more than 10 tested. [Figure 2B]Antibody MF4327xMF5196 mediates CLEC12A-specific activation of T cells and redirects these T cells to induce lysis of CLEC12APOSHL60 cells. Resting T cells from healthy donors were purified by negative selection and co-cultured with CFSE-labeled HL60 cells in the presence of antibody MF4327xMF5196, MockxCD3, control IgG or PBS. Cells were co-incubated at an IgG concentration of 1000ng / mL with the indicated IgGs (all with WTFc effector function) at an E:T ratio of 5:1. The ability of antibody MF4327xMF5196 prototypes with WTFc effector function to induce target cell lysis was quantified after 24 / 48 / 72 hours by flow cytometry compared to PBS conditions. Data shown for 2 healthy donors representative of more than 10 tested. [Figure 2C] Antibody MF4327xMF5196 mediates CLEC12A-specific activation of T cells and redirects these T cells to induce lysis of CLEC12APOSHL60 cells. Resting T cells derived from healthy donors were purified by negative selection and co-cultured with CFSE-labeled HL60 cells in the presence of antibody MF4327xMF5196, MockxCD3, control IgG or PBS. Cells were co-incubated at a range of IgG concentrations with the indicated IgGs (all with silenced Fc effector function) at an E:T ratio of 5:1. The ability of antibody MF4327xMF5196 (with silenced Fc effector function) to induce activation of CD4 was quantified after 48 hours by flow cytometry compared to PBS conditions. Data shown are from a representative donor, with a total of six donors tested in three independent assays. [Figure 2D]Antibody MF4327xMF5196 mediates CLEC12A-specific activation of T cells and redirects these T cells to induce lysis of CLEC12APOSHL60 cells. Resting T cells derived from healthy donors were purified by negative selection and co-cultured with CFSE-labeled HL60 cells in the presence of antibody MF4327xMF5196, MockxCD3, control IgG or PBS. Cells were co-incubated at a range of IgG concentrations with the indicated IgGs (all with silenced Fc effector function) at an E:T ratio of 5:1. The ability of antibody MF4327xMF5196 (with silenced Fc effector function) to induce activation of CD8 T cells was quantified after 48 hours by flow cytometry compared to PBS conditions. Data shown are from a representative donor, with a total of six donors tested in three independent assays. [Figure 2E] Antibody MF4327xMF5196 mediates CLEC12A-specific activation of T cells and redirects these T cells to induce lysis of CLEC12APOSHL60 cells. Resting T cells derived from healthy donors were purified by negative selection and co-cultured with CFSE-labeled HL60 cells in the presence of antibody MF4327xMF5196, MockxCD3, control IgG or PBS. Cells were co-incubated at a range of IgG concentrations with the indicated IgGs (all with silenced Fc effector function) at an E:T ratio of 5:1. The ability of antibody MF4327xMF5196 (with silenced Fc effector function) to induce target cell lysis was quantified after 48 hours by flow cytometry compared to PBS conditions. Data shown are from a representative donor, with a total of six donors tested in three independent assays. [Figure 3A]Antibody MF4327xMF5196 has the ability to induce CLEC12A-mediated lysis of monocytes and inflammatory cytokine release, and induce CLEC12A antigen-specific T cell proliferation. PBMC samples from healthy donors were co-cultured with antibody MF4327xMF5196 or MockxCD3 antibody at the indicated concentration range and compared to PBS conditions. T cell activation was assessed 48 hours later using flow cytometry. Data shown are from one donor and are representative of four independent donors from multiple independent experiments. [Figure 3B] Antibody MF4327xMF5196 has the ability to induce CLEC12A-mediated lysis of monocytes, inflammatory cytokine release, and induce CLEC12A antigen-specific T cell proliferation. PBMC samples from healthy donors were co-cultured with antibody MF4327xMF5196 or MockxCD3 antibody at the indicated concentration range and compared to PBS conditions. Monocyte lysis by autologous T cells was assessed after 48 hours using flow cytometry. Data shown are from one donor and are representative of four independent donors from multiple independent experiments. [Figure 3C] Antibody MF4327xMF5196 has the ability to induce CLEC12A-mediated lysis of monocytes and inflammatory cytokine release, and induce CLEC12A antigen-specific T cell proliferation. PBMC samples from healthy donors were co-cultured with antibody MF4327xMF5196 or MockxCD3 antibody at the indicated concentration range and compared to PBS conditions. B cell lysis was assessed after 48 hours using flow cytometry. Data shown are from one donor and are representative of four independent donors from multiple independent experiments. [Figure 3D]Antibody MF4327xMF5196 has the ability to induce CLEC12A-mediated lysis of monocytes and inflammatory cytokine release, and induce CLEC12A antigen-specific T cell proliferation. Monocytes from healthy donors were purified by negative selection and co-cultured with CFSE-labeled resting autologous T cells at a 5:1 E:T ratio with 1,000 ng / mL of antibody MF4327xMF5196 and MockxCD3 antibody (anti-CD3 IgG formatted with WTFc effector function) for 5 days. T cell proliferation was demonstrated by a reduction in CFSE-positive T cells. Data shown is from one donor and is representative of 4 independent donors from multiple independent experiments. [Figure 3E] Antibody MF4327xMF5196 has the ability to induce CLEC12A-mediated lysis of monocytes and inflammatory cytokine release, and induce CLEC12A antigen-specific T cell proliferation. PBMCs from healthy donors were cultured with 1,000 ng / mL of antibody MF4327xMF5196, MockxCD3, or anti-CD3 (CD3) antibody for 48 hours. Supernatants were subsequently collected and human cytokine levels were measured in these supernatants using a human 10-plex Luminex setup. Cytokine levels were detectable for the six cytokines shown. Data shown are from two representative healthy PBMC donors, with duplicates for each donor, and PBMCs from eight healthy donors were tested. [Figure 4A] Antibody MF4327xMF5196 preserves the potential of normal bone marrow progenitors to develop complete myeloid and erythroid lineages. CD34POS hematopoietic progenitors from healthy donor bone marrow were co-cultured with autologous preactivated T cells in the presence of 200ng / mL antibody MF4327xMF5196 or MockxCD3 at an E:T ratio of 10:1 for 16 hours. Antibody MF4327xMF5196-induced lysis was measured in comparison to non-IgG conditions for the indicated cell fractions using quantified flow cytometry settings. Each data point represents the average of three independent bone marrow donors tested (n=4). The gating strategy is shown in FIG. 10. [Figure 4B]Antibody MF4327xMF5196 preserves the potential of normal bone marrow progenitors to develop complete myeloid and erythroid lineages. CD34POS hematopoietic progenitors from healthy donor bone marrow were co-cultured with autologous preactivated T cells in the presence of 200ng / mL antibody MF4327xMF5196 or MockxCD3 at an E:T ratio of 10:1 for 16 hours. Analysis of the effect of antibody MF4327xMF5196 on the different CD34POS bone marrow populations after a 16 hour cytotoxicity assay, absolute numbers for three donors are shown. Gating of CD34POS progenitor cells is shown in FIG. 11. Quantification of absolute numbers in the different test conditions was calculated by integral of the frequency and absolute number of total CD34POS cells measured by flow count fluorocytometer. Data represent the mean of duplicate measurements. [Figure 4C] Antibody MF4327xMF5196 preserves the potential of normal bone marrow progenitors to develop complete myeloid and erythroid lineages. CD34POS hematopoietic progenitors from healthy donor bone marrow were co-cultured with autologous preactivated T cells in the presence of 200ng / mL antibody MF4327xMF5196 or MockxCD3 at an E:T ratio of 10:1 for 16 hours. Scatter plot showing the effect of antibody MF4327xMF5196 on CLEC12A and / or CD123 expressing subsets after 16 hours of cytotoxicity assay in the indicated conditions, gated on CD34POSLINNEG cells. One representative donor out of two is shown. [Figure 4D]Antibody MF4327xMF5196 preserves the potential of normal bone marrow progenitors to develop complete myeloid and erythroid lineages. CD34POS hematopoietic progenitors from healthy donor bone marrow were co-cultured with autologous preactivated T cells in the presence of 200ng / mL antibody MF4327xMF5196 or MockxCD3 at an E:T ratio of 10:1 for 16 hours. The ability of CD34POS cells to give rise to granulocyte macrophage colonies (CFU-GM), burst forming unit erythroid (BFU-E) and megakaryocyte colonies (CFU-Mk) in a 16 hour cytotoxicity assay with antibody MF4327xMF5196 was quantified after 2 weeks of culture in semi-solid medium from four different donors. Control cultures were either co-cultures not treated with IgG or CD34POS cells inoculated alone. Each data point represents the mean of triplicate cultures (CFU-GM and BFU-E) or quadruplicate cultures (CFU-Mk) per donor. [Figure 4E] Antibody MF4327xMF5196 preserves the potential of normal bone marrow progenitors to develop complete myeloid and erythroid lineages. CD34POS hematopoietic progenitors from healthy donor bone marrow were co-cultured with autologous preactivated T cells in the presence of 200ng / mL antibody MF4327xMF5196 or MockxCD3 at an E:T ratio of 10:1 for 16 hours. Flow cytometric analysis of CD15POSCD33POS and CD14POSCD36POSCD33POS cell content of 2-week MethoCult cultures depicted in Figure 4D. Data show the frequency of monocytic and myeloid cells among total CD33POS cells of three different donors. HSC: hematopoietic stem cell; MPP: multipotent progenitor; CMP: common myeloid progenitor; GMP: granulocyte-macrophage progenitor; MEP: megakaryocyte-erythroid progenitor; CLP: common lymphoid progenitor. Statistical analysis was performed using Student's t-test (two-tailed). *p<0.05, **p<0.01, ***p<0.001. [Figure 5A]Antibody MF4327xMF5196 efficiently induces redirected lysis of CLEC12APOS primary AML blasts by autologous T cells. Resting CD3POS T cells isolated from peripheral blood of AML patients in clinical remission were co-cultured for 48 h with CFSE-labeled CLEC12APOS autologous primary AML blasts collected at the time of AML diagnosis. The E:T ratio was 5:1 and IgG was added at a concentration of 1,000 ng / mL: antibody MF4327xMF5196, MockxCD3, or control IgG (all with WTFc effector function). The extent of AML blast lysis induced by each IgG compared to PBS conditions was quantified using flow cytometry. Diagram showing the stage of AML from which AML blast samples and autologous T cells were obtained. [Figure 5B] Antibody MF4327xMF5196 efficiently induces redirected lysis of CLEC12APOS primary AML blasts by autologous T cells. Resting CD3+ T cells isolated from peripheral blood of AML patients in clinical remission were cocultured for 48 h with CFSE-labeled CLEC12APOS autologous primary AML blasts collected at the time of AML diagnosis. The E:T ratio was 5:1, and IgG was added at a concentration of 1,000 ng / mL: antibody MF4327xMF5196, MockxCD3, or control IgG (all with WTFc effector function). The degree of AML blast lysis induced by each IgG compared to PBS conditions was quantified using flow cytometry. Expression of CLEC12A antigen in primary AML blast samples (AML blasts defined as CD45±SSC±). Histograms show anti-CLEC12A IgG binding for each patient sample, with lymphocytes shown in grey and AML blasts in black. The CLEC12A positive gate was set based on the CLEC12A negative lymphocyte fraction. The percentage of CLEC12A positive blasts is shown. [Figure 5C]Antibody MF4327xMF5196 efficiently induces redirected lysis of CLEC12APOS primary AML blasts by autologous T cells. Resting CD3POS T cells isolated from peripheral blood of AML patients in clinical remission were co-cultured for 48 h with CFSE-labeled CLEC12APOS autologous primary AML blasts collected at the time of AML diagnosis. The E:T ratio was 5:1 and IgG was added at a concentration of 1,000 ng / mL: antibody MF4327xMF5196, MockxCD3, or control IgG (all with WTFc effector function). The extent of AML blast lysis induced by each IgG compared to PBS conditions was quantified using flow cytometry. The activation status of CD4POS in AML patients. [Figure 5D] Antibody MF4327xMF5196 efficiently induces redirected lysis of CLEC12APOS primary AML blasts by autologous T cells. Resting CD3POS T cells isolated from peripheral blood of AML patients in clinical remission were cocultured for 48 h with CFSE-labeled CLEC12APOS autologous primary AML blasts collected at the time of AML diagnosis. The E:T ratio was 5:1, and IgG was added at a concentration of 1,000 ng / mL: antibody MF4327xMF5196, MockxCD3, or control IgG (all with WTFc effector function). The extent of AML blast lysis induced by each IgG compared to PBS conditions was quantified using flow cytometry. Activation status of CD8POS T cells. [Figure 5E]Antibody MF4327xMF5196 efficiently induces redirected lysis of CLEC12APOS primary AML blasts by autologous T cells. Resting CD3+ T cells isolated from peripheral blood of AML patients in clinical remission were cocultured for 48 h with CFSE-labeled CLEC12APOS autologous primary AML blasts collected at the time of AML diagnosis. The E:T ratio was 5:1, and IgG was added at a concentration of 1,000 ng / mL: antibody MF4327xMF5196, MockxCD3, or control IgG (all with WTFc effector function). The degree of AML blast lysis induced by each IgG compared to PBS conditions was quantified using flow cytometry. Cytotoxicity of CLEC12APOS primary AML blasts mediated by autologous primary T cells when incubated with the indicated IgG (compared to PBS). Data shown are the mean ± SE of three AML patient samples (patients 7–9, see Table III) tested in triplicate. Data were analyzed by ANOVA compared to PBS: **P<0.01, ***P<0.001. [Figure 6] Antibody MF4327xMF5196 induces the death of AML blasts in primary AML samples. Primary AML patient samples obtained at the time of AML diagnosis were cultured for 10 days in medium supplemented with a cytokine cocktail to support the survival of AML blasts. Antibody MF4327xMF5196 or MockxCD3 antibody was added at 200ng / mL. For each patient, CD45xCD33 plots show the live gates for each IgG on days 0 and 10. T cells are shown in red, AML blasts are shown in blue (defined as CD45±SSC±), and the percentages of T cells and AML blasts are shown. Histograms on day 0 show the CLEC12A expression levels of AML blast cells in each patient. Staining with anti-CLEC12A antibody is shown in black, and staining with isotype control antibody is shown in grey. [Figure 7]Gating strategy used to quantify HL60 target cell lysis by flow cytometry. A fixed amount of cells were measured by flow cytometry. Viable cells were gated based on FSC-SSC profile (left plot). Within the live gate, CFSEPOS HL60 cells were then gated (right plot). The absolute number of CFSEPOS viable HL60 target cells within that gate was used to calculate the percentage of HL60 target cell lysis as described in Materials and Methods. The top plot shows the PBS condition with no cytotoxicity, and the bottom plot shows the antibody MF4327xMF5196 condition where HL60 cytotoxicity was observed. [Figure 8-1] Gating strategy used to quantify cytotoxicity in the PBMC-based monocyte cytotoxicity assay. Monocytes (CD14POS), CD4 (CD4POSCD3POS) and CD8 (CD8POSCD3POS) T cells, and B cells (CD19POS) were gated out as indicated. Absolute numbers of monocytes and B cells measured in a fixed volume of assay medium were quantified and used to calculate the percentage of cytotoxicity induced by the test IgG conditions (Figure 8-2) compared to the PBS control condition (Figure 8-1) as described in Materials and Methods. CD4POS and CD8POS T cell subsets were identified within the T cell population and their activation status was determined using the activation marker CD69 compared to the PBS control condition. [Figure 8-2]Gating strategy used to quantify cytotoxicity in the PBMC-based monocyte cytotoxicity assay. Monocytes (CD14POS), CD4 (CD4POSCD3POS) and CD8 (CD8POSCD3POS) T cells, and B cells (CD19POS) were gated out as indicated. Absolute numbers of monocytes and B cells measured in a fixed volume of assay medium were quantified and used to calculate the percentage of cytotoxicity induced by the test IgG conditions (Figure 8-2) compared to the PBS control condition (Figure 8-1 above) as described in Materials and Methods. CD4POS and CD8POS T cell subsets were identified within the T cell population and their activation status was determined using the activation marker CD69 compared to the PBS control condition. [Figure 9] The activity of antibody MF4327xMF5196 bispecific IgG depends on the E:T ratio. Resting T cells from healthy donors were co-cultured with CFSE-labeled HL60 cells in the presence of antibody MF4327xMF5196, MockxCD3, or control IgG. The effect of increasing E:T ratios on HL60 target cell lysis with 1,000 ng / mL IgG was measured after 48 hours of co-culture. [Figure 10A] Gating strategy used to quantify recovery of healthy donor bone marrow CD34POS progenitor cells. Total CD34POS cells, CLEC12APOS, CLEC12ANEGCD10NEG, and CLEC12ANEGCD10NEG cells were gated as indicated. Single cells were first gated based on FSC and SSC area and width, then viable cells were defined based on FSC-SSC profile and 7AAD negativity. Within viable cells, CD34 and CD5 were used to gate CD34POS cells from co-cultured T cells. Within total CD34POS cells, cells were divided into three populations based on expression of CLEC12A and CD10. For quantification of CD34POS populations, a fixed amount of flow-counting fluorospheres of known concentration was added to each sample prior to acquisition. The absolute number of cells per mL was calculated based on the ratio of fluorospheres to cells. [Figure 10B]Gating strategy used to quantify recovery of healthy donor bone marrow CD34POS progenitor cells. Scatter plots showing the various CD34POS populations in all tested conditions from one representative donor. [Figure 11-1] Gating strategy used to determine the identity and relative proportions of different CD34POS progenitors. First single cells were gated based on FSC and SSC area and width, then viable cells were defined based on FSC-SSC profile and 7AAD negativity. Within viable cells, lineage-negative CD34POS cells were gated, followed by FSC-SSC backgating. Cells were then divided into CD38POS and CD38NEG compartments. Within the CD34POSCD38POS compartment, CLPs were gated based on CD10 expression. Cells lacking CD10 expression were further separated into CMPs, MEPs, and GMPs based on CD123 and CD45RA expression. For some donors, CD135 was used instead of CD123 to define these three populations. Within the CD34POSCD38NEG compartment, HSCs and MPPs were defined according to CD90 and CD45RA expression. Gates were confirmed with fluorescence-minus one controls. HSC: hematopoietic stem cell; MPP: multipotent progenitor; CMP: common myeloid progenitor; GMP: granulocyte-macrophage progenitor; MEP: megakaryocyte-erythroid progenitor; CLP: common lymphoid progenitor. [Figure 11-2]Gating strategy used to determine the identity and relative proportions of different CD34POS progenitors. First single cells were gated based on FSC and SSC area and width, then viable cells were defined based on FSC-SSC profile and 7AAD negativity. Within viable cells, lineage-negative CD34POS cells were gated, followed by FSC-SSC backgating. Cells were then divided into CD38POS and CD38NEG compartments. Within the CD34POSCD38POS compartment, CLPs were gated based on CD10 expression. Cells lacking CD10 expression were further separated into CMPs, MEPs, and GMPs based on CD123 and CD45RA expression. For some donors, CD135 was used instead of CD123 to define these three populations. Within the CD34POSCD38NEG compartment, HSCs and MPPs were defined according to CD90 and CD45RA expression. Gates were confirmed with fluorescence-minus one controls. HSC: hematopoietic stem cell; MPP: multipotent progenitor; CMP: common myeloid progenitor; GMP: granulocyte-macrophage progenitor; MEP: megakaryocyte-erythroid progenitor; CLP: common lymphoid progenitor. [Figure 11-3]Gating strategy used to determine the identity and relative proportions of different CD34POS progenitors. First single cells were gated based on FSC and SSC area and width, then viable cells were defined based on FSC-SSC profile and 7AAD negativity. Within viable cells, lineage-negative CD34POS cells were gated, followed by FSC-SSC backgating. Cells were then divided into CD38POS and CD38NEG compartments. Within the CD34POSCD38POS compartment, CLPs were gated based on CD10 expression. Cells lacking CD10 expression were further separated into CMPs, MEPs, and GMPs based on CD123 and CD45RA expression. For some donors, CD135 was used instead of CD123 to define these three populations. Within the CD34POSCD38NEG compartment, HSCs and MPPs were defined according to CD90 and CD45RA expression. Gates were confirmed with fluorescence-minus one controls. HSC: hematopoietic stem cell; MPP: multipotent progenitor; CMP: common myeloid progenitor; GMP: granulocyte-macrophage progenitor; MEP: megakaryocyte-erythroid progenitor; CLP: common lymphoid progenitor. [Figure 12]Antibody MF4327xMF5196 redirects AML patient T cells and healthy donor T cells to specifically lyse the CLEC12APOS AML cell line with comparable efficiency. Resting CD3POS T cells were isolated from peripheral blood of six healthy donors or six AML patients in clinical remission (patients 1-6; see Table III for patient characteristics). Isolated T cells were co-cultured with CFSE-labeled HL60 target cells at a 5:1 E:T ratio in the presence of 1,000 ng / mL of antibody MF4327xMF5196, MockxCD3IgG, or control IgG (all with WTFc effector function) for 48 h. (AB) Flow cytometry to analyze the subset composition of CD4POS T cells (A) and CD8POS T cells (B) within the CD3POS fraction of T cells obtained from healthy donors (HD) or AML patients, which were subsequently used for HL60 target cell lysis. TCM: central memory T cells; TNaive: naive T cells; TEMRA+: effector memory cells with reacquired CD45RA. TEM: effector memory T cells. (CE) Activation of CD4POS T cells (C) and CD8POS T cells (D) and HL60 target cell lysis (E) analyzed by flow cytometry and compared to PBS conditions for the indicated IgG and T cell sources. Each circle represents the mean value of a single T cell donor tested in triplicate. Data were analyzed by ANOVA compared to PBS: *P<0.05, **P<0.01, ***P<0.001. [Figure 13] Morphology and JAK2V617F mutation status of cells generated by secondary plating of SSClowCD45dimCD34+CD38-CLEC12A± cells. May-Giemsa staining of re-plated CLEC12A positive cells was performed and individual colonies were picked and tested for JAK2 driver mutations. [Figure 14A]IL-6 levels in patients treated with antibody MF4327xMF5196, measured in pg / ml pre-dose, 4 hours and 24 hours after the end of infusion. Dosing occurred on days 1, 4, 8 and 22. Patients received a priming dose of 1 mg on day 1, followed by step-up doses of 3 mg on day 4 and 15 mg on day 8, and a full dose of 25 mg on day 15. Cytokine levels were also measured at the end of the treatment cycle (day 28). [Figure 14B] IL-8 levels in patients treated with antibody MF4327xMF5196, measured in pg / ml pre-dose, 4 hours and 24 hours after end of infusion. Dosing occurred on days 1, 4, 8 and 22. Patients received a priming dose of 1 mg on day 1, followed by step-up doses of 3 mg on day 4 and 15 mg on day 8, and a full dose of 25 mg on day 15. Cytokine levels were also measured at the end of the treatment cycle (day 28). [Figure 14C] TNFα levels in patients treated with antibody MF4327xMF5196, measured in pg / ml pre-dose, 4 hours and 24 hours after end of infusion. Dosing occurred on days 1, 4, 8 and 22. Patients received a priming dose of 1 mg on day 1, followed by step-up doses of 3 mg on day 4 and 15 mg on day 8, and a full dose of 25 mg on day 15. Cytokine levels were also measured at the end of the treatment cycle (day 28). [Figure 14D] IFNγ levels in patients treated with antibody MF4327xMF5196, measured in pg / ml pre-dose, 4 hours and 24 hours after end of infusion. Dosing occurred on days 1, 4, 8 and 22. Patients received a priming dose of 1 mg on day 1, followed by step-up doses of 3 mg on day 4 and 15 mg on day 8, and a full dose of 25 mg on day 15. Cytokine levels were also measured at the end of the treatment cycle (day 28). [Figure 15A]IL-6 levels in patients treated with antibody MF4327xMF5196, measured in pg / ml pre-dose, 4 hours and 24 hours after end of infusion. Dosing occurred on days 1, 4, 8 and 22. Patients received a priming dose of 3 mg on day 1, followed by step-up doses of 10 mg on day 4 and 25 mg on day 8, and a full dose of 40 mg on day 15. Cytokine levels were also measured at the end of the treatment cycle (day 28). [Figure 15B] IL-8 levels in patients treated with antibody MF4327xMF5196, measured in pg / ml pre-dose, 4 hours and 24 hours after the end of infusion. Dosing occurred on days 1, 4, 8 and 22. Patients received a priming dose of 3 mg on day 1, followed by step-up doses of 10 mg on day 4 and 25 mg on day 8, and a full dose of 40 mg on day 15. Cytokine levels were also measured at the end of the treatment cycle (day 28). [Figure 15C] TNFα levels in patients treated with antibody MF4327xMF5196, measured in pg / ml pre-dose, 4 hours and 24 hours after end of infusion. Dosing occurred on days 1, 4, 8 and 22. Patients received a priming dose of 3 mg on day 1, followed by step-up doses of 10 mg on day 4 and 25 mg on day 8, and a full dose of 40 mg on day 15. Cytokine levels were also measured at the end of the treatment cycle (day 28). [Figure 15D] IFNγ levels in patients treated with antibody MF4327xMF5196, measured in pg / ml pre-dose, 4 hours and 24 hours after end of infusion. Dosing occurred on days 1, 4, 8 and 22. Patients received a priming dose of 3 mg on day 1, followed by step-up doses of 10 mg on day 4 and 25 mg on day 8, and a full dose of 40 mg on day 15. Cytokine levels were also measured at the end of the treatment cycle (day 28). [Figure 16A]IL-6 levels in patients treated with antibody MF4327xMF5196, measured in pg / ml pre-dose, 4 hours and 24 hours after the end of infusion. Dosing occurred on days 1, 4, 8 and 22. Patients received a priming dose of 5 mg on day 1, followed by step-up doses of 15 mg on day 4 and 25 mg on day 8, and a full dose of 60 mg on day 15. Cytokine levels were also measured at the end of the treatment cycle (day 28). [Figure 16B] IL-8 levels in patients treated with antibody MF4327xMF5196, measured in pg / ml pre-dose, 4 hours and 24 hours after end of infusion. Dosing occurred on days 1, 4, 8 and 22. Patients received a priming dose of 5 mg on day 1, followed by step-up doses of 15 mg on day 4 and 25 mg on day 8, and a full dose of 60 mg on day 15. Cytokine levels were also measured at the end of the treatment cycle (day 28). [Figure 16C] TNFα levels in patients treated with antibody MF4327xMF5196, measured in pg / ml pre-dose, 4 hours and 24 hours after the end of infusion. Dosing occurred on days 1, 4, 8 and 22. Patients received a priming dose of 5 mg on day 1, followed by step-up doses of 15 mg on day 4 and 25 mg on day 8, and a full dose of 60 mg on day 15. Cytokine levels were also measured at the end of the treatment cycle (day 28). [Figure 16D] IFNγ levels in patients treated with antibody MF4327xMF5196, measured in pg / ml pre-dose, 4 hours and 24 hours after end of infusion. Dosing occurred on days 1, 4, 8 and 22. Patients received a priming dose of 5 mg on day 1, followed by step-up doses of 15 mg on day 4 and 25 mg on day 8, and a full dose of 60 mg on day 15. Cytokine levels were also measured at the end of the treatment cycle (day 28). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Dosing of therapeutic monoclonal antibodies (mAbs) is often based on body size. This is due to the idea that dosing based on body size reduces intersubject variability in drug exposure. However, most mAbs are target specific, have a relatively large therapeutic window, and generally the contribution of body size to pharmacokinetic variability is small. On the other hand, depending on the type of antibody, the target specificity of the antibody, and other factors, antibodies can exhibit severe on- and off-target side effects. Some of the more serious of them are conditions collectively referred to as cytokine release syndrome (CRS). Cytokine release syndrome is a type of systemic inflammatory response syndrome that occurs as a complication of some diseases or infections, and is known to occur as an adverse effect of some antibody drugs and adoptive T cell therapies. Severe cases are also called "cytokine storm".

