JP2018511311A - Compositions and methods targeting CD99 in hematopoietic tumors and lymphomas - Google Patents

Abstract

(a) binding to the extracellular domain of CD99, (b) linking CD99 expressed on the surface of myeloid or lymphoid malignant cells, (c) capping / clustering CD99 expressed on the surface of myeloid or lymphoid malignant cells Promotes aggregation and (d) hematological disorders, in particular CD99 + acute myeloid, including one or more antibodies that induce apoptosis in CD99 + myeloid or lymphoid malignant cells linked to antibody and result in cytotoxicity Compositions and methods for treating hematopoietic tumors and lymphomas including leukemia (AML), myelodysplastic syndrome (MDS) and T cell neoplasms are provided. The disclosed methods are affected by hematopoietic tumors or lymphomas that are sensitive to treatment with anti-CD99 antibodies by detecting increased expression of CD99 in patient-derived tissue samples or myeloid or lymphoid malignant cells Methods for identifying patients and anti-CD99 antibodies alone or in combination with one or more additional components such as mobilizing agents, transmigration blocking agents and chemotherapeutic agents such as daunorubicin, idarubicin, cytarabine, 5-azacytidine and decitabine A method of treating a patient suffering from a hematopoietic tumor or lymphoma that exhibits increased CD99 gene and / or cell surface protein expression by administering a composition comprising it.

Description

The present disclosure relates generally to the treatment of hematopoietic tumors and lymphomas such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and T cell malignant neoplasms. More specifically, the present disclosure includes (i) anti-CD99 antibodies and one or more anti-CD99 antibodies for the treatment of hematopoietic tumors and lymphomas such as acute myeloid leukemia, myelodysplastic syndrome and T cell neoplasms. A composition comprising an antibody; (ii) a method of making and identifying an anti-CD99 antibody suitable for the treatment of hematopoietic tumors and lymphomas; (iii) a composition comprising an anti-CD99 antibody and / or one or more anti-CD99 antibodies. Methods for identifying patients with hematopoietic tumors and lymphomas, such as acute myeloid leukemia, myelodysplastic syndrome and / or T cell lymphoma, sensitive to the treatment used; (iv) CD99 ⁺ leukemia stem cells and / or hematopoietic stem cells, etc. the method for inducing apoptosis in acute myeloid leukemia and / or myelodysplastic syndrome and / or hematologic malignancies and lymphomas related to CD99 ⁺ cells, such as T cell lymphoma; and (v) CD99 ⁺ Sex myeloid leukemia, relates to a method of treating a CD99 ⁺ myelodysplastic syndrome and / or CD99 ⁺ T-cell lymphomas.

Description of prior art
CD99 is a 32 kDa transmembrane protein that regulates T cell maturation and transendothelial migration of leukocytes. CD99 is also used as a biomarker to help diagnose acute T-cell lymphoblastic lymphoma / leukemia (T-ALL), Ewing sarcoma and neuroendocrine tumors. However, it remains unclear whether CD99 is a viable therapeutic target in various hematopoietic tumors and lymphomas.
T cell neoplasms, including T acute lymphoblastic leukemia (T-LL) / lymphocytic leukemia (T-ALL) and anaplastic large cell lymphoma (ALCL) are cancers of T cell lineage blood cells. T-LL and T-ALL are characterized by rapid proliferation of abnormal immature T progenitors (blasts) that accumulate in the bone marrow and prevent normal blood cell production, with a blast cut-off value of 20-25 in the bone marrow Arbitrarily classified by%. T-ALL accounts for approximately 15% and 25% of ALL in pediatric and adult cohorts, and T-LL represents approximately 2% of non-Hodgkin lymphoma (NHL) patients in adults. In contrast, ALCL is a rare type of NHL (approximately 3% of patients) but is one of the more common types of T-cell lymphoma.
Clinical signs and symptoms of T-LL / T-ALL are caused by the replacement of normal bone marrow with leukemia cells, which causes a decrease in red blood cells, platelets and normal white blood cells (cytopenia). These signs and symptoms include fatigue, shortness of breath, ease of bruising and bleeding, and also increased risk of infection, splenomegaly or hepatomegaly, and other symptoms caused by leukemic infiltration of tissue. May be. Several risk factors, chromosomal abnormalities and other somatic mutations have been identified, but the specific cause is not clear. T-ALL progresses rapidly as an acute leukemia and generally causes death within a few weeks or months if left untreated.

Acute myeloid leukemia (AML), also known as acute myeloid leukemia or acute nonlymphocytic leukemia (ANLL), is myeloid blood characterized by rapid proliferation of abnormal white blood cells that accumulate in the bone marrow and prevent the production of normal blood cells It is cancer of the cell. AML is the most common acute leukemia that affects adults, and its prevalence increases with age. Although AML is a relatively rare disease that accounts for approximately 1.2% of cancer deaths in the United States, its prevalence is expected to increase with the age of the population.
Clinical signs and symptoms of AML are caused by the replacement of normal bone marrow with leukemia cells, which results in a decrease in red blood cells, platelets and normal white blood cells (cytopenia). These signs and symptoms include fatigue, shortness of breath, ease of bruising and bleeding, increased risk of infection, splenomegaly or hepatomegaly, and other symptoms caused by leukemic infiltration of tissue There is a case. Several risk factors, chromosomal abnormalities and other somatic mutations have been identified, but the specific cause is not clear. T-ALL progresses rapidly as an acute leukemia and generally causes death within a few weeks or months if left untreated.
There are several subtypes of AML. Treatment and prognosis vary between subtypes. Depending on the subtype, the 5-year survival rate is 15-70% and the recurrence rate is 33-78%. AML is initially treated with chemotherapy with the goal of introducing remission. Patients can continue to receive additional chemotherapy or can receive allogeneic hematopoietic stem cell transplantation.
Treatment of patients with acute myeloid leukemia (AML) has not changed for more than 20 years, and AML survival is well below 50% in adults and approximately 60-70% in children. Even if the patient's disease is cured, there are often significant pathologies from conventional chemotherapy regimens and bone marrow transplants. Clearly, there is a need for more effective and less toxic therapies.

Myelodysplastic syndrome (MDS, formerly known as pro-leukemia) is a diverse collection of blood (blood-related) pathologies that include ineffective production (and dysplasia) of myeloid blood cells. Patients with MDS may develop severe anemia and require blood transfusions. The disease may worsen and the patient may develop cytopenia (low blood count) caused by progressive bone marrow failure.
The prognosis for MDS depends on its type and severity. Many people live a normal lifespan with MDS. In many cases, people are asymptomatic and do not even realize they have MDS until their identity is revealed by routine blood tests.
Myelodysplastic syndrome refers to any disease that occurs in hematopoietic stem cells of the bone marrow. In MDS, hematopoiesis (blood production) is impaired and ineffective. The number and characteristics of hematopoietic cells are irreversibly reduced, which further impairs blood production.
Myelodysplastic syndrome (MDS) is a related group of clonal blood diseases characterized by peripheral cytopenia due to ineffective hematopoiesis. This syndrome may occur de novo or secondary after treatment with chemotherapy and / or radiation therapy for other diseases. Secondary myelodysplasia usually has a poorer prognosis than de novo myelodysplasia. At various intervals after diagnosis, MDS changes to acute myeloid leukemia (AML) in approximately 30% of patients.
MDS occurs mainly in older patients, but a 2-year-old patient has also been reported. Anemia, bleeding, bruising and fatigue are common early findings. Splenomegaly or hepatosplenomegaly may be associated with a common myeloproliferative disorder. Approximately 50% of patients have detectable cytogenetic abnormalities, usually a deletion of all or part of chromosome 5 or 7, or trisomy 8. The bone marrow is usually cytological at the time of diagnosis, but 15% to 20% of patients exhibit bone marrow hypoplasia. Patients with hypoplastic myelodysplasia tend to show significant cytopenias and can often respond to immunosuppressive therapy.

  Both AML and MDS are initiated and maintained by self-renewing stem cells. Lapidot et al., Nature 367: 645-648 (1994); Bonnet and Dick, Nat. Med. 3: 730-737 (1997); Nilsson et al., Blood 100: 259-267 (2002); Nilsson et al ., Blood 110: 3005-3014 (2007); Tehranchi et al., New Engl. J. Med. 363: 1025-1037 (2010); Nilsson et al., Blood 96: 2012-2021 (2000); and Pang et al., Proc. Natl. Acad. Sci. USA 110: 3011-3016 (2013). These disease-initiating cells share the immunophenotypic characteristics of normal hematopoietic stem cells and hematopoietic progenitor cells (HSPC), but abnormal expression of cell surface proteins allows for predictive isolation of diseased stem cells and an attractive treatment Is the target. The identification of CD99 as a cell surface protein that is strongly expressed in AML leukemia stem cells (LSC) and MDS hematopoietic stem cells (HSC) has been described. Strong expression of CD99 is associated with disease malignancy in AML xenografts. In contrast, patients with high CD99 transcript expression show improved prognosis. It may be due to increased chemosensitivity imparted by transendothelial migration of leukemia cells and enhanced recruitment to peripheral blood (PB). Finally, targeting of CD99 based on a monoclonal antibody (mAb) directly induces cytotoxicity in the absence of immune effector cells or complement, while relatively preserving normal HSCs and endothelial cells. Taken together, these data indicate that CD99 as a cell surface protein preferentially expressed in AML and MDS stem cells, as a mediator of transendothelial migration and mobilization of leukemia blasts, and for direct targeting of mAbs Established as a promising therapeutic target.

The present disclosure provides compositions and methods for treating hematological malignancies and lymphomas, such as blood diseases, particularly acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and T cell lymphoma. As described in detail herein, hematopoietic tumors and lymphomas, such as AML, MDS and T cell malignancies, can be effectively treated with one or more compounds that promote the aggregation of cellular CD99. In particular, the disclosure provides a composition comprising one or more anti-CD99 antibodies, wherein each anti-CD99 antibody binds to (i) the extracellular domain of CD99; (ii) CD99 aggregation, clustering and / or capping. And (iii) providing the composition, which induces cell death of AML and / or MDS and / or T cell malignant neoplastic cells bound by the anti-CD99 antibody. Furthermore, as evidenced herein, the induction of cell death is closely correlated with the combination of binding and one or more of aggregation, clustering or capping, the latter predicting the occurrence of cell death. Can be helpful.
In some embodiments, (i) a composition comprising an anti-CD99 antibody and one or more anti-CD99 antibodies for the treatment of a T cell neoplasm; (ii) an anti-CD99 suitable for the treatment of a T cell neoplasm. (Iii) a method of identifying patients with T cell neoplasia that are sensitive to treatment with an anti-CD99 antibody and / or a composition comprising one or more anti-CD99 antibodies; (iv) Providing a method of inducing apoptosis in CD99 ⁺ cells associated with T cell neoplasms such as CD99 ⁺ leukemia stem cells and / or hematopoietic stem cells; and (v) a method of treating CD99 ⁺ T cell neoplasms, A method of treating a T cell neoplasm is disclosed.

In one embodiment, a hematopoietic comprising administering to the patient a therapeutically effective amount of an antibody capable of recognizing or specifically binding to the extracellular domain of CD99 in a patient in need thereof. A method of treating a tumor or lymphoma is provided. In one embodiment, a patient affected with a hematopoietic tumor or lymphoma has CD99 ⁺ myeloid or lymphoid malignant cells. In some embodiments, the malignant cells are from mature T cell lymphoma cells, T acute lymphocytic leukemia (T-LL), T acute lymphocytic leukemia (T-ALL) cells or anaplastic large cell lymphoma (ALCL) cells. Selected. In other embodiments, the malignant cells are selected from the group consisting of primary AML blast cells, leukemia stem cells (LSC), primary MDS blast cells and MDS hematopoietic stem cells (HSC). In some embodiments, the malignant cells are cells from a patient affected by T lymphoblastic lymphoma, hemangioimmunoblastic T cell lymphoma or anaplastic large cell lymphoma (ALCL).
In one embodiment, a method of treating a hematopoietic tumor or lymphoma in a patient in need thereof, wherein the malignant tumor is T lymphoblastic lymphoma, hemangioblastic T cell lymphoma, undifferentiated large Said method is provided selected from cell lymphoma (ALCL), NK / T cell lymphoma, acute myeloid leukemia and / or myelodysplastic syndrome. In one embodiment, the ALCL is an anaplastic lymphoma kinase (ALK) positive anaplastic large cell lymphoma (ALCL) or an ALK negative anaplastic large cell lymphoma. In one embodiment, the malignant tumor is non-Hodgkin lymphoma. In one embodiment, the malignancy is selected from acute myeloid leukemia and / or myelodysplastic syndrome.
In one embodiment, the present disclosure provides an anti-CD99 antibody and a composition comprising one or more anti-CD99 antibodies for the treatment of acute myeloid leukemia and myelodysplastic syndrome.
In other embodiments, the present disclosure provides methods for making and identifying anti-CD99 antibodies suitable for the treatment of acute myeloid leukemia and / or myelodysplastic syndrome.
In further embodiments, the present disclosure provides hematopoiesis comprising acute myeloid leukemia, myelodysplastic syndrome or T cell malignancy that is sensitive to treatment with an anti-CD99 antibody and / or a composition comprising one or more anti-CD99 antibodies. Methods for identifying patients with organ tumors or lymphomas are provided.

In other embodiments, the present disclosure induces cell death in CD99 ⁺ cells associated with hematopoietic tumors or lymphomas, including acute myeloid leukemia and / or myelodysplastic syndromes such as CD99 ⁺ leukemia stem cells and / or hematopoietic stem cells Provide a way to do it.
In still other embodiments, the present disclosure provides methods of treating CD99 ⁺ hematopoietic tumors or lymphomas, such as acute myeloid leukemia and / or CD99 ⁺ myelodysplastic syndrome, which show elevated CD99 levels.

