CN114829399A - Methods of depleting hematopoietic stem/progenitor cells (HSC/HP) in a patient using bispecific antibodies - Google Patents

Abstract

Said invention provides compositions comprising bispecific antibodies that bind to the human tyrosine kinase receptor FLT3/FLK2 receptor protein and the CD3 receptor protein expressed on T-cells, and the use of compositions comprising said bispecific antibodies in the manufacture of a medicament for depleting hematopoietic stem/progenitor cells (HSC/HP) in a patient.

Description

Methods of depleting hematopoietic stem/progenitor cells (HSC/HP) in patients using bispecific antibodies

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. patent application No. 16/506,764 filed on 9/7/2019, said application is a continuation-in-part application of U.S. patent application No. 16/091,139 filed on 4.10.2018, said U.S. patent application No. 16/091,139 is 35 u.s.c. § 371, national phase filing of international application No. PCT/US2017/025951 filed 4/2017, the international application No. PCT/US2017/025951 claims priority to U.S. provisional application No. 62/317,906, entitled "Method of Eliminating Hematopoietic Stem/progenitor Cells (HSC/HP) in patients Using bispecific Antibodies" (Method of Eliminating Hematopoietic Stem Cells/Hematopoietic prognosticators (HSC/HP) in a Patient Using Bi-Specific Antibodies) ", filed on 4.4.2016. The entire contents of each of the foregoing applications are incorporated herein by reference in their entirety.

Sequence listing

This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on 7/2020/is named 128557-.

Technical Field

The invention described relates generally to hematopoietic cell transplantation, therapeutic antibody preparations and their uses.

Background

Hematopoietic stem cells

Hematopoietic stem cells are a common ancestor of all blood cells. As pluripotent cells, they can differentiate into multiple cell lineages, but cannot differentiate into all lineages derived from three germ layers. Hematopoietic stem cell differentiation leads to Lymphoid and myeloid cell lineages, the two major branches of hematopoiesis (Kondo, m. "Lymphoid and myeloid linkage administration in multipotent hematopoietic prognostics," immunol. rev. 2010, 11 months; 238(1): 37-46). Lymphoid lineage cells include T, B and Natural Killer (NK) cells. The Myeloid lineage includes megakaryocytes and erythrocytes (MegE) and different subsets of granulocytes (neutrophils, eosinophils and basophils), monocytes, macrophages and mast cells (GM) which belong to the Myeloid lineage (supra, cited Kondo M, et al. Biology of biochemical stem and precursors: oligonucleotides for clinical application. Ann. Rev Immunol. 2003;21: 759. 19. weisan IL. transforming stem and promoter cell Biology to the clinical: Science and antigens. Science (New York, N.2000, 25.287; 5457: 1442-6; see also Iwaskin, H. and assessment, K., and diagnosis, K. molecular cell, 40. th. 40. this publication).

HSCs have self-renewal potential and the ability to differentiate into blood lineages; that is, when stem cells divide, on average 50% of the daughter cells are committed to a cell lineage, while the remaining 50% are undifferentiated. The process maintains the same number of stem cells through asymmetric cell division, so that each dividing stem cell produces one new stem cell and one differentiated cell. In contrast, in symmetric division, stem cells produce 100% of the same stem cells (Gordon, M.Stem cells and haemopoiesis. See: hoffbrand, V., Catovsky, D., Tuddenham, E.G., Blackwell Publishing, 5 th edition (2005): Differential niche and Wnt requirements along with ingredient muscle leukomia, pages 1-12. New York.).

Lymphoid and myeloid lineages are separable at the ancestral level. Common lymphoid progenitor Cells (CLPs) can differentiate under physiological conditions into all types of lymphocytes without significant myeloid potential (Kondo M, Scherer DC, Miyamoto T, King AG, Akashi K, Sugamura K, et al. Cell-quality conversion of lymphoid-conditioned prognostics by induced activities of cytokines. Nature. 2000, 9.21. day; 407(6802):383-6), although some bone marrow-related genes may be detected in CLPs, depending on experimental conditions (Delogu A, Schebesta A, Sun Q, Archenbrenner K, Perlot T, Busslinger M. Gene expression by Pax 5B cells is used for cells and 269. 24. mu.269.3. 269.269.81).