[0016] CRS has been known as a potential adverse effect of antibody treatment since the approval of muromonab-CD3, the first monoclonal antibody drug to cause CRS. However, the problem became recognized as serious after a phase I clinical trial in 2006 with the agonistic CD28-binding antibody TGN1412. All six healthy volunteers showed severe CRS and became seriously ill.

[0017] More recently, in relation to CD3 antibodies, it has been highlighted that clinical trials with CD3 / CD19 and CD3 / CD123 bispecific antibodies have been postponed (Johnson A.Oct 9,2018 and Sheridan C,2016).

[0018] In the present invention, it has been found that a priming dose administered as a first administration, followed by a full dose of an antibody that binds to CD3 and CLEC12A, is effective and well tolerated by subjects treated for CLEC12A-positive cancer. Without being bound by theory, it is believed that priming with a low dose, gradually increasing to the full dose in one or more steps, is useful for this. The priming dose is typically a single administration, but can be administered twice or more times. The priming dose can be followed by step-up doses, which are incrementally increased in dose until the full dose is reached. The first amount of antibody is preferably a priming dose. The antibody doses are typically administered at regular intervals. The interval between the priming administration and the subsequent priming or step-up administration is typically one or three days, although if more than one day, the interval between subsequent administrations of the step-up dose can vary between one and seven days, although if more than one day, the interval is typically three days. The interval between administrations of the full dose is typically one week. The interval between the first priming dose and the subsequent step-up doses is typically 3 days, if at all. The interval between the last step-up dose and the subsequent full doses is typically 3 days, if at all, although it may vary between 3-7 days. The interval between the first dose and the subsequent full doses is typically 7 days. The dose administration is typically calculated per day. The priming dose, step-up dose or full dose is the amount administered in a day. The actual administration may take some time. It typically does not need to be an infusion over an hour or more.

[0019] In the prior art, dose escalation studies are mainly conducted in clinical trials to quickly find effective doses and avoid unnecessary exposure of patients to subtherapeutic doses (Tourneau, Michiels et al. 2009). In the present invention, a priming dose is not administered to quickly find therapeutic doses and avoid exposure of patients to subtherapeutic doses. Instead, when the full dose is reached, it is administered to the subject as part of a dosing regimen designed to treat the subject while simultaneously reducing the incidence and / or severity of side effects in the treated population.

[0020] The present invention provides a method of treating a subject with CLEC12A-positive cancer. The method comprises treating a subject in need thereof with two or more doses of a bispecific antibody that binds CD3 and CLEC12A, where in a first dose, a first amount of the bispecific antibody is administered and in subsequent doses, the amount of the bispecific antibody is greater than the amount of the bispecific antibody in the first dose. The amount of the second administered bispecific antibody in the two or more doses is preferably greater than the amount of the bispecific antibody in the first dose and, in the case of more than two doses, less than the amount of the third bispecific antibody in the two or more doses if the second dose is accompanied by a step-up dose. If the second dose is a full dose, the amounts of the bispecific antibody administered in the second and third doses are equal. Preferably, the amount of the third administered bispecific antibody in the two or more doses is greater than the amount of the second bispecific antibody in the two or more doses and, in the case of the third dose is accompanied by a step-up dose, less than the fourth amount in the two or more doses. After administration of increasing amounts of the bispecific antibody, i.e., after the priming and step-up phases, the bispecific antibody is preferably administered at an essentially constant dose in each subsequent administration, also referred to as a total dose.

[0021] The essentially constant dose in each subsequent administration is preferably at least 15 mg of the bispecific antibody. The essentially constant dose is preferably at least 20, 25, 30, 35, 40, 45, 50, 55 mg, preferably 60-600 mg, for example 60-120; 60-180; 60-200; 60-240; 60-480; 120-240; 120-480; 240-480; or 240-600 mg of the bispecific antibody per subsequent administration.

[0022] The first amount of the bispecific antibody is preferably 9 mg or less, for example 1 to 9 mg or 1 to 5 mg, preferably 3 mg or less, for example 1 to 3 mg, more preferably about 1 mg, even more preferably about 3 mg, and most preferably about 5 mg.

[0023] The administration of increasing amounts of antibody is preferably performed with an interval of 3 to 4 days between doses. The interval between days of administration of an essentially constant amount of antibody is preferably 6 to 8 days, preferably 7 days.

[0024] The first amount of the bispecific antibody is preferably a sub-therapeutic amount of the bispecific antibody. A sub-therapeutic amount of a bispecific antibody is an amount of antibody that typically does not provide a therapeutic effect when administered in a single weekly dose.

[0025] In one embodiment, a first amount of the bispecific antibody of 1-9 mg is administered on day 1 of treatment. On day 3, 4, or 5 of treatment, a first step-up dose of 3-20 mg, preferably 10-20 mg, is administered. On day 7, 8, or 9 of treatment, a second step-up dose of 15-30 mg, preferably 20-30 mg, is administered, and on day 14, 15, or 16 of treatment, a total dose of 25-600 mg is administered.

[0026] In one embodiment, a first amount of the bispecific antibody of 1 mg is administered on day 1 of treatment. A first step-up dose of 3 mg is administered on day 4 of treatment. A second step-up dose of 15 mg is administered on day 8 of treatment. And a total dose of 25 mg is administered on day 15 of treatment.

[0027] In one embodiment, a first amount of 3 mg of the bispecific antibody is administered on day 1 of treatment. A step-up dose of 15 mg is administered on day 4 of treatment. And a full dose of 25 mg is administered on day 8 of treatment.

[0028] In one embodiment, a first amount of bispecific antibody of 5 mg is administered on day 1 of treatment. A step-up dose of 15 mg is administered on day 4 of treatment. And a full dose of 25 mg is administered on day 8 of treatment.

[0029] In one embodiment, a first amount of the bispecific antibody of 3 mg is administered on day 1 of treatment. A first step-up dose of 10 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment. And a total dose of 40 mg is administered on day 15 of treatment.

[0030] In one embodiment, a first amount of the bispecific antibody of 3 mg is administered on day 1 of treatment. A first step-up dose of 15 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 40 mg is administered on day 15 of treatment.

[0031] In another embodiment, a first amount of bispecific antibody of 5 mg is administered on day 1 of treatment. A first step-up dose of 10 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment. And a total dose of 40 mg is administered on day 15 of treatment.

[0032] In another embodiment, a first amount of bispecific antibody of 5 mg is administered on day 1 of treatment. A first step-up dose of 15 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment. And a total dose of 40 mg is administered on day 15 of treatment.

[0033] In another embodiment, a first amount of bispecific antibody of 3 mg is administered on day 1 of treatment. A first step-up dose of 10 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment. And a total dose of 60 mg is administered on day 15 of treatment.

[0034] In another embodiment, a first amount of bispecific antibody of 3 mg is administered on day 1 of treatment. A first step-up dose of 15 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment. And a total dose of 60 mg is administered on day 15 of treatment.

[0035] In another embodiment, a first amount of bispecific antibody of 5 mg is administered on day 1 of treatment. A first step-up dose of 10 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment. And a total dose of 60 mg is administered on day 15 of treatment.

[0036] In another embodiment, a first amount of bispecific antibody of 5 mg is administered on day 1 of treatment. A first step-up dose of 15 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment. And a total dose of 60 mg is administered on day 15 of treatment.

[0037] In another embodiment, a first amount of bispecific antibody of 3 mg is administered on day 1 of treatment. A first step-up dose of 10 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 120 mg is administered on day 15 of treatment.

[0038] In another embodiment, a first amount of bispecific antibody of 3 mg is administered on day 1 of treatment. A first step-up dose of 15 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 120 mg is administered on day 15 of treatment.