CD99 on the MDS cell line MDS92 (Tohyama et al., Br. J. Haematol. 91: 795 (1995)) and an anti-CD99 antibody called 12E7 (Levy et al., Proc. Natl. Acad. Sci. USA 76 : 6552 (1979)) Ligation with 20 μg / ml is demonstrated by a 128-fold decrease in the number of MDS92 cells (p <0.001) after 72 hours of 20 μg / ml isotype control antibody. It is a bar graph which shows that the cytotoxicity with respect to these MDS92 cells is shown compared with the number of MDS92 cells in presence. Plot of fluorescence values of 7-AAD vs. Annexin V in evidence of effector cell-independent apoptosis (no apoptosis of MDS92 cells was observed after 72 hours incubation in the presence of 20 μg / ml isotype control antibody In contrast, annexin V positivity (p <0.001) of MDS92 cells increased by 77% at 72 hours after ligation of 10 μg / ml 12E7 and CD99). This is a bar graph showing 12E7-mediated cytotoxicity, and the number of MDS92 cells at 22 hours after ligation of anti-CD99 antibody 12E7 and CD99 compared to the number of MDS92 cells in the presence of isotype control antibody. And a 128-fold decrease (p <0.001). FIG. 2B is a log plot showing the time-dependent decrease in cell surface expression of CD99 on MDS92 cells after ligation of anti-CD99 antibody 12E7 and CD99. FIG. 2C is a plot of fluorescence values of myeloid differentiation marker CD11b vs. CD14. This plot shows a time-dependent decrease in cell surface expression of both markers after ligation of anti-CD99 antibody 12E7 and CD99. Ligation of CD99 and 12E7 anti-CD99 antibody on primary CD34 ⁺ MDS cells is a bar graph showing cytotoxicity against primary MDS cells, 10 times the number of cells compared to no antibody after 48 hours This was evidenced by a reduction and an 8-fold reduction in cell number relative to the isotype (IgG) control antibody. Ligation of 12E7 anti-CD99 antibody with CD99 on CD99 ⁺ AML cell lines HL60 and MOLM13 (ligation) shows cytotoxicity, showing cell number against isotype control antibody after 72 hours Proven by a 49-fold decrease (HL60; p <0.001) and a 70-fold decrease (MOLM13; p <0.001). FIG. 5 is a log plot showing increased CD99 expression levels on MOLM13 and HL60 cells compared to isotype control antibodies. Ligation of CD99 with 12E7 anti-CD99 antibody on primary AML1520 blast cells expressing CD99, as evidenced by a 57-fold decrease in cell number after 48 hours (p <0.001) It is a bar graph which shows showing cytotoxicity. The ligation of CD99 and 12E7 anti-CD99 antibody on primary AML890 blast cells expressing CD99 as evidenced by a 48-fold decrease in cell number after 48 hours (p <0.001) It is a bar graph which shows showing injury. FIG. 6 is a bar graph showing that 12E7 anti-CD99 antibody shows only slight cytotoxicity against normal spinal blood HSC, as evidenced by a 1.4-fold decrease in HSC cell number after 80 hours. Figures 6A-6F show that CD99 is overexpressed on disease-initiating stem cells in MDS and AML. FIG. It is a figure which shows the analysis result by the flow cytometry (FC) of ⁺ CD38-CD90 + CD45RA-). Figures 6A-6F show that CD99 is overexpressed on disease-initiating stem cells in MDS and AML. FIG. 6B is a plot of unfractionated AML blasts (CD19-CD3-CD45lowSSClow) (n = 63) and normal CB control analyzed by FC for CD99 expression. Error bars represent ± SD. The asterisk represents p <0.0001 (unpaired t test). Figures 6A-6F show that CD99 is overexpressed on disease-initiating stem cells in MDS and AML. FIG. 6C shows the results of CD99 expression evaluated by flow cytometry in CD3-CD19-CD34 + CD38− cells from AML specimen UPenn 1956. By immunophenotype, “low CD99” cells are mainly similar to HSCs and pluripotent progenitor cells (MPP, LN CD34 + CD38-CD90-CD45RA-), and “high CD99” cells are lymphoid MPP ( LMPP, LN CD34 + CD38-CD90-CD45RA +). Figures 6A-6F show that CD99 is overexpressed on disease-initiating stem cells in MDS and AML. FIG. 6D shows that “low CD99” when the “low CD99” and “high CD99” cell populations were double-sorted to a purity of 95% or higher by FACS and plated on the methylcellulose assay (1600 cells). FIG. 5 is a bar graph showing that many normal myeloid colonies formed on day 14 from the fraction but not from the “high CD99” population. Figures 6A-6F show that CD99 is overexpressed on disease-initiating stem cells in MDS and AML. FIG. 6E shows an agarose gel showing that colony-derived “low CD99” cells lacked FLT3-ITD molecular abnormalities present in UPenn 1956 AML specimens (B-bulk AML blasts, 1-7-low CD99 colonies). FIG. Figures 6A-6F show that CD99 is overexpressed on disease-initiating stem cells in MDS and AML. FIG. 6F is a graph showing that CD99 expression was high in the LSC enriched CD34 + CD38− fraction in CD34 + expressing AML (n = 54). Error bars represent ± SD. * Represents p <0.0001 (paired t-test). 7A-7E show that CD99 promotes disease malignancy in vivo but improves patient outcome in relation to chemotherapy. Figure 7A is a graph showing that knockdown of CD99 in HL60 cells by two shRNAs (2.3-fold and 8.3-fold with # 61 and # 59, respectively) did not significantly alter growth kinetics in vitro. It is. Error bars represent ± SD. 7A-7E show that CD99 promotes disease malignancy in vivo but improves patient outcome in relation to chemotherapy. FIG. 7B shows that the survival (OS) of sub-lethal dose irradiated NSG mice transplanted with MOLM13 cells transduced with shRNA # 59 showed a significant improvement compared to the vector control ( 58 and 34 days, respectively, p = 0.0033, n = 5 per group, independent set of 3 grafts). 7A-7E show that CD99 promotes disease malignancy in vivo but improves patient outcome in relation to chemotherapy. FIG. 7C shows that CD99 surface expression is related to disease burden as measured by% blasts in peripheral blood of AML patients (n = 31, R2 = 0.2476, p = 0.0051, Pearson correlation) It is a graph. 7A-7E show that CD99 promotes disease malignancy in vivo but improves patient outcome in relation to chemotherapy. Figure 7D shows that in 358 AML patients randomized to standard dose or high dose daunorubicin (DNR) in the Eastern Cooperative Oncology Group (ECOG) 1900 clinical trial, high expression of CD99 transcripts was observed in the standard dose DNR group. Survival curves showing an association with improved OS (25.0 and 10.4 months for high CD99 and low CD99, p = 0.000585, upper left panel, respectively). Enhanced DNR alleviated the poor prognosis of patients with low CD99 expression (23.3 months for high CD99 versus 21.3 months, p = 0.755, lower left panel). 7A-7E show that CD99 promotes disease malignancy in vivo but improves patient outcome in relation to chemotherapy. FIG. 7E shows that in DNMT3a or NPM1 mutations or MLL rearrangement subgroups where extra benefit has been described by DNR enhancement, DNR enhancement improved OS in the low CD99 group (for 12.3 to 20.2 months, p = 0.007, Upper right panel), survival curve showing no improvement in high CD99 group (18.1 to 17.5 months, p = 0.701, lower right panel). Log rank test was used for all survival comparisons. Figures 8A-8D show that CD99 promotes transendothelial migration and mobilization of leukemia blasts. FIG.8A shows human umbilical vein endothelial cells (HUVEC) transduced to overexpress CD99 (7-fold) using a tetracycline-inducible lentiviral vector and grown to confluence on a transwell insert (pore size 8 μM). It is a bar graph of HL60 cells seeded with it. These data indicate that overexpression of CD99 led to a significant increase in migration efficiency as measured at 4 and 28 hours. Figures 8A-8D show that CD99 promotes transendothelial migration and mobilization of leukemia blasts. FIG. 8B is a bar graph showing that after 72 hours, the remaining non-migrated cells showed significantly lower levels of CD99 expression compared to the migrated cells. Figures 8A-8D show that CD99 promotes transendothelial migration and mobilization of leukemia blasts. FIG. 8C is a bar graph showing that primary AML specimens (n = 9) collected from peripheral blood show significantly higher CD99 expression levels compared to bone marrow (n = 31). Figures 8A-8D show that CD99 promotes transendothelial migration and mobilization of leukemia blasts. FIG. 8D is a graph of flow cytometry data showing CD99 expression on transplanted cells 10 weeks after xenotransplantation of human AML sample UPenn 2741 to NSG mice irradiated with 10 quasi-lethal doses. These data show that in a paired analysis, CD99 expression was significantly higher on transplanted tumor cells circulating in peripheral blood compared to bone marrow. Error bars represent ± SD. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 (paired t-test for panel d, unpaired t-test for all others). FIGS. 9A-9H show that anti-CD99 monoclonal antibody (mAb) exhibits direct cytotoxicity against AML and MDS cells, and such antibody-mediated cytotoxicity is effector and complement independent. It shows that. FIG. 9A (left panel) shows a bar graph of relative cell number after incubation of leukemic blasts from AML specimen AU 5.1 with anti-CD99 mAb clone 12E7 for 72 hours at a dose of 10 μg / ml or 20 μg / ml. These data show a significant decrease in the number of cells after incubation with the 12E7 antibody compared to incubation with the IgG1 isotype control antibody. FIG. 9A (right panel) shows a bar graph of relative cell number after 72 hours incubation of lineage negative CD34 + CD38− cells from primary MDS patient samples with anti-CD99 mAb clone 12E7. These data show a significant decrease in the number of cells after incubation with the 12E7 antibody compared to incubation with the IgG1 isotype control antibody. FIGS. 9A-9H show that anti-CD99 monoclonal antibody (mAb) exhibits direct cytotoxicity against AML and MDS cells, and such antibody-mediated cytotoxicity is effector and complement independent. It shows that. FIG. 9B is a graph of relative cell numbers of MOLM13 cells after 72 hours incubation with anti-CD99 mAb H036-1.1 (Abcam, Cambridge, Mass.). These data show a dose-dependent decrease in cell number at IC50 = 186 ng / ml. FIGS. 9A-9H show that anti-CD99 monoclonal antibody (mAb) exhibits direct cytotoxicity against AML and MDS cells, and such antibody-mediated cytotoxicity is effector and complement independent. It shows that. FIG. 9C is a bar graph of activated caspase 3 (aCaspase 3) after incubation of MOLM13 cells with 5 μg / mL (microgram / mL) with H036-1.1. These data show an increase in aCaspase 3, which indicates H036-1.1-mediated induction of cell death by apoptosis. FIGS. 9A-9H show that anti-CD99 monoclonal antibody (mAb) exhibits direct cytotoxicity against AML and MDS cells, and such antibody-mediated cytotoxicity is effector and complement independent. It shows that. FIG. 9D is a graph of relative cell numbers of MOLM13 cells, spinal cord blood (CB) HSCs and HUVECs after 72 hours incubation with anti-CD99 mAb H036-1.1. These data indicate that the IC50 is 186 ng / ml for MOLM13 cells and 5201 ng / ml for CB HSC, which is the basis for a substantial therapeutic concentration range. In HUVEC, IC50 was not reached at doses up to 20,000 ng / ml. This experiment was repeated with MSK MDS-001 CD34 ⁺ CD38- cells after exposure to anti-CD99 mAb H036-1.1 and compared to non-ML adult bone marrow CD34 ⁺ CD38- cells and HUVEC cells, as shown in FIG. . Similarly, the dramatic decrease in relative cell numbers showed a broad therapeutic window for MSK MDS-001 CD34 ⁺ CD38-, but much less effect on adult BM CD34 ⁺ CD38- and HUVEC cells. FIGS. 9A-9H show that anti-CD99 monoclonal antibody (mAb) exhibits direct cytotoxicity against AML and MDS cells, and such antibody-mediated cytotoxicity is effector and complement independent. It shows that. FIG. 9E is a graph of human chimerism in peripheral blood (PB) after 45 minutes ex vivo treatment of AML specimen UPenn 2741 with H036-1.1 prior to transplantation into sub-lethal dose irradiated NSG mice. At 8 weeks, these data show a significant reduction in tumor engraftment as measured by human chimerism in PB. FIGS. 9A-9H show that anti-CD99 monoclonal antibody (mAb) exhibits direct cytotoxicity against AML and MDS cells, and such antibody-mediated cytotoxicity is effector and complement independent. It shows that. FIG. 9F (left panel) shows the results of immunofluorescence analysis (CD99-green, DAPI-blue) of HL60 cells after treatment with anti-CD99 mAb (12E7). At 105 minutes, significant cell surface capping of CD99 was revealed. FIG. 9F (right panel) is a bar graph of relative cell numbers after 72 hours incubation of MOLM13 cells in the presence of cross-linking anti-IgG secondary mAb. This graph shows a significant enhancement of anti-CD99 mAb (IgG-isotype) cytotoxicity, consistent with IgG-mediated promotion of anti-CD99-mediated aggregation of cell surface CD99, followed by promotion of MOLM13 cell death. FIGS. 9A-9H show that anti-CD99 monoclonal antibody (mAb) exhibits direct cytotoxicity against AML and MDS cells, and such antibody-mediated cytotoxicity is effector and complement independent. It shows that. FIG. 9G is an autoradiograph showing activation of Src family kinases (SFKs, pSrc [Y416]) following incubation of MOLM13 cells with anti-CD99 mAb H036-1.1. FIGS. 9A-9H show that anti-CD99 monoclonal antibody (mAb) exhibits direct cytotoxicity against AML and MDS cells, and such antibody-mediated cytotoxicity is effector and complement independent. It shows that. FIG. 9H is a graph of relative numbers after MOLM13 cells were preincubated with SFK anti-CD99 antibody PP2 (20 μM) for 3 hours and then with H036-1.1 for 48 hours. These data show a significant reduction in cytotoxicity in the absence of PP2 (IC50 476 ng / ml at 48 hours). IC50 was not obtained in the presence of PP2. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 (unpaired Student t test)). This is consistent with the down-regulation of cell surface CD99 during the preincubation step resulting in reduced H036-1.1 mediated CD99 aggregation and cell death. FIG. 6 is a bar graph showing relative cell numbers for 15 AML cell lines after 48 hours incubation with anti-CD99 antibody 12E7. Of the 15 AML cell lines tested, AML5Q, NB4, KCL22, MOLM13, HL60, MOLM14, NOMO1, U937, MonoMac1, KG1a and AML14 are sensitive to growth inhibition after ligation of 12E7 antibody Was found to show. The four cell lines tested, KBM5, SET2, KU812, and K562, were found to be insensitive to CD99 ligation with 12E7 antibody. Flow cytometry plot (upper panel) showing relative expression of c-kit and CD99 in AML cell lines HL60, K562, KBM5, SET2 and KU812 cells and relative after 48 hours incubation with IgG1κ or anti-CD9912E7 antibody It is a bar graph (lower panel) which shows a cell number. HL60 is a typical “sensitive” cell line that exhibits high expression of CD99 and low expression of c-kit. “Resistant” cell lines do not express CD99 (K562 and KBM5) or express high levels of c-kit (SEM2 and KU812) and are Bcr-abl positive (K562 / KBM5 / KU812), or JAK2 is positive (SEM2). These data indicate that low expression of CD99 or high expression of c-kit predicts resistance to cytotoxicity mediated by anti-CD99 antibody 12E7. FIG. 6 is a graph of transendothelial migration kinetics of CD99 ⁺ AML cells in the presence of anti-CD99 antibodies 12E7, HEC2, DN16 and H036. This graph shows that in the absence of secondary antibodies, certain anti-CD99 antibodies do not suppress transendothelial migration of AML cells known to be mediated by CD99. The upper panel shows a schematic diagram of a combination of ex vivo pre-treatment and in vivo treatment with anti-CD99 mAb H036-1.1 in AML mice xenografted with UPenn 2522. The lower panel shows an assessment of human chimerism after 5 months in the bone marrow of mice xenografted with AML blast UPenn 2522. In vivo treatment of mice xenografted with AML blasts or anti-CD99 mAb pre-coated with anti-CD99 mAb prior to xenotransplantation removed AML xenografts, whereas 4/5 of control animals inherited leukemia It is. FIG. 6 is a graph of the relative cell numbers of MSK MDS-001 CD34 ⁺ CD38 − cells, adult BM CD34 + CD38 − cells and HUVEC after 72 hours incubation with anti-CD99 mAb H036-1.1. Anti-CD99 mAb was significantly more toxic to MDS-001CD34 ⁺ CD38- cells, indicating a good therapeutic window for the treatment of leukemia cells. Table 4 shows the results of screening 264 NHL patients for CD99 expression by staining tissue microarrays by immunohistochemistry. 4/13 (31%) ALCL, 11/20 (55%) T lymphoblastic lymphoma, 7/16 (44%) hemangioblastoblastic T cell lymphoma with 5% cutoff, 10 / 63 (16%) peripheral T cell lymphoma (non-specific), 0/3 (0%) NK / T cell lymphoma, 2/17 (11.7%) follicular lymphoma, 2/24 (8%) mantle cell lymphoma , 4/22 (18%) chronic lymphocytic leukemia, 3/16 (18.8%) marginal zone lymphoma and 1/70 (1.4%) diffuse large B cell lymphoma (DLBCL) positive for CD99 expression Met. For samples,% positive cells and staining intensity were also recorded. When showing weak to moderate staining, mainly a cytoplasmic pattern was shown, but when it was strong, membrane staining was shown. It is a figure which shows the typical staining pattern of CD99 by immunohistochemistry. A) T lymphoblastic lymphoma (membrane, strong), B) Mantle cell lymphoma (cytoplasm, moderate), C) Marginal zone lymphoma (cytoplasm, weak), D) Undifferentiated large cell lymphoma (cytoplasm, moderate) ). FIG. 6 shows CD99 (mRNA) expression in normal bone marrow (n = 7) and bone marrow (n = 117) of pediatric T-ALL patients. The results show that CD99 is upregulated in T-ALL versus normal BM (p <.0001). The raw data used for this analysis was obtained from published transcription profiles in BioGPS. Patients in this study were enrolled in the German Co-operative study group for childhood ALL (COALL) and Dutch Childhood Oncology Group (DCOG) protocols. FIG. 6 shows CD99 expression (by flow cytometry) between matched diagnosis-relapse pairs of T-ALL patients. CD99 has been shown to be expressed at comparable levels at diagnosis and at recurrence. Taken together, these data suggest that CD99 in T-ALL may be targeted at diagnosis and at recurrence. The raw data used for this analysis was obtained from published transcription profiles in BioGPS. Patients in this study were enrolled in the Dutch Childhood Oncology Group protocol. It is a figure which shows the expression level of CD99 on the lymphoid neoplasm (cell line) with respect to normal peripheral blood B and T cell. As a positive control, HL60, an AML cell line, was used. CD99 expression was normalized to 293T cells as well as individual cell line isotype controls. According to this experiment, CD99 was up-regulated on Karpas-299 (ALCL cell line) and KoptK1 (T-ALL cell line) against normal T cells, and Granta-519 (leukemia transformation [B line]). It becomes clear that mantle cell lymphomas with cell lines express CD99 at higher levels than normal B cells. In contrast, Mac2A (ALCL), Hut78 (Sezary syndrome), Hut102 (mycosis fungoides), Ly8 (diffuse large B-cell lymphoma, DLBCL), WSU (DLBCL), Daudi (Burkitt lymphoma), DOHH2 (immunoblastic B cell lymphoma), SKO-007 (multiple myeloma), SKMM1 (multiple myeloma), SKMM2 (multiple myeloma), LICR2 (plasma cell leukemia) and RPMI8226 (plasmacytoma) The expression of CD99 on (multiple myeloma) is lower than their normal B and T cell counterparts. Despite this, 4/4 cutaneous T-cell lymphoma, 3 / 3DLBCL, 0/1 Burkitt lymphoma and 1/1 leukemic mantle cell lymphoma were CD99 positive by FC (data not shown). FIG. 4 is a graph showing DN16-mediated cytotoxicity in the 4KOPTK1 cell line after 72 hours incubation with 40 ug / ml DN16 and 50-100 ug / ml anti-IgG versus 0 ug / ml IgG and 50-100 ug / ml anti-IgG control. This is evidenced by a 70-81% reduction in cell number (p ≦ .0002). FIG. 4 is a graph showing DN16-mediated cytotoxicity in Karpas-299 cell line, 40 ug / ml DN16 and 50-100 ug / ml anti-40 ug / ml IgG and 50-100 ug / ml anti-IgG control (p ≦ .0021). This is evidenced by a 38-43% reduction in cell number after 72 hours incubation with IgG. 72 hours incubation with 40ug / ml DN16 and 50-100ug / ml anti-IgG cannot induce cytotoxicity in Mac2A (ALCL cell line expressing low levels of CD99) and therefore serve as a suitable negative control It is a graph which shows. FIG. 4 is a graph showing 12E7-mediated cytotoxicity in the KOPTK1 cell line, cells after 72 hours incubation with 40 ug / ml IgG and 50-100 ug / ml anti-IgG control with 40 ug / ml 12E7 and 50-100 ug / ml anti-IgG This is evidenced by a 42-78% reduction in numbers (p ≦ .0003). FIG. 5 is a graph showing O13-mediated cytotoxicity in Karpas-299 cell line, cell number after 72 hours incubation with 40 ug / ml O13 and 75 ug / ml anti-IgG versus 40 ug / ml IgG and 75 ug / ml anti-IgG control Proven by a 25% decrease in (p <.0001). Figure 5 shows a summary of the maximum change in cell numbers identified in Karpas-299, KOPTK1 and Mac2A cell lines by treatment with CD99mAb (F8, O13, 12E7, DN16) for 72 hours in the presence of anti-IgG. is there. These findings suggest that mAb-mediated cytotoxicity of CD99 is epitope specific.

DETAILED DESCRIPTION OF THE INVENTION This disclosure is based, in part, on the results of transcriptome analysis of hematopoietic stem cells (HSC) from low-risk myelodysplastic syndrome (MDS) patients and normal bone marrow (BM) cells from normal adult donors. . Among the 3,258 transcripts with different expression levels identified in the transcriptome analysis, 25 cell surface transcripts expressed differentially were detected. Flow cytometric analysis of 37 de novo MDS patients confirmed dysregulated cell surface protein expression of CD99 in 73% of patient samples. In 26 treatment-related MDS patient samples, flow cytometric analysis of the same 25 cell surface proteins revealed that CD99 was overexpressed in 85% of the patient samples.
These reserves that the expression of certain cell surface proteins, especially the cell surface marker CD99, is elevated not only in hematopoietic stem cells (HSC) from patients with low-risk myelodysplastic syndrome (MDS) but also in treatment-related MDS patient samples Based on experimental observations, the potential therapeutic effects of antibodies with binding specificity for the extracellular domain of CD99 were evaluated not only in MDS and primary cells, but also in AML and primary AML blasts. In our experiments, CD99 was essentially the most frequently overexpressed marker in both MDS and AML, and was significantly overexpressed (average about 7 times over controls). .
As described herein, ligation of certain anti-CD99 antibodies, such as anti-CD99 antibody 12E7, with CD99 on MDS cells (i) reduces the cell number in a time-dependent manner ( ii) death of MDS cells ligated with anti-CD99 antibody is induced (as evidenced by increased annexin V positivity); (iii) cell surface expression level of CD99 is decreased; (iv) CD14 and CD11b As such, the levels of certain myeloid specific differentiation markers were reduced. Similarly, ligation of certain anti-CD99 antibodies, such as anti-CD99 antibody 12E7, with CD99 on primary CD34 ⁺ MDS cells showed cytotoxicity to primary MDS cells, which is due to the absence of antibody. This was evidenced by a time-dependent decrease in the number of cells in the culture grown in the presence of anti-CD99 antibody versus the number of cells in the culture grown under or in the presence of an isotype control antibody.

Ligation of certain anti-CD99 antibodies such as anti-CD99 antibody 12E7 with CD99 on AML cells expressing high levels of CD99 was also observed to be cytotoxic to AML cells, This is due to the time-dependent decrease in the number of cells in the culture grown in the presence of anti-CD99 antibody versus the number of cells in the culture grown in the absence of antibody or in the presence of an isotype control antibody. Proved. Similarly, ligation of certain anti-CD99 antibodies, such as anti-CD99 antibody 12E7, with CD99 on primary AML blasts expressing CD99 is cytotoxic to primary AML blasts, which Only a moderate time-dependent effect on normal spinal cord blood HSC cell counts was seen, as evidenced by a time-dependent decrease in the number.
Moreover, as described herein, cytotoxicity due to anti-CD99 antibody ligation of CD99 on MDS cell lines, MDS primary cells, AML cell lines and primary AML blasts expressing CD99 is complement, as described herein. It was further demonstrated that it occurs in the absence of antibody effector functions, including in the absence of dependent cell cytotoxicity (CDC) and / or antibody dependent cell mediated cytotoxicity (ADCC).
These studies found that a subset of the tested anti-CD99 antibodies showed cytotoxicity against CD99 ⁺ cell proliferation, which was due to induction of cell death such as apoptosis. It was due to. These cytotoxic anti-CD99 antibodies share the property of linking to cell surface CD99, thereby promoting cell surface aggregation of antibody-bound CD99, thereby its clustering within a localized region of the cell surface Or capping occurs. Furthermore, the cytotoxicity of specific anti-CD99 antibodies and the apoptosis induced thereby was directly related to each antibody's ability to promote CD99 aggregation, clustering and capping. These results indicate that anti-CD99 antibodies effective to suppress CD99 ⁺ AML and CD99 ⁺ MDS desirably (i) induce CD99 aggregation; (ii) induce CD99 clustering; and (iii) CD99 ⁺ It was shown to have one or more of the properties that induce CD99 capping on the surface of MDS and AML cells.