Similarly, common myeloid progenitor Cells (CMP) can give rise to all classes of myeloid cells with no or sufficiently low levels of B-cell potential (Akashi K, Traver D, Miyamoto T, Weissman IL. A clonal common myeloid species promoter that is derived from all myeloid species lines, Nature 9.3.2000; 404(6774): 193-7). Another cell type, Dendritic Cells (DCs), has not been clearly classified into lymphoid or myeloid lineages because DCs can be derived from CLP or CMP (Manz MG, Traver D, Miyamoto T, Weissman IL, Akashi K. Dendritic cell potentials of early lymphoid and myelid progenisors. blood, 6.2001, 1; 97(11), 3333-41, Traver D, Akashi K, Manz M, Merad M, Miyamoto T, Engleman EG, et al. Development of CD8alpha-positive Dendritic cells from a mony myoid progenior Science, New York, N.2000, NY.15; 290: 5499), and can be differentiated into proliferating megakaryocytes (GM-E) which produce further proliferation of cells, Granulocytes, monocytes and other cells (Iwasaki H, Akashi K. Myeloid linkage administration from the hematotic stem cell. immunity. 2007;26: 726-.

Differences in the expression levels of transcription factors may determine lineage membership (affiliation) of differentiated cells. Transcription factors pu.1 and GATA-1 have been implicated in myeloid and erythroid/megakaryocytic lineage differentiation (Gordon, M), respectively.Stem cells and haemopoiesis. See: hoffbrand, V., Catovsky, D., Tuddenham, E.G., Blackwell Publishing, 5 th edition (2005): Differential niche and Wnt requirements along with ingredient muscle leukomia, pages 1-12. New York.).

Characterization of HSC

HSCs are undifferentiated and resemble small lymphocytes. Most HSCs are dormant, at the G0 phase of the cell cycle, protecting them from the effects of cell cycle dependent drugs. The dormant state of stem cells is maintained by transforming growth factor-beta (TGF- β). The activity of TGF- β is mediated by p53, a tumor suppressor gene that regulates cell proliferation and targets the cyclin-dependent kinase inhibitor p21 (Gordon, M).Stem cells and haemopoiesis. See: hoffbrand, V., Catovsky, D., Tuddenham, E.G., Blackwell Publishing, 5 th edition (2005): Differential niche and Wnt requirements along with ingredient muscle leukomia, pages 1-12. New York). Dormancy of HSCs is not only critical for the protection of the stem cell compartment and maintenance of the stem cell pool during a long period of time, but also forIt is critical to minimize the accumulation of replication-related mutations. Many intrinsic transcription factors that maintain HSC dormancy have been found to be associated with leukemia. For example, chromosomal translocations that lead to fusion of FoxOs with Myeloid/lymphoid or mixed lineage leukemias have been reported in Acute Myeloid Leukemia (see, e.g., Srgio Paulo Bydowski and Felipe de Lara Janz (2012). Hematotoic Stem Cell in enzyme Melloid Leukoid Leukomia Development, Advances in Hematotoic Stem Cell Research, Dr. Rosa Pelayo (eds.), ISBN: 978-.

Most normal HSCs are present in the CD34+/CD38-/CD90+ bone marrow cell fraction, and some HSCs are also observed in CD 34-/Lin-cells. The CD34+/CD38+ cell fraction contains some HSCs with short-term regenerative (repopulating) activity. Other accepted markers include the tyrosine kinase receptor c-kit (CD117) conjugated to a deficiency in terminal differentiation markers such as CD4 and CD8 (Rossi et al, Methods in Molecular Biology (2011) 750(2): 47-59).