[0039] In another embodiment, a first amount of bispecific antibody of 5 mg is administered on day 1 of treatment. A first step-up dose of 10 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 120 mg is administered on day 15 of treatment.

[0040] In another embodiment, a first amount of bispecific antibody of 5 mg is administered on day 1 of treatment. A first step-up dose of 15 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 120 mg is administered on day 15 of treatment.

[0041] In another embodiment, a first amount of bispecific antibody of 3 mg is administered on day 1 of treatment. A first step-up dose of 10 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 240 mg is administered on day 15 of treatment.

[0042] In another embodiment, a first amount of bispecific antibody of 3 mg is administered on day 1 of treatment. A first step-up dose of 15 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 240 mg is administered on day 15 of treatment.

[0043] In another embodiment, a first amount of bispecific antibody of 5 mg is administered on day 1 of treatment. A first step-up dose of 10 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 240 mg is administered on day 15 of treatment.

[0044] In another embodiment, a first amount of bispecific antibody of 5 mg is administered on day 1 of treatment. A first step-up dose of 15 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 240 mg is administered on day 15 of treatment.

[0045] In another embodiment, a first amount of bispecific antibody of 3 mg is administered on day 1 of treatment. A first step-up dose of 10 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 400 mg is administered on day 15 of treatment.

[0046] In another embodiment, a first amount of bispecific antibody of 3 mg is administered on day 1 of treatment. A first step-up dose of 15 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 400 mg is administered on day 15 of treatment.

[0047] In another embodiment, a first amount of bispecific antibody of 5 mg is administered on day 1 of treatment. A first step-up dose of 10 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 400 mg is administered on day 15 of treatment.

[0048] In another embodiment, a first amount of bispecific antibody of 5 mg is administered on day 1 of treatment. A first step-up dose of 15 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 400 mg is administered on day 15 of treatment.

[0049] In another embodiment, a first amount of bispecific antibody of 3 mg is administered on day 1 of treatment. A first step-up dose of 10 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 600 mg is administered on day 15 of treatment.

[0050] In another embodiment, a first amount of bispecific antibody of 3 mg is administered on day 1 of treatment. A first step-up dose of 15 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 600 mg is administered on day 15 of treatment.

[0051] In another embodiment, a first amount of bispecific antibody of 5 mg is administered on day 1 of treatment. A first step-up dose of 10 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 600 mg is administered on day 15 of treatment.

[0052] In another embodiment, a first amount of bispecific antibody of 5 mg is administered on day 1 of treatment. A first step-up dose of 15 mg is administered on day 4 of treatment. A second step-up dose of 25 mg is administered on day 8 of treatment, and a total dose of 600 mg is administered on day 15 of treatment.

[0053] If a step from one dose to another, e.g., from 25 mg to 240 mg, is deemed to cause CRS symptoms, the dosing regimen can be adapted to include one or more further step-up doses, e.g., of 120 mg. Also, an extension of the interval between step-up doses and / or total doses can be determined. These principles can be applied to any of the dosing regimens disclosed herein.

[0054] The present invention uses a specific dosing method, preferably at discrete time intervals. The dosing regimen is designed to administer an effective and safe total dose. In one aspect, the safe dose is intended to prevent and minimize the severity and incidence of cytokine release syndrome (CRS).

[0055] A preferred dosing regimen includes administering a priming dose in one or more doses and administering one or more step-up doses, where the dose increases with dose increments until the full dose is reached. The increments are preferably stepwise. In one aspect, the increments are equal increments until the full dose or maintenance dose is reached. However, it has been found that starting with a higher priming dose and / or a higher first step-up dose results in less severe side effects, such as cytokine release, at the full dose, compared to starting with a low or very low priming dose and / or a first step-up dose. In other words, starting with a higher priming dose reduces cytokine release more effectively than starting with a lower priming dose. An exemplary higher priming dose is, for example, 3 or 5 mg. A lower priming dose is, for example, 1 mg.

[0056] The total dose is typically fixed over the course of treatment, but can be adjusted up or down depending on the subject's individual therapeutic and / or side effect response.

[0057] One or more step-up doses are preferably administered one or two weeks after the first dose. In one embodiment, the first priming dose and the step-up dose are administered once every 3 days (D1-D4-D8), followed by a full dose once every 7 days (once a week). The dose on day 8 (D8) can be equal to the full dose. The full dose is preferably administered starting from day 8-15, depending on the number of step-up doses.

[0058] In one aspect of the invention, one or more administrations of the antibody are accompanied by pre- and / or concomitant treatment with acetaminophen and an antihistamine. The antihistamine is preferably an H1 antagonist. 1It inhibits the action of histamine at the receptor. The acetaminophen and antihistamine treatment is preferably administered on the same day as the antibody administration, preferably prior to the antibody administration. The acetaminophen and antihistamine treatment is preferably administered with each antibody administration.

[0059] A bispecific antibody that binds CD3 and CLEC12A is provided for use in a method of treating a CLEC12A-positive cancer in a subject, the treatment comprising two or more administrations of a bispecific antibody that binds CD3 and CLEC12A, wherein in a first administration a first amount of the bispecific antibody is administered and in each subsequent administration the amount of the bispecific antibody is greater than the amount of the bispecific antibody in the first administration. The invention further provides the use of a bispecific antibody that binds CD3 and CLEC12A for the manufacture of a medicament for treating a CLEC12A-positive cancer in a subject, wherein in a first administration a first amount of the bispecific antibody is administered and in each subsequent administration the amount of the bispecific antibody is greater than the amount of the bispecific antibody in the first administration.

[0060] Further provided is a method for purging CLEC12A-positive hematopoietic cells from a subject and repopulating the hematopoietic system of the subject with normal CLEC12A-positive cells. The method comprises administering to a subject in need thereof a bispecific antibody that binds to CD3 and CLEC12A at intervals, thereby stimulating the hematopoietic stem cells of the subject to repopulate the hematopoietic system with newly formed hematopoietic stem cell-derived cells that contain CLEC12A-positive cells. The subject is preferably a subject with AML, myeloproliferative neoplasm blast stage (MPN-BP), myelofibrosis (MF), or myelodysplastic syndrome (MDS). In a preferred embodiment, the subject has myeloproliferative neoplasm blast stage (MPN-BP), myelofibrosis (MF), or myelodysplastic syndrome (MDS). Normal hematopoietic stem cells have been found to be predominantly CLEC12A-negative. In the progenitor cell fraction, CLEC12A expression is restricted to granulocyte-macrophage progenitor (GMP) cells and common myeloid progenitor (CMP) and megakaryocyte-erythroid progenitor (MEP) cell fractions. After purging of CLEC12A-positive cells, unpurged CMPs and MEPs can still give rise to GMPs and BFU-E (erythrocytes) and CFU-Mk (megakaryocytes). Treatment with the CD3xCLEC12A bispecific antibody described herein can eliminate malignant cells while maintaining intact the potential of hematopoietic stem and progenitor cells. These are stimulated upon treatment with the antibodies described herein to repopulate the hematopoietic system with new healthy cells, including CLEC12A-positive cells. The stimulated cells also produce newly differentiated CLEC12A-negative cells. This can be achieved by antibody therapy, which at least reduces the immunological impact of depleting CLEC12A positive cells from the hematopoietic system when compared to CD3 engager approaches using CD19 or CD123 or other targets. Repopulation times are at least shorter than other CD3 engager approaches such as CD19 and CD123. Furthermore, because CFU-Mk and BFU-E progenitor cells are preserved in the treatment of the present invention, the platelet- and erythropoietic capacity of the hematopoietic system is also preserved when compared to other CD3 engager formats.

[0061] Also provided is a method for purging CLEC12A-positive hematopoietic cells from a subject and repopulating the hematopoietic system of the subject with normal CLEC12A-positive cells, comprising administering to a subject in need thereof, at intervals, a bispecific antibody that binds CD3 and CLEC12A, thereby inducing the subject's hematopoietic stem and / or progenitor cells to repopulate the hematopoietic system with CLEC12A-positive cells derived from newly formed hematopoietic stem cells.

[0062] Further provided is a method for stimulating granulocyte and monocyte development in a subject, the method comprising administering at intervals to a subject in need thereof a bispecific antibody that binds CD3 and CLEC12A, thereby inducing granulocyte and monocyte development from CLEC12A-negative hematopoietic stem or progenitor cells. The spaced administration is preferably according to the intervals as described herein above. The administration schedule is preferably as described herein.

[0063] The subject is preferably a subject with AML, myeloproliferative neoplasms blast stage (MPN-BP), myelofibrosis (MF), or myelodysplastic syndrome (MDS). In a preferred embodiment, the subject has myeloproliferative neoplasms blast stage (MPN-BP), myelofibrosis (MF), or myelodysplastic syndrome (MDS).

[0064] Also provided is a method of preparing an autologous bone marrow cell graft for a subject undergoing treatment for a CLEC12A-positive cancer, comprising incubating a bone marrow cell preparation of the subject with a bispecific antibody that binds CD3 and CLEC12A under conditions suitable for killing CLEC12A-positive cells, and subsequently harvesting the bone marrow cells from the incubation. Further provided is a method of treating a CLEC12A-positive cancer in a subject, comprising incubating a bone marrow cell preparation of the subject with a bispecific antibody that binds CD3 and CLEC12A under conditions suitable for killing CLEC12A-positive cells, treating the subject with hematopoietic ablative therapy, and providing the subject with a bone marrow cell graft comprising the incubated bone marrow cells. Autologous T cells derived from the bone marrow or blood in the graft are retargeted to eliminate CLEC12A-positive cells from the autologous graft. The CD3 and CLEC12A bispecific antibodies disclosed herein are effective even at low effector target cell ratios, for example in the range of 1:3 to 1:97, and at levels reflected in FIG. 6 and Table II. Conditions suitable for killing CLEC12A positive cells as referred to herein involve the presence of T cells, such as autologous T cells or preactivated T cells. Such cells are usually present in bone marrow preparations as a result of the presence of a significant amount of blood, which is also typically collected in many current bone marrow harvesting methods. Ablative therapies are treatments that result in the destruction of cells as a result of the therapy. Many chemotherapeutic and other antitumor therapies also kill normal cells. One of the organs that is more susceptible to damage is the bone marrow. Hematopoietic system ablative therapy in the context of the present invention is a therapy (or treatment) that has additional effects or side effects, in which a large portion of the "normal" or "healthy" hematopoietic system is killed by the therapy or treatment. Often, after the bone marrow is harvested, the patient is treated with hematopoietic system ablative therapy. This bone marrow is transplanted into the subject after completion of the therapy to stimulate repopulation of the subject's hematopoietic system. To prevent diseased cells from also being transplanted, such bone marrow preparations are typically purged for diseased cells. Selection of CD34 positive cells is one method when the diseased cells are not CD34 positive.Such purging methods often remove more cells than necessary and result in slower than potential repopulation of the subject's hematopoietic system. The purging method of the present invention efficiently removes abnormal cells from bone marrow preparations, allowing for more rapid repopulation.

[0065] In some embodiments of the invention, a method is provided for providing a hematopoietic stem cell sparing cancer treatment to a subject, the method comprising administering a bispecific antibody that binds CD3 and CLEC12A to a subject in need thereof. In some embodiments, the subject has a CLEC12A positive cancer.

[0066] The bispecific antibodies disclosed herein are preferably administered as an intravenous infusion. Infusions typically take about 2-4 hours, although longer and shorter administrations are possible. Infusions usually do not extend for a period of more than 12 hours.

[0067] Incrementing the amount of antibody to reach an essentially constant amount per administration is also referred to as a single intrapatient dose escalation regimen, or intrapatient dose escalation.

[0068] In order to make this specification easier to understand, certain terms are defined at the beginning. Additional definitions are described throughout the detailed description. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, and conventional methods of immunology, protein chemistry, biochemistry, recombinant DNA technology and pharmacology are employed.

[0069] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0070] As used herein, the term "CLEC12A" refers to C-type lectin domain family 12 member A. CLEC12A is also known as C-type lectin protein CLL-1; MICL, dendritic cell-associated lectin 2, C-type lectin superfamily, myeloid inhibitory C-type lectin-like receptor, C-type lectin-like molecule-1, DCAL2; CLL1; C-type lectin-like molecule 1, DCAL-2, Killer cell lectin like receptor subfamily L, member 1 (KLRL1), CD371 (cluster of differentiation 371) (Bakker, van den Oudenrijn et al. 2004; GenBank™ Accession No.: AY547296; Zhang W. et al. GenBank™ Accession No.: AF247788; Marshall, Willment et al. 2004; GenBank™ Accession No.: AY498550; Han, Zhang et al. 2004; Chen, Floyd et al. (Chen, Floyd et al. 2006. GenBank™ Accession Numbers: AY426759; Chen, Floyd et al. 2006. Ids; HGNC: 31713; Entrez Gene: 160364; Ensembl: ENSG00000172322; OMIM: 612088; UniProtKB: Q5QGZ9).

[0071] CLEC12A is an antigen expressed on leukemic blast cells and leukemic stem cells, e.g., CD34-negative or CD34-low expressing leukemic stem cells (side population) in acute myeloid leukemia (AML) (Bakker, van den Oudenrijn et al. 2004; van Rhenen, van Dongen et al. 2007; Moshaver, van Rhenen et al. 2008), as well as in myelodysplastic syndromes (MDS) (Bakker, van den Oudenrijn et al. 2004, and Toft-Peterson, Nederby et al. 2016). Expression of CLEC12A is otherwise thought to be restricted to cells of the hematopoietic lineage, particularly the myeloid lineage in peripheral blood and bone marrow, i.e., granulocyte, monocyte, and dendritic cell precursors. More importantly, CLEC12A is absent from normal hematopoietic stem cells. As used herein, when reference is made to CLEC12A (SEQ ID NO:1), it refers to human CLEC12A, unless otherwise specified.

[0072] The term "CLEC12A" refers to all variants (such as splices and mutations) and isoforms thereof referred to herein that retain the myeloid expression profile (both at the surface expression level and at the mRNA level), including those described in Bakker, van den Oudenrijn et al. 2004 and Marshall, Willment et al. 2004. The accession numbers are provided primarily as a further method of identification, although the actual sequence of the protein may vary due to mutations in the encoding gene, such as those that occur in some cancers.

[0073] The term "CD3" (cluster of differentiation 3) refers to a protein complex composed of the CD3 gamma chain (SwissProt P09693), the CD3 delta chain (SwissProt P04234), the CD3 epsilon chain (SwissProt P07766), and the CD3 zeta chain homodimer (SwissProt P20963). CD3 epsilon is known under various aliases, some of which are "CD3e molecule, epsilon (CD3-TCR complex)", "CD3e antigen, epsilon polypeptide (TiT3 complex)"; T cell surface antigen T3 / Leu-4 epsilon chain; T3E; T cell antigen receptor complex, epsilon subunit of T3; CD3e antigen; CD3-epsilon 3; IMD18; TCRE. The Ids for the CD3E gene are HGNC:1674; Entrez Gene:916; Ensembl:ENSG00000198851; OMIM:186830, and UniProtKB:P07766. These chains associate with the T cell receptor (TCR) and the ζ chain to generate activation signals in T lymphocytes. The TCR, ζ chain, and CD3 molecule together constitute the TCR complex. CD3 is expressed on T cells. In this specification, when CD3 is mentioned, it refers to human CD3 (SEQ ID NOs:2-5) unless otherwise specified.

[0074] As used herein, the term "antibody" refers to a protein molecule belonging to the immunoglobulin class of proteins that contains one or more domains that bind to an epitope on an antigen, such domains being derived from or sharing sequence homology with the variable region of the antibody. Antibodies are typically composed of a basic structural unit, each having two heavy chains and two light chains. Antibodies for therapeutic use are preferably as close as possible to the natural antibodies of the subject to be treated (e.g., human antibodies of a human subject). The antibodies according to the present invention are not limited to any particular format or method of producing them.

[0075] A "bispecific antibody" is an antibody as described herein in which one domain of the antibody binds to a first antigen while a second domain of the antibody binds to a second antigen, the first and second antigens being non-identical. The term "bispecific antibody" also encompasses antibodies in which one heavy chain variable region / light chain variable region (VH / VL) combination binds to a first epitope on an antigen and a second VH / VL combination binds to a second epitope. The term further includes antibodies in which the VH can specifically recognize a first antigen and the VL paired with the VH in an immunoglobulin variable region can specifically recognize a second antigen. The resulting VH / VL pair binds either antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in WO 2008 / 027236, WO 2010 / 108127, and Schaefer, Haber et al, 2011. The bispecific antibodies according to the invention are not limited to any particular bispecific format or method of producing them.

[0076] The term "common light chain" as used herein refers to the two light chains (or VL portions thereof) in a bispecific antibody. The two light chains (or VL portions thereof) may be identical or may have some amino acid sequence differences, although the binding specificity of the full-length antibody is not affected. "Common" refers to functional equivalents of light chains that are not identical in amino acid sequence. Many variants of the light chain exist, and there are mutations (deletions, substitutions, insertions and / or additions) that do not affect the formation of a functional binding region. The light chain of the present invention may also be a light chain having 0-10, preferably 0-5, amino acid insertions, deletions, substitutions, additions or combinations thereof, as specified herein above. It is within the scope of the definition of common light chain used herein to prepare or find light chains that are not identical but are still functionally equivalent, for example by introducing and testing conservative amino acid changes, amino acid changes in regions that do not contribute or only partially contribute to the binding specificity when paired with the heavy chain, etc.