Based on these and other discoveries (described in further detail herein), this disclosure
(1) A composition comprising an anti-CD99 antibody and one or more anti-CD99 antibodies for the treatment of acute myeloid leukemia and myelodysplastic syndrome, wherein an appropriate anti-CD99 antibody is expressed on the surface Link (ie, bind) to CD99; inhibit proliferation of CD99 ⁺ AML and / or MDS cells; facilitate aggregation, clustering and / or capping of ligated CD99; and apoptosis, necrosis / necroptosis, Accelerate cell death of AML and / or MDS cells by direct induction of cell death by other cell death mechanisms such as autophagy cell death, endoplasmic reticulum stress-related cytotoxicity or cell death, paratosis, pyrotosis, pyronecrosis entosis A composition comprising said anti-CD99 antibody and one or more anti-CD99 antibodies;
(2) a method for producing and identifying an anti-CD99 antibody suitable for the treatment of acute myeloid leukemia and / or myelodysplastic syndrome;
(3) a method of identifying a patient with acute myeloid leukemia and / or myelodysplastic syndrome that is susceptible to treatment with an anti-CD99 antibody and / or a composition comprising one or more anti-CD99 antibodies;
(4) a method of inducing apoptosis in CD99 ⁺ cells associated with acute myeloid leukemia and / or myelodysplastic syndrome, such as CD99 ⁺ leukemia stem cells and / or hematopoietic stem cells; and (5) CD99 exhibiting elevated CD99 levels. ⁺ Provide a method of treating acute myeloid leukemia and / or CD99 ⁺ myelodysplastic syndrome.

As described in further detail herein, these antibodies, compositions and methods of treating acute myeloid leukemia and / or myelodysplastic syndrome include (1) acute myeloid leukemia (primary AML cells, blasts and In tissues and / or cells associated with leukemia stem cells (including LSC) and / or associated with de novo or therapy-related myelodysplastic syndromes (including primary MDS cells, blasts and / or hematopoietic stem cells (HSC)) Increased expression of CD99; (2) ligation with specific anti-CD99 antibodies and the resulting CD99 ⁺ AML and MDS for cell CD99 aggregation, clustering and capping resulting from ligation with such anti-CD99 antibodies. Tissue and cell sensitivity; and (3) anti-CD99 ligation and resulting aggregation, clustering and capping (cell surface Derived from the newly disclosed relationship between the newly discovered cell damage (mediated by anti-CD99 binding to expressed CD99) and cell death caused by its CD99 aggregation (ie, apoptosis) To do.
Anti-CD99 antibodies for ligation-mediated clustering of CD99 on CD99 ⁺ AML and MDS cells This disclosure is suitable for methods of the present disclosure that inhibit proliferation of CD99 ⁺ AML and / or MDS cells or induce their apoptosis A cytotoxic anti-CD99 antibody suitable for the treatment of patients suffering from AML and / or MDS composed of cells exhibiting elevated CD99 expression levels I will provide a.

The cytotoxic anti-CD99 antibodies disclosed herein have the following properties:
1． Binding affinity for the extracellular domain of CD99 when expressed on the surface of CD99 ⁺ AML and / or MDS cells;
2． Promotion or further promotion of aggregation, clustering and capping of antibody-bound CD99 on the surface of CD99 ⁺ AML and / or MDS cells; and
3． CD99 of ⁺ AML and / or MDS when bound to CD99 on the surface of cells, the induction of cell death in CD99 ⁺ AML and / or MDS cells,
Indicates. Promotion of aggregation, clustering and / or capping by CD99-bound antibodies on the surface of CD99 ⁺ AML and / or MDS cells is responsible for cell damage as well as apoptosis, necrosis / necroptosis, autophagy cell death, endoplasmic reticulum It will be appreciated that the ability to induce cell death, whether or not by cell death mechanisms such as stress-related cell injury or mitotic death, paratosis, pyrotosis, pyronecrosis and tosis, will be understood. See Kroemer et al., Cell Death Differ. 16 (1): 3-11 (2009). In any case, cell death can be observed directly with or without intermediate assessment of aggregation, clustering and / or capping.

Exemplary antibodies that can be suitably used in these methods include mouse IgG1 antibody 12E7 (Levy et al., Proc. Natl. Acad. Sci. 76: 6552 (1979)) and O13 (Dracopoli et al., Am. J. Hum. Genet. 37: 199 (1985)) (both of these are epitopes “DGEN” (SEQ ID NO: 7) of the extracellular domain of CD99 (which are amino acids 61 to 64 of SEQ ID NO: 2, SEQ ID NO: 4 A mouse IgM H036-1.1 antibody (which binds to the epitope DDPRPPNPPK (SEQ ID NO: 12) of the extracellular domain of CD99) and a mouse IgM H036-1.1 antibody (defined by amino acids 45-48 of SEQ ID NO: 6 and amino acids 61-64 of SEQ ID NO: 6) It is.
In contrast, in some embodiments, the mouse IgG1 antibody DN16 (Hahn et al., J. Immunol. 159: 2250 (1997) (incorporated herein by reference) (the epitope “LPDNENKK” of the extracellular domain of CD99) Binds to (SEQ ID NO: 8) (defined by amino acids 32-39 of SEQ ID NO: 2 and 6), but includes SEQ ID NO: 4 (wherein amino acids 32-39 comprise the amino acid sequence “LPGDDFDL” (SEQ ID NO: 9)) Is not suitable for the disclosed method because the binding of CD99 to the extracellular domain is due to aggregation of antibody-bound CD99 and correspondingly low levels of cytotoxicity. Show only limited ability to promote and its antibody-mediated aggregation, cytotoxicity and apoptosis can be enhanced by combining anti-CD99 antibody-related CD99 with a second anti-IgG antibody, which Because of its bivalent nature, two anti-CD99 antibodies This is because the combined CD99 molecules are brought close together.

To further identify potential epitopes required for anti-CD99 mAb cytotoxicity, overlapping 15-mer peptide peptide scanning experiments in the CD99 extracellular domain were used. The results are shown in Table 1. Both mAbs 12E7 and F8 recognized overlapping peptides containing the epitope DAVVDGEND (SEQ ID NO: 10). mAb O13 recognized an overlapping peptide containing the epitope AVVDGEN (SEQ ID NO: 11). mAb H036-1.1 recognized an overlapping peptide containing the epitope DDPRPPNPPK (SEQ ID NO: 12). MAb 3B2 recognized an overlapping peptide containing the epitope LPD (SEQ ID NO: 13). mAb recognized an overlapping peptide containing the epitope DALPDN (SEQ ID NO: 14). The sequence of the extracellular domain having this 15mer peptide as a fragment is SEQ ID NO: 15.
Similarly, in some embodiments, mouse IgG antibodies 3B2 and F8 are not suitable for the methods of the present disclosure. This is because antibody-bound AML and / or MDS cells bind to the extracellular domain of CD99 but do not promote aggregation of antibody-bound CD99, cannot mediate aggregation, and cannot induce cell death. This is because it does not show cytotoxicity against the bacterium. Moreover, the binding of a second anti-IgG antibody to CD99 bound by 3B2 and F8 antibodies is ineffective in promoting anti-CD99 antibody-mediated aggregation or cytotoxicity.

Table 1 shows the characteristics of 12E7, O13 and H036-1.1 anti-CD99 antibodies. Each (1) binds to the extracellular domain of CD99 when expressed in AML and / or MDS cells; (2) aggregation, clustering and capping of CD99 expressed on the surface of AML and / or MDS cells Exhibit the desired property of promoting at least one of These antibodies show cytotoxicity to CD99 ⁺ AML and / or MDS cells, to induce cell death in CD99 ⁺ AML and / or MDS cells has been demonstrated.
Table 1 also shows the characteristics of DN16, 3B2 and F8 anti-CD99 antibodies. These antibodies exhibit the desired property of binding to the extracellular domain of CD99 when expressed on the surface of AML and / or MDS cells, but of CD99 expressed on the surface of AML and / or MDS cells. Aggregation, clustering and capping are not promoted. These antibodies, the CD99 ⁺ AML and / or MDS cells showed no cytotoxicity, lack the ability to induce cell death in CD99 ⁺ AML and / or MDS cells.
Thus, cells that share the ability to promote aggregation / clustering / capping with the 12E7, O13 and H036-1.1 anti-CD99 antibodies, and consequently share the ability to induce cell death in CD99 ⁺ AML and / or MDS cells Toxic antibodies are within the scope of this disclosure and are intended to induce cytotoxicity in CD99 ⁺ AML and / or MDS cells, or AML and / or MDS-related cells that exhibit elevated CD99 expression levels and / or It will be appreciated that the presently disclosed methods for treating MDS patients can be suitably used.

In contrast, DN16, 3B2, and F8 anti-CD99 antibodies have limited ability to promote aggregation / clustering / capping or induce cell death in CD99 ⁺ AML and / or MDS cells or It will also be appreciated that non-cytotoxic antibodies that share inability are outside the scope of the present disclosure and methods.

Table 1 Characteristics of anti-CD99 antibodies

Anti-CD99 antibodies that may be suitable for use in the methods disclosed herein are from about 100 ng / ml to about 10 μg / ml or from about 250 ng / ml to about 5 μg / ml or from about 500 ng / ml to about 1 μg. Binds to the extracellular domain of human CD99 with an IC50 of / ml.
Suitable anti-CD99 antibodies for practicing the methods of the present disclosure are preferably monoclonal antibodies, whether previously described or newly developed, these anti-CD99 antibodies are 12E7, O13 and a H036-1.1 anti CD99 antibody, (1) when expressed in CD99 ⁺ AML and / or MDS surface of the cell, binding affinity to the extracellular domain of CD99; (2) CD99 ⁺ AML and / or MDS cells Promote aggregation, clustering and / or capping of antibody-bound CD99 on the surface of CD99 ⁺ ; and (3) CD99 ⁺ AML and / or MDS cells when binding to CD99 on the surface of CD99 ⁺ AML and / or MDS cells As long as they share the induction of cell death in the 12E7, O13 and H036-1.1 anti-CD99 antibodies described herein or any one of the other antibodies, Fab fragments, F (ab ′) fragments By Fab expression library It can be a human, humanized or chimeric monoclonal antibody comprising the fragments produced and / or binding fragments. Due to the close correlation between one or more of aggregation, clustering and capping and cytotoxicity, aggregation, clustering and capping can serve as markers of cytotoxicity, more specifically cell death. Thus, antibodies previously identified to cause aggregation, clustering and capping can be used to promote cell injury by inducing cell death.

The anti-CD99 antibody of the present disclosure can be produced by a conventional means or can be produced by a recombinant technique. Monoclonal antibodies are obtained from individual antibodies that comprise a substantially homogeneous population of antibodies, ie, a population that may be present in small amounts but that are identical except for naturally occurring and likely mutations. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
The anti-CD99 monoclonal antibodies of the present disclosure can be produced using the hybridoma method first described by Kohler et al., Nature 256: 495 (1975) or by recombinant DNA methods (US Pat. No. 4,816,567). No.).
In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized to produce or produce an antibody that specifically binds to the protein utilized for immunization. Induces possible lymphocytes. Antibodies against CD99 can be produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of CD99 and an adjuvant. CD99 can be prepared using methods known in the art, some of which are further described herein. For example, the recombinant production of human and mouse CD99 is described below. In one embodiment, animals are immunized with CD99 fused to the Fc portion of an immunoglobulin heavy chain. In other embodiments, animals are immunized with a CD99-IgG1 fusion protein. Usually, animals are immunized against an immunogenic complex of CD99 or a derivative thereof and monophosphoryl lipid A (MPL) / trehalose diclinomicolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, Mont.) This solution is injected intradermally at multiple sites. The animal is boosted after 2 weeks. After 7-14 days, the animals are bled and the serum is assayed for anti-CD99 titer. Animals are boosted until the titer reaches a plateau.

Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused to myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986 )).
The hybridoma cells thus prepared are seeded and preferably grown in a suitable medium containing one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma medium typically contains hypoxanthine, aminopterin and thymidine (HAT medium), but these The substance inhibits the growth of HGPRT-deficient cells.
Myeloma cells can be efficiently fused, maintain a stable high level of antibody production by selected antibody-producing cells, and be sensitive to a medium such as HAT medium. Myeloma cell lines are available from mouse myeloma lines such as MOPC-21 and MPC-11 mouse tumors available from Salk Institute Cell Distribution Center, San Diego, Calif. USA and American Type Culture Collection, Rockville, Md. USA It can be a mouse myeloma line derived from SP-2 or X63-Ag8-653 cells. Human myeloma and mouse human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies against CD99. The binding specificity of monoclonal antibodies produced by hybridoma cells can be measured by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked antibody immunosorbent assay (ELISA).
The binding affinity of a monoclonal antibody can be measured, for example, by the Scatchard analysis of Munson et al., Anal. Biochem. 107: 220 (1980). After identifying hybridoma cells that produce antibodies of the desired specificity, affinity and / or activity, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals.
Monoclonal antibodies secreted by subclones are suitably obtained by conventional immunoglobulin purification methods such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, in medium, ascites or serum. Separated from.

The anti-CD99 antibodies of the present disclosure can be produced using a combinatorial library for screening synthetic antibody clones having the desired activity (s). In principle, synthetic antibody clones are selected by screening a phage library containing phage that display various fragments of antibody variable regions (Fv) fused to a phage coat protein. Such phage libraries are panned by affinity chromatography against the desired antigen. A clone expressing an Fv fragment capable of binding to the desired antigen is adsorbed to that antigen and is therefore selected from unbound clones in the library. The binding clones are then eluted from the antigen and can be further enriched by further cycles of antigen adsorption / elution. All of the anti-CD99 antibodies of the present disclosure designed an appropriate antigen screening method to select the phage clone of interest, and then the Fv sequence from the phage clone of interest and Kabat et al., Sequences of Proteins of Construction of full-length anti-CD99 antibody clones using appropriate constant region (Fc) sequences as described in Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3 Can be obtained by:
The antigen-binding domain of an antibody consists of two approximately 110 amino acids, one from each of the L (VL) and H (VH) chains, both of which have three hypervariable loops or complementarity determining regions (CDRs). It is formed from a variable (V) region. The variable domain is a single chain Fv in which VH and VL are covalently linked via a short mobile peptide, as described in Winter et al., Ann. Rev. Immunol. 12: 433-455 (1994). It can be functionally presented as a (scFv) fragment or as a Fab fragment that is fused non-covalently, each fused to a constant domain. In this specification, scFv encoding a phage clone and Fab encoding a phage clone are collectively referred to as "Fv phage clone" or "Fv clone".

Repertoires of VH and VL genes were cloned separately by polymerase chain reaction (PCR) as described in Winter et al., Ann. Rev. Immunol. 12: 433-455 (1994) Can be randomly recombined and then antigen-binding clones can be searched. Libraries from immunogens provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, human antibodies against a wide range of non-self and self-antigens can be cloned and cloned without immunization as described in Griffiths et al., EMBO J. 12: 725-734 (1993) A single source of can be provided. Finally, as described in Hoogenboom and Winter, J. Mol. Biol. 227: 381-388 (1992), the naive library also cloned unrecombined V gene segments from stem cells. It can be synthetically produced using PCR primers containing random sequences, encoding a highly variable CDR3 region, and achieving in vitro rearrangements.
Filamentous phage is used to display antibody fragments by fusing to the minor coat protein pIII. An antibody fragment is one in which the VH and VL domains are linked to the same polypeptide chain by a mobile polypeptide spacer, as described, for example, in Marks et al., J. Mol. Biol. 222: 581-597 (1991). Single-chain Fv fragments, or a single strand fused to pIII as described in Hoogenboom et al., Nucl. Acids Res. 19: 4133-4137 (1991). Another chain is secreted into the bacterial host cell periplasm, where the associated form of the Fab coat protein structure is also presented as a Fab fragment that is displayed on the phage surface by replacing a portion of the wild type coat protein. it can.

In general, nucleic acids encoding antibody gene fragments are obtained from immune cells taken from humans or animals. If a library biased towards anti-CD99 clones is desired, individuals are immunized with CD99 to generate an antibody response, and other peripheral blood lymphocytes (PBLs) that are spleen cells and / or circulating B cells are Recovered for construction. CD99 immunization produces an anti-CD99 antibody response in transgenic mice with a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system), such that B cells producing human antibodies against CD99 are generated. To obtain a human antibody gene fragment library biased to anti-CD99 clones. The production of human antibody-producing transgenic mice is described below.
Further enrichment of the anti-CD99 reactive cell population can be achieved using appropriate screening methods to separate B cells expressing CD99-specific membrane-bound antibodies, for example by cell separation by CD99 affinity chromatography or by fluorescent dyes. It can be obtained by adsorption of cells onto labeled CD99 followed by flow activated cell sorting (FACS).
Alternatively, the use of spleen cells and / or B cells or other PBLs from non-immunized donors provides a better presentation of possible antibody repertoires, and any animal (human or non-human) species where CD99 is not antigenic It is possible to construct an antibody library using For libraries that incorporate in vitro antibody gene construction, stem cells are harvested from individuals and nucleic acids encoding non-recombinant antibody gene segments are obtained. The immune cells of interest can be obtained from various animal species such as humans, mice, rats, rabbits, luprine, dogs, cats, pigs, cows, horses and birds.

  Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered from the cell of interest and amplified. For reconstituted VH and VL gene libraries, genomic DNA or mRNA can be expressed as described in Orlandi et al., Proc. Natl. Acad. Sci. USA 86: 3833-3837 (1989). The desired DNA can be obtained by performing a polymerase chain reaction (PCR) with primers that are isolated from the sphere and then matched at the 5 ′ and 3 ′ ends of the reconstituted VH and VL genes, thereby A diverse V gene repertoire for expression is created. The V gene is a back primer at the 5 'end of the exon encoding the mature V domain, as described in Orlando et al. (1989) and Ward et al., Nature 341: 544-546 (1989). ) And forward primers based within the J segment can be used to amplify from cDNA and genomic DNA. However, to amplify from cDNA, the back primer can be based on a leader exon as described in Jones et al., Biotechnol. 9: 88-89 (1991), and the forward primer can be et al., Proc. Natl. Acad. Sci. USA 86: 5728-5732 (1989). As described in Orlandi et al. (1989) or Sastry et al. (1989), degeneracy can be incorporated into the primers to maximize complementarity. For example, as described in the method of Marks et al., J. Mol. Biol. 222: 581-597 (1991) or in Orum et al., Nucleic Acids Res. 21: 4491-4498 (1993). As described in the method, the diversity of the library allows PCR primers to target each V gene family to amplify all available VH and VL sequences present in the nucleic acid sample of immune cells. Can be maximized by using. For cloning of amplified DNA into expression vectors, as described in Orlandi et al. (1989), as a single-ended tag or in Clackson et al., Nature 352: 624-628 (1991). As described, additional PCR amplification using tagged primers can be performed to introduce rare restriction sites within the PCR primers.

  A repertoire of synthetically rearranged V genes can be derived from V gene segments in vitro. Most of the human VH gene segments have been cloned, sequenced (reported in Tomlinson et al., J. Mol. Biol. 227: 776-798 (1992)) and mapped (Matsuda et al. , Nature Genet. 3: 88-94 (1993)). As described in Winter, J. Mol. Biol. 227: 381-388 (1992), these cloned segments (including all of the major conformations of the H1 and H2 loops) can be PCR primers encoding the H3 loop of length and can be used to create a diverse VH gene repertoire. Natl. Acad. Sci. USA 89: 4457-4461 (1992), the VH repertoire is focused on all sequence diversity into a single long H3 loop. Can also be produced. Human Vκ and Vλ segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol. 23: 1456-1461 (1993)) to produce a synthetic light chain repertoire. Can be used. The synthetic V gene repertoire encodes antibodies of considerable structural diversity based on a series of VH and VL folds and the lengths of L3 and H3. Following amplification of the V gene encoding DNA, germline V gene segments can be reconstituted in vitro according to the method of Hoogenboom and Winter, J. Mol. Biol. 227: 381-388 (1992).

In several ways, a repertoire of antibody fragments can be constructed by combining the VH and VL gene repertoires. Each repertoire is made with a different vector and those vectors are generated in vitro, e.g. as described in Hogrefeet al, Gene, 128: 119-126 (1993) or combinatorial infections such as Waterhouse et al. , Nucl. Acids Res. 21: 2265-2266 (1993). This in vivo recombination approach utilizes double-stranded species of Fab fragments to overcome library size limitations imposed by E. coli transformation efficiency. The naive VH and VL repertoires are cloned separately, one into a phagemid and the other into a phage vector. The two libraries are then combined by phage infection of phagemid-containing bacteria, where each cell contains a different combination and the library size is limited only by the number of cells present (about 10 ¹² clones). Both vectors contain an in vivo recombination signal so that the VH and VL genes are recombined into a single replicon and packaged together into a phage virion. These large libraries provide a large number of diverse antibodies with excellent affinities (K _d ⁻¹ of about 10 ⁻⁸ M).