Classification of HSC

Hematopoietic stem cell banks can be subdivided into three main groups: (1) short-term HSCs, which are capable of producing clones of differentiated cells for only 4-6 weeks; (2) metaphase HSCs, which are capable of maintaining differentiated cell progeny for 6-8 months before becoming extinct; and (3) long-term HSCs capable of indefinitely maintaining hematopoiesis (Testa U. Annals of Hematology (2011) 90(3): 245-.

Hematopoiesis

Hematopoiesis is a highly coordinated process in which HSCs differentiate into mature blood cells supported by a specialized regulatory microenvironment, consisting of components that control the fate instructions (specification) of stem and progenitor cells, and maintain their development by supplying the necessary factors ("niches"). The term "Bone Marrow (BM) niche" as used herein refers to a well-organized construct composed of elements that play an essential role in the survival, growth and differentiation of different blood cell lineages, such as osteoblasts, osteoclasts, bone marrow endothelial cells, stromal cells, adipocytes and extracellular matrix proteins (ECMs). The bone marrow niche is an important postnatal microenvironment in which HSCs proliferate, mature and produce myeloid and lymphoid progenitor cells.

Bone Marrow (BM) is present in the medullary cavity of all animal bones. It consists of a variety of precursors and mature cell types, including hematopoietic cells (precursors of mature blood cells) and stromal cells (precursors of a wide range of connective tissue cells), both of which appear to be capable of differentiating into other cell types. The mononuclear fraction of bone marrow contains stromal cells, hematopoietic precursors, and endothelial precursors.

Unlike secondary lymphoid organs such as the spleen, which have unique macroscopic structures (including red and white marrow), BM has no distinct structural features except for the endosteum, which contains osteoblasts. The endosteal region comes into contact with calcified hard bone and provides the special microenvironment necessary for the maintenance of HSC activity (Kondo M, Immunology Reviews (2010) 238(1): 37-46; srgio Paulo Bydowski and Felipe de Lara Janz (2012), Hematographic Stem Cell in enzyme myoid Leukemedia Development, Advances in Hematographic Stem Cell Research, Dr. Rosa Pelayo (eds.), ISBN: 978-.

Within the niche, HSCs are believed to receive support and growth signals from several sources including: fibroblasts, endothelial and reticulocytes, adipocytes, osteoblasts, and Mesenchymal Stem Cells (MSCs). The primary functions of the niches are to integrate local changes in nutrient, oxygen, paracrine and autocrine signals, and to alter HSC dormancy, trafficking and/or expansion in response to signals from the systemic circulation (Broner, f. and Carson, M c. Topics in bone biology. springer. 2009; 4: pages 2-4. New York, USA).

Although the properties of authentic MSCs remain misled, CD146 MSCs expressing CXC chemokine ligand 12 (CXCL12) have recently been reported to be self-renewing progenitors that reside on sinus surfaces and contribute to the organization of sinus wall structures, produce Angiopoietin (Angiopoietin) -1 (Ang-1), and are capable of producing osteoblasts that form endosteal niches (konopova, MY, and Jordan, CT, Biology and Therapeutic Targeting (2011) 9 (5: 591) -599). These CXCL12 reticulocytes can serve as a delivery pathway for shuttling HSCs between osteoblast niches and vascular niches where basic but distinct maintenance signals are provided.

Cytokines and chemokines produced by bone marrow MSCs are concentrated in specific niches, sequentially to altered local production and through the action of cytokine-binding glycosaminoglycans. Among these, CXCL 12/stromal Cell derived factor-1 α positively regulates HSC homing, while transforming growth factor FMS-like tyrosine kinase 3 (Flt3) ligand and Ang-1 act as dormancy factors (see, e.g., Srgio Paulo Bydlowski and Felipe de Lara Janz (2012), Hematotoitic Stem Cell in enzyme Myeloid Leukemia Development, Advances in Hematotoitic Stem Cell Research, Dr. Rosa Pelayo (eds.), ISBN: 978-. CXCL12-CXCR4 signaling is involved in HSC homing into the BM during ontogeny as well as survival and proliferation of colony forming progenitor cells. CXCR 4-selective antagonists induce HSC mobilization into the peripheral blood, which further indicates a role for CXCL12 in retaining HSCs within hematopoietic organs.