[0077] The term "full-length IgG" or "full-length antibody" according to the present invention is defined as including essentially a complete IgG, but not necessarily having all the functions of a complete IgG. For the avoidance of doubt, a full-length IgG comprises two heavy chains and two light chains. Each chain contains a constant (C) region and a variable (V) region, which can be broken down into domains designated CH1, CH2, CH3, VH and CL, VL. IgG antibodies bind to antigens via the variable region domains contained in the Fab portion, and after binding can interact with molecules and cells of the immune system via the constant domains, primarily the Fc portion. Full-length antibodies according to the present invention encompass IgG molecules in which mutations may be present that provide desired properties. Full-length IgG should not have a deletion of a substantial portion of any of the regions. However, IgG molecules in which one or several amino acid residues have been deleted without essentially altering the binding properties of the resulting IgG molecule are included in the term "full-length IgG". For example, such IgG molecules can have a deletion of 1-10 amino acid residues, preferably in the non-CDR regions, where the deleted amino acids are not essential for the binding specificity of the IgG.

[0078] "Percent (%) identity" as referred to herein in reference to amino acid sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a selected sequence after aligning the sequences for optimal comparison purposes. Gaps may be introduced in either of the two sequences being compared to optimize alignment between the two sequences. Such alignments can be performed over the entire length of the sequences being compared. Alternatively, alignments may be performed over shorter lengths, such as about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Sequence identity is the percentage of identical matches between the two sequences over the reported alignment region.

[0079] The comparison of sequences and the determination of the percentage of sequence identity between two sequences can be accomplished using mathematical algorithms. Those skilled in the art will recognize the fact that several different computer programs are available for aligning two sequences and determining the identity between the two sequences (Kruskal, JB, 1983). The percent sequence identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm for alignment of two sequences. (Needleman and Wunsch, 1970). The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. For the purposes of the present invention, the NEEDLE program from the EMBOSS package can be used (version 2.8.0 or later, Rice, Longden et al, 2000; (http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 is used for the substitution matrix. Optional parameters used are a gap open penalty of 10 and a gap extension penalty of 0.5.

[0080] After alignment with the NEEDLE program as described above, the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: the number of corresponding positions in the alignment that show identical amino acids or identical nucleotides in both sequences, after subtracting the total number of gaps in the alignment, is divided by the total length of the alignment.

[0081] An antibody typically recognizes a part of an antigen, and such epitopes may be present in other compounds as well, and an antibody according to the invention that "specifically recognizes" an antigen, e.g., CLEC12A or CD3, may also recognize other compounds as well, if such other compounds contain the same type of epitope. Thus, the term "specifically recognizes" in relation to the interaction of an antigen and an antibody does not exclude the binding of the antibody to other compounds that contain the same type of epitope.

[0082] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both from contiguous amino acids juxtaposed by tertiary folding of a protein (so-called linear and conformational epitopes) or from non-contiguous amino acids. Epitopes formed from contiguous linear amino acids are typically retained on exposure to denaturing solvents, while epitopes formed by tertiary folding typically lose conformation on treatment with denaturing solvents. Epitopes may typically include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining spatial conformation of epitopes are known to those skilled in the art and include techniques in the art, such as x-ray crystallography, HDX-MS and 2-dimensional nuclear magnetic resonance, pep scan, and alanine scan, depending on the nature of the epitope (see, e.g., Morris GE, 1996).

[0083] The term "abnormal cell" as used herein includes tumor cells, more specifically tumor cells of hematological origin, including preleukemic cells, such as those causing myelodysplastic syndromes (MDS), and leukemic cells, such as acute myeloid leukemia (AML) tumor cells or chronic myeloid leukemia (CML) cells.

[0084] As used herein, the term "immune effector cell" or "effector cell" refers to a cell in the natural repertoire of cells in the mammalian immune system that can be activated to affect the viability of a target cell. Immune effector cells include lymphoid cells, such as natural killer (NK) cells, T cells, including cytotoxic T cells, or B cells, and myeloid lineage cells, such as monocytes or macrophages, dendritic cells, and neutrophil granulocytes. Thus, the effector cells are preferably NK cells, T cells, B cells, monocytes, macrophages, dendritic cells, or neutrophil granulocytes. Recruitment of effector cells to abnormal cells means that the immune effector cells are brought into close proximity to the abnormal target cells, and the effector cells can initiate direct or indirect killing of the abnormal cells.

[0085] As used herein, the terms "subject" and "patient" are used interchangeably and refer to a mammal, such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, etc. (e.g., a patient suffering from cancer, such as a human patient).

[0086] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention or procedure performed or the administration of an active agent or combination of active agents to a subject with the goal of reversing, ameliorating, ameliorating, inhibiting, or slowing or preventing the progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical manifestation associated with a disease.

[0087] As used herein, "effective treatment" or "positive therapeutic response" refers to a treatment that results in a beneficial effect, e.g., amelioration of at least one symptom of a disease or disorder, such as cancer. A beneficial effect can result in an improved state over a baseline, such as an improvement over a measurement or observation made before the initiation of therapy according to the method. For example, a beneficial effect can result in a state that slows, stabilizes, stops, or reverses the progression of cancer in a subject at any clinical stage, as evidenced by a reduction or elimination of clinical or diagnostic symptoms of the disease, or a reduction or elimination of a marker of cancer. An effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent metastasis of tumors, slow or prevent tumor growth, and / or prevent or delay tumor recurrence or relapse.

[0088] The term "therapeutic amount" refers to an amount of a drug or combination of drugs that provides a desired biological, therapeutic, and / or prophylactic result. The result may be reduction, amelioration, remission, reduction, delay, and / or alleviation of one or more signs, symptoms, or causes of a disease, or other desired changes in biology. In some embodiments, a therapeutic amount is an amount sufficient to delay the development of a tumor. In some embodiments, a therapeutic amount is an amount sufficient to prevent or delay the recurrence of a tumor. A therapeutic amount may be administered in one or more administrations. A therapeutic amount of a drug or composition may (i) reduce the number of cancer cells; (ii) reduce the size of a tumor; (iii) inhibit, prevent, slow, or stop to some extent cancer cell invasion into peripheral organs; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the onset and / or recurrence of tumors; and / or (vii) relieve to some extent one or more symptoms associated with cancer. In one example, a "therapeutic amount" is the amount of CLEC12A / CD3 bispecific antibody that results in a reduction in cancer (e.g., a reduction in cancer cell count) or a slowing of progression of cancer, such as acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia.

[0089] As disclosed herein, bispecific antibodies for use in the methods provided herein include bispecific antibodies comprising one heavy chain variable region / light chain variable region (VH / VL) combination that binds CLEC12A and a second VH / VL combination that binds CD3.

[0090] Suitable CLEC12A heavy chain variable (VH) regions for use in CLEC12A / CD3 bispecific antibodies include those that bind to CLEC12A. In a preferred embodiment, the CLEC12A VH region of the CLEC12A / CD3 bispecific antibody binds to CLEC12A expressed on tumor cells. Exemplary CLEC12A VH regions for use in CLEC12A / CD3 bispecific antibodies are disclosed, for example, in WO 2017 / 010874, WO 2014 / 051433, and WO 2005 / 000894 (each of which is incorporated herein by reference).

[0091] In some embodiments, the binding affinity of the CLEC12A / CD3 bispecific antibody to CLEC12A on tumor cells is at least 2-fold, 4-fold, 6-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold higher than the affinity of binding to CD3. In certain embodiments, the CLEC12A binding affinity of the CLEC12A / CD3 bispecific antibody to CLEC12A is about 1x10e-6M to 1x10e-10M, about 1x10e-7M to 1x10e-10M, or about 1x10e-8 to 1x10e-10. In some embodiments, the CLEC12A binding affinity of the CLEC12A / CD3 bispecific antibody to CLEC12A is at least 1x10e8M, preferably at least 1x10e9M. In certain embodiments, the binding affinity of the CLEC12A / CD3 bispecific antibody to CLEC12A is between about 1x10e-8M and 1x10e-9M, such as about 2x10e-9M, about 3x10e-9M, about 4x10e-9M, about 5x10e-9M, about 6x10e-9M, about 7x10e-9M, 8x10e-9M or about 9x10e-9M, and the binding affinity of the CLEC12A / CD3 bispecific antibody to CD3 is at least 30 times less, at least 40 times less, or at least 50 times less.

[0092] In certain embodiments, the CD3 VH region of the CLEC12A / CD3 bispecific antibody binds to cell surface expressed CD3 / TCR on a human T cell line with an affinity (KD) significantly lower than that of the mouse anti-CD3 antibody, mOKT3. In some embodiments, the CD3 VH region of the CLEC12A / CD3 bispecific antibody binds to CD3 with a binding affinity of at least 1x10-e6M. In some embodiments, the CD3 binding affinity of the bispecific antibody is between about 1x10e-6M and 1x10e-10M. In some embodiments, the CD3 binding affinity of the CD3 VH region of the bispecific antibody is between about 1x10e-7M and 1x10e-8M.

[0093] In some embodiments, the CLEC12A / CD3 bispecific antibody comprises a first heavy chain variable region that binds human CLEC12A, the heavy chain variable region comprising: (a) a heavy chain CDR1 comprising the amino acid sequence SGYTFTGY (SEQ ID NO: 9), a heavy chain CDR2 comprising the amino acid sequence IINPSGGS (SEQ ID NO: 10), and a heavy chain CDR3 comprising the amino acid sequence GTTGDWFDY (SEQ ID NO: 11); (b) a heavy chain CDR1 comprising the amino acid sequence SGYTFTSY (SEQ ID NO: 13), a heavy chain CDR2 comprising the amino acid sequence IINPSGGS (SEQ ID NO: 14), and a heavy chain CDR3 comprising the amino acid sequence GNYGDEFDY (SEQ ID NO: 15); or (c) a heavy chain CDR1 comprising the amino acid sequence SGYTFTGY (SEQ ID NO: 17), a heavy chain CDR2 comprising the amino acid sequence WINPNSGG (SEQ ID NO: 18), and a heavy chain CDR3 comprising the amino acid sequence DGYFADAFDY (SEQ ID NO: 19).

[0094] Conservative variations of 1, 2, or 3 amino acid residues from the listed CDR sequences are possible while retaining the same binding activity in kind (essentially equivalent, but not necessarily in amount). Thus, the heavy chain CDR1, 2, and 3 sequences preferably contain sequences that deviate from the listed CDR sequences by no more than 3 amino acids, preferably no more than 2, more preferably no more than 1 amino acid. In certain embodiments, the heavy chain CDR1, 2, and 3 sequences are identical to the listed CDR sequences.

[0095] In some embodiments, the CLEC12A / CD3 bispecific antibody comprises a heavy chain variable region that binds human CLEC12A, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the VH region shown in SEQ ID NO: 12, 16 or 20.

[0096] In some embodiments, the CLEC12A / CD3 bispecific antibody comprises a heavy chain variable region that binds human CLEC12A, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical or 100% identical to the amino acid sequence set forth in SEQ ID NO: 12, 16 or 20.

[0097] For example, in some embodiments, the heavy chain variable region of a bispecific antibody that binds to human CLEC12A may have 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof in the heavy chain variable region sequence outside of the three CDR sequences. In some embodiments, the heavy chain variable region comprises 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, preferably 0 amino acid insertions, deletions, substitutions, additions, or a combination thereof relative to the amino acid sequence shown.

[0098] In certain embodiments, the CLEC12A / CD3 bispecific antibody comprises a heavy chain variable region that binds human CLEC12A, wherein the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 12, 16 and 20.

[0099] CD3 heavy chain variable (VH) regions suitable for use in CLEC12A / CD3 bispecific antibodies include those capable of binding to the CD3 gamma chain, the CD3 delta chain, the CD3 epsilon chain, or a combination of CD3 delta / CD3 epsilon or CD3 gamma / CD3 epsilon. In some embodiments, the CD3 VH region of the bispecific antibody binds to the CD3 epsilon chain. In some embodiments, the CD3 VH region binds to human CD3. In some embodiments, the CD3 VH region binds to the human CD3 epsilon chain.

[0100] Exemplary CD3 binding regions for use in CLEC12A / CD3 bispecific antibodies are disclosed in WO 2017 / 010874, WO 2014 / 051433, and WO 2005 / 118635, each of which is incorporated by reference herein.

[0101] In certain embodiments, the CLEC12A / CD3 bispecific antibody comprises a heavy chain variable region that binds CD3, the heavy chain variable region comprising: (a) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 comprising the amino acid sequence IWYNGRKQ (SEQ ID NO: 22), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); (b) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 comprising the amino acid sequence IWYSGSKKN (SEQ ID NO: 30), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); (c) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 comprising the amino acid sequence IWYHGRKQ (SEQ ID NO: 32), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); (d) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 comprising the amino acid sequence IWYHARKQ (SEQ ID NO: 34), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); (e) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 comprising the amino acid sequence IWYNARKQ (SEQ ID NO: 36), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); (g) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 comprising the amino acid sequence IWYNTRKQ (SEQ ID NO: 45), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); (h) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 comprising the amino acid sequence IWYDGKNT (SEQ ID NO: 47), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); (i) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 sequence comprising the amino acid sequence IYYDGSRT (SEQ ID NO: 49), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); or (j) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 sequence comprising the amino acid sequence IWHDGRKT (SEQ ID NO: 51), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23).

[0102] CDR1 comprises the amino acid sequence GFTFSSYG (SEQ ID NO: 21) as defined according to IMGT. CDR1 may comprise the amino acid sequence SYGMH (SEQ ID NO: 60) as defined according to Kabat.

[0103] Mutation of one, two or three amino acid residues from the listed CDR sequences is possible while retaining the same binding activity in kind (essentially the same, but not necessarily the same amount). Thus, the heavy chain CDR1, 2 and 3 sequences preferably contain sequences that deviate from the listed CDR sequences by no more than three amino acids, preferably no more than two, more preferably no more than one amino acid. In a particular embodiment, the heavy chain CDR1, 2 and 3 sequences are identical to the listed CDR sequences.

[0104] In some embodiments, the CLEC12A / CD3 bispecific antibody comprises a heavy chain variable region that binds to human CD3, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the VH region shown in SEQ ID NOs: 24 to 29, 31, 33, 35, 37 to 44, 46, 48, 50 and 52.

[0105] In some embodiments, the CLEC12A / CD3 bispecific antibody comprises a heavy chain variable region that binds to human CD3, and the heavy chain variable region comprises an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical or 100% identical to one of the VH region sequences set forth in SEQ ID NOs: 24 to 29, 31, 33, 35, 37 to 44, 46, 48, 50 and 52.

[0106] In a specific embodiment, the CLEC12A / CD3 bispecific antibody comprises a heavy chain variable region that binds to human CD3, and the heavy chain variable region is at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical or 100% identical to one of the amino acid sequences of the VH region of the sequences set forth in SEQ ID NOs: 37 to 44.

[0107] For example, in some embodiments, the heavy chain variable region of a bispecific antibody that binds to human CD3 may have 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof in the sequence of the heavy chain variable region outside of the three CDR sequences. In some embodiments, the heavy chain variable region comprises 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, preferably 0 amino acid insertions, deletions, substitutions, additions, or a combination thereof relative to the amino acid sequence shown.

[0108] Further variants of the disclosed amino acid sequences that retain CLEC12A or CD3 binding can be obtained, for example, from phage display libraries containing reshaped human IGKV1-39 / IGKJ1 VL regions (de Kruif, Kramer et al. 2010) and collections of VH regions that incorporate amino acid substitutions in the amino acid sequences of the CLEC12A or CD3 VH regions disclosed herein (e.g., US Patent Publication No. 2016 / 0368988). Phages encoding Fab regions that bind CLEC12A or CD3 can be selected, analyzed by flow cytometry, and sequenced to identify variants with amino acid substitutions, insertions, deletions, or additions that retain antigen binding. For example, as described in US Patent Application Publication No. 2016 / 0368988, the CD3 VH region may be substituted at position A50 and modified with S, Y, M or Q; D59 may be substituted with L, I, V, F, R, A, N, H, S, T, Y or E, preferably Y or E; A61 may be substituted with N, I, H, Q, L, R, Y, E, S, T, D, K, V; F105 may be substituted with Y or M. Acceptable amino acid substitutions can be readily found.

[0109] In a specific embodiment, the CLEC12A / CD3 bispecific antibody comprises a heavy chain variable region that binds to human CD3, and the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 24 to 29, 31, 33, 35, 37 to 44, 46, 48, 50, or 52.

[0110] In a specific embodiment, the CLEC12A / CD3 bispecific antibody comprises a heavy chain variable region that binds to human CD3, and the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 37-44.

[0111] In a specific embodiment, the CLEC12A / CD3 bispecific antibody comprises a first heavy chain variable region that binds to human CLEC12A, wherein the first VH region comprises an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical or 100% identical to the amino acid sequence of the VH region set forth in SEQ ID NOs: 12, 16 and 20; and a second heavy chain variable region that binds to human CD3, wherein the second VH region comprises an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical or 100% identical to the amino acid sequence of one of the VH region sequences set forth in SEQ ID NOs: 37 to 44.