  Alternatively, the repertoire can be sequentially cloned into the same vector, for example as described in Barbas et al., Proc. Natl. Acad. Sci. USA 88: 7978-7982 (1991). For example, as described in Clackson et al., Nature 352: 624-628 (1991), it can also be cloned after assembly by PCR. PCR assembly can also be used to join DNA encoding a mobile peptide spacer with VH and VL DNA to form a single-stranded Fv (scFv) repertoire. In yet another technique, as described in Embleton et al., Nucl. Acids Res. 20: 3831-3837 (1992), “intracellular PCR assembly” can be performed by PCR to detect VH and lymphocytes in lymphocytes. The VL genes are linked and then used to clone the repertoire of linked genes.

Antibodies produced by naive libraries (either natural or synthetic) can be those moderate affinity (K _d ^-1 of about ^{10 6 ~10 7 M -1),} Winter et As described in al. (1994) (supra), affinity maturation can be mimicked in vitro by constructing and reselecting from a second library. For example, in the method of Hawkins et al., J. Mol. Biol. 226: 889-896 (1992) or Gram et al., Proc. Natl. Acad. Sci. USA 89: 3576-3580 (1992), By using error-prone polymerase (reported in Leung et al., Technique 1: 11-15 (1989)), mutations can be randomly introduced in vitro. In addition, by randomly mutating one or more CDRs, for example by screening higher affinity clones in selected individual Fv clones using PCR with primers containing random sequences spanning the CDRs of interest. Affinity maturation can be performed. PCT Patent Publication WO1996 / 07754 (published on March 14, 1996) describes a method for producing a library of L chain genes by inducing mutagenesis in complementarity determining regions of immunoglobulin L chains. Yes. Another effective approach uses a repertoire of naturally occurring V domain variants obtained from non-immunized donors as described in Marks et al .. Biotechnol. 10: 779-783 (1992) Recombining VH or VL domains selected by phage display, screening for higher affinity with several rounds of chain reshuffling. This technique allows the production of antibodies and antibody fragments with a ^10-9 M region affinity. The nucleic acid and amino acid sequences for CD99 are known in the art and are shown in Table 2A herein. A nucleic acid sequence encoding CD99 can be designed using the amino acid sequence of a desired region of CD99.

A nucleic acid encoding CD99 can be prepared by various methods known in the art. These methods include, but are not limited to, Engels et al., Agnew. Chem. Int. Ed. Engl. 28, such as triester method, phosphite method, phosphoramidite method and H-phosphonate method. : 716-734 (1989), including chemical synthesis by any of the methods described. In one embodiment, codons preferred for expression host cells are used in the design of DNA encoding CD99. Alternatively, DNA encoding CD99 can be isolated from a genomic library or a cDNA library.
Following construction of a DNA molecule encoding CD99, the DNA molecule is operably linked to expression control sequences in an expression vector, such as a plasmid, which regulatory sequences are expressed by host cells transformed with the vector. Be recognized. In general, plasmid vectors contain replication and regulatory sequences which are derived from species compatible with the host cell. This vector usually contains a replication site as well as a sequence encoding a protein capable of conferring phenotypic selection in transformed cells. Suitable vectors for expression in prokaryotic and eukaryotic host cells are known in the art, some of which are further described herein. Cells derived from eukaryotes such as yeast or multicellular organisms such as mammals can be used.
In some cases, the DNA encoding CD99 is operably linked to a secretory leader sequence to allow secretion of the expression product into the medium by the host cell. Examples of secretory leader sequences include stII, ecotin, lamB, herpes GD, lpp, alkaline phosphatase, invertase and alpha factor. The 36 amino acid leader sequence of protein A is also suitable for use herein (Abrahmsen et al., EMBO J. 4: 3901 (1985)).

Host cells are transfected, preferably transformed, with the above-described expression or cloning vectors of the present disclosure, and if necessary, modify the inducible promoter, select for transformed cells, or encode the desired sequence. The gene is amplified and cultured in normal nutrient media.
Transfection refers to the uptake of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed. Many methods of transfection are known to those skilled in the art, such as CaPO ₄ precipitation and electroporation. In general, successful transfection is recognized when any sign of operation of the vector is seen in the host cell. Methods of transfection are known in the art, some of which are further described herein.
Transformation means that DNA is introduced into an organism, thereby allowing the DNA to replicate with either extrachromosomal factors or chromosomal integrants. Depending on the host cell used, transformation is done using standard methods appropriate to such cells. Methods of transformation are known in the art, some of which are further described herein.
Prokaryotic host cells used for the production of CD99 can be cultured as generally described in Sambrook et al. (Supra). Mammalian host cells used for production of CD99 can be cultured in a variety of media, which are known in the art, some of which are described herein. Host cells referred to in this disclosure include not only cells in in vitro culture, but also cells in the host animal.

Purification of CD99 can be accomplished using methods apparent to those skilled in the art, some of which are described herein. Purified CD99 is agarose beads, acrylamide beads, glass beads, cellulose, various acrylic acid copolymers, hydroxymethacrylate gels, polyacrylic acid copolymers, for use in affinity chromatography separation of phage display clones, It can be adsorbed on a suitable matrix such as polymethacrylic acid copolymer, nylon, neutral carrier, ionic carrier. Adsorption of CD99 protein to the matrix can be achieved by the method described in Methods in Enzymology, vol. 44 (1976). A commonly used technique for adsorbing protein ligands to polysaccharide matrices such as agarose, dextran or cellulose is the activation of the carrier with cyanogen halide followed by the primary aliphatic amine or fragrance of the peptide ligand to the activated matrix. Includes coupling of group amines.
Alternatively, CD99 expressed on host cells affixed to the adsorption plate can be used to coat the wells of the adsorption plate, can be used for cell sorting, and capture of streptavidin-coated beads. Can be conjugated to biotin and used in any other method known in the art for panning phage display libraries.

The phage library sample is contacted with immobilized CD99 under conditions suitable for binding of the adsorbent to at least a portion of the phage particles. Usually, conditions including pH, ionic strength, temperature, etc. are selected to mimic physiological conditions. The phage bound to the solid phase is washed and then eluted with acid as described, for example, in Barbas et al., Proc. Natl. Acad. Sci. USA 88: 7978-7982 (1991) Or, for example, eluted by alkali as described in Marks et al., J. Mol. Biol. 222: 581-597 (1991) or, for example, Clackson et al., Nature 352: 624- Elution by CD99 antigen competition in a procedure similar to the antigen competition method of 628 (1991). Phages can be enriched 20-1,000 times in a single round of selection. Moreover, the enriched phage can be grown in bacterial culture and subjected to further rounds of selection.
The efficiency of selection depends on many factors, including the dissociation kinetics during washing and whether multiple antibody fragments on a single phage can simultaneously engage the antigen. Antibodies with fast dissociation kinetics (and weak binding affinity) can be retained by short washes, multivalent phage display and high coating density of antigen in the solid phase. The high density not only stabilizes the phage through multivalent interactions, but also supports rebinding of dissociated phage. The selection of antibodies with slow dissociation kinetics (and excellent binding affinity) has been described by Bass et al., Proteins, 8: 309-314 (1990 and WO 92/09690 as described in The use of phage display can be further facilitated by the use of a low antigen coating density as described in Marks et al., Biotechnol. 10: 779-783 (1992).

  Selection is possible among phage antibodies with different affinities for CD99, even if the affinity difference is slight. However, random mutations of selected antibodies (such as those used in some of the affinity maturation techniques described above) are prone to many variants, mostly binding antigens, but higher affinity. There are few things that have sex. If CD99 is restricted, rare high affinity phage may not be able to compete. To retain all higher affinity variants, the phage can be incubated with a concentration of biotinylated CD99 that is an excess of biotinylated CD99 but at a lower molar concentration than the target molar affinity constant of CD99. . High affinity binding phage can then be captured on streptavidin coated paramagnetic beads. Such “equilibrium capture” allows antibodies to be isolated according to their binding affinity with a sensitivity that allows isolation of mutant clones with only twice the affinity from a large excess of low affinity phage. It becomes possible to select. The conditions used to wash the phage bound to the solid phase can also be manipulated to distinguish based on dissociation kinetics.

Fv clones corresponding to the CD99 antibody were (1) isolated from the phage library as described above from the CD99 clone and optionally isolated from the phage clone by growing the population in a suitable bacterial host. Amplify the population; (2) select the CD99 and second protein for which blocking and non-blocking activities are desired, respectively; (3) adsorb the anti-CD99 phage clone to the immobilized CD99; (4) excess second The protein is used to recognize overlapping CD99 binding determinants or elute any unwanted clones in common with the second protein binding determinants; (5) Following step (4), the adsorbed It is possible to select by eluting the remaining clones. In some cases, clones having the desired blocking / non-blocking properties can be further enriched by repeating the selection procedure described herein one or more times.
The DNA encoding the hybridoma-derived monoclonal antibody or phage display Fv clone of the present disclosure is designed using conventional methods (e.g., to specifically amplify the heavy and light chain coding regions of interest from the hybridoma or phage DNA template). Can be easily isolated and sequenced). Once isolated, the DNA can be inserted into an expression vector, which is then E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that would normally not produce immunoglobulin proteins. In the recombinant host cell to obtain the desired monoclonal antibody composite. Review of recombinant expression in bacteria of DNA encoding the antibody includes Skerra et al., Curr. Opinion in Immunol. 5: 256 (1993) and Pluckthun, Immunol. Revs. 130: 151 (1992) It is.

  DNA encoding the Fv clone of the present disclosure is ligated to a known DNA sequence encoding a heavy and / or light chain constant region (eg, suitable DNA sequences can be obtained from Kabat et al., Supra). Thus, a clone encoding a full-length or partial-length heavy chain and / or light chain can be formed. For this purpose, constant regions of any isotype can be used, including IgG, IgM, IgA, IgD and IgE constant regions, and such constant regions can be obtained from any human or animal species It will be clear that it can. Derived from the variable domain DNA of one animal (such as human) species and then fused to the constant region DNA of another animal species to produce a "hybrid" full-length heavy and / or light chain coding sequence (single Fv clones that form (or more) are included herein in the definition of “chimeric” and “hybrid” antibodies. In a preferred embodiment, Fv clones derived from human variable DNA are fused to human constant region DNA to form all human full-length or partial-length heavy and / or light chain coding sequence (s). The

The DNA encoding the hybridoma-derived anti-CD99 antibody of the present disclosure can also be modified, for example, by replacing human heavy and light chain constant domain coding sequences in place of homologous mouse sequences from hybridoma clones. (For example, by the method of Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)). DNA encoding an antibody or fragment derived from a hybridoma or Fv clone can be further modified by covalently linking all or part of the coding sequence of the non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this way, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of an Fv clone or hybridoma clone-derived antibody of the present disclosure.
The present disclosure further contemplates a chimeric derivative of an anti-CD99 antibody comprising a non-human animal variable region and a human constant region. A chimeric antibody molecule can comprise, for example, an antigen binding domain from a mouse, rat, camel or other species of antibody comprising a human constant region. Various approaches for preparing chimeric antibodies have been described and can be used to prepare chimeric antibodies comprising immunoglobulin variable regions that recognize the extracellular domain of CD99 on the surface of AML and / or MDS cells. . For example, Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851 (1985); Takeda et al., Nature 314: 452 (1985); Cabilly et al., US Patent No. 4,816,567; Boss et al., US Patent No. 4,816,397; Tanaguchi et al., European Patent Application EP171496, European Patent Application 100173494, British Patent GB2177096B and PCT Patent Publication WO2013 / 064700, each incorporated herein by reference.

In certain aspects, anti-CD99 antibodies within the scope of this disclosure include Fab, Fab ′ and F (ab ′) 2 ′, Fd, single chain Fv (scFv), single chain antibodies, disulfide linked Fv (sdFv) and Includes human antigen-binding antibody fragments, such as fragments containing either VL or VH domains. CD99 binding antibody fragments, including single chain antibodies, can be variable alone or in combination with the following: hinge region, C _H1 , C _H2 , C _H3 and C _L domain all or part, variable region (s) Can be included. Also included are CD99 binding fragments comprising any combination of the hinge region, C _H1 , C _H2 , C _H3 and C _L domains and variable region (s). The antibody can be a human, mouse, rat, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken antibody. As used herein, a “human” antibody includes an antibody comprising the amino acid sequence of a human immunoglobulin, and is an antibody isolated from a human immunoglobulin library, an antibody isolated from a human B cell, or one or more humans Antibodies isolated from transgenic animals that express immunoglobulins are included. The production of camel antibodies is described, for example, in PCT patent publication WO2013 / 064700.

As described herein, an anti-CD99 antibody that can be suitably used in the method comprises aggregation, clustering and / or capping of antibody-bound CD99 on the surface of AML and / or MDS cells. An anti-CD99 antibody that can confirm at least one of the promoting properties. Thus, it will be apparent that properties such as aggregation, clustering and / or capping of antibody bound CD99 are facilitated by using bispecific or multispecific anti-CD99 antibodies.
As used herein, each of the terms “bispecific antibody” and “bifunctional antibody” refers to at least one first antigen binding site specific for a first epitope, antigen or hapten and a second epitope, antigen Alternatively, it refers to an antibody that recognizes two different antigenic epitopes of the CD99 extracellular domain by having at least one second antigen binding site specific for a hapten.

  Such antibodies can be prepared by recombinant DNA methods and can include, but are not limited to, antibodies that are chemically produced by methods known in the art. Bispecific antibodies include all antibodies or antibody conjugates or antibody polymers that are capable of recognizing two different CD99 epitopes. Bispecific antibodies include antibodies that have been reduced and modified to retain their bivalency and antibodies that are chemically conjugated so that they can have multiple antigen recognition sites for CD99.

By way of non-limiting example, the bispecific antibody used in the method can bind to at least one of the CD99 epitopes described herein and can bind to more than one of such CD99 epitopes. it can. In some embodiments, one of the antibody specificities has a low affinity of, for example, a binding constant of less than about 10 ⁻⁹ , typically a binding constant of less than about 10 ⁻⁸ , and greater than about 10 ⁻⁷ . It can be a coupling constant.
Suitable antibodies for performing the methods of the present disclosure can be bispecific, trispecific or more multispecific. Furthermore, the antibodies of the present disclosure can have low risk toxicity against granulocytes (neutrophils), NK cells and CD4 + cells as bystander cells.
Methods for producing bispecific antibodies are known in the art. Traditional methods for producing full-length bispecific antibodies are based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities. Millstein et al., Nature 305: 537-539 (1983). These chains can be linked by a linker as described below. A commonly used suitable linker is (GGGGS) ₃ .

Because of the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a mixture of various antibody molecules. Purification of antibodies having the desired combination of anti-CD99 epitope binding specificities can be performed by an affinity chromatography step. Related separation methods are disclosed in International Publication No. WO1993 / 08829 and EMBO J. 10: 3655-3659 (1991).
Bispecific antibodies are designed to aggregate two variable regions (a first variable region specific for the first CD99 epitope and a second variable specific for the second CD99 epitope) to aggregate CD99 expressed on the surface of AML and / or MDS cells. 2 variable regions). Such bispecific antibodies are similar to the single-chain bispecific T cell engager (BiTE®) antibody described in Bassan, Blood 120 (26): 5094-5095 (2012) The breakthrough described in Topp et al., Blood 120 (26): 5185-5187 (2012) and Loffler et al., Blood 95 (6): 2098-2103 (2000) The typical pharmaceutical CD19x CD3 BiTE antibody is typically shown by Blinatumomab.

BiTE® antibodies consist of two different single chain Fv fragments having the structure V _L1 -V _H1 -V _H2 -V _L2 on a single peptide chain, linked by a glycine-serine linker. BiTE antibody constructs are generally on the order of 60 kDa in size, expressed in mammalian cells such as Chinese hamster ovary (CHO) cells, and secreted in a fully active form and in high yield without the need for reconstitution. It can be purified by terminal histidine tag. BiTE® antibodies can be designed to exhibit high levels of cytotoxic activity at concentrations as low as 10-100 pg / ml and at a 2: 1 effector to target cell ratio.
Antibody VL chain (VL) and VH chain (VH) domains can be cloned by standard polymerase chain reaction (PCR) methods. cDNA can be synthesized with oligo dT primers and reverse transcriptase as described in Dubel et al., J. Immunol. Methods 175: 89-95 (1994), where the V domain is Amplification can be achieved by PCR with adjacent primers 5'L1 and 3'K and primers 5'H1 and 3'G based on the H chain.

The VL and VH regions are placed on separate plasmid vectors as templates for VL-specific and VH-specific PCR. It can be cloned using 5'VHGS15 (GGCGGCGGCG GCTCCGGTGG TGGTGGTTCT CAGGT (GC) (AC) A (AG) C TGCAG (GC) AGTC (AT) GG) / 3'VHBspE1 (AATCCGGAGG AGACGGTGAC CGTGGTCCCT TGGCCCCAG). In subsequent fusion PCR, a complementary sequence that overlaps the PCR product can be introduced to bind and form the coding sequence for the 15 amino acid (G4S1) ₃ linker. This amplification step can be performed using the primer pair 5'VLB5RRV / 3'VHBspEa, and the resulting fusion product (scFv fragment) is cleaved with restriction enzymes (e.g., EcoRV and BspE1) to produce a single bispecific It can be cloned into a plasmid vector (eg, bluescript KS vector; Stratagene, La Jolla, Calif.) Containing the coding sequence of the chain antibody. The resulting bispecific single chain antibody can be subcloned into an expression vector (eg, pEF-DHFR) and transfected, eg, by electroporation and selection. Bispecific antibodies can be prepared using a nickel-nitrilotriacetic acid (Ni-NTA) column (Qiagen) as described in Mack et al., Proc. Natl. Acad. Sci. USA 92: 7021-7025 (1995). Can be purified by its C-terminal H-tail using affinity chromatography (Hilden, Germany).

According to another approach described in International Publication No. WO1996 / 27011, the interface between a pair of antibody molecules is manipulated to maximize the percentage of heterodimers recovered from recombinant cell culture. Can do. Such surfactants can include at least a portion of the C _H3 domain of an antibody constant domain. By this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (eg tyrosine or tryptophan). By replacing a smaller amino acid side chain (eg alanine or threonine) with a larger amino acid side chain, the compensatory “cavity” of the same or smaller size for the larger side chain (s) at the interface of the second antibody molecule. Is made. This provides a mechanism to increase the yield of the heterodimer over other undesirable end products such as homodimers. An alternative method binds two different single chain variable regions to a heat stable antigen (HSA). The use of HSA as a linker increases the serum half-life, and therefore has the advantage of low immunogenicity.

Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be linked to avidin and the other linked to biotin. Such antibodies can be used, for example, to target unwanted cells to immune system cells (US Pat. No. 4,676,980) and to treat HIV infection (International Publication Nos. WO1991 / 100360 and WO1992 / 200373). European patent EP03089) has also been proposed. Heteroconjugate antibodies can be produced using any convenient cross-linking method. Suitable crosslinking agents are known in the art and are disclosed in US Pat. No. 4,676,980, along with a number of crosslinking techniques.
Techniques for making bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Breunanet et al., Science 229: 81 (1985) describes a procedure in which intact antibodies are proteolytically cleaved to generate F (ab ′) 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The produced Fab ′ fragment is then converted to a thionitrobenzoate (TNB) derivative. One of the Fab′-TNB derivatives is then converted to Fab′-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of other Fab′-TNB derivatives to produce bispecific antibodies.

Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins were linked to the Fab ′ portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers.
The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provides an alternative mechanism for producing bispecific antibody fragments. The fragment contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites.
Other strategies for the production of bispecific antibody fragments using single chain Fv (sFv) dimers have also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994). Alternatively, the antibody can be a “linear antibody” as described in Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem F _d segments (VH "C _H1 -VH" C _H1 ) that form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

Within the context of this disclosure, anti-CD99 antibodies include monoclonal and polyclonal antibodies, antibody fragments (e.g., Fab and F (ab ') 2), chimeric antibodies, bifunctional or bispecific antibodies and tetrameric antibody conjugates. It is known to contain the body.
An antibody is considered to be reactive to CD99 on the surface of a cell if it binds with an appropriate affinity (association constant), eg, an association constant greater than 10 ⁷ M ⁻¹ . Furthermore, antibodies that can be used in the methods of the present disclosure are also 5x10 ^-2 M, 10 ^-2 M, 5x10 ^-3 M, 10 ^-3 M, 5x10 ^-4 M, 10 ^-4 M, 5x10 ^-5 M , 10 ^-5 M, 5x10 ^-6 M, 10 ^-6 M, 5x10 ^-7 M, 10 ^-7 M, 5x10 ^-8 M, 10 ^-8 M, 5x10 ^-9 M, 10 ^-9 M, 5x10 ^-10 M , 10 ^-10 M, 5x10 ^-11 M, 10 ^-11 M, 5x10 ^-12 M, 10 ^-12 M, 5x10 ^-13 M, 10 ^-13 M, 5x10 ^-14 M, 10 ^-14 M, 5x10 ^-15 M And their binding affinities, including binding affinities with dissociation constants or K _d of less than 10 ⁻¹⁵ M.
Antibodies are fragmented using conventional methods, and the fragments can be screened for binding activity in the same manner as described above for full-length antibodies. For example, F (ab ′) 2 fragments can be generated by treating antibody with pepsin. The F (ab ′) 2 fragment obtained to reduce disulfide bridges can be processed to generate Fab ′ fragments.
Chemical linkage is based on the use of E-amino groups or hinge region thiol groups and homo- and heterobifunctional reagents. Homobifunctional reagents such as 5,5'-dithiobis (2-nitrobenzoic acid) (DNTB) produce a disulfide bond between two Fabs, and O-phenylene dimaleimide (O-PDM) A thioether bond is formed between Fabs. Breuner et al., (1985) and Glennie et al., (1987). Heterobifunctional reagents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), bind the exposed amino group of the antibody to the Fab fragment, regardless of class or isotype. Van Dijk et al., (1989).

In particular, but not limited to, peptide or polypeptide linkers that mimic the hinge region of the Fc domain of an antibody can also be used. See, for example, US Pat. No. 7,928,072, which is incorporated by reference in its entirety for the disclosure of the appropriate class of linkers. Anti-CD99 antibodies of the present disclosure, i.e., antibodies useful for treating AML and / or MDS as well as AML and / or MDS stem cells, such as leukemic and hematopoietic stem cells, expressing CD99 are covalently bound. Including derivatives modified by covalent attachment of any type of molecule to the antibody, so long as the antibody does not prevent binding to its cognate epitope (s) on the extracellular domain of CD99. For example, antibody derivatives are modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage, binding to cellular ligands or other proteins, etc. Antibodies may be included. Any of a number of chemical modifications can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. Furthermore, the derivative can comprise one or more non-classical amino acids.
The anti-CD99 antibodies described in this disclosure can be conjugated to one or more cytotoxic compounds to further promote antibody cytotoxicity. For example, antibody drug conjugates (ADCs) are antibodies (such as whole mAbs or single chain antibodies [scFv]) linked by a chemically stable linker with a cytotoxic payload or a labile bond to the drug. Fragment) is one type of bioconjugate / immunoconjugate. Antibody-drug conjugates promote the overall cytotoxic activity and target specificity of the cytotoxicity by combining the anti-CD99 antibody's binding and cytotoxic activity with CD99 and the cytotoxic ability of the cytotoxic agent . Due to this targeting, this cytotoxin shows reduced side effects, and this antibody shows a further improvement in the therapeutic concentration range.

The cytotoxin can be linked to the antibody by a stable bond between the anti-CD99 antibody and the cytotoxin. Linkers can be based on chemical motifs including disulfides, hydrazones or peptides (cleavable) or thioethers (non-cleavable), which regulate the distribution and delivery of cytotoxic agents to target cells. The cleavable and non-cleavable types of linkers have proven safe in preclinical and clinical trials. For example, brentuximab vedotin can be cleaved with enzyme sensitivity to deliver the potent and highly toxic microtubule inhibitor monomethyl auristatin E (MMAE), a synthetic antineoplastic agent against human-specific CD30 positive malignant cells A simple linker. Due to its high toxicity of inhibiting cell division by inhibiting tubulin polymerization, MMAE cannot be used as a single agent chemotherapeutic agent. The combination of MMAE conjugated to anti-CD30 monoclonal antibody is stable in extracellular fluid, cleavable by cathepsin in vivo, and thus safe for treatment. Trastuzumab emtansine is another approved ADC, the microtubule formation inhibitor meltansine (DM-1) (a derivative of maytansine) and the antibody trastuzumab (Herceptin®) joined by a stable, non-cleavable linker / Genentech / Roche).
The type of linker, whether cleavable or non-cleavable, confers specific properties on the cytotoxin. For example, a non-cleavable linker retains the drug within the cell. As a result, whole antibodies, linkers and cytotoxins can enter target cells where they are broken down into their constituent amino acids. The resulting amino acids, linkers and cytotoxins are thereby activated. In contrast, cleavable linkers are cleaved enzymatically in vivo to release cytotoxins. This cytotoxin can exit the target cell and kill neighboring cells.

Detection of anti-CD99-mediated aggregation, clustering and / or capping of CD99 expressed on the surface of AML and / or MDS cells In certain aspects, the disclosure provides for CD99 expressed on the surface of AML and / or MDS cells. Imaging methods for detecting anti-CD99 mediated aggregation, clustering and / or capping are provided.
For example, immunofluorescence can be adapted for use to detect and localize CD99 on cell surfaces. AML or MDS cells are incubated with anti-CD99 monoclonal antibody for 3-6 hours, then fixed with paraformaldehyde, and a secondary antibody labeled with a fluorescent dye that binds to the constant domain of the anti-CD99 antibody by its isotype is added. The cells are then placed on a slide and images are obtained by confocal microscopy or wide field microscopy. Using this technique, cell surface CD99 molecules can be evaluated for aggregation, clustering and / or capping by addition of various anti-CD99 antibodies.
Other methods that can be adapted for use in the detection of anti-CD99-mediated aggregation, clustering and / or capping of antibody-bound CD99 are also readily available in the art, for example, flow cytometry methods that bind antibody It can be used for CD99 confocal imaging. For example, Amnis (Seattle, WA) offers a high-performance system (FlowSight®) that includes 12 standard detection channels that simultaneously produce bright and dark fields and 10 channels of fluorescent images of individual cells. ing.

Detection of anti-CD99-mediated cytotoxicity and apoptosis of AML and / or MDS cells exhibiting increased expression of CD99 In other aspects, the present disclosure provides AML by detecting anti-CD99-mediated apoptotic AML and / or MDS cells And / or a method of assessing cytotoxicity of anti-CD99 antibodies in MDS cells. For example, in vitro, serum-free media (for primary patient samples) or media with serum inactivated complement to test cytotoxicity of anti-CD99 antibodies against AML or MDS cell lines or primary patient samples Cells can be incubated with anti-CD99 antibody in it. For primary patient samples, the medium can be supplemented with cytokines including stem cell factor, thrombopoietin, IL-3 and IL-6. The amount of antibody can be titrated in the range of 100 ng / ml to 20 μg / ml. After 72 hours, the absolute number of viable cells can be determined by manual cell counting with a hemacytometer using trypan blue as the viability exclusion dye, or single bead enhancement using propidium iodide or DAPI as the viability exclusion dye. -enhanced) can be counted by cytofluorimetry (Montes et al., J. Immunol. Methods 317: 45 (2006)).

  To evaluate in vitro apoptosis, experimental conditions similar to those described above can be used, except for a shorter time course (8-24 hours). During this time frame, induction of apoptosis by anti-CD99 antibodies can be assessed by annexin V / 7-AAD staining when assessed by flow cytometry, whereas annexin V + 7-AAD- cells are early apoptotic cells. Annexin V + 7-AAD + cells are late apoptotic cells. Apoptotic cells can also be detected by cell fixation and permeabilization followed by intracellular labeling using a fluorochrome-labeled antibody that detects activated caspase-3.

Compositions and formulations comprising anti-CD99 antibodies The present disclosure provides compositions comprising therapeutic compositions comprising one or more anti-CD99 antibodies for the treatment of AML and / or MDS associated with elevated CD99 gene expression levels To do. One or more anti-CD99 antibodies are administered to a human patient as a pharmaceutical composition in which each antibody is mixed with an appropriate carrier or excipient at a dose to treat or alleviate AML and / or MDS as described herein. Can be done. As a pharmaceutical composition, a mixture of anti-CD99 antibodies can also be administered to a patient.
Compositions within the scope of the present disclosure include therapeutic agents (1) ligated to the extracellular domain of CD99 displayed on the cell surface associated with AML and / or MDS, and (2) on AML and / or MDS cells. Mediate clustering and aggregation of ligated CD99, and (3) promote ligated CD99 cytotoxicity of antibodies on AML and / or MDS cells, for example, by inducing apoptosis of cells ligated with antibodies A composition that is an effective amount of an anti-CD99 antibody.
Determination of the optimal range of effective amounts of each component is within the skill of those in the art. The effective amount is a function of several factors including the age and clinical status of the patient, as well as the particular anti-CD99 antibody used. The dosage will depend on the age, health and weight of the recipient, the type of combination treatment (if any), the frequency of treatment and the nature of the desired effect.

A composition comprising an anti-CD99 antibody can be administered parenterally. As used herein, the term “parenteral administration” refers to administration methods other than enteral administration and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal. Including intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, epidermal, intraarticular, subcapsular, intrathecal, intrathecal and intrasternal injection and infusion.
A composition comprising an anti-CD99 antibody can be administered intravenously, for example, by intravenous push or bolus. Alternatively, a composition comprising an anti-CD99 antibody can be administered by intravenous infusion.
Suitable doses for intravenous infusion of a composition comprising an anti-CD99 antibody are at least about 2 mg anti-CD99 antibody / m2 / day or at least about 10 mg anti-CD99 antibody / m2 / day or at least about 20 mg anti-CD99 antibody / m2 / A dose of at least about 50 mg anti-CD99 antibody / m2 / day or at least about 100 mg anti-CD99 antibody / m2 / day or at least about 200 mg anti-CD99 antibody / m2 / day or at least about 500 mg anti-CD99 antibody / m2 / day.

The composition comprising one or more anti-CD99 antibodies can further comprise one or more additional compounds such as, for example, chemotherapeutic compounds for the treatment of AML and / or MDS. For example, induction chemotherapy for AML can include cytarabine (ara-C) and an anthracycline such as daunorubicin or idarubicin. Cytarabine can be administered by continuous intravenous infusion for 7 consecutive days, whereas anthracycline is generally administered as an IV push for 3 consecutive days. Acute promyelocytic leukemia, an AML subtype, is often treated with all-trans retinoic acid (ATRA) in combination with anthracycline and / or arsenic trioxide. The compositions of the present disclosure can also include an anti-CD99 antibody immunoconjugate crosslinked to a cytotoxin similar to gemtuzumab ozogamicin (Myrotag).
For MDS, one or more anti-CD99 antibodies may require erythrocyte transfusion in patients with hematopoietic factors (such as erythropoietin) or, in particular, MDS subtypes lacking chromosome 5q, as well as other low-risk subtypes of MDS Three drugs (5-azacytidine, decitabine (alone or in combination with valproic acid) and lenalidomide approved by the US Food and Drug Administration (FDA) for the treatment of MDS that are effective in reducing sex One of these can be used in combination.

A composition comprising an anti-CD99 antibody generally comprises a therapeutically effective amount of the antibody and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable” is approved for use in animals, more specifically in humans, by federal or state regulatory authorities, or in the United States Pharmacopoeia or Means described in other commonly known pharmacopoeias. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene , Glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting or emulsifying agents or pH buffering agents as required.
These compositions can take the form of solutions, suspensions, emulsions, powders, and the like. These compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate. Such compositions comprise a therapeutically effective amount of the anti-CD99 antibody, preferably as a purified product, with an appropriate amount of carrier, in order to provide a form suitable for administration to a patient. The formulation must be suitable for the method of administration.

The composition can be formulated according to a conventional method as a pharmaceutical composition adapted for intravenous administration to humans. In general, compositions for intravenous administration are solutions of sterile isotonic aqueous buffer or other physiologically acceptable buffer. If necessary, the composition can also include a solubilizer and a local anesthetic such as lignocaine to reduce pain at the site of the injection. In general, the ingredients are supplied in unit dosage form separately or mixed as a lyophilized powder or anhydrous concentrate, for example, in a sealed container such as an ampoule or sachet in which the amount of active agent is indicated. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
The anti-CD99 antibodies disclosed herein can be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine , Salts formed with cations derived from 2-ethylaminoethanol, histidine, procaine and the like.

Many of the anti-CD99 antibodies of the present disclosure can be provided as salts with pharmaceutically compatible counterions (ie, pharmaceutically acceptable salts). “Pharmaceutically acceptable salt” refers to any non-toxic salt that can provide, directly or indirectly, an antibody or composition of the present disclosure upon administration to a recipient.
A “pharmaceutically acceptable counterion” is an ionic portion of a salt that is non-toxic when released from the salt upon administration to a subject. Pharmaceutically compatible salts can be formed with many acids, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, and succinic acid. Salts tend to be more soluble in water or other protic solvents than their corresponding free base forms. The compositions of the present disclosure can include such salts.
The compositions described in this disclosure can be used ex vivo according to known methods, for example, to remove autologous bone marrow CD99 ⁺ tumor cells (and CD99 ⁺ tumor stem cells). See, for example, Robertson et al., 79: 2229-2236 (1992).

Methods of Detecting Increased CD99 Expression in AML and MDS Cells The present disclosure is directed to cytotoxic activity against AML and / or MDS tissues or cells exhibiting elevated CD99 cell surface expression as well as antibodies and compositions thereof And / or methods of using one or more antibodies or antibody compositions exhibiting apoptosis-inducing activity. Thus, the present disclosure addresses the extent of CD99 gene expression, including the relative number of CD99 protein molecules exposed on the surface of AML and / or MDS tissues or cells, as well as the relative activity of CD99 gene transcription and CD99 transcript levels. A method for assessing an increase in CD99 expression based also on quantification of the protein is provided. An exemplary method for assessing elevated CD99 expression is presented in this section.

MRNA from AML and / or MDS patient tissue samples or cells and non-AML and / or MDS control tissue samples or cells to identify AML and / or MDS patient tissue samples or cells exhibiting elevated CD99 gene expression MRNA can be measured, the level of expression of a given mRNA can be measured, and mRNA measured for tissue samples or cells of AML and / or MDS patients and non-AML and / or MDS control tissue samples or cells By comparing levels, an increase in gene expression can be assessed.
Alternatively, an AML and / or MDS patient tissue sample or cell exhibiting elevated CD99 gene expression isolates mRNA from the AML and / or MDS patient tissue sample or cell and measures the level of CD99 mRNA and control mRNA; CD99 mRNA relative to control mRNA levels can be identified by assessing elevated gene expression by comparing CD99 mRNA levels in leukemia tissue samples or cells with control mRNA levels and determining the ratio of mRNA expression. An increase in the ratio of levels indicates an increase in the CD99 gene expression level. As used in this context, control mRNA refers to mRNA from a gene that does not show elevated levels of expression of AML and / or MDS in tissues or cells. Suitable control mRNAs include, for example, β-actin, GAPDH and cyclophilin.

Suitable AML and / or MDS tissue samples include, for example, blood, lymph node, bone marrow and / or tumor biopsy samples from AML and / or MDS patients. Suitable non-AML and / or MDS control tissue samples include, for example, blood, lymph node and / or bone marrow samples from non-AML and / or MDS donors such as disease-free healthy donors. Such blood, lymph node and / or bone marrow samples from non-AML and / or MDS donors typically contain CD34 ⁺ cells. It will be appreciated that, regardless of the exact nature or origin of the donor tissue sample or cells, it is essential that the donor tissue or cells are known not to exhibit increased CD99 gene expression.
Suitable AML and / or MDS cells include, for example, myeloid progenitor cells. Suitable non-AML and / or MDS control cells, particularly non-AML and / or MDS CD34 ⁺ control cells are, for example, myeloid progenitor cells from non-AML and / or MDS donors (such as disease-free healthy donors) or, for example, Kasumi-1 Includes one or more cell lines such as the CD34 ⁺ cell line including cell lines. It will be understood that regardless of its origin or identity, suitable non-AML and / or MDS control tissue samples or cells are characterized by no increase in CD99 gene levels.

AML cell lines Almost all AML cell lines tested were sensitive to anti-CD99 mAbs. In some embodiments, one or more AML cell lines are used in the disclosed methods, wherein the one or more cell lines are Kg1a, SET2, Meg1a, THP-1, NB4, AML14, KBM5, AML5Q, KCL22. , MOLM14, HL60, K562, U937, KU812, MOLM13, Monomac and NOMO1.
FIG. 10A is a bar graph showing the relative cell number for the 15 AML cell lines tested for cell growth inhibition after 48 hours incubation with anti-CD99 antibody 12E7. Eleven of the 15 AML cell lines tested, AML5Q, NB4, KCL22, MOLM13, HL60, MOLM14, NOMO1, U937, MonoMac1, KG1a and AML14 are sensitive to growth inhibition after CD99 ligation with 12E7 antibody. It was found to show. The four cell lines tested, KBM5, SET2, KU812 and K562, were characterized as insensitive to CD99 ligation with the 12E7 antibody.

FIG. A bar graph (lower panel) showing the relative cell number after incubation is shown. HL60 is a typical “sensitive” cell line that exhibits high expression of CD99 and low expression of c-kit. “Resistant” cell lines that do not express CD99 (K562 and KBM5) or express high levels of c-kit (SEM2 and KU812) are Bcr-abl positive (K562 / KBM5 / KU812), or JAK2 is positive (SEM2). These data indicate that low expression of CD99 or high expression of c-kit is predicted to be resistant to cytotoxicity mediated by anti-CD99 antibody 12E7.
FIG. 10C is a graph of transendothelial migration kinetics of CD99 ⁺ AML cells in the presence of anti-CD99 antibodies 12E7, HEC2, DN16 and H036. This graph shows that in the absence of secondary antibodies, certain anti-CD99 antibodies do not suppress transendothelial migration of AML cells known to be mediated by CD99.
In another experiment, 17 AML cell lines were exposed to anti-CD99 mAb H036-1.1. All 17 AML cell lines responded to the anti-CD99 monoclonal antibody H036-1.1, and for the majority (11/17) the response was complete at the dose tested. The decrease in the number of cells after antibody exposure ranged from 2.74 to 370.5, as shown in Table 2B.

Table 2B AML cell lines sensitive to anti-CD99 mAb H036-1.1

In this experiment, the 17 AML cell lines tested (Kg1a, SET2, Meg1a, THP-1, NB4, AML14, KBM5, AML5Q, KCL22, MOLM14, HL60, K562, U937, KU812, MOLM13, Monomac and NOMO1) All were found to be sensitive to growth inhibition after CD99 ligation with anti-CD99H036-1.1 antibody (Abcam). The four cell lines tested (Kg1a, SET2, KCL22 and K562) were found to be somewhat sensitive to growth inhibition by CD99 ligation with the H036-1.1 antibody.
Next, the gene expression level quantification method that can be easily adapted to detect the increase in the expression of the CD99 gene will be described in more detail.

Microarray analysis
Increased CD99 gene expression can be detected and quantified by microarray analysis of RNA isolated from tissue samples or cells of AML and / or MDS patients and / or control donors. However, due to its limited sensitivity, the microarray method cannot accurately measure the absolute tissue distribution of low-abundance genes and underestimates the extent of CD99 gene expression increase due to signal saturation. There is also a risk of doing. For cells that show increased CD99 expression by microarray expression profiling, provide greater dynamic range of sensitivity, such as RT-PCR based on Taqman® probe detection (Invitrogen Life Sciences, Carlsbad, Calif.) Further analysis can be performed using one or more of the quantitative PCR methods.
Briefly, microarray analysis involves AML and / or MDS patients or controls with primer pairs that hybridize to coding sequences in the CD99 gene and / or coding sequences in non-CD99 genes whose expression is detected and / or quantified. Includes PCR amplification of RNA extracted from donor tissue samples or cells. PCR products are scattered on an array format slide, and each PCR product occupies a specific position in the array. The RNA is then reverse transcribed to produce a fluorescently labeled cDNA probe. The microarray is probed with a fluorescently labeled cDNA probe, the slide is scanned, and the fluorescence intensity is measured. The level of fluorescence intensity correlates with hybridization intensity, which correlates with the relative level of gene expression.