BM engraftment involves subsequent cell-cell interactions through the complex extracellular matrix produced by BMSCs. Thus, vascular cell adhesion molecule-1 (VCAM-1) or fibronectin is critical for adhesion to BM-derived MSCs. In this way, control of the kinetics of hematopoietic stem cell proliferation is very important for the regulation of proper hematopoietic cell production. These control mechanisms can be classified as either intrinsic or extrinsic to the Stem Cell, or a combination of both (see, e.g., Srgio Paulo Bydlowski and Felipe de Lara Janz (2012). Hematoptic Stem Cell in acid Myeloid Leukoidea Development, Advances in Hematoptic Stem Cell Research, Dr. Rosa Pelayo, ISBN: 978-.

HSC self-renewal and differentiation can be controlled by external factors (extrinsic control), such as cell-cell interactions or cytokines in the hematopoietic microenvironment, such as SCF (stem cell factor) and its receptors c-kit, Flt-3 ligand, TGF- β, TNF- α, and the like. Cytokines regulate a variety of hematopoietic cell functions through activation of a variety of signal transduction pathways. The major pathways involved in Cell proliferation and differentiation are the Janus kinase (Jak)/Signal Transducer and Activator of Transcription (STAT), mitogen-activated protein (MAP) kinase and Phosphatidylinositol (PI) 3-kinase pathways (Sorgio Paulo Bydlowski and Felipe de Lara Janz (2012), Hematotoic Stem Cell in enzyme Myeloid Leukoidea Development, Advances in Hematotoic Stem Cell Research, Dr. Rosa Pelayo (eds.), ISBN: 978-.

In addition, expression of other transcription factors (e.g., Stem Cell Leukemia (SCL) Hematopoietic transcription factors; GATA-2; and gene products involved in Cell cycle control, such as cyclin-dependent kinase inhibitors (CKI) pl6, p21, and p 27) have been shown to be essential (intrinsic control) for Hematopoietic Cell Development from the earliest stages (S orgio Paulo Bydlski and Felipe de Lara Janz (2012). Hematodiotic Stem Cell in enzyme Myeloid Leukomia Development, Advances in Hematodiotic Stem Cell Research, Dr. Rosana Pelayo (eds.), ISBN: 978-307-953-.

The Notch-l-Jagged pathway can be used to integrate extracellular signals with intracellular signaling and cell cycle control. Notch-1 is a surface receptor on the membrane of hematopoietic stem cells that binds to its ligand, Jagged on stromal cells. This results in cleavage of the cytoplasmic portion of Notch-1, which can then act as a transcription factor (Gordon, M.Stem cells and haemopoiesis. See: hoffbrand, V., Catovsky, D., Tuddenham, E.G., Blackwell Publishing, 5 th edition (2005): Differential niche and Wnt requirements along with ingredient muscle leukomia, pages 1-12. New York.).

Disorders treated using Bone Marrow (BM)/Hematopoietic Stem Cell (HSC) transplantation

Disorders treated using Bone Marrow (BM)/Hematopoietic Stem Cell (HSC) transplantation include, but are not limited to, Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML), peripheral T-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, neuroblastoma, non-malignant genetic and acquired bone marrow disorders (e.g., sickle cell anemia, β -thalassemia major, refractory Diamond-Blackfan anemia, myelodysplastic syndrome, severe idiopathic aplastic anemia, paroxysmal nocturnal hemoglobinuria, monocytic aplasia, fanconi anemia, megakaryocyte deficiency (megakaryosis), or congenital thrombocytopenia), multiple myeloma, and