[0112] In certain embodiments, the CLEC12A / CD3 bispecific antibody comprises a first heavy chain variable region that binds human CLEC12A and a second heavy chain variable region that binds human CD3, (a) the first heavy chain variable region: (i) a heavy chain CDR1 comprising the amino acid sequence SGYTFTGY (SEQ ID NO: 9), a heavy chain CDR2 comprising the amino acid sequence IINPSGGS (SEQ ID NO: 10), and a heavy chain CDR3 comprising the amino acid sequence GTTGDWFDY (SEQ ID NO: 11); (ii) a heavy chain CDR1 comprising the amino acid sequence SGYTFTSY (SEQ ID NO: 13), a heavy chain CDR2 comprising the amino acid sequence IINPSGGS (SEQ ID NO: 14), and a heavy chain CDR3 comprising the amino acid sequence GNYGDEFDY (SEQ ID NO: 15); or (iii) a heavy chain CDR1 comprising the amino acid sequence SGYTFTGY (SEQ ID NO: 17), a heavy chain CDR2 comprising the amino acid sequence WINPNSGG (SEQ ID NO: 18), and a heavy chain CDR3 comprising the amino acid sequence DGYFADAFDY (SEQ ID NO: 19); and (b) the second heavy chain variable region (i) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 comprising the amino acid sequence IWYNGRKQ (SEQ ID NO: 22), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); (ii) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 comprising the amino acid sequence IWYSGSKKN (SEQ ID NO: 30), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); (iii) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 comprising the amino acid sequence IWYHGRKQ (SEQ ID NO: 32), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); (iv) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 comprising the amino acid sequence IWYHARKQ (SEQ ID NO: 34), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); (v) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 sequence comprising the amino acid sequence IWYNARKQ (SEQ ID NO: 36), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); (vi) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 comprising the amino acid sequence IWYNTRKQ (SEQ ID NO: 45), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); (vii) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 sequence comprising the amino acid sequence IWYDGKNT (SEQ ID NO: 47), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); (viii) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 sequence comprising the amino acid sequence IYYDGSRT (SEQ ID NO: 49), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23); or (ix) a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 comprising the amino acid sequence IWHDGRKT (SEQ ID NO: 51), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23).

[0113] In a particular embodiment, the CLEC12A / CD3 bispecific antibody comprises a first heavy chain variable region that binds human CLEC12A, the first VH region comprising a heavy chain CDR1 comprising the amino acid sequence SGYTFTGY (SEQ ID NO: 9), a heavy chain CDR2 comprising the amino acid sequence IINPSGGS (SEQ ID NO: 10), and a heavy chain CDR3 comprising the amino acid sequence GTTGDWFDY (SEQ ID NO: 11); and a second heavy chain variable region, the second VH region comprising a heavy chain CDR1 comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 21), a heavy chain CDR2 comprising the amino acid sequence IWYNARKQ (SEQ ID NO: 36), and a heavy chain CDR3 comprising the amino acid sequence GTGYNWFDP (SEQ ID NO: 23).

[0114] In a specific embodiment, the CLEC12A / CD3 bispecific antibody comprises a first heavy chain variable region that binds to human CLEC12A, wherein the amino acid sequence of the first VH region is selected from SEQ ID NOs: 12, 16, and 20; and a second heavy chain variable region that binds to human CD3, wherein the amino acid sequence of the second VH region is selected from SEQ ID NOs: 24 to 29, 31, 33, 35, 37 to 44, 46, 48, 50, and 52.

[0115] In a specific embodiment, the CLEC12A / CD3 bispecific antibody comprises a first heavy chain variable region that binds to human CLEC12A, wherein the amino acid sequence of the first VH region is SEQ ID NO: 12; and a second heavy chain variable region that binds to human CD3, wherein the amino acid sequence of the second VH region is selected from SEQ ID NOs: 37 to 44.

[0116] In one embodiment, the CLEC12A / CD3 bispecific antibody comprises a first heavy chain variable region that binds human CLEC12A, wherein the amino acid sequence of the first VH region is set forth in SEQ ID NO: 12; and a second heavy chain variable region that binds human, wherein the amino acid sequence of the second VH region is set forth in SEQ ID NO: 37.

[0117] The light chain variable regions of the VH / VL binding domains of the CLEC12A / CD3 bispecific antibody may be the same as the VL domain of the parent CLEC12A monospecific antibody and / or the VL domain of the parent CD3 monospecific antibody, or alternative VL domains may be used for one or both VH / VL domain combinations, so long as the bispecific antibody retains binding to both the CLEC12A and CD3 antigens.

[0118] In some embodiments, the VL region of the VH / VL CLEC12A binding domain of a CLEC12A / CD3 bispecific antibody is similar to the VL region of the VH / VL CD3 binding domain, in certain embodiments, the VL regions of the first and second VH / VL domain combinations are identical.

[0119] In certain embodiments, the light chain variable region of one or both VH / VL binding regions of the CLEC12A / CD3 bispecific antibody comprises a common light chain. In some embodiments, the common light chain variable region of one or both VH / VL binding regions comprises a germline O12 variable region V-segment. In certain embodiments, the light chain variable region of one or both VH / VL binding regions comprises a kappa light chain V-segment IgVκ1-39. *01. IgVκ1-39 is the short form for immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39, IGKV139, IGKV1-39, O12a, or O12. The external Ids for this genetic element are HGNC:5740, Entrez Gene:28930, Ensembl:ENSG00000242371. The amino acid sequence of the V region is provided in SEQ ID NO:56. The V region can be combined with one of five J regions. The preferred J regions are jk1 and jk5, with the combined sequences designated IGKV1-39 / jk1 and IGKV1-39 / jk5. Alternative names include IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * 01 (nomenclature according to the worldwide web of the IMGT database at imgt.org). In a specific embodiment, the light chain variable region of one or both VH / VL joining regions is the kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ1 * 05 (SEQ ID NO:57 and SEQ ID NO:58, respectively).

[0120] In some embodiments, the light chain variable region of one or both VH / VL binding domains of a CLEC12A / CD3 bispecific antibody comprises an LCDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 53), an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence QQSYSTP (SEQ ID NO: 55) (i.e., the CDRs of IGKV1-39 according to IMGT). In some embodiments, the light chain variable region of one or both VH / VL binding domains of a CLEC12A / CD3 bispecific antibody comprises an LCDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 53), an LCDR2 comprising the amino acid sequence AASLQS (SEQ ID NO: 54), and an LCDR3 comprising the amino acid sequence QQSYSTP (SEQ ID NO: 55).

[0121] In some embodiments, one or both of the VH / VL binding regions of the CLEC12A / CD3 bispecific antibody comprise a light chain variable region comprising an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical or 100% identical to the amino acid sequence set forth in SEQ ID NO: 57. In some embodiments, one or both of the VH / VL binding regions of the CLEC12A / CD3 bispecific antibody comprise a light chain variable region comprising an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical or 100% identical to the amino acid sequence set forth in SEQ ID NO:58.

[0122] For example, in some embodiments, the variable light chain of one or both VH / VL binding regions of a CLEC12A / CD3 bispecific antibody may have 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof, relative to SEQ ID NO: 57 or SEQ ID NO: 58. In some embodiments, the light chain variable region of one or both VH / VL binding regions of a CLEC12A / CD3 bispecific antibody comprises 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, preferably 0 amino acid insertions, deletions, substitutions, additions, or a combination thereof, relative to the amino acid sequence shown.

[0123] In other embodiments, the light chain variable region of one or both VH / VL binding regions of the CLEC12A / CD3 bispecific antibody comprises the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 58. In a specific embodiment, both VH / VL binding regions of the CLEC12A / CD3 bispecific antibody comprise identical VL regions. In one embodiment, both VL of the VH / VL binding regions of the CLEC12A / CD3 bispecific antibody comprise the amino acid sequence set forth in SEQ ID NO: 57. In one embodiment, both VL of the VH / VL binding regions of the CLEC12A / CD3 bispecific antibody comprise the amino acid sequence set forth in SEQ ID NO: 58.

[0124] CLEC12A / CD3 bispecific antibodies for use in the methods disclosed herein can be provided in a number of formats. Many different formats of bispecific antibodies are known in the art and are reviewed by Kontermann, Brinkmann, 2015, 2018, and Spiess, Zhai et al., 2015, each of which is incorporated herein by reference. For example, non-traditional antibody bispecific antibody formats with two VH / VL combinations have at least a variable domain comprising a heavy chain variable region and a light chain variable region. This variable domain may be linked to a single chain Fv fragment, a monobody, a VH, and a Fab fragment that provides a second binding activity.

[0125] In some embodiments, the CLEC12A / CD3 bispecific antibodies used in the methods provided herein are generally of the human IgG subclass (e.g., IgG1, IgG2, IgG3, IgG4). In certain embodiments, the antibodies are of the human IgG1 subclass. Full-length IgG antibodies are preferred due to their advantageous half-life and reduced immunogenicity. Thus, in certain embodiments, the CLEC12A / CD3 bispecific antibodies are full-length IgG molecules. In one embodiment, the CLEC12A / CD3 bispecific antibodies are full-length IgG1 molecules.

[0126] Thus, in certain embodiments, the CLEC12A / CD3 bispecific antibody comprises a fragment crystallizable (Fc). The Fc of the CLEC12A / CD3 bispecific antibody is preferably composed of a human constant region. The constant region or Fc of the CLEC12A / CD3 bispecific antibody may contain one or more, preferably no more than 10, preferably no more than 5 amino acids that differ from the constant region of a naturally occurring human antibody. For example, in certain embodiments, each Fab arm of the bispecific antibody may further comprise an Fc region that includes modifications that facilitate bispecific antibody formation, modifications that affect Fc-mediated effector functions, and / or other features described herein.

[0127] In a preferred embodiment, the CLEC12A / CD3 bispecific full-length IgG antibody has a mutated lower hinge and / or CH2 domain such that the bispecific IgG antibody has a reduced interaction with Fc gamma (Fcγ) receptors. As used herein, the term "such that the bispecific IgG antibody has a reduced interaction with Fc gamma receptors" means that the interaction of the CLEC12A / CD3 bispecific antibody with Fc gamma receptors is reduced when such Fc gamma receptors are present in the vicinity of the antibody. In certain embodiments, the interaction of the CLEC12A / CD3 bispecific antibody with Fc receptors is essentially abolished. Bispecific antibodies with reduced Fcγ receptor binding have been previously described (US 2014 / 0120096, incorporated herein by reference).

[0128] In a particular embodiment, the CLEC12A / CD3 bispecific antibody comprises a mutated lower hinge and / or CH2 domain with at least one substitution at amino acid positions 235 and / or 236 (EU numbering). Preferably, both amino acid positions 235 and 236 are substituted. As described in US Patent Application Publication No. 2014 / 0120096, substitutions at these sites can essentially prevent the interaction between the antibody and Fc receptors present on tumor cells or effector cells. Thus, in a particular embodiment, the CLEC12A / CD3 bispecific antibody comprises a mutated CH2 and / or lower hinge domain comprising a L235G and / or G236R substitution. Preferably, both L235G and G236R are substituted. Alternatively, the skilled person can introduce lower hinge and / or CH2 domain mutations comprising substitutions 234F, 235E and / or 331S (Oganesyan, Gao et al. 2008). Preferably, all three substitutions are introduced in this substitution.

[0129] Bispecific antibodies are typically produced by cells expressing nucleic acids encoding the antibodies. Thus, in some embodiments, the bispecific CLEC12A / CD3 antibodies disclosed herein are produced by providing a cell comprising one or more nucleic acids encoding the heavy and light chain variable and constant regions of a bispecific CLEC12A / CD3 antibody. The cell is preferably an animal cell, more preferably a mammalian cell, more preferably a primate cell, and most preferably a human cell. A suitable cell is any cell capable of comprising and preferably producing a CLEC12A / CD3 bispecific antibody.

[0130] Suitable cells for antibody production are known in the art and include hybridoma cells, Chinese Hamster Ovary (CHO) cells, NS0 cells, or PER-C6 cells. Various institutions and companies have developed cell lines for large-scale production of antibodies, for example for clinical use. Non-limiting examples of such cell lines are CHO cells, NS0 cells, or PER.C6 cells. In a particularly preferred embodiment, the cells are human cells. Preferably, cells transformed by adenovirus E1 region or functional equivalents thereof. A preferred example of such a cell line is the PER.C6 cell line or its equivalent. In a particularly preferred embodiment, the cells are CHO cells or variants thereof. Preferably, variants utilizing the glutamine synthetase (GS) vector system for the expression of antibodies. In a preferred embodiment, the cells are CHO cells.

[0131] In some embodiments, the cells express different light and heavy chains that make up the CLEC12A / CD3 bispecific antibody. In certain embodiments, the cells express two different heavy chains and at least one light chain. In one preferred embodiment, the cells express a "common light chain" as described herein to reduce the number of different antibody species (different heavy and light chain combinations). For example, each VH region is cloned into an expression vector together with a reshaped human IGKV139 / IGKJ1 (huVκ139) light chain using methods known in the art for the production of bispecific IgG (WO 2013 / 157954; incorporated herein by reference). huVκ139 has previously been shown to pair with multiple heavy chains, thereby generating antibodies with diverse specificities and facilitating the generation of bispecific molecules (de Kruif, Kramer et al. 2009; WO 2009 / 157771).

[0132] Antibody-producing cells expressing a common light chain and equal amounts of two heavy chains typically produce 50% bispecific antibodies and 25% each of monospecific antibodies (i.e., with the same heavy and light chain combination). Several methods have been published that favor the production of bispecific antibodies over the production of each monospecific antibody. This is typically achieved by modifying the constant regions of the heavy chains such that they favor heterodimerization (i.e., dimerization with the heavy chains of other heavy / light chain combinations) over homodimerization. In a preferred embodiment, the bispecific antibody of the invention comprises two different immunoglobulin heavy chains with compatible heterodimerization domains. Various compatible heterodimerization domains have been described in the art. The compatible heterodimerization domains are preferably compatible immunoglobulin heavy chain CH3 heterodimerization domains. Various methods have been described in the art by which such heterodimerization of heavy chains can be achieved.

[0133] One preferred method for producing CLEC12A / CD3 bispecific antibodies is disclosed in US Pat. Nos. 9,248,181 and 9,358,286. Specifically, the preferred mutations for producing essentially only bispecific full-length IgG molecules are the amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain ("KK-mutant" heavy chain) and L351D and L368E in the second domain ("DE-mutant" heavy chain), or vice versa. As mentioned above, the DE-mutant and the KK-mutant preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE-mutant heavy chains (DEDE homodimers) or KK-mutant heavy chains (KKKK homodimers) rarely occurs due to the strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0134] Thus, in one embodiment, the heavy / light chain combination comprising a variable domain that binds CLEC12A comprises a DE mutant of the heavy chain, in this embodiment, the heavy / light chain combination comprising a variable domain that binds CD3 comprises a KK mutant of the heavy chain.

[0135] Candidate CLEC12A / CD3IgG bispecific antibodies can be tested for binding using any suitable assay. For example, binding to membrane-expressed CD3 on HPB-ALL cells can be assessed by flow cytometry (following the FACS procedure previously described in WO 2014 / 051433). In one embodiment, binding of candidate CLEC12A / CD3 bispecific antibodies to CD3 on HPB ALL cells is demonstrated by flow cytometry performed according to standard procedures known in the art. Binding to cell-expressed CD3 is confirmed using CHO cells transfected with CD3δ / ε or CD3γ / ε. Binding of candidate bispecific IgG1 to CLEC12A is determined using CHO cells transfected with a CLEC12A expression construct. CD3 and CLEC12A monospecific antibodies as well as an irrelevant IgG1 isotype control mAb are included in the assay as controls (e.g., an antibody that binds CD3 and another antigen such as tetanus toxoid (TT)).

[0136] The affinity of CD3 and CLEC12AFab of candidate CLEC12A / CD3 bispecific antibodies to the target can be measured by surface plasmon resonance (SPR) technique using BIAcore T100. Briefly, anti-human IgG mouse monoclonal antibody (Becton and Dickinson, cat.Nr.555784) is coupled to the surface of a CM5 sensor chip using free amine chemistry (NHS / EDC). The bsAb is then captured on the sensor surface. Recombinant purified antigen human CLEC12A (Sino Biological Inc, cat.Nr.11896-H07H) and human CD3δε-Fc protein are then flowed over the sensor surface in a range of concentrations to measure the on / off rates. After each cycle, the sensor surface is regenerated by a pulse of HCl and the bsAb is captured again. From the resulting sensorgrams, on / off rates and affinity values ​​for binding to human CD3 and CLEC12A are measured using BIAevaluation software as previously described (e.g., US 2016 / 0368988).

[0137] The T cell stimulatory capacity of CLEC12A / CD3 bispecific antibodies can be measured in an assay using resting healthy donor T cells obtained according to the procedures described in WO 2014 / 051433 and US 2016 / 0368988. For example, candidate CLEC12A / CD3 bispecific antibodies are tested on purified resting healthy donor T cells incubated with cells of the leukemia-derived HL60 cell line in 10% fetal bovine serum (FBS) or 10% normal human serum (HS) at an effector:target cell ratio of 10:1 or 5:1 for 2 days. Results are expressed as the percentage of CD69- or CD25-positive cells within the CD4- or CD8-positive T cell population.

[0138] The ability of CLEC12A / CD3 bispecific antibodies to induce target cell lysis can be tested in an assay using leukemic cells (such as HL-60 cells) labeled with carboxyfluorescein diacetate succinidyl ester (CFSE) and co-cultured with T cells from healthy donors according to the procedures described in WO 2014 / 051433 and US 2016 / 0368988. Surviving CFSE-positive cells are quantified by flow cytometry and results are expressed as a percentage of specific lysis relative to PBS control conditions.

[0139] In one aspect, a pharmaceutical composition is provided that includes a CLEC12A / CD3 bispecific antibody and a pharma- ceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved by a government regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, particularly humans, and includes any and all physiologically compatible solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, glycerol polyethylene glycol ricinoleate, and the like. Water or saline solutions, as well as aqueous dextrose and glycerol solutions, can be used as carriers, particularly for injectable solutions. Liquid compositions for parenteral administration can be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravesical, intratumoral, intravenous, intraperitoneal, intramuscular, intrathecal, and subcutaneous. Depending on the route of administration (e.g., intravenous, subcutaneous, intraarticular, etc.), the active compound can be coated with a material to protect it from the action of acids and other natural conditions that may inactivate the compound.

[0140] Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, for example, in a liquid carrier, or suitable for reconstitution into a liquid solution or suspension for intravenous administration. The compositions can be formulated in dosage unit form for ease of administration and uniformity of dosage.

[0141] Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions, and emulsions.