Analysis of CD99 gene expression can be performed using commercially available microarrays (eg U133A chip; Affymetrix, Santa Clara, Calif.) Or custom microarrays. Alternatively, CD99 gene expression was increased using a Synteni microarray (Palo Alto, Calif.) According to the manufacturer's instructions, using Schena et al., Proc. Natl. Acad. Sci. USA 93: 10614-10619 ( 1996) and Heller et al., Proc. Natl. Acad. Sci. USA 94: 2150-2155 (1997). Microarray hybridization can be performed according to the method described in Abraham et al., Blood 105: 794-803 (2005).
Probe level data can be normalized using commercially available algorithms (eg, Affymetrix Microarray Suite 5.0 algorithm) or custom algorithms. CD99 gene expression intensity values and non-CD99 gene expression intensity values are log-converted using a commercially available program (e.g., GeneSpring 7.3.1 GX; Agilent Technologies, Santa Clara, Calif.) Or a custom algorithm, and centered. The value can be placed and / or analyzed.

Several factors can be used to assess the quality of the CD99 gene expression analysis, such as the 3 ′: 5 ′ ratio of GAPDH and the 3 ′: 5 ′ ratio of actin. Samples that show poor quality results can be defined as having a 3 ′: 5 ′ ratio of GAPDH greater than about 1.25 and / or having a 3 ′: 5 ′ ratio of actin greater than about 3.0.
Increased CD99 gene expression is determined using Welch's ANOVA with variance values calculated by applying a cross-gene error model based on deviation from 1 available within GeneSpring be able to. This overcomes the lack of repetition and dispersion associated with individual samples, which can in principle be considered similar to dispersion filtering. Unsupervised clustering can be performed using a hierarchical aggregation algorithm. Pearson's correlation coefficient and centroid connection can be used as the similarity method and the connection method, respectively.
Statistically with a corrected P value of 0.05 by Student's t-test using a gene with a 1.5-fold differential expression between individual samples and / or Benjamini-Hochberg multiple test correction to detect possible differences between samples Can be extracted from the data set. Genes with different expression levels can be evaluated for gene ontology (GO) enrichment (eg, using GeneSpring).

Quantitative PCR
CD99 gene based on factors such as the relative number of AML and / or MDS cells present in the AML and / or MDS tissue sample and / or the level of CD99 gene expression in each AML and / or MDS cell in the tissue sample It may be preferable to perform quantitative PCR analysis to detect and / or quantify the level of expression.
For example, CD99 gene mRNA and / or non-HOX cluster / non-CD99 gene mRNA or corresponding cDNA from AML and / or MDS tissue samples or cells and / or non-AML and / or MDS control donor tissue samples or cells At least two oligonucleotide primers can be used in a PCR-based assay to amplify at least a portion of. At least one of the oligonucleotide primers is specific for and hybridizes to the mRNA encoded by the CD99 gene. In some cases, the amplified cDNA can be subjected to a fractionation step such as gel electrophoresis before detection.
RT-PCR is a quantitative PCR method in which PCR amplification is performed in combination with reverse transcription. RNA is extracted from tissue samples or cells such as blood, lymph nodes, bone marrow and / or tumor biopsy samples and it is reverse transcribed to produce cDNA molecules. The cDNA molecules can be amplified by at least one specific primer PCR amplification, which can be separated and visualized using, for example, gel electrophoresis. Amplification can be performed on tissue samples or cells taken from patients and controls not affected by cancer. Amplification reactions can be performed on several dilutions of cDNA spanning two orders of magnitude. At least about 3-fold, at least about 5-fold, at least about 10-fold in some test dilutions of AML and / or MDS patient samples compared to the same dilution of non-AML and / or MDS control donor sample Thus, an increase in expression of at least about 15 fold or more, at least about 20 fold or more, or at least about 25 fold or more is generally considered positive.

As used herein, the term “amplification” refers to the production of multiple copies of a target nucleic acid containing at least a portion of the intended specific target nucleic acid sequence. Multiple copies are used interchangeably with amplicon or amplification product. In certain aspects of the present disclosure, the target to be amplified is a complete target mRNA sequence (ie, spliced transcripts of exons and flanking untranslated sequences) and / or target genomic sequences (including introns and / or exons) Smaller part. For example, a particular amplicon can be prepared by amplifying a portion of a target polynucleotide using an amplification primer that hybridizes to an internal location of the target polynucleotide and initiates polymerization therefrom. The portion to be amplified includes a detectable target sequence that can be detected using any of a variety of known methods.
Many of the known nucleic acid amplification methods require a thermal cycle in which double-stranded nucleic acid denaturation and primer hybridization are alternately performed. However, other known methods of nucleic acid amplification are isothermal. Polymerase chain reaction (PCR; described in U.S. Pat.Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188) is an exponential function of denaturation, annealing of primer pairs to reverse strands and copy number of target sequences. Use multiple cycles of primer extension to increase the efficiency. In a variation called RT-PCR, reverse transcriptase (RT) is used to produce complementary DNA (cDNA) from mRNA, which is then amplified by PCR to produce multiple copies of the DNA.

CD99 gene expression can be further characterized or uniquely detected and / or quantified using quantitative real-time PCR methods. Gibson et al., Genome Research 6: 995-1001 (1996) and Heid et al., Genome Research 6: 986-994 (1996). Real-time PCR is a technique for evaluating the accumulation level of PCR products during the amplification process. This technique allows quantitative assessment of mRNA levels in multiple samples. By this method, AML and / or MDS tissue samples or cells can be obtained from corresponding non-AML and / or MDS control donor samples or cells and / or unrelated normal non-AML and / or MDS tissue samples or cells. Can be tested with the panel.
Real-time PCR can be performed, for example, by either ABI 7700 Prism or GeneAmp® 5700 sequence detection system (Applied Biosystems, Foster City, Calif.). The 7700 system uses forward and reverse primers in combination with a specific probe (Taqman®) with a 5 ′ fluorescent reporter dye at one end and a 3 ′ quencher dye at the other end. When real-time PCR is performed using Taq DNA polymerase having 5′-3 ′ nuclease activity, the probe is cleaved and starts to emit fluorescence, so that the reaction can be monitored by increasing fluorescence (real time). The 5700 system is a primer expression program (Applied Biosystems, Foster City, Calif.) That uses SYBR® green, a fluorescent dye that binds only to double-stranded DNA, and the same forward and reverse primers as the 7700 instrument. ), Matching primers and fluorescent probes can be designed. Optimal concentrations of primers and probes are initially determined by those skilled in the art. Control (eg, β-actin specific) primers and probes are commercially available from, for example, Perkin Elmer / Applied Biosystems (Foster City, Calif.).

In order to quantify the amount of CD99 gene expression in the sample, a standard curve is prepared using a plasmid containing the gene of interest. A standard curve is generated using Ct values measured by real-time PCR, which are related to the initial cDNA concentration used in the assay. If standard dilutions ranging from the gene of interest 10 to 10 ⁶ copies, is generally sufficient. In addition, a calibration curve is generated for the control sample sequence. This makes it possible to normalize the initial RNA content of AML and / or MDS tissue samples or cells to the amount of control tissue samples or cells for comparison purposes.
As described herein, Trizol reagent can be used to extract total RNA from AML and / or MDS tissue samples or cells and non-AML and / or MDS control tissue samples or cells. First strand synthesis can be performed using 1-2 μg of total RNA and using SuperScript II reverse transcriptase (Life Technologies, Carlsbad, Calif.) For 1 hour at 42 ° C. Then amplify the cDNA by PCR using CD99 gene specific primers designed based on CD99 mRNA or other sequences presented in Table 2A or other known CD99 gene specific primers readily available to those skilled in the art. Can do.

Households such as β-actin as an internal control for each of the tissue samples and / or cells of the AML and / or MDS patients being tested and non-AML and / or MDS control donors to ensure RT-PCR quantification A keeping gene can be used. Prepare serial dilutions of first strand cDNA and perform RT-PCR assay using β-actin specific primers. A dilution is then selected that exhibits a sensitivity that allows linear range amplification of the β-actin template and sufficiently reflects the difference in the initial copy number. Using these conditions, β-actin levels are measured for reverse transcription reactions originating from each tissue. DNA contamination is minimized by DNase treatment and by confirming negative PCR results using first strand cDNA prepared without the addition of reverse transcriptase.
In an exemplary RT-PCR reaction using Dynabeads mRNA direct microkit (Invitrogen, Life Sciences Technologies, Carlsbad, Calif.), H ₂ O14.25 μl; BSA 1.5 μl; first strand buffer 6 μl; 10 mM dNTP mix 0.75 mL Samples containing 10 ⁵ cells or less are tested in a total reaction volume of 30 μl containing; Rnasin 3 μl; 0.1 M dTT 3 μl; and Superscript II 1.5 μl. The resulting solution is incubated at 42 ° C. for 1 hour, diluted 1: 5 with H ₂ O and heated at 80 ° C. for 2 minutes to separate the cDNA from the beads and immediately placed on the MPS. Transfer the supernatant containing the cDNA to a new tube and store at -20 ° C.

RNA sequencing
Increased expression of the CD99 gene can be measured by direct sequencing of mRNA in AML and / or MDS patient tissue samples or cells and / or non-AML and / or MDS donor control tissue samples or cells. Alternatively, the increase in CD99 gene expression can be measured after conversion of mRNA into cDNA by reverse transcription.
True Single Molecule Sequencing (tSMS®) and / or Direct RNA Sequencing (DRS®) quantifies gene expression, which can be easily adapted to detect and quantify CD99 gene expression Is a useful technique for. These synthetic sequencing techniques can be performed on mRNA from tissue samples or cells without the need for prior reverse transcription or PCR amplification.
Transcriptome profiling using direct RNA sequencing technology (Helicos BioSciences Corporation, Cambridge, Mass.) And single molecule direct RNA sequencing are described by Ozsolak et al., Nature 461 (7265): 814-818 (2009) and Ozsolak. and Milos, Methods Mol Biol 733: 51-61 (2011). True Single Molecule Sequencing and Direct RNA Sequencing technologies are further described in U.S. Patent Publication Nos. 2008/0081330, 2009/0163366, 2008/0213770, 2010/0184045, 2010/0173363, 2010 / No. 0227321, 2008/0213770 and 2008/0103058 and U.S. Pat.Nos. 7,666,593; 7,767,400; 7,501,245; and 7,593,109, all of which are hereby incorporated by reference in their entirety. Embedded in the book.

a CD99 gene encoded by the mRNA is a specific sequencing primer designed based on the CD99 mRNA sequence (eg, the sequence presented in Table 2A or otherwise known and readily available to those of skill in the art) Can be directly sequenced by using True Single Molecule and Direct RNA Sequencing techniques.
Methods for inhibiting proliferation and / or survival of CD99 ⁺ cells associated with AML and / or MDS and methods for treating patients affected by AML and / or MDS exhibiting elevated CD99 ⁺ expression. And / or a therapeutic method comprising administering a composition comprising one or more anti-CD99 antibodies to a human patient to treat AML and / or MDS wherein MDS exhibits increased expression of CD99.
The amount of anti-CD99 antibody effective for the treatment of AML and / or MDS characterized by elevated CD99 expression can be determined by standard clinical techniques. In some cases, in vitro assays can be used to help identify optimal dosage ranges. The exact dose used in the formulation will depend on the route of administration and the severity of the disease or disorder. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

  The compounds or pharmaceutical compositions of the invention can be tested in vitro and then in vivo for the desired therapeutic or prophylactic effect prior to use in humans. For example, in vitro assays to determine the therapeutic or prophylactic effect of a compound or pharmaceutical composition include the effect of the compound on cell lines or patient tissue samples. The effect of the compound or composition on the cell line and / or tissue sample can be determined using techniques known to those skilled in the art including, but not limited to, proliferation assays and apoptosis assays. According to the present disclosure, in vitro assays that can be used to determine whether administration of a particular compound is required are those in which patient tissue samples are grown in culture and exposed to the compound or other In vitro cell culture assays in which a compound is administered in a method and the effect of the compound on a tissue sample is observed.

The disclosure provides methods of treatment and suppression by administering to a subject an effective amount of an anti-CD99 antibody or pharmaceutical composition, as described herein. In one aspect, the compound is substantially purified such that the compound is substantially free of substances that limit its effect or cause undesirable side effects.
Therapeutic and inhibitory methods using one or more anti-CD99 antibodies can further include, for example, administration of one or more additional compounds, such as chemotherapeutic compounds for the treatment of AML and / or MDS. Induction chemotherapy for AML can also include cytarabine (ara-C) and anthracyclines such as daunorubicin. Cytarabine can be administered by continuous intravenous infusion for 7 consecutive days, whereas anthracycline is generally administered as an IV push for 3 consecutive days. Acute promyelocytic leukemia, an AML subtype, is often treated with all-trans retinoic acid (ATRA) in combination with anthracycline and / or arsenic trioxide.
For patients at high risk of recurrence (eg, patients with high-risk cytogenetics, previous history of MDS or treatment-related MDS / AML), if the patient can tolerate a graft and has an appropriate donor, Allogeneic stem cell transplantation is recommended. For leukemias with a good prognosis (i.e. inv (16), t (8; 21) and t (15; 17)), patients generally receive 3-4 additional intensive chemotherapy, known as consolidation chemotherapy. Do the course. In patients with relapsed AML, anti-CD99 antibodies can be administered in combination with hematopoietic stem cell grafts as a pre-transplant cell loss or post-transplant prevention.

Various delivery systems, such as encapsulation in liposomes, microparticles, microcapsules, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1987) Etc.) are known and can be used to administer the compositions of the present disclosure. These will be known to those skilled in the art.
Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The anti-CD99 antibody or composition may be administered by any convenient route, such as by injection or bolus injection, or by absorption from epithelial or dermal mucosal coated cells (such as oral mucosa, rectal and intestinal mucosa). They can be administered with other bioactive agents. Administration can be systemic or local. Furthermore, it may be preferable to introduce the anti-CD99 antibody or composition into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection can be assisted, for example, by an intraventricular catheter attached to a reservoir, such as an on-maya reservoir. Transpulmonary administration can also be accomplished, for example, by use of a formulation comprising an inhaler or nebulizer and an aerosol propellant.
It may be preferable to administer the anti-CD99 antibody or composition locally to the site in need of treatment. This can be achieved, for example, by local injection during surgery, local injection by injection, catheter, suppository or implant, said implant comprising porous, non-porous, including membranes, shear membranes or fibers etc. Or a gelatinous material.

Anti-CD99 antibodies can be delivered in vesicles or sustained release systems such as liposomes (Langer, Science 249: 1527-1533 (1990)). Sustained release systems can be placed close to the therapeutic target and therefore require only a portion of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, Vol. 2, pp. 115- 138 (1984)).
Intravenous infusion of a composition comprising an anti-CD99 antibody is at least about 1 day, or at least about 3 days, or at least about 7 days, or at least about 14 days, or at least about 21 days, or at least about 28 days, or at least It can last for a period of about 42 days, or at least about 56 days, or at least about 84 days, or at least about 112 days.
Continuous intravenous infusion of a composition comprising an anti-CD99 antibody can continue for a specified period of time followed by another period of rest. For example, the continuous infusion period can be about 1 day to about 7 days, about 14 days, about 21 days, about 28 days, about 42 days, about 56 days, about 84 days, or about 112 days. The continuous infusion can be followed by a rest period of about 1 to about 2 days, about 3 days, about 7 days, about 14 days or about 28 days. The continuous infusion as described above can then be repeated, followed by another rest period.
Regardless of the exact continuous infusion protocol employed, continuous infusion of a composition comprising an anti-CD99 antibody will not continue until the desired efficacy is achieved or an unacceptable level of toxicity is manifested. Will be understood.

In some aspects of the present disclosure, methods for inhibiting proliferation and / or survival of CD99 ⁺ cells associated with AML and / or MDS and patients affected by AML and / or MDS exhibiting elevated CD99 ⁺ expression The method of treatment further comprises treating the cell with a compound that promotes mobilization of AML and / or MDS cells and associated hematopoietic and / or leukemic stem cells from the bone marrow to the peripheral blood, respectively, or administering it to a patient. Can be included. Exemplary agents for accomplishing such mobilization are described in the art and include G-CSF (Pabst et al., Blood 119 (23): 5367-5373 (2012)) and Plerixafor (Uy et al., Blood 119 (17): 3917-3924 (2012).

Of course, unless stated otherwise, the term shall be “open” (eg, the term “including” should be interpreted as “including but not limited to”). The term “having” should be interpreted as “having at least” and the term “includes” should be interpreted as “including but not limited to”. Should be). Phrases such as “at least one” and “one or more” and terms such as “one (a)” or “an” include both the singular and plural.
It is further understood that where a feature or aspect of the present disclosure is described by a Markush group, the disclosure shall also be described with respect to any individual member or subgroup of members of the Markush group. I will. Similarly, all of the ranges disclosed herein also include all possible subranges and combinations of subranges, eg, “between”, “up to”, “at least”, “to” Terms such as “super”, “less than” include the stated number in the range and also include individual members.
This specification, including but not limited to, including, but not limited to, patents, patent applications, and patent publications and all technical and / or scientific publications, whether or not US, PCT, non-US All references cited in are incorporated herein by reference.

While various embodiments have been disclosed herein, other embodiments will be apparent to those skilled in the art. The various embodiments disclosed herein are for purposes of illustration and not limitation, and the true scope and spirit is indicated by the following claims.
The present disclosure will be further described with reference to the following non-limiting examples. The teachings of all patents, patent applications and all other publications cited herein are hereby incorporated by reference in their entirety.
The antibodies used in the following experiments are all commercially available (eg, from Abcam, Cambridge, Mass.).

Anti-CD9912E7 antibody is cytotoxic to CD99 ⁺ MDS cell lines, MDS primary cells, AML cell lines and primary AML blasts and induces apoptosis in them In this example, MDS expressing CD99 Reveals that anti-CD99 antibody 12E7-mediated ligation of CD99 on cell lines, MDS primary cells, AML cell lines and primary AML blasts is cytotoxic to these cells and induces apoptosis in these cells To do. Moreover, cytotoxicity due to CD99 ligation occurs in the absence of antibody effector functions. Thus, 12E7 mediated cytotoxicity and apoptosis are independent of complement dependent cytotoxicity (CDC) and / or antibody dependent cellular mediated cytotoxicity (ADCC).

CD99 on the MDS cell line MDS92 (Tohyama et al., Br. J. Haematol. 91: 795 (1995)) and an anti-CD99 antibody called 12E7 (Levy et al., Proc. Natl. Acad. Sci. USA 76 : 6552 (1979)) Ligation with 20 μg / ml is 128 times the number of MDS92 cells after 72 hours (p <0.001) compared to the number of MDS92 cells in the presence of 20 μg / ml isotype control antibody. Cytotoxicity against these MDS92 cells is demonstrated as evidenced by a decrease (FIG. 1A). Analysis of fluorescence values of AAD vs. Annexin V revealed effector-independent apoptosis (compared to the absence of MDS92 cell apoptosis after 72 hours in the presence of 20 μg / ml isotype control antibody). This was evidenced by a 77% increase in annexin V positivity (p <0.001) in MDS92 cells 72 hours after ligation of 10 μg / ml 12E7 and CD99 (FIG. 1B).
12E7-mediated cytotoxicity against CD99 ⁺ MDS92 cells was dependent on the time of MDS92 cell count at 22 hours after ligation of anti-CD99 antibody 12E7 with CD99 compared to MDS92 cell count in the presence of isotype control antibody. Further confirmation by a fold reduction (p <0.001) (FIG. 2A). Cell surface expression of CD99 on MDS92 cells showed a time-dependent decrease after ligation of anti-CD99 antibodies 12E7 and CD99 (FIG. 2B). Cell surface expression of myeloid differentiation markers CD11b and CD14 showed a time-dependent decrease after ligation of anti-CD99 antibody 12E with CD99 (FIG. 2C).

CD99 and 12E7 anti-CD99 antibodies on primary CD34 ⁺ MDS cells, as evidenced by a 10-fold decrease in cell number versus no antibody control at 48 hours and an 8-fold decrease in cell number relative to isotype (IgG) control antibody Ligation with and shows cytotoxicity against primary MDS cells (Figure 3).
Ligation of CD99 and 12E7 anti-CD99 antibodies on AML cell lines expressing high levels of CD99, such as HL60 and MOLM13, resulted in a 49-fold reduction in cell numbers relative to isotype control antibodies after 72 hours (HL60; p It shows cytotoxicity against AML cell lines as evidenced by <0.001) and a 70-fold reduction (MOLM13; p <0.001) (FIG. 4A). Compared to the isotype control, CD99 is expressed at high levels on MOLM13 and HL60 cells.
Ligation of CD99 with 12E7 anti-CD99 antibody on primary AML1520 blast cells expressing CD99 was confirmed by primary AML buds as evidenced by a 57-fold reduction in cell number after 48 hours (p <0.001). It shows cytotoxicity against sphere cells (FIG. 5A). Ligation of CD99 and 12E7 anti-CD99 antibody on primary AML890 blast cells expressing CD99 was confirmed by primary AML buds, as evidenced by a 48-fold reduction in cell number after 48 hours (p <0.001). It shows cytotoxicity against sphere cells (FIG. 5B). The 12E7 anti-CD99 antibody shows only slight cytotoxicity against normal spinal blood HSC, as evidenced by a 1.4-fold decrease in HSC cell number after 80 hours (FIG. 5C). This indicates that anti-CD99 has a substantial therapeutic window.

Comparative transcriptome analysis of highly purified lineage marker negative [Lin-] CD38 + CD34-CD90 + CD45RA-hematopoietic stem cells This example demonstrates that CD99 is expressed on disease-initiating stem cells in MDS and AML To clarify. In AML, LSCs have been shown to have the ability to self-replicate and differentiate into non-self-replicating progeny that contain the majority of diseased cells (Lapidot et al., Nature 367: 645-648 (1994) And Bonnet and Dick, Nat. Med. 3: 730-737 (1997)), this is the first malignancy that met the criteria devised by the cancer stem cell hypothesis. Therefore, it is expected that disease-initiating stem cells in AML will be attacked by targeting LSCs with anti-CD99 antibodies.
MDS has been shown to be initiated in HSCs, indicating that MDS HSCs contain disease-related cytogenetic abnormalities and are able to engraft disease in immunodeficient animals. Nilsson et al., Blood 100: 259-267 (2002); Nilsson et al., Blood 110: 3005-3014 (2007); Tehranchi et al., New Engl. J. Med. 363: 1025-1037 (2010) Nilsson et al., Blood 96: 2012-2021 (2000); Pang et al., Proc. Natl. Acad. Sci. USA 110: 3011-3016 (2013); and Will et al., Blood 120: 2076- 2086 (2012). Therefore, it is expected that disease-initiating stem cells in MDS will be attacked by targeting HDS of MDS with anti-CD99 antibodies.

Many researchers have shown that cell surface proteins are preferentially expressed on AML LSCs compared to normal HSCs (Jin et al., Nat. Med. 12: 1167-1174 ( 2006); Majeti et al., Cell 138: 286-299 (2009); Jan et al., Proc. Natl. Acad. Sci. USA 108: 5009-5014 (2011); Jin et al., Cell Stem Cell 5 : 31-42 (2009); van Rhenen et al., Blood 110: 2659-2666 (2007); Saito et al., Sci. Transl. Med. 2: 17ra19 (2010)). These are promising targets for treatments that selectively target AML LSCs while helping HSC. To date, cell surface protein expression in MDS HSCs has not been carefully characterized and treatments targeting stem cells in this disease have not been evaluated.
8 patients with myelodysplastic syndrome (MDS), 7 low-risk patients and 1 intermediate-risk patient (Greenberg et al., Blood 89: 2079-2088 (1997)) and age-matched normal controls (McGowan et al. , Blood 118: 3622-3633 (2011)), a comparative transcriptome analysis of highly purified HSC (strain negative [Lin-] CD38 + CD34-CD90 + CD45RA-) was performed and 25 dysregulated cell surface proteins A transcript (FDR <0.1) was identified. Using flow cytometry (FC) to identify cell surface markers specific to MDS HSCs, spinal cord blood (CB) HSC controls in a validation cohort of 26 treatment-related MDS specimens and 37 de novo MDS specimens In comparison, cell surface expression of these markers was confirmed and CD99 was identified as the most frequently overexpressed (85% and 73% of patients, respectively, FIG. 6A). The high frequency of CD99 expression among these randomly selected MDS patients suggests that anti-CD99 antibodies are effective against a wide range of MDS subtypes.

To examine the validity of CD99 as a marker for other myeloid malignancies, leukemia blasts from 78 diagnostic and recurrent AML samples were evaluated for expression of CD99 by FC, 81% of diagnostic samples and relapse An increase in the expression level of CD99 (average 9.2-fold increase, p <0.0001, FIG. 6B) was observed in 83% of the samples, which revealed that CD99 was overexpressed not only in AML but also in MDS. CD99 expression was stable between diagnosis and recurrence, suggesting that anti-CD99 antibodies are effective in many disease states (both early diagnosis and recurrence). In one experiment, surface expression of CD99 was significantly increased in recurrence compared to diagnosis (p = 0.047).
To test whether CD99 expression can distinguish leukemia cells from normal hematopoietic stem cells and hematopoietic progenitor cells (HSPCs), cell surface expression of CD99 in the CD34 ⁺ CD38- fraction of stem cells enriched AML evaluated. Within this fraction, “CD99 high” cells become lymphocyte pluripotent progenitor cells (LMPP, CD34 ⁺ CD38-CD90-CD45RA +) consistent with the immunophenotype found in most human AML. Similar (Goardon et al., Cancer Cell 19: 138-152 (2011)), while “low CD99 (CD99 low)” cells are HSC / pluripotent progenitor cells (MPPs) that are dominant in normal hematopoiesis. It was similar (Figure 6C). Majeti et al., Cell Stem Cell 1: 635-645 (2007). When “low CD99” cells were purified into methylcellulose medium using FACS, they formed robust myeloid colonies characteristic of normal HSCs, but no LSCs were seen (FIG. 6D). Picking these colonies and performing allele-specific PCR reveals that they lack a corresponding molecular marker associated with AML (e.g., FLT3-ITD), indicating that the remaining normal or It was consistent with their induction from preleukemia HSCs (Figure 6E). Jan et al., Sci. Transl. Med. 4: 149ra118 (2012). This reveals the specificity of leukemia stem cell (LSC) CD99 expression compared to the remaining normal hematopoietic stem cells (HSC) in the same patient, suggesting that anti-CD99 antibodies have a wide therapeutic concentration range. did.

The CD34 ⁺ CD38 ⁻ fraction of AML has been shown to be enriched in LSC (Bonnet and Dick, Nat. Med. 3: 730-737 (1997) and Goardon et al., Cancer Cell 19: 138- 152 (2011)), this fraction was found to consistently show higher levels of CD99 expression in AML compared to more differentiated AML blasts (FIG. 6F). To test whether functional LSCs have increased CD99 expression, the upper and lower 10% of CD99-expressing bodies in the primary AML LSC-enriched CD34 ⁺ CD38-CD90-CD45RA-fractions were subdivided at the limiting dilution. Transplanted into lethal dose irradiated (185cGy) NSG mice and found that leukemia initiation ability (LIC) was limited to high CD99 cells (1 for 24,401 cells, 10% for the top 10%) (See Table 3A). The upper and lower 10% of CD99-expressing bodies were purified from the “LMPP-like” LSC enriched fraction of the AML specimen UPenn 2741 and transplanted to NSG mice (185cGy) irradiated with quasi-lethal doses at limiting dilution. Leukemia engraftment (cut-off value> 0.1% human CD45 + cells) was observed only in mice transplanted with “top 10%” cells. This validates CD99 expression as a specific marker of functionally related leukemia-initiating cells, suggesting that anti-CD99 antibodies preferentially kill this disease-initiating cell population.

Table 3A Leukemia initiation ability (LIC) is limited to high CD99 cells

  Similar dilution analysis experiments were performed using another independent AML sample UPenn 2522. Table 3B shows a limiting dilution analysis of transplantation of UPenn 2522, a CD19-CD3-CD45 (dim) SSC (low) leukemia blast that exhibits high or low CD99 expression. In Table 3B, the upper and lower 15% of “LMPP-like” LSC enriched blasts expressing CD99 from the AML specimen UPenn 2522 were similarly purified and transplanted into NSG mice at limiting dilution. Leukemia engraftment was observed only in mice transplanted with “top 15%” CD99-expressing blasts.

Table 3B Limit dilution analysis of transplantation of UPenn 2522, a CD19-CD3-CD45 (dim) SSC (low) leukemia blast with high or low CD99 expression

  Thus, CD99 is not only strongly expressed in AML, but is thought to be enriched in functional LSCs. This is the first LSC marker reported to have this feature. Other LSC markers were found to be expressed at approximately equivalent levels on LSCs and more differentiated AML blasts (eg, CD44, CD47 and TIM3; data not shown). Jin et al., Nat. Med. 12: 1167-1174 (2006); Majeti et al., Cell 138: 286-299 (2009); and Jan et al., Proc. Natl. Acad. Sci. USA 108: 5009-5014 (2011).

To further study, we evaluated the diverse expression of CD99 in patient AML blasts. Patient AML blasts were fractionated. Unfractionated cells were compared to fractionated CD34 ⁺ CD38-AML cells to assess the expression level of selected possible LSC markers including CD99, CD44, CD123, CD47 and TIM3. In patient AML blasts, among several AML markers tested, only CD99 was found to be significantly enriched in fractionated leukemic stem cells (CD34 ⁺ , CD38-), which is statistically relevant Admitted. No such statistically relevant increase in expression was observed for CD44, CD123, CD47 or TIM3.
The distribution of CD99 on leukemic cells is much higher than that of normal HSPC. Based on this observation, a predictive separation of the remaining normal HSCs from leukemia cells was performed. An AML patient sample (MSK AML 003) was obtained. CD99 expression in CD3-CD19-CD34 + CD38- cells from AML specimen MSK AML-003 was assessed by FC. The cell population was sorted by FACS into “low CD99 group” and “high CD99 group” with a purity of 95% or more. Two groups were plated on methylcellulose supplemented with cytokines (750 cells in triplicate). Normal myeloid / erythroid colonies are formed only from the “low CD99” fraction and show the expected distribution of different types of hematopoietic cells (CFU-E, BFU-E, GEMM, CFU-M and CFU-G). Had. Furthermore, “low CD99” derived colonies lacked homozygous FLT3-ITD abnormalities present in MSK AML-003.
In another experiment, fractions from AML samples (MSK-003) were separated into “high CD99” (Lin-CD34 + CD38-CD99 +) and “low CD99” (Lin-CD34 + CD38-CD99-) fractions, These were xenografted into NSG mice. Engraftment was assessed after 12 weeks. As shown in Table 3C, “low CD99” cells showed normal lymphoid myeloid engraftment, consistent with the activity of the remaining normal hematopoietic stem cells (HSC). In contrast, as shown in Table 3C, “high CD99” cells produced leukemic engraftment at high cell mass in 4 out of 4 mice.

Table 3C Engraftment of high and low CD99 cells 12 weeks after xenotransplantation into NSG mice

  To characterize the function of CD99 in AML, shRNA targeting CD99 was stably transduced into MOLM13 AML cells (8.0-fold knockdown). Knockdown of CD99 in HL60 cells by two shRNAs (2.3-fold and 8.3-fold with # 61 and # 59, respectively) did not significantly alter proliferation kinetics in vitro. Error bars represent ± SD. Although this did not result in a significant difference in in vitro growth (Figure 7A), NSG mice implanted with sub-lethal doses of these cells were significantly different compared to the vector control. CD99 is able to promote more invasive disease by modulating its interaction with the microenvironment, as it showed improved survival (58 vs 34 days, p = 0.02, Figure 7B). It was suggested that it can be done. Thus, high CD99 surface protein expression in human AML samples was significantly associated with increased BM disease burden (FIG. 7C). CD99 high expression is associated with more invasive proliferation of AML in both mice and humans.

Gene expression traits enriched in LSC have been shown to be a precursor to poor clinical outcome (Majeti, Proc. Natl. Acad. Sci. USA 106: 3396-3401 (2009) and Eppert, et al. Nature Medicine 17 : 1086-1093 (2011)), overexpression of LSC markers such as CD123 and CD47 correlate with increased leukemia burden and OS deterioration, respectively. Therefore, it was assumed that CD99 mRNA expression in AML increases “stemness” and a worse prognosis is expected. Analysis of transcriptome data available from 358 AML patients enrolled in the Eastern Cooperative Oncology Group (ECOG) clinical trial (E1900) by comparing two doses of daunorubicin (DNR) for the treatment of early disease did. Surprisingly, low CD99 expression was associated with a worse prognosis, which was thought to be alleviated by the high dose DNR regimen (FIG. 7D).
Moreover, within the molecular subtype of AML (NPM1 mutation, DNMT3a mutation or MLL rearrangement; Patel et al., New Engl. J. Med. 366: 1079-1089 (2012)), where benefits from DNR enhancement are expected to be The benefit was limited to CD99 low expressers (FIG. 7E). Given the defined role of CD99 in monocyte and neutrophil transendothelial migration in response to inflammation (Schenkel et al., Nat. Immunol. 3: 143-150 (2002) and Lou et al., J Immunol. 178: 1136-1143 (2007)), in relation to chemotherapy, high levels of CD99 mRNA expression through the endothelium (in PB, it has been shown to be more sensitive to chemotherapy). Uy et al., Blood 119: 3917-3924 (2012) is expected to result in improved clinical outcome by promoting the recruitment of leukemia blasts to

CD99 promotes transendothelial migration and mobilization of leukemia blasts. To test the effect of CD99 expression on transendothelial migration of leukemia blasts, it is stable to the AML cell line HL60 to overexpress (6.7-fold) CD99. It was seeded onto human umbilical vein endothelial cells (HUVECs) that had been transduced and grown to confluence on transwell membranes and migrated through HUVEC towards the chemoattractant SDF-1 (data shown) ) Overexpression of CD99 led to a significant increase in the efficiency of transendothelial migration (3.72 fold increase at 4 hours (p <0.0009), 2.93 fold increase at 28 hours (p = 0.0065), FIG. 8A). The efficiency of this migration could be variously promoted or suppressed by the addition of various anti-CD99 mAbs (data not shown). Anti-CD99 antibodies that block transendothelial migration are not only sensitive to peripheral blood (as described herein, but tumor cells are not only sensitive to cytotoxic anti-CD99 antibodies but also enhanced chemosensitivity. Can be used in combination with a mobilizing agent such as G-CSF or Plerixafor to “capture” leukemia cells mobilized in (which may be shown).
After 72 hours, the remaining non-migrated cells showed significantly lower levels of CD99 expression compared to the migrated cells (3.65 fold reduction, p = 0.028, FIG. 8B). There was no correlation between CD99 expression and SDF-1 receptor CXCR4 expression (data not shown). The surface expression of CD99 was found to be significantly higher on AML samples taken from peripheral blood compared to BM (1.65 times higher, p = 0.028, Figure 8C) and taken simultaneously from primary AML xenografts In a paired analysis of BM and PB, CD99 surface expression was significantly higher on leukemic blasts circulating in PB compared to BM (2.32 fold increase, p <0.0001, Figure 8D) . Finally, overexpression of CD99 in the AML cell line led to increased adhesion to tissue culture plates coated with substrate components such as fibronectin and collagen (FIG. 8E).
Taken together, these findings suggest that CD99 promotes leukemia blast recruitment by promoting transendothelial migration and reducing adhesion to substrate components in BM.

Anti-CD99 monoclonal antibody (mAb) shows direct cytotoxicity against AML and MDS cells
The ability of anti-CD99 mAb to induce cell death was tested in AML and MDS. Several anti-CD99 mAb clones (12E7, O13, H036-1.1) were generated in vitro against primary AML blasts and MDS CD34 ⁺ cells (Figure 9A) and 11 of 15 AML cell lines (Figure 9B). Have been found to be cytotoxic. Induction of apoptosis in MOLM13 cells incubated with anti-CD99 mAb 5 μg / mL (Fig. Suggested injury. Apoptosis was induced independent of cell cycle status (data not shown) and could be suppressed by the pan-caspase anti-CD99 antibody QVD (data not shown).

  CD99 is expressed at low and intermediate levels on normal HSC (Figure 6A) and endothelial cells, respectively (data not shown), whereas anti-CD99 mAb is very poor in these cell types in vitro. Only an effect is shown, indicating a potentially wide therapeutic window (FIG. 9D). When measured at 8 weeks in PB, ex vivo treatment of primary AML cells with anti-CD99 mAb (H036-1.1, 20 μg / ml) prior to xenotransplantation into sub-lethal doses of NSG mice. A significant decrease in arrival was derived (20.5% vs. 67.4%, p = 0.006, Figure 9E). These experiments show that ex vivo treatment of autologous bone marrow transplants from AML patients with anti-CD99 antibodies is beneficial in helping to deplete transplant disease-initiating cells and reduce the risk of recurrence after transplantation. It suggests.

Remarkable cell surface CD99 capping induced by cytotoxic anti-CD99 mAb and the addition of a second cross-linked antibody to IgG isotype anti-CD99 mAb (e.g. clone O13) repeats this capping effect and promotes cytotoxicity. This suggests that multimerization of CD99 on the cell surface is the key to induce cell death (FIG. 9F). Cytotoxic anti-CD99 antibodies (eg 12E7) also induced CD99 cell surface aggregation and clustering, whereas non-cytotoxic anti-CD99 antibodies (eg 3B2) clearly induced CD99 surface expression redistribution. I did not.
CD99 has been described to physically bind to and suppress the activity of Src family kinases (SFK) in osteosarcoma cells. Scotlandi et al., Oncogene 26: 6604-6618 (2007). CD99 was co-immunoprecipitated with SFK in AML (data not shown), confirming that cytotoxic anti-CD99 mAb induces robust SFK activation (FIG. 9G). Pharmacological inhibition of SFK by small molecule anti-CD99 antibody PP2 significantly reduces cytotoxicity induced by anti-CD99 mAb (FIG. 9H). Thus, anti-CD99 mAbs can promote cell death through apoptosis induced by oncogenes by promoting activation of dysregulated SFK.

Anti-CD99 monoclonal antibody (mAb) removes AML xenografts
The ability of anti-CD99 mAb to remove AML xenografts was tested. The combined treatment of xenograft AML specimen UPenn 2522 in NSG mice with H036-1.1 ex vivo and in vivo was performed. A schematic diagram of the experimental protocol is shown in the upper panel of FIG. 450,000 AML blasts (UPenn 2522) were precoated with H036-1.1 anti-CD99 mAb (20 microgram / mL) or isotype (20 microgram / mL) for 45 minutes. The blasts were xenografted into NSG mice irradiated with quasi-lethal doses. Two weeks later, the mice were treated with H036-1.1 or isotype (15 micrograms). After 5 months, mice were sacrificed and bone marrow (BM) was evaluated. As shown in the lower panel of FIG. 11, in the evaluation of mouse bone marrow (BM) for human chimerism at 5 months, 0/6 of animals administered blasts precoated with H036-1.1, in vivo H036-1.1 0/5 of the treated animals showed less than 0.1% human engraftment to the BM (threshold boundary is indicated by a gray dotted line), while 4/5 of the transplanted control animals showed human engraftment . Error bars represent ± SD. As shown in FIG. 11, AML xenografts were removed by pre-coating AML blasts with anti-CD99 mAb prior to xenotransplantation or by treating xenografted mice with anti-CD99 mAb in vivo. 4/5 of the control animals showed leukemic engraftment. These data indicate that ex vivo pretreatment of blasts or in vivo treatment of transplanted grafts with H036-1.1 anti-CD99 mAb resulted in the removal of AML xenografts in NSG mice.