[0142] The compositions and methods provided herein are particularly useful for activating a T cell or T cells in a patient. The patient is suffering from or at risk of suffering from cancer. The latter is a patient who is in remission and at risk of cancer recurrence, relapse, or metastasis. Thus, the compositions and methods can be used to treat various myeloid malignancies, such as myeloid leukemia and pre-leukemia diseases derived from bone marrow. Thus, diseases that can be treated according to the methods provided herein include myeloid leukemia (e.g., AML and CML), or pre-leukemia diseases, such as myelodysplastic syndrome (MDS) (and progression to AML), myelofibrosis (MF), or myeloproliferative neoplasms blast stage (MPN-BP).

[0143] As used herein, the CLEC12A / CD3 bispecific antibody, which is a bispecific antibody, can be combined with another therapeutic molecule. Such combined administration (co-administration) includes simultaneous, separate or sequential administration of the CLEC12A / CD3 bispecific antibody and the IL-15 moiety in the same or different dosage forms. Thus, in some embodiments, the CLEC12A / CD3 bispecific antibody can be used in a method for activating T cells in a subject, where the CLEC12A / CD3 bispecific antibody is administered simultaneously, separately or sequentially with the IL-15 moiety. In other embodiments, the CLEC12A / CD3 bispecific antibody can be used to treat cancer in a subject, where the CLEC12A / CD3 bispecific antibody can be administered simultaneously, separately or sequentially with the IL-15 moiety.

[0144] In other embodiments, the CLEC12A / CD3 bispecific antibody may be used in the manufacture of a medicament for activating T cells in a subject, where the CLEC12A / CD3 bispecific antibody is administered simultaneously, separately or sequentially with the IL-15 moiety. A product comprising the CLEC12A / CD3 bispecific antibody and the IL-15 moiety may be a combined preparation for simultaneous, separate or sequential use in activating T cells in a subject. In other embodiments, the CLEC12A / CD3 bispecific antibody may be used in the manufacture of a medicament for treating cancer in a subject, where the CLEC12A / CD3 bispecific antibody may be administered simultaneously, separately or sequentially with the IL-15 moiety. A product comprising the CLEC12A / CD3 bispecific antibody and the IL-15 moiety may be a combined preparation for simultaneous, separate or sequential use in treating cancer in a subject.

[0145] The IL-15 moiety can be administered according to a suitable dosage and route (eg, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously).

[0146] The CLEC12A / CD3 bispecific antibody and the IL-15 moiety can be administered simultaneously in a single formulation. Alternatively, the CLEC12A / CD3 bispecific antibody and the IL-15 moiety can be formulated for separate administration, administered simultaneously or sequentially.

[0147] In some embodiments, the IL-15 portion and the CLEC12A / CD3 bispecific antibody are administered simultaneously.

[0148] In one embodiment, a subject is administered a single dose of the IL-15 moiety and a single dose of the CLEC12A / CD3 bispecific antibody. In some embodiments, the CLEC12A / CD3 bispecific antibody and the IL-15 moiety are administered repeatedly over the course of treatment. For example, in certain embodiments, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses of the IL-15 moiety and multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses of the CLEC12A / CD3 bispecific antibody are administered to a subject in need of treatment.

[0149] In some embodiments, administration of the IL-15 moiety and the CLEC12A / CD3 bispecific antibody may occur once a week, and in that regimen may be administered on the same day (e.g., simultaneously) or sequentially (e.g., one or more minutes, hours, or days before or after). When administered separately, the CLEC12A / CD3 bispecific antibody and the IL-15 moiety may, but do not necessarily, follow the same administration (i.e., dosage) protocol. A therapeutically effective amount of the IL-15 moiety may be administered more or less frequently than the CLEC12A / CD3 bispecific antibody. In certain embodiments, doses of the IL-15 moiety and the CLEC12A / CD3 bispecific antibody may be administered on the same day, or the IL-15 moiety may be administered one or more days before or after the CLEC12A / CD3 antibody.

[0150] For convenience, the total daily dosage may be divided and administered in portions during the day as required. Intermittent treatment (e.g., 1 week out of 3 or 3 weeks out of 4) may also be employed.

[0151] In some embodiments, the IL-15 moiety used in the methods provided herein is recombinant human IL-15 (rhIL-15). In certain embodiments, rhIL-15 is administered at a dose of about 0.125 μg / kg / day to 2.0 μg / kg / day. In certain embodiments, rhIL-15 is administered at 0.125, 0.25, 0.5, 1.0, or 2.0 μg / kg / day. In certain embodiments, rhIL-15 is administered by intravenous bolus administration. In other embodiments, rhIL-15 is administered by continuous intravenous infusion (CIV). In certain embodiments, rhIL-15 is administered by CIV for 2-10 days, 5-10 days, or 7-10 days. In one embodiment, rhIL-15 is administered by CIV for 10 days. In a related embodiment, rhIL-15 is administered in treatment cycles of about 30-60 days, where rhIL-15 is administered by CIV for the first 5-10 days of each cycle.

[0152] In some embodiments, the IL-15 moiety administered according to the methods provided herein is a soluble IL-15 receptor or receptor fragment (sIL-15Ra). In other embodiments, the IL-15 moiety is a complex comprising IL-15 and sIL-15Ra, e.g., as described in U.S. Pat. Nos. 9,255,141 and 9,328,159. In certain embodiments, the IL-15 / IL-15Ra complex is administered to the subject at a dose of about 0.1 μg / kg to about 20 μg / kg, about 10 μg / kg to about 20 μg / kg, about 20 μg / kg to about 40 μg / kg, or about 25 μg / kg to 50 μg / kg.

[0153] In certain embodiments, the IL-15 moiety is IL-15 / IL-15Ra administered subcutaneously. In some embodiments, the IL-15 / IL-15Ra complex is administered once a day, once every two days, once every three, four, five, six, or seven days. In certain embodiments, the IL-15 / IL-15Ra is administered for one, two, three, four, five, six, or seven days per week. In certain embodiments, the dose for the first cycle and each subsequent cycle is between 0.1 μg / kg and 1 μg / kg, between 1 μg / kg and 5 μg / kg, or between 5 μg / kg and 10 μg / kg. In another embodiment, the first dose of the first cycle and each subsequent cycle is 0.1 μg / kg to 0.5 μg / kg, 1 μg / kg to 2 μg / kg, 1 μg / kg to 3 μg / kg, 2 μg / kg to 5 μg / kg, or 2 μg / kg to 4 μg / kg. In another embodiment, the dose for the first cycle and each subsequent cycle is 0.1 μg / kg, 0.25 μg / kg, 0.5 μg / kg, 1 μg / kg, 1.25 μg / kg, 1.5 μg / kg, 1.75 μg / kg, 2 μg / kg, 2.25 μg / kg, 2.5 μg / kg, 2.75 μg / kg, 3 μg / kg, 3.25 μg / kg, 3.5 μg / kg, 4 μg / kg, 4.25 μg / kg, 4.5 μg / kg, 4.75 μg / kg, or 5 μg / kg. In one embodiment, the IL-15 / IL-15Ra complex is administered according to a 28 day cycle in which the IL-15 / IL-15Ra complex is administered subcutaneously three times a week for two consecutive weeks.

[0154] In yet other embodiments, the IL-15 moiety used in the methods provided herein is a long-acting IL-15, e.g., a conjugate of IL-15 and a water-soluble polymer, e.g., as described in WO2015815373. One of skill in the art can determine the amount of a long-acting IL-15 agonist sufficient to provide clinically relevant agonist activity at the IL-15 receptor ("IL-15R"). In some embodiments, the IL-15 polymer conjugate is administered at a dose of about 0.001 mg / kg to about 10 mg / kg, preferably about 0.001 to about 5 mg / kg. In certain embodiments, the IL-15 polymer conjugate is administered at a dose of about 0.03 mg / kg to about 3 mg / kg. In certain embodiments, the IL-15 polymer conjugate is administered at a dose of about 0.03 mg / kg, about 0.1 mg / kg, about 0.3 mg / kg, or about 3.0 mg / kg. In other embodiments, the IL-15 polymer conjugate is administered at a dose of about 0.0025 mg / kg, about 0.008 mg / kg, about 0.01 mg / kg, about 0.025 mg / kg, 0.05 mg / kg, or about 0.25 mg / kg.

[0155] The therapeutic method described herein is typically continued as long as the clinician supervising the patient's care considers the therapeutic method to be effective, i.e., the patient is responding to the treatment.Non-limiting parameters that indicate that the therapeutic method is effective can include one or more of the following: reduction in tumor cells; inhibition of tumor cell proliferation; elimination of tumor cells; progression-free survival; appropriate response by suitable tumor markers (if applicable); increase in the number of NK (natural killer) cells; increase in the number of CLEC12A-specific T cells; and increase in the number of CLEC12A-specific memory T cells.

[0156] Regarding the frequency of administration of the CLEC12A / CD3 bispecific antibody, a person skilled in the art will be able to determine the appropriate frequency. For example, a clinician may decide to administer the CLEC12A / CD3 bispecific antibody relatively infrequently (e.g., once every two weeks) and gradually reduce the administration period as tolerated by the patient. If an IL-15 moiety is administered, the frequency of these agents can be determined in a similar manner. Exemplary lengths of time associated with a course of treatment according to the claimed methods include about 1 week, 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 30 months, about 3 years, about 4 years, about 5 years, or indefinitely (e.g., continued maintenance therapy). The aforementioned time periods may relate to one or more treatment rounds / cycles.

[0157] The efficacy of the therapeutic methods provided herein can be evaluated using any suitable means. In one embodiment, the clinical efficacy of the combination treatment is analyzed using AML blast reduction in bone marrow as an objective response criterion. A patient, e.g., a human, treated according to the methods disclosed herein preferably experiences an improvement in at least one symptom of cancer. In some embodiments, one or more of the following may occur: the number of cancer cells may be reduced; recurrence of the cancer may be prevented or delayed; one or more symptoms associated with the cancer may be alleviated to some extent. Furthermore, an in vitro assay for determining T cell-mediated target cell lysis (as described in WO2017 / 010874) may be performed using AML tumor blasts isolated from a subject.

[0158] In some embodiments, tumor cells are no longer detectable after treatment as described herein. In some embodiments, the subject is in partial or complete remission. In certain embodiments, the subject has increased overall survival, median survival, and / or progression-free survival.

[0159] The treatments of the invention (e.g., CLEC12A / CD3 bispecific antibodies) or combination treatments with IL-15 moieties may also be used in conjunction with other well-known therapies selected for their particular usefulness against the cancer being treated. Alternatively, the combinations of the invention may be used sequentially with known pharmacologic acceptable agents, where appropriate.

[0160] Methods for the safe and effective administration of chemotherapeutic agents are known to those skilled in the art. Moreover, their administration is described in standard texts. For example, the administration of many chemotherapeutic agents is described in the Physicians' Desk Reference (PDR), e.g., the 1996 edition (Medical Economics Company, Montvale, NJ 07645-1742, USA), the disclosure of which is incorporated herein by reference.

[0161] It will be apparent to one skilled in the art that the administration of chemotherapeutic agents and / or radiation therapy may vary depending on the disease being treated and the known effects of chemotherapeutic agents and / or radiation therapy on that disease, and treatment protocols (e.g., dosage and time of administration) may be altered in accordance with the knowledge of the skilled artisan, taking into account the observed effects of the administered therapeutic agent on the patient, and taking into account the observed response of the disease to the administered therapeutic agent.

[0162] All documents and references mentioned herein, including Genbank entries, patents and published patent applications, and websites, are expressly incorporated by reference herein to the same extent as if fully or partially set forth herein.

[0163] The present invention will now be described by reference to the following examples, which are illustrative only and are not intended to limit the invention. Although the present invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

[0164] Although features are described herein as part of the same or separate embodiments for clarity and conciseness of description, it will be understood that the scope of the invention may include embodiments having all or a partial combination of the described features. EXAMPLES

[0165] Example 1 Materials and Methods Flow cytometry Flow cytometry was performed using a Coulter FC500, Cyan-ADP, Gallios or NAVIOS™ flow cytometer (Beckman Coulter) or a FACS CantoA (BD Biosciences). Data were analyzed using Flowjo software (TreeStar Inc) or Kaluza software® (Beckman Coulter). Data were analyzed based on CD45 / SSC and CD3 (for T cells), SSC ± CD45DIM and / or CD45 (for lymphocytes) excluding lymphocytes. POS The cells were defined as blast cells.

[0166] 1. The specific binding pattern of antibody MF4327xMF5196 versus isotype control IgG on cell lines was assessed with unconjugated antibody visualized with anti-human IgG-PE antibody.

[0167] 2. The specific binding pattern of antibody MF4327xMF5196 bispecific antibody versus isotype control IgG in peripheral blood samples from healthy donors was tested using Oregon Green conjugated IgG batches in combination with a) CD1c-PE, CD19-ECD, CD3-PECY5, CD56-PECY7 (Beckman Coulter), CD8-AF700, CD4-Pacific blue, CD45-Krome orange; or b) CD303-PE, CD304-PE, CD14-ECD, CD33-PECY5, CD16-PECY7, and CD45-Krome orange.

[0168] 3. CD34 in bone marrow samples from healthy donors POS The specific binding pattern of antibody MF4327xMF5196 bispecific antibody versus isotype control IgG in fractions was tested using PE-conjugated IgG batches in combination with the series (CD3, CD14, CD16, CD19, CD20, CD56)-FITC, CD38-ECD, 7AAD, CD34-PECY7, CD10-APC, CD45RA-AF700, CD135a-BV421, CD90-BV510, and CLEC12A-PE (R&D Systems).

[0169] 4. CD25-PE, 7AAD, CD3-PECY7, CD69-APC, CD8-AF700, and CD4-BV421 were used to determine T cell activation in a cytotoxicity assay using HL60 cells.

[0170] 5. Monocyte cytotoxicity was quantified using CD14-FITC, CD16-PE, CD19-ECD, 7AAD, CD3-PECY7, CD69-APC, and CD8-AF700.

[0171] 6. To quantify T cell proliferation, T cells were visualized using 7AAD, CD3-PECY7, CD8-AF700, and CD4-BV421.

[0172] 7. CD34 using a) CLEC12A-PE (Biolegend), 7AAD, CD34-PECY7, CD10-APC-AF750, and CD5-BV421; and b) CD90-FITC, CLEC12A-PE (Biolegend) / IgG2a-PE, CD38-ECD, 7AAD, CD34-PECY7, CD10-APC, CD45RA-AF700, CD123-BV421, or CD135a-BV421, and lineage (CD3, CD14, CD16, CD19, CD20, CD56)-BV510. POS Cytotoxicity of bone marrow progenitor cells was quantified.

[0173] 8. CLEC12A expression on AML blasts used in cytotoxicity assays with AML blasts and purified T cells was quantified using a) CLEC12A-PE (R&D Systems), CD14-ECD, CD33-PECY5, CD38-PECY7, CD117-APC, CD19-AF700, CD34-BV421, and CD45-krome orange; or b) CLEC12A-PE (R&D Systems), CD19-ECD, CD3-PECY5, CD56-PECY7 (Biolegend), CD8-AF700, CD4-BV421, and CD45-krome orange.

[0174] 9. The efficacy of antibody MF4327xMF5196 in cytotoxicity assays using AML blasts and purified T cells was quantified using a) CD197-AF488, CD4-PE, CD45RO-ECD, 7AAD, CD3-PECY7, CD8-AF700, and CD45RA-Pacific blue; and b) CD25-PE, CD4-ECD, 7AAD, CD3-PECY7, CD117-APC, CD8-AF700, CD34-BV421, and CD45-krome orange.

[0175] 10. CLEC12A expression on AML blasts was determined using a) (1,000 ng / mL) CD14-FITC, CLEC12A-PE (R&D Systems) / IgG2b-PE, CD34-ECD, 7AAD, CD33-PECY7, CD8-AF700, CD4-BV421, and CD45-krome orange; or b) (200 ng / mL) CD14-FITC, CLEC12A-PE (R&D Systems), CD34-ECD, CD4-PECY5.5, CD117-APC, CD8-AF700, CD33-APC AF750, and CD45-krome orange.

[0176] 11. The efficacy of antibody MF4327xMF5196 in primary AML blast samples was evaluated using a) (1,000ng / mL) CD14-FITC, CD34-ECD, 7AAD, CD33-PECY7, CD3-APC, CD8-AF700, CD4-BV421, and CD45-krome orange; or b) (200ng / mL) CD3-FITC, 7AAD, CD4-PECY7, CD8-AF647, CD45RA-AF700, CD33-APC AF750, and CD45-krome orange.

[0177] bispecific antibody The antibody MF4327xMF5196 used in the examples and the figures referred to in the examples corresponds to IgVk1-39 in SEQ ID NO: 56 in the sequence listing. *MF4327XMF5196 is a full-length human IgG1 bispecific antibody specific for CLEC12A and CD3 with a common light chain variable region of MF4327XMF5196. It has the heavy chain variable region of MF4327 and the heavy chain variable region of MF5196 as outlined in the sequence listing. The constant parts of the antibody may or may not have effector function silenced as indicated. The negative control antibodies were a cLC monospecific antibody against tetanus toxin (TTxTTIgG, isotype control (de Kruif, Kramer et al. 2009)) and a cLC bispecific antibody against tetanus toxin and CD3 (MockxCD3 or TTxCD3IgG, using the same CD3Fab as in antibody MF4327XMF5196); the positive control antibody was a bivalent cLC monospecific antibody against CD3 (CD3xCD3, using the same CD3Fab as in antibody MF4327XMF5196). In the initial batches of bispecific antibodies, electrostatically engineered heavy chains were used for transient production, resulting in bispecific IgG1s with purity >95% (Gunasekaran, Pentony et al. 2010). Subsequent batches were generated using heavy chain Fc engineering in which unique mutations involving charged residues at the CH3 interface were introduced (De Nardis, Hendriks et al. 2017). Unless otherwise stated, the IgG batches of antibodies MF4327xMF5196, MockxCD3, and TTxTTIgG are deficient in Fc effector function upon introduction of changes in the CH2 region.

[0178] Affinity measurement Antibody MF4327xMF5196 was immobilized on a CM5 chip (GE Healthcare) via a mouse anti-human IgG monoclonal antibody (BD Biosciences). Recombinant purified CLEC12A-His (Sino Biologicals) or recombinant CD3δε-Fc protein was then flowed over the surface using a Biacore T100 instrument (GE Healthcare).

[0179] In vivo pharmacokinetic studies C57Bl / 6J mice were given a single intravenous dose of 1 mg / kg of antibody MF4327xMF5196. Antibody MF4327xMF5196 serum levels were quantified at various time points using an anti-human IgG ELISA (ZeptoMetrix).

[0180] cell line HL60 cells (DSMZ) were cultured in IMDM (GIBCO Invitrogen, 21980-065) containing 2 mM l-glutamine and 10% FBS (Integro). CHO-K1 (DSMZ), Jurkat E6.1 (LGC), and J.RT-T3.5 (LGC) cell lines were maintained in DMEM / F12 (GIBCO Invitrogen, 11320-033) supplemented with 2 mM l-glutamine and 10% FBS (ThermoFisher Scientific). CLEC12A-CHO-K1 cell lines were generated by stable integration of the pcDNA3.1+ vector (ThermoFisher Scientific) encoding human CLEC12A.

[0181] Clinical samples AML patient samples (Table III) and healthy donor bone marrow were obtained in accordance with the Declaration of Helsinki and RUMC institutional guidelines and regulations. For storage in liquid nitrogen, cells were resuspended in IMDM medium containing 7% dimethyl sulfoxide (DMSO; Sigma-Aldrich, W387509) and 10% fetal bovine serum (FBS, Integro). After thawing, cells were resuspended in IMDM medium containing 250 μg / mL DNAse (Roche, 11284932001) and 1.25 mM MgCl. 2 The cells were incubated for 10 minutes with 10% human serum (HS, PAA Laboratories) containing 10% HS. The cells were then washed with an excess amount of medium.

[0182] Cytotoxicity assay using purified T cells Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation from peripheral blood (PB) collected from healthy donors or AML patients (see Table III for patient characteristics). CD3 POS T cells were purified. Cytotoxicity assays were performed in IMDM supplemented with 10% AB+HS (Sanquin) in 96-well plates (Nunc™, ThermoFisher Scientific). 5 T cells were used. CFSE-labeled HL60 or primary AML blast target cells were used (ThermoFisher Scientific, C11-57). Prior to CFSE labeling, AML blasts were precultured overnight in assay medium supplemented with 100 ng / mL GM-CSF (Immunotools, 11343125), 100 ng / mL G-CSF (Amgen Inc, Neupogen®), 50 ng / mL IL-3 (Cellgenix), 25 ng / mL SCF (Immunotools, 11343325), and 20 ng / mL Flt3L (Immunotools, 11343305). T cells and target cells were then cocultured in assay medium supplemented with the same cytokines in the presence of antibody MF4327xMF5196 or control IgG. After 24-72 hours, T cell activation and remaining CFSE were assessed by flow cytometry. POS The number of target cells was quantified (see FIG. 7 for gating strategy). The percentage of specific lysis was calculated as follows: specific lysis (%)=100-[(100×number of target cells in IgG-containing condition) / (average number of target cells in IgG-free condition)].

[0183] PBMC-based monocyte cytotoxicity assay PBMCs (2 × 10 5) were incubated for 48 h. Prior to harvesting, the assay plates were centrifuged (500 g, 5 min) to collect the supernatant for cytokine quantification. After a subsequent 1 h incubation at 2–8 °C, cells were stained with assay medium without washing. Specific lysis of monocytes, B cells, and natural killer (NK) cells was quantified by flow cytometry in a fixed amount collected from each assay well (Figure 8).

[0184] T cell proliferation assay CFSE labeled CD3 POS T cells, CD14 POS MACS-purified (Miltenyi, 130-050-201) autologous monocytes were co-cultured in IMDM with 10% AB+HS with or without test IgG at an E:T ratio of 5:1. After 5 days, the fraction of proliferating T cells was identified by flow cytometry for CFSE. LOW T cells were visualized.

[0185] Cytokine analysis Levels of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, GM-CSF, TNF-α, and IFN-γ in the culture supernatants were measured using the Luminex® platform (Life Technologies, LHC0001).

[0186] Cytotoxicity assay using bone marrow cells from healthy donors CD3 POS T cells and CD34 POSCells were isolated from healthy donor bone marrow mononuclear cells (obtained from orthopedic patients or purchased from Lonza, 2M-125C) by positive selection using MACS (Miltenyi, 130-046-702 and 130-050-101, respectively). Preactivated T cells were generated by activating purified T cells for 6 days on plates precoated with anti-CD3 (1 μg / mL, BD Bioscience, 555329) and anti-CD28 (2 μg / mL, BD Bioscience, 555725) in IMDM supplemented with 10% HS A+ (Sanquin Blood Bank) and 30 IU / mL IL-2 (Chiron, Proleukin®), followed by overnight placement on plates with 30 IU / mL IL-2. The following day, preactivated T cells were cloned into autologous CD34 POS The cells were co-cultured at an E:T ratio of 10:1 in IMDM supplemented with 10% A+HS in 96-well plates (Corning Costar, 3799) in the presence of antibody MF4327xMF5196 or in control conditions. After 16 hours, the effect of antibody MF4327xMF5196 was assessed on various CD34 POS Progenitor cell populations and colony forming unit (CFU) assays were quantified by flow cytometry. Flow Count Fluorospheres (Beckman Coulter, 7547053) were used for flow cytometric quantification. See Figures 10 and 11 for gating strategies.

[0187] Colony forming unit (CFU) assay Upon washing according to the manufacturer's instructions, T cells and CD34 cells were cultured in MethoCult GF H84435 (Stemcell Technologies, 84435) for analysis of erythroid and monomyelocyte colony growth, and MegaCult™-C (complete kit supplemented with cytokines (04971) and human low density lipoprotein (40 μg / mL, 02698), both from Stemcell Technologies) for analysis of megakaryocyte colony formation. POSCo-cultures of cells were seeded. Both MethoCult and MegaCult™-C assays showed CD34 POS Based on cell number, co-cultures were inoculated at two different concentrations, with equal amounts of cells in the co-cultures treated with antibody MF4327xMF5196 and control IgG. In the MethoCult assay, 1 × 10 3 and 5 x 10 2 CD34 POS Cells were seeded in triplicate in 35 mm culture dishes (Corning, 430165) and 7.5 × 10 cells were seeded per chamber in a double-chamber culture slide and two slides per condition for the MegaCult-C assay. 3 and 2.5 x 10 3 CD34 POS Cells were seeded and the number of erythroid, monomyelocyte and megakaryocyte colonies were recorded after 13-15 days of culture, and MethoCult cultures were subsequently harvested and analyzed for monocytic and myeloid markers by flow cytometry.

[0188] Cytotoxicity assay using primary AML samples Primary AML patient samples (5 × 10 5 Cells) were cultured. After 24 hours, 20 ng / mL IL-15 (Miltenyi, 130-095-766) was added to the cultures. After 10 days, AML blast lysis and T cell proliferation were quantified by flow cytometry.

[0189] Antibody MF4327xMF5196 dosing regimen in human AML patients. The MF4327xMF5196 antibody will be studied as a single agent in a first-in-human study, using dose escalation in cohorts of 3-6 patients. Patients were administered the antibody in a dosing regimen designed to provide an effective and safe dose. The antibody was administered as a 2-4 hour infusion. Treatments were scheduled at time intervals. The dosing regimen was designed to prevent and minimize the severity and incidence of cytokine release syndrome (CRS).

[0190] The treatment regimen has components that are applied in the first cycle (first 28 days): a priming dose (C1D1); step-up and fixed total doses (C1D4, C1D8, C1D15), as well as the use of reserve medication. Priming dose: the first dose (C1D1) is administered at a low dose (1 mg, 3 mg, 5 mg). Step-up dose: subsequent doses after C1D1 are administered at increasing doses until the planned full dose is administered. Step-up doses include more frequent administration during the first week. In the first week, treatment is administered once every 3 days (D1-D4-D8), after which treatment is administered once every 7 days (once a week). Fixed total dose: the full dose is administered on the 15th day, from which point treatment is administered at a fixed total dose. Pre-medication: those administered prior to each MF4327xMF5196 antibody dose include acetaminophen and an antihistamine (anti-H1).

[0191] After the first cycle, treatment continues with the full dose administered in a once-weekly regimen (days 1-8-15-22). Infusions are administered over 2-4 hours, always with premedication.

[0192] In one cohort, patients received a priming dose of 1 mg on day 1, followed by step-up doses of 3 mg on day 4 and 15 mg on day 8, and a total dose of 25 mg on day 15.

[0193] In another cohort, patients received a priming dose of 3 mg on day 1, followed by step-up doses of 10 mg on day 4 and 25 mg on day 8, and a total dose of 40 mg on day 15.

[0194] In a further cohort, patients received a priming dose of 5 mg on day 1, followed by step-up doses of 15 mg on day 4 and 25 mg on day 8, and a total dose of 60 mg on day 15.

[0195] Patients' responses to treatment were tracked, including analysis of cytokine levels before each dose and 4 and 24 hours after the end of the infusion.

[0196] result Antibodies MF4327xMF5196 inhibit CLEC12A in the normal hematopoietic compartment POS and CD3 POS Binds specifically to cells. The antibody MF4327xMF5196 is a T cell-targeting bispecific antibody developed to treat CLEC12a-positive cancer cells, such as those present in AML. It binds to CD3 on T cells and to CLEC12A on AML blasts and LSCs. The antibody MF4327xMF5196 is a human full-length IgG1 bispecific antibody based on a common light chain (cLC) bispecific IgG format. Flow cytometry using tumor cell lines showed that the antibody MF4327xMF5196 specifically binds to CLEC12A and CD3 (Figure 1A). It binds to CLEC12A via its CLEC12A arm. POS It bound to HL60 cells, as well as CHO-K1 cells transfected with human CLEC12A, but not to parental CHO-K1 cells. It binds to CD3 through its CD3 arm. POSJurkatE6.1 cells but not CD3NEGJurkat J.RT-T3.5 cells. The affinity of antibody MF4327xMF5196 for its target proteins was 3 nM for CLEC12A and 177 nM for CD3, as determined by surface plasmon resonance techniques using recombinant proteins. To investigate whether the engineered Fc region of antibody MF4327xMF5196 has a potential effect on pharmacokinetics (PK), the in vivo half-life of antibody MF4327xMF5196 was measured in C57BL / 6J mice and found to be 226 hours (9.4 days). This half-life is within the range of in vivo half-lives typically observed for human IgG1 antibodies in rodents, indicating that the specific design of the human cLC bispecific antibody does not affect nonspecific clearance pathways of IgG. Analysis of the binding profile of antibody MF4327xMF5196 in normal peripheral blood demonstrated that antibody MF4327xMF5196 inhibits CD3-expressing CD4 POS and CD8 POS T cells and CD3 POS CD56 POS We showed that MF4327xMF5196 bound to NK T (NKT) cells (Figure 1B). In accordance with the previously reported expression profile of CLEC12A (Bakker, van den Oudenrijn et al. 2004; Marshall, Willment et al. 2004; Lahoud, Proietto et al. 2009), antibody MF4327xMF5196 also bound to monocytes, myeloid dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), and granulocytes, but not to natural killer (NK) cells or B cells (Figure 1B and data not shown). In normal bone marrow, CD34 POSThe binding profile of antibody MF4327xMF5196 within the progenitor compartment (Figure 1C) also resembled the reported CLEC12A expression profile (van Rhenen, van Dongen et al. 2007). Antibody MF4327xMF5196 bound homogeneously to granulocyte-macrophage progenitor (GMP) cells, whereas it bound only to a small proportion of common myeloid progenitor (CMP) and megakaryocyte-erythroid progenitor (MEP) subsets. Notably, antibody MF4327xMF5196 binding was restricted to CD34 POS CD38 NEG They are not observed in the compartment that contains multipotent progenitor cells (MPPs) and multipotent hematopoietic stem cells (HSCs).

[0197] Antibody MF4327xMF5196 inhibits CLEC12A-specific T cell activation and CLEC12A POS Induces lysis of HL60 cells. Next, in a cytotoxicity assay, the CLEC12AxCD3 bispecific IgG antibody inhibited CLEC12A antigen-specific T cell activation and CLEC12A POS The ability to induce lysis of target cells was measured. Effector cells were resting T cells from healthy donors and target cells were CLEC12A POSThe target cells were HL60 cells. In this assay, the MF4327xMF5196 bispecific antibody with a wild-type (WT) Fc region (intact Fc effector function) was compared to a MockxCD3 control IgG formatted with a WT Fc region. The MF4327xMF5196 bispecific antibody induced upregulation of CD69 (Figure 2A) and CD25 (data not shown) on CD4 and CD8 T cells after 24 and 48 hours. T cell-mediated lysis of HL60 cells was already evident after 24 hours, and more than 95% cytotoxicity of HL60 cells was measured after 48 hours (Figure 2B). Lysis of target cells correlated with the effector-to-target ratio (E:T ratio), with maximal lysis seen at E:T ratios of 5:1 or higher (Figure 9). Notably, CLEC12AxCD3-induced lytic activity was CLEC12A antigen-mediated (Figure 2A,B); only at later time points was some T cell activation and HL60 target cell lysis observed for the MockxCD3 control. Nevertheless, this revealed that the bispecific IgG format with WTFc can induce Fcγ receptor-mediated T cell activation without the need for CLEC12A binding (Figure 2A and B, 72 hours).

[0198] Antibody MF4327xMF5196 with silenced Fc effector function inhibits CLEC12A POS Induces specific lysis of AML cell lines. To prevent the bispecific antibody MF4327xMF5196 from inducing non-specific Fcγ receptor-mediated cell lysis, a bispecific IgG format lacking Fc effector function was generated by introducing changes in the CH2 region of both IgG1 heavy chains. Various means of reducing Fc effector function are known in the art. Binding to CD16, CD32, and C1q, as well as binding to CD64 reduced by more than 200-fold, was achieved through Fc engineering modifying residues in the CH2 domain. Normal resting T cells and CLEC12A POSIn a cytotoxicity assay using HL60 cells, antibody MF4327xMF5196 induced upregulation of CD69 (Figure 2C) and CD25 (Figure 2D) in CD4 and CD8 T cells. The most sensitive readout of antibody MF4327xMF5196-induced activity was a mean half-maximal effective concentration (EC) of 44 ng / mL. 50 ) was upregulation of CD69 on CD8 T cells with IgG1 (Table I). More importantly, antibody MF4327xMF5196 induced >80% lysis at concentrations of 100 ng / mL or higher, with a mean EC 50 MF4327xMF5196 induced T cell-mediated lysis of HL60 cells bearing MockxCD3 antibody (Figure 2E, Table I). Whereas activation of CD4 and CD8 T cells by MockxCD3 antibody with wild-type Fc effector function was already seen on day 2 (Figure 2A, 1000 ng / mL), no T cell activation was observed with Fcγ receptor-silencing MockxCD3 antibody after 48 hours, even at 8000 ng / mL (Figure 2C-D). Taken together, this indicates that the T cell cytotoxicity induced by the Fcγ receptor-silencing antibody MF4327xMF5196 (Figure 2C-E) is CLEC12A antigen-specific. Taken together, these data indicate that the antibody MF4327xMF5196 with reduced Fc effector function is able to mediate CLEC12A-specific T cell activation and effectively redirect these T cells to target CLEC12A. POS The ability to induce lysis of HL60 cells is demonstrated.

[0199] Antibody MF4327xMF5196 inhibits autologous T cell-mediated CLEC12A POS Induces specific lysis of monocytes. Antibody MF4327xMF5196 inhibits CLEC12A by resting autologous T cells POSThe ability to induce redirected lysis of monocytes was determined in PBMC cultures. Antibody MF4327xMF5196 induced activation of CD4 and CD8 T cells (Figure 3A) and lysis of monocytes (from 100 ng / mL, Figure 3B). Importantly, antibody MF4327xMF5196-induced activity was CLEC12A-specific, since the MockxCD3 control did not induce observable T cell activation or monocyte lysis at concentrations up to 10,000 ng / mL (Figures 3A-B). Furthermore, the lytic activity of cytotoxic T cells activated with antibody MF4327xMF5196 was selective for CLEC12A antigen-expressing cells, since it did not affect B cell and NK cell fractions, even at the highest antibody MF4327xMF5196 concentrations tested (Figure 3C, data not shown).

[0200] These data suggest that antibody MF4327xMF5196 inhibits CLEC12A through the redirection and activation of autologous T cells. POS We demonstrate that we can efficiently induce CLEC12A-restricted redirected lysis of monocytes.

[0201] CLEC12A-specific induction of cytokine release and T cell proliferation. The ability of antibody MF4327xMF5196 to induce T cell proliferation was then tested in co-incubations with monocytes and autologous resting T cells. Antibody MF4327xMF5196 efficiently induced CLEC12A-specific proliferation of both CD4 and CD8 T cells at 100 and 1,000 ng / mL (Figure 3D). After 5 days of co-culture, the MockxCD3 control did not induce any T cell proliferation, once again proving that the function of antibody MF4327xMF5196 is CLEC12A-specific. Cytokine levels were then measured in these PBMC cultures after 48 hours of exposure to antibody MF4327xMF5196 at 1,000 ng / mL. Antibody MF4327xMF5196 induced the release of IL-1β, TNF-α, IFN-γ, IL-6, and IL-10 in the range of 10-200 pg / mL, and IL-8 in the range of 10,000-30,000 pg / mL (Figure 3E). In general, antibody MF4327xMF5196-induced cytokine levels were lower than those obtained after stimulation with anti-CD3 bivalent monospecific antibody. Antibody MF4327xMF5196-induced cytokine release was CLEC12A specific, since cytokine release in the presence of MockxCD3 control was absent or limited. These data indicate that antibody MF4327xMF5196 inhibits the release of CLEC12A POS We demonstrate that autologous T cells can be induced to proliferate and release cytokines upon co-incubation with monocytes.