De novo antibody production, humanization and validation De novo anti-CD99 mAb production was performed by standard means. Mice were immunized with recombinant CD99 purified by vendor (Thermo Fisher) using conventional methods. Based on in vitro potency and efficacy, 85 candidate lead anti-CD99 mAbs were screened. Hybridoma supernatants containing Ab were evaluated compared to IgG isotype negative controls. The MOLM13 relative cell number was determined after 72 hours incubation with the hybridoma supernatant. Three supernatants with markedly reduced relative cell numbers were identified. One of the candidates, supernatant 13, was particularly effective in reducing the relative cell number. MOLM13 cells exposed to supernatant 13 also showed decreased viability and decreased SSC with PI staining compared to MOLM13 cells treated with IgG isotype (data not shown). Future candidate assessments include criteria that are equivalent or better than 12E7 / H036-1.1 / O13 mAb. Further characterization of these lead compounds will be done to ensure that their pro-apoptotic activity does not induce killing in human endothelial cell lines and HSCs. Pro-apoptotic lead Abs will be assessed in vitro for ADCC / CDC activity using human AML cell lines, primary AML cells and LSCs. Affinity optimization of lead candidate Abs to increase efficacy and efficacy and in vivo validation of lead humanized mAb candidates will be performed.

Targeting CD99 in T cell malignant neoplasms
CD99 is a 32 kDa transmembrane protein that regulates T cell maturation and transendothelial migration of leukocytes. CD99 is also used as a biomarker to help diagnose acute T-cell lymphoblastic lymphoma / leukemia (T-ALL), Ewing sarcoma and neuroendocrine tumors. Here we show that a monoclonal antibody (mAb) against CD99 effectively induces cytotoxicity in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Further experiments were conducted to determine whether CD99 is a viable therapeutic target in T cell neoplasms.
In this experiment, tissue microarray was used to evaluate CD99 protein expression on 115 lymphoma patient samples by immunohistochemistry (IHC). CD99 expression was also assessed by flow cytometry on mature T cell lymphoma cell lines, T-ALL cell lines and peripheral blood (PB) T cells. The cell line showing the highest level of CD99 expression is used in the in vitro cytotoxicity assay and anti-CD99 mAb (DN16, O13 or 12E7) in the presence of increasing concentrations of anti-IgG cross-linking antibody (Ab) (50-100 μg / ml) Incubated with 40 μg / ml. At 72 hours after incubation with anti-CD99 mAb, cell viability and morphology were assessed by flow cytometry and light microscopy. The results are shown in FIGS.

By IHC, various T cell neoplasms showed CD99 expression based on a 5% cut-off value: 11/20 (55%) T lymphoblastic lymphoma, 7/16 (44%) hemangioblastic T cell lymphoma, 4/13 (31%) anaplastic large cell lymphoma (ALCL), 10/63 (16%) peripheral T cell lymphoma and 0/3 (0%) NK / T cell lymphoma. Flow cytometric analysis revealed that CD99 was up-regulated on Karpas-299 (ALCL) and Kopt-K1 (T-ALL) cell lines against PB T cells (normalized) MFI values: 56.1, 39.5, 20.5), Mac2A (ALCL), Hut78 (Sezary syndrome) and Hut102 (mycosis fungoides) cell lines expressed low levels of CD99 (normalized MFI values, respectively) : 0.19, 10.9, 0.5).
Incubation of DN16 or 12E7 with Kopt-K1 cells induced significant cell death in the presence of increasing concentrations of anti-IgG Ab relative to the IgG isotype control (DN16: 70-81% cell death, p ≦ .0002; 12E7: 42-78% cell death, p ≦ .0003). Incubation of DN16 or O13 with Karpas-299 also resulted in a significant decrease in cell number in the presence of anti-IgG Ab (O13: 25% cell death with 75 μg / ml anti-IgG Ab, p <.0001; DN16: 38-43% cell death with 50-100 μg / ml anti-IgG Ab, p ≦ .0021). Consistent with previous findings in B-ALL, cell death was associated with significant cell aggregation.

  This experiment shows that CD99 is strongly expressed on T-ALL blasts and a significant percentage of cells in T-cell lymphomas, especially ALCL. Without being bound by theory, anti-CD99 mAb is cytotoxic to T-ALL and ALCL cell lines in the presence of anti-IgG Ab, which is a cross-linked CD99 to induce cell death. Or it suggests that multimerization is necessary. In summary, as a general rule, these studies suggest that targeting CD99 with therapeutic mAbs is a viable new strategy in the treatment of multiple T cell neoplasms.

  Viewed individually or collectively, these studies established that CD99 is a promising prognostic marker and therapeutic target enriched on disease initiating cells in AML, MDS and T cell neoplasms. Moreover, dysregulation of SFK activity by anti-CD99 mAbs is a novel therapeutic vulnerability in AML and MDS that can be developed using other modulators of this pathway. By directly inducing apoptosis and / or enhancing chemosensitivity, CD99-directed therapy can allow eradication of diseased stem cells leading to long-term remission in AML and MDS.

Claims (51)

A method for identifying a patient's susceptibility to treatment with an anti-CD99 antibody in a patient having a hematopoietic tumor or lymphoma, comprising:
a. quantifying CD99 mRNA levels in said patient-derived cells by amplifying said cellular RNA using a primer pair specific for the CD99 gene sequence;
b. quantifying CD99 mRNA levels in control cells from healthy subjects by amplifying RNA of said cells using a primer pair specific for the CD99 gene sequence; and
c. comparing the CD99 mRNA level of the cell from the patient with the CD99 mRNA level of the cell from the healthy subject,
Including
The CD99 mRNA level of the patient cell as compared to the CD99 mRNA level of the healthy subject cell is an indicator of the patient's sensitivity to treatment with an anti-CD99 antibody;
The anti-CD99 antibody is
(i) binds to the extracellular domain of CD99 on the surface of CD99 ⁺ myeloid or lymphoid malignant cells;
(ii) promote one or more of aggregation, clustering and capping on the surface of the myeloid or lymphoid malignant cells of CD99 bound by the antibody;
The method, wherein the antibody induces cell death of the myeloid or lymphoid malignant cell when bound to CD99 on the surface of the myeloid or lymphoid malignant cell.   The method of claim 1, wherein the primer pair comprises a forward primer and a reverse primer, the forward primer hybridizes to the 5 'end of CD99 mRNA, and the reverse primer hybridizes to the 3' end of the CD99 mRNA. .   3. The method of claim 2, wherein the CD99 mRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5.   2. The method of claim 1, wherein the cells are selected from the group consisting of primary AML blast cells, leukemia stem cells (LSC), primary MDS blast cells and MDS hematopoietic stem cells (HSC).   The antibody binds to an epitope of CD99 comprising an amino acid sequence selected from the group consisting of DGEN (SEQ ID NO: 7), DAVVDGEND (SEQ ID NO: 10), AVVDGEN (SEQ ID NO: 11) and DDPRPPNPPK (SEQ ID NO: 12). Item 2. The method according to Item 1.   6. The method of claim 5, wherein the antibody is an IgG monoclonal antibody.   The method according to claim 6, wherein the IgG monoclonal antibody is a mouse antibody selected from the group consisting of 12E7 and O13.   2. The method of claim 1, wherein the antibody is IgM.   9. The method of claim 8, wherein the antibody is IgM monoclonal antibody H036-1.1.   2. The method of claim 1, wherein the antibody binds to a second antibody.   11. The method of claim 10, wherein the second antibody binds to the extracellular domain of CD99.   The extracellular domain of CD99, wherein the second antibody comprises an amino acid sequence selected from the group consisting of DGEN (SEQ ID NO: 7), DAVVDGEND (SEQ ID NO: 10), AVVDGEN (SEQ ID NO: 11) and DDPRPPNPPK (SEQ ID NO: 12) 12. The method of claim 11, wherein the method binds to an epitope of.   The extracellular domain of CD99, wherein the second antibody does not contain an amino acid sequence selected from the group consisting of DGEN (SEQ ID NO: 7), DAVVDGEND (SEQ ID NO: 10), AVVDGEN (SEQ ID NO: 11) and DDPRPPNPPK (SEQ ID NO: 12) 12. The method of claim 11, wherein the method binds to an epitope of. A method of inducing cell death of a CD99 ⁺ myeloid or lymphoid malignant cell associated with a hematopoietic tumor or lymphoma, comprising contacting a CD99 ⁺ myeloid or lymphoid malignant cell with an anti-CD99 antibody, comprising: The antibody binds to the extracellular domain of CD99 on the surface of the CD99 ⁺ myeloid or lymphoid malignant cells, and when the antibody binds to CD99 on the surface of the myeloid or lymphoid malignant cells, Said method of inducing cell death of malignant cells.   15. The method of claim 14, wherein the antibody promotes one or more of aggregation, clustering and capping of CD99 to which the antibody is bound on a myeloid or lymphoid malignant surface. A method of treating a patient affected by a hematopoietic tumor or lymphoma associated with a myeloid or lymphoid malignant cell exhibiting elevated CD99 levels comprising administering to the patient a composition comprising an anti-CD99 antibody. Administering to the patient a composition comprising an anti-CD99 antibody,
The antibody is
binds to the extracellular domain of CD99 on the surface of said CD99 ⁺ myeloid or lymphoid malignant cells;
b. the antibody promotes aggregation, clustering and / or capping of the antibody-bound CD99 on the surface of the myeloid or lymphoid malignant cells;
The method, wherein the antibody induces cell death of the myeloid or lymphoid malignant cell when bound to CD99 on the surface of the myeloid or lymphoid malignant cell. A composition for treating a patient affected by a hematopoietic tumor or lymphoma associated with cells exhibiting elevated CD99 levels, comprising:
a first anti-CD99 antibody comprising:
i. binds to the extracellular domain of CD99 on the surface of said CD99 ⁺ myeloid or lymphoid malignant cells;
ii. promote aggregation, clustering and / or capping of the antibody-bound CD99 on the surface of the myeloid or lymphoid malignant cells; and
iii. induces cell death of the myeloid or lymphoid malignant cells when bound to CD99 on the surface of the myeloid or lymphoid malignant cells,
Said first anti-CD99 antibody, and
b. a second anti-CD99 antibody that inhibits transendothelial migration of said CD99 ⁺ myeloid or lymphoid malignant cells,
Said composition comprising.   17. The composition of claim 16, wherein the second antibody is selected from the group consisting of 12E7, HEC2, DN16, and H036. A method of inhibiting the growth of CD99 ⁺ myeloid or lymphoid malignant cells, comprising contacting CD99 ⁺ myeloid or lymphoid malignant cells with an anti-CD99 antibody comprising:
The antibody is
binds to the extracellular domain of CD99 on the surface of said CD99 ⁺ myeloid or lymphoid malignant cells;
b. the antibody promotes aggregation, clustering and / or capping of CD99 bound to the antibody, CD99 on the surface of the myeloid or lymphoid malignant cells;
The method, wherein the antibody induces cell death of the myeloid or lymphoid malignant cell when bound to CD99 on the surface of the myeloid or lymphoid malignant cell. 19. The method of claim 18, wherein the anti-CD99 antibody binds to the CD99 extracellular domain with a _Kd of about 100 nM to about 10 [mu] M or about 250 nM to about 5 [mu] M or about 500 nM to about 1 [mu] M. 19. The method of claim 18, further comprising contacting CD99 ⁺ myeloid or lymphoid malignant cells with a compound selected from the group consisting of daunorubicin, idarubicin, cytarabine, azacitidine and decitabine.   21. The method of claim 20, wherein the compound is administered concurrently with the anti-CD99 antibody.   21. The method of claim 20, wherein the compound and the anti-CD99 antibody are administered sequentially.   A method of treating a patient's hematopoietic tumor or lymphoma comprising administering to the patient an anti-CD99 antibody in an amount effective to induce cytotoxicity of myeloid or lymphoid malignant cells, said patient comprising: The method as described above, wherein said method is affected by a hematopoietic tumor or lymphoma that exhibits elevated CD99 expression levels compared to CD99 expression levels in control myeloid or lymphoid non-malignant cells.   25. The method of claim 24, further comprising administering to the patient a chemotherapeutic effective amount of a compound selected from the group consisting of daunorubicin, idarubicin, cytarabine, azacitidine and decitabine.   25. The method of claim 24, wherein the compound is administered concurrently with the anti-CD99 antibody.   25. The method of claim 24, wherein the compound is administered subsequent to the anti-CD99 antibody.   24. The method of claim 23, further comprising administering a mobilization agent to the AML and / or MDS patient.   28. The method of claim 27, wherein the mobilizing agent is prilixaphor or G-CSF or a combination thereof.   28. The method of claim 27, wherein the mobilizing agent is administered to the AML and / or MDS patient prior to administering the anti-CD99 antibody.   A method of treating a hematopoietic tumor or lymphoma that exhibits elevated CD99 gene expression levels in a patient previously confirmed to be affected by myeloid or lymphoid malignant cells, comprising myeloid or lymphoid malignant cells Wherein said anti-CD99 antibody is administered to said patient in an amount effective to induce cytotoxicity.   32. The method of claim 31, further comprising administering to the AML and / or MDS patient a compound selected from the group consisting of daunorubicin, idarubicin, cytarabine, azacitidine and decitabine.   32. The method of claim 31, wherein the compound is administered concurrently with the anti-CD99 antibody.   32. The method of claim 31, wherein the compound is administered subsequent to the anti-CD99 antibody.   32. The method of claim 30, further comprising administering a mobilization agent to the patient.   35. A method according to claim 34, wherein the mobilizing agent is plurixefore or G-CSF or a combination thereof.   35. The method of claim 34, wherein the mobilizing agent is administered to the patient prior to administering the anti-CD99 antibody. measuring CD99 gene expression levels in myeloid or lymphoid malignant cells from patients affected by hematopoietic tumors or lymphomas;
b. measuring said CD99 gene expression level in non-malignant donor control myeloid or lymphoid cell-derived cells;
c. comparing the CD99 gene level in patient cells to the gene expression level in corresponding control cells;
A method of measuring the sensitivity of said patient to treatment with an anti-CD99 antibody comprising:
A therapeutic effect of anti-CD99 antibody is predicted by a CD99 gene expression level in the myeloid or lymphoid malignant cells that is at least about 2 times greater than the corresponding gene expression level in the control cell,
The method, wherein the anti-CD99 antibody can promote at least one of aggregation, clustering or capping of the CD99 when bound to CD99 on the surface of the myeloid or lymphoid malignant cell.   A method for predicting sensitivity of a patient to treatment with an anti-CD99 antibody, comprising testing myeloid or lymphoid malignant cells of a patient affected by a hematopoietic tumor or lymphoma for increased expression of CD99. Thus, the increased expression of CD99 predicts a therapeutic effect of an anti-CD99 antibody that promotes at least one of CD99 aggregation, clustering or capping when binding to the CD99 on the surface of myeloid or lymphoid malignant cells, Said method.   further testing of the patient's myeloid or lymphoid malignant cells for c-kit gene expression, and the absence of c-kit gene expression or low levels of c-kit gene expression to treatment with anti-CD99 antibodies 40. The method of claim 38, wherein the patient's susceptibility is predicted.   Further comprising testing the patient's myeloid or lymphoid malignant cells for the presence of a BCR-ABL translocation, wherein the presence of BCR-ABL predicts the patient's sensitivity to treatment with an anti-CD99 antibody. 39. The method according to item 38.   Further testing the patient's myeloid or lymphoid malignant cells for the presence of the JAK2 variant, wherein the presence of the constitutively activated JAK2 mutation predicts the patient's susceptibility to treatment with an anti-CD99 antibody, 40. The method of claim 38.   A method of predicting the sensitivity of a patient to treatment with an anti-CD99 antibody, comprising examining myeloid or lymphoid malignant cells of a patient affected by a hematopoietic tumor or lymphoma for increased expression of CD99. Thus, the method wherein the increased expression of CD99 predicts the therapeutic effect of an anti-CD99 antibody exhibiting cytotoxicity when bound to CD99 on the surface of myeloid or lymphoid malignant cells.   A method for predicting the sensitivity of a patient affected by a hematopoietic tumor or lymphoma to treatment with an anti-CD99 antibody, comprising testing myeloid or lymphoid malignant patient cells for increased expression of CD99, The method, wherein an increase in the expression of CD99 predicts the therapeutic effect of an anti-CD99 antibody that induces cell death when bound to CD99 on the surface of myeloid or lymphoid malignant cells. A method of making a candidate anti-CD99 antibody for the treatment of a hematopoietic tumor or lymphoma in a patient comprising:
producing an antibody that binds to the extracellular domain of CD99,
b. said anti-CD99 antibody,
i. antibody-mediated aggregation, clustering and / or capping of CD99 expressed on the surface of myeloid or lymphoid malignant cells, and
ii. antibody-mediated induction of cell death of CD99 expressed on the surface of myeloid or lymphoid malignant cells,
Testing for,
Wherein the anti-CD99 antibody that mediates CD99 aggregation, clustering and / or capping and induces cell death is a candidate anti-CD99 antibody for the treatment of hematopoietic tumor or lymphoma in a patient. A method for determining whether an anti-CD99 antibody is a candidate for use in the treatment of hematopoietic tumors or lymphomas in patients whose Aml or MDs cells show elevated levels of CD99 expression respectively.
providing an anti-CD99 antibody;
b. subjecting said anti-CD99 antibody to one or more tests to determine whether it results in antibody-mediated aggregation, clustering and / or capping of CD99 expressed on the surface of myeloid or lymphoid malignant cells;
And the antibody is cytotoxic to CD99 ⁺ myeloid or lymphoid malignant cells and is itself a candidate for the treatment of hematopoietic tumors or lymphomas when said aggregation, clustering and / or capping occurs And if the aggregation, clustering and / or capping does not occur, the antibody is not a candidate for the therapeutic agent.   A method for treating a hematopoietic tumor or lymphoma in a patient exhibiting elevated CD99 levels on the surface of myeloid or lymphoid malignant cells, comprising one or more anti-CD99 antibodies, a composition or formulation comprising one or more anti-CD99 antibodies And / or administering to said patient a composition or formulation comprising one or more anti-CD99 antibodies in combination with one or more other agents effective for the treatment of AML and / or MDS, wherein the anti-CD99 antibody comprises: The method, wherein cell death is induced when bound to the surface of the cell. A method for identifying a patient's susceptibility to treatment with an anti-CD99 antibody in a patient affected with a hematopoietic tumor or lymphoma, comprising:
quantifying CD99 mRNA levels in cells from said patient;
b. quantifying the level of non-CD99 control RNA in cells from said patient;
c. comparing the level of CD99 mRNA in the cell with the level of the control RNA in the cell, thereby obtaining a ratio of gene expression of the CD99 gene and the control gene, wherein the CD99 gene and non-AML and Anti-CD99 induces apoptosis when the ratio of gene expression of CD99 gene and control gene that exceeds a predetermined threshold ratio of control gene in non-MDS cells binds to CD99 on the surface of myeloid or lymphoid malignant cells Said method being indicative of the patient's sensitivity to treatment with an antibody.   48. The method of claim 47, wherein the control mRNA is selected from the group consisting of β-actin, GAPDH and cyclophilin. A method for identifying a patient's susceptibility to treatment with an anti-CD99 antibody in a patient affected with a hematopoietic tumor or lymphoma, comprising:
quantifying CD99 mRNA levels in myeloid or lymphoid malignant cells from said patient by amplifying said cellular RNA using a primer pair specific for the CD99 gene sequence;
b. quantifying CD99 mRNA levels in control cells from healthy subjects by amplifying said cellular RNA using a primer pair specific for the CD99 gene sequence;
c. comparing the CD99 mRNA level in the myeloid or lymphoid malignant cells from the patient with the CD99 mRNA level in the control cells from the healthy subject;
Including
The level of the CD99 mRNA in the patient's myeloid or lymphoid malignant cells as compared to the CD99 mRNA in the control cells of the healthy subject is an indicator of the patient's sensitivity to treatment with an anti-CD99 antibody. ;
The anti-CD99 antibody induces cell death in the myeloid or lymphoid malignant cells when bound to CD99 on the surface of the myeloid or lymphoid malignant cells;
Said method.   The cell is selected from the group consisting of mature T cell lymphoma cells, T acute lymphoblastic leukemia (T-LL), T acute lymphoblastic leukemia (T-ALL) cells and anaplastic large cell lymphoma (ALCL) cells Item 51. The method according to any one of Items 1, 14, 16, 19, 24, 31, 38, 39, 43, 44, 45, 46, 47, 48, or 50.