[0202] Antibody MF4327xMF5196-induced targeting of normal CD34+ cells preserves erythroid and megakaryocytic differentiation and retains the potential to develop monomyeloid lineages. CLEC12A is a marker for normal CD34 POS We aimed to evaluate the impact of the antibody MF4327xMF5196 on normal hematopoiesis, since it is expressed in the monomyeloid progenitor compartment in the hematopoietic compartment. To this end, we used bone marrow-derived CD34 POS Cytotoxicity assays using autologous T cells and preactivated autologous T cells were performed. Preactivated T cells were used to assess the effect of CD34 on the outcome of the assay. POSWe obtained a short cytotoxicity assay window of 16 hours, which minimizes the effect of in vitro differentiation of cells. POS CD34 POS Specifically lysed over 86% of cells and inhibited CLEC12A NEG CD10 POS or CD10 NEG The cell fraction was observed to be unaffected (Figure 4A). POS Detailed analysis of the progenitor cell fraction revealed that antibody MF4327xMF5196 targets GMPs expressing CLEC12A (3-fold reduction, FIG. 4B). Antibody MF4327xMF5196 had no significant effect on cell fractions partially expressing CLEC12A (CMPs and MEPs), nor on CLEC12A-negative HSCs, MPPs, and common lymphoid progenitors (CLPs). Interestingly, antibody MF4327xMF5196 downregulated CD123 POS Only a fraction of cells was lysed, which expressed CD123 POS CLEC12A NEG We show that a large subset of cells is unaffected by antibody MF4327xMF5196, the subset that contains CMP (Figure 4C).

[0203] Second, upon antibody MF4327xMF5196-induced redirected lysis, normal CD34 POS The ability of the cell fractions to give rise to monocytes, granulocytes, erythrocytes, and megakaryocytes was assessed at the functional level using CFU assays. POSThe growth of erythroid (BFU-E; burst forming unit-erythroid) or megakaryocytic (CFU-Mk; colony forming unit-megakaryocyte) colonies from hematopoietic progenitors was not affected by antibody MF4327xMF5196 (Figure 4D). Notably, even though antibody MF4327xMF5196-induced redirected lysis caused a significant reduction in GMPs, only a modest reduction of 24% in the growth of granulocyte-macrophage colonies (CFU-GM) was observed at the functional level. Interestingly, GM progenitors unaffected by antibody MF4327xMF5196 were CD14-positive, as measured by flow cytometric analysis of CFU-GM colonies. POS CD36 POS CD33 POS Monocytes and CD15 POS CD33 POS The cells retained the ability to develop both myeloid and myeloid cells (Figure 4E).

[0204] Taken together, these data indicate that antibody MF4327xMF5196 does not affect erythro-megakaryocyte development. POS CD34 POS While effectively eradicating progenitor cells, antibody MF4327xMF5196 treatment preserves the developmental potential of granulocytes and monocytes.

[0205] Antibodies MF4327xMF5196 induce redirected lysis of primary AML blasts by autologous T cells. Next, the antibody MF4327xMF5196 was used to induce T cells from AML patients to express CLEC12A POS The ability of T cells to lyse target cells was measured. The T cell compartment of AML patients in morphological clinical remission showed a normal composition of naive, central memory (CM), effector memory (EM), and effector memory cells with reacquired CD45RA (EMRA+) (Figure 12A, Figure 12B and Table III, patients 1-6). AML patient T cells expressed CLEC12A POS The antibody MF4327xMF5196-mediated T cell activation and CLEC12A were analyzed by analyzing the ability of target cells to mediate antibody MF4327xMF5196 cytotoxicity.POS We found no difference in the ability of T cells from AML patients to induce cell lysis compared with T cells from healthy donors (Figures 12C to 12E).

[0206] We measured the ability of antibody MF4327xMF5196 to induce CLEC12A-specific lysis of primary AML blasts by resting autologous T cells collected from the same AML patients at a later time point (Figure 5A, patients listed in Table III, patients 7-9). Primary AML blast samples obtained at the time of diagnosis contained a high percentage (>80%) of blasts expressing CLEC12A (Figure 5B). AML blasts were co-cultured for 48 hours with autologous resting T cells obtained during clinical remission at an E:T ratio of 5:1. Antibody MF4327xMF5196 induced potent CLEC12A-specific activation of CD4 and CD8 T cells and lysis of >70% of AML blasts (Figures 5C-E). These data indicate that antibody MF4327xMF5196 inhibits CLEC12A activation by autologous T cells. POS We have demonstrated that it can efficiently induce lysis of primary AML blasts.

[0207] Antibody MF4327xMF5196 redirects lysis of primary AML blasts in diagnostic samples with low E:T ratios. We next measured the ability of antibody MF4327xMF5196 to induce lysis of AML blasts by autologous T cells in primary AML samples collected at diagnosis. In the primary AML samples tested (listed in Table III, patients 10-19), the number of T cells was relatively low compared to the number of AML blasts, resulting in a low effective E:T ratio (1:3-1:97, Table II). FACS analysis revealed patient heterogeneity with respect to CLEC12A expression (Table II). Primary AML samples were cultured for 10 days in the presence of a cytokine cocktail to support the survival of AML blasts and T cells during the assay. After 10 days of culture, the median AML blast recovery in the absence of antibody MF4327xMF5196 was 41-1591%. Antibody MF4327xMF5196 and MockxCD3 control IgG were added once at the start of culture. T cell proliferation and AML blast lysis were quantified by flow cytometry on days 7 and 10. Antibody MF4327xMF5196 not only efficiently induced CLEC12A-mediated T cell proliferation (fold expansion range 6-157), but also induced AML blast lysis on days 7 and 10 (Figure 6). On day 10, antibody MF4327xMF5196 efficiently induced AML blast lysis in 10 / 11 patient samples (23-98%). MockxCD3 control antibody also reduced the number of tumor cells in some samples, but in those samples the level of T cell activation was much lower than that observed with antibody MF4327xMF5196 (median 48 vs. 2-fold T cell proliferation based on antibody MF4327xMF5196 vs. MockxCD3). Notably, antibody MF4327xMF5196 was active even in AML samples with very low effector-to-target ratios (Figure 6 and Table II). In the one AML sample (patient 16) where no blast lysis was observed, the level of T-cell proliferation was also only 15-fold compared to a median 48-fold T-cell proliferation observed in 10 antibody MF4327xMF5196-responsive samples.In conclusion, antibody MF4327xMF5196 efficiently induced CLEC12A-mediated lysis of AML blasts by T cells present in bone marrow samples from AML patients, even at very low E:T ratios, and also triggered vigorous T-cell proliferation.

[0208] The antibody MF4327xMF5196 dosing regimen in human AML patients leads to reduced cytokine levels. Patient cytokine levels were measured upon treatment with antibody MF437xMF5196.

[0209] FIG. 14 shows data from a patient who received a priming dose of 1 mg on day 1, followed by step-up doses of 3 mg on day 4 and 15 mg on day 8, and a total dose of 25 mg on day 15.

[0210] FIG. 15 shows data from a patient who received a priming dose of 3 mg on day 1, followed by step-up doses of 10 mg on day 4 and 25 mg on day 8, and a total dose of 40 mg on day 15.

[0211] FIG. 16 shows data obtained from a patient who received a priming dose of 5 mg on day 1, followed by step-up doses of 15 mg on day 4 and 25 mg on day 8, and a total dose of 60 mg on day 15.

[0212] Overall, the data indicate that in selected patients, administration of priming and step-dose doses mitigates cytokine release when the full dose is administered. In certain patients in various cohorts, limited cytokine release was observed throughout the priming, step-up, and full doses (data not shown).

[0213] Discussion The antibody MF4327xMF5196 mentioned in the examples is a full-length antibody. An Fc effector-silenced version of the CLEC12AxCD3 bispecific human IgG1 antibody has been shown to be able to treat all subtypes of AML by targeting CLEC12A on AML blasts and leukemia stem cells (LSCs). The antibody MF4327xMF5196 is a potent bispecific antibody that efficiently activates and redirects T cells to lyse CLEC12A-expressing cells such as AML cells. In 10 / 11 primary AML patient samples, this CLEC12AxCD3 T cell engager efficiently induced CLEC12A-mediated lysis of AML blasts by redirecting resident autologous T cells. In this ex vivo cytokine support system, AML samples were cultured in the presence of the antibody MF4327xMF5196 for 7-10 days. This assay confirms the efficacy of the antibody MF4327xMF5196 in two ways. That is, we demonstrated that antibody MF4327xMF5196 has the ability to induce proliferation of AML patient T cells in these samples, and show that those antibody MF4327xMF5196 redirected T cells can redirect cytolytic activity to AML blasts via cytolysis (shown in Table II). Antibody MF4327xMF5196 was able to induce significant T cell proliferation and blast lysis even in AML samples with low initial E:T ratios (1:45 to 1:97) or relatively low levels of CLEC12A expression. Comparison of antibody MF4327xMF5196 vs. MockxCD3 conditions in ex vivo assays revealed that antibody MF4327xMF5196-induced activity was CLEC12A antigen-mediated; vigorous T cell proliferation and AML blast lysis were observed with antibody MF4327xMF5196 compared to minimal activity with MockxCD3 in 9 / 10 primary AML samples. One non-responsive sample (patient 16), which lacked both AML blast lysis and vigorous antibody MF4327xMF5196-induced T-cell proliferation, likely had severe T-cell dysfunction.

[0214] Although healthy donor T cells were used in many initial experiments, cytotoxicity assays were also performed using AML patient-derived T cells to test the ability of antibody MF4327xMF5196 to redirect T cells in the disease setting. Certain defects have been reported for AML T cells, such as impaired immune synapse formation and reduced T cell costimulatory capacity (Wendelbo, Nesthus et al. 2004, Le Dieu, Taussig et al. 2009). Here, we demonstrate that antibody MF4327xMF5196 redirects healthy donor-derived T cells to upregulate CLEC12A. POS It has been shown that AML patient-derived T cells obtained in morphological clinical remission (n=6) can be redirected as efficiently as inducing CLEC12A-specific lysis of tumor cells. Moreover, in 10 / 11 AML samples, it was found that the antibody MF4327xMF5196 can effectively redirect autologous T cells in primary de novo AML samples to lyse AML blasts. Here, it is demonstrated that AML patient-derived T cells can be functionally activated by full-length IgG CD3-engaging bispecific IgG, which results in T cell activation and proliferation in a HSC-sparing manner and at a low E:T ratio. Such functional activation of autologous T cells via the antibody MF4327xMF5196 effectively expands the effector T cell compartment, thus promoting a more favorable E:T ratio for efficient eradication of the patient's AML blasts and LSCs.

[0215] In normal bone marrow, the binding of antibody MF4327xMF5196 is restricted to the myeloid compartment, as antibody MF4327xMF5196 is able to spare other areas of the bone marrow by specifically targeting CLEC12A. Here, it is shown that antibody MF4327xMF5196 binds homogeneously to GMP cells in the bone marrow, but only to a small fraction of the CMP and MEP subsets. More importantly, antibody MF4327xMF5196 binds to CD34 cells, including multipotent HSCs. POS CD38 NEGSimilar to the trace expression of CLEC12A in the CMP and MEP fractions, antibody MF4327xMF5196 has been shown not to affect erythroid or megakaryocytic lineage development in in vitro CFU assays. Furthermore, antibody MF4327xMF5196 inhibits CLEC12A in ex vivo cytotoxicity assays. POS Although the number of GMP cells was reduced, follow-up CFU assays showed that the remaining CD34 POS It was observed that bone marrow cells were able to give rise to both monocytic and myeloid lineages, which indicates that CD34+, including HSCs and MPPs, were not observably affected by antibody MF4327xMF5196 treatment. POS CLEC12A NEG It allows the outgrowth of monomyelocytes arising from progenitor cells (van Rhenen, van Dongen et al. 2007, Notta, Zandi et al. 2016, Bill, van Kooten Niekerk et al. 2018).

[0216] It is expected that hematological toxicity after administration of antibody MF4327xMF5196 to patients may be limited to neutropenia and a decrease in monocyte counts. The present results indicate that upon antibody MF4327xMF5196 redirected lysis, neutrophils and monocytes are able to differentiate into the remaining CD34 POS These results demonstrate that hematopoietic cells can repopulate the host from hematopoietic stem cells. The potential for hematopoietic cell repopulation following treatment with the antibody MF4327xMF5196 contrasts with the likely situation of therapeutic targeting CD33 or CD123 (Aigner, Feulner et al. 2013, Al-Hussaini, Rettig et al. 2016). Compared to CLEC12A, CD33 and CD123 are more abundant than normal CD34, including CMPs, GMPs, and multipotent HSCs. POS It is more widely expressed in progenitor cells (Taussig, Pearce et al. 2005). The present invention shows that targeting CLEC12A inhibits normal CD123, which is the target for CD123-directed therapy. POSIn vivo studies in humanized mice have shown that CD33 and CD123 targeting of chimeric antigen receptor (CAR)-transduced T cells is associated with a higher level of CD34 targeting than CD34. POS CD38 NEG It has been shown to induce lysis in the stem cell compartment (Pizzitola, Anjos-Afonso et al. 2014; Kenderian, Ruella et al. 2015). As a result, concerns about hematologic toxicity have been raised about therapies targeting CD33 and CD123: these therapies may induce thrombocytopenia, neutropenia, and anemia, in addition to the risk of HSC eradication, which prevents efficient resumption of the normal hematopoietic compartment.

[0217] The Fc region of antibody MF4327xMF5196 has been modified to prevent binding to FcγR and C1q without affecting binding to the FcRn receptor. Results from monocyte assays demonstrated that antibody MF4327xMF5196-induced T cell activation and associated cytokine release were restricted to CLEC12A-expressing cells. Fc silencing inhibits CD3 POS and / or CLEC12A POS The data suggest that T cells activated with the antibody MF4327xMF5196 suppress the typical antibody-dependent cellular cytotoxicity (ADCC) and C1q-mediated complement-dependent cytotoxicity (CDC) of CLEC12A. POS It has been shown to selectively lyse cells (eg, B cells and NK cells were unaffected in PBMC cultures).

[0218] Studies in mice confirmed that the normal FcRn-mediated in vivo half-life of the full-length IgG1 antibody MF4327xMF5196 is 9-10 days, which means that, unlike other T cell engager concepts, effective systemic levels of antibody MF4327xMF5196 in patients can be achieved by intravenous administration once a week.

[0219] Antibody MF4327xMF5196 selectively targets myeloblasts while sparing normal HSCs. Based on its potential to eradicate residual LSCs, antibody MF4327xMF5196 is being considered for the management of minimal residual disease (MRD) in AML.

[0220] Example 2 Study of CLEC12A-negative and CLEC12A-positive CD34+ stem and progenitor cells from patients with myeloproliferative neoplasms blast phase (MPN-BP).

[0221] Cells were harvested from the bone marrow of MPN-BP patients. CD34+ cells were selected based on forward scatter (FSC), side scatter (SSC), and expression of CD45, CD34, CD38, and CLEC12A. dim , CD34 + , CD38 -The cells were sorted into two fractions based on CLEC12A expression. Cells were plated on MethoCult™ H4435 Enriched (STEMCELL technologies) supplemented with SCF, IL-3, IL-6, EPO, G-CSF, and GM-CSF and incubated for 12-14 days. Colonies were picked and replated on MethoCult™ H4435 Enriched (STEMCELL technologies) supplemented with SCF, IL-3, IL-6, EPO, G-CSF, and GM-CSF and incubated for 12-14 days. After the first plating, the CLEC12A negative fraction had 20 large colonies (>40 cells) and 10 small colonies (<40 cells per colony) per 1500 plated cells. The CLEC12A positive fraction had no large colonies and 32 small colonies per 1500 plated cells. Colonies from plates of the CLEC12A negative fraction failed to generate secondary colonies in the replating experiment, with zero large or small colonies per 1500 inoculated cells. In contrast, replating of cells from the first plating of CLEC12A gave rise to 44 small colonies per 1500 cells, with approximately 80% of these being positive for JAK2 driver mutations (see FIG. 13).

[0222] summary CD34 from MPN-BP patients pos CD38-CLEC12A- cells can generate hematopoietic colonies (CFU-GM) only in primary culture, which are predominantly CFU-GM. In contrast, CD34-CD38- stem cells from MPN-PB patients expressing CLEC12A can generate blast colonies that can be serially re-plated and thus contain leukemia-initiating potential.

[0223] Differential CLEC12A expression can be used to identify, eradicate, and / or isolate leukemia-initiating cells (LICs) from patients with MPN-BP. The CD3-CLEC12A bispecific antibodies disclosed herein can be used to eradicate these LICs in MPN-PB and reduce the number of MPN blast-stage cells, thereby alleviating disease in MPN-BP patients.

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[0225] [Table 1-1] [Table 1-2] [Table 1-3]

[0226] [Table 2]

[0227] Antibody MF4327xMF5196EC 50 Values ​​were determined in the HL60 cytotoxicity assay and data are from six healthy donors.

[0228] [Table 3]

[0229] [Table 4-1] [Table 4-2]

[0230]

Table 5

Claims

[Claim 1] 1. A method of treating a subject with CLEC12A positive cancer, comprising treating the subject in need thereof with two or more administrations of a bispecific antibody that binds CD3 and CLEC12A, wherein in a first administration, a first amount of the bispecific antibody is administered, and in a subsequent administration, the amount of bispecific antibody is greater than the amount of bispecific antibody in the first administration.

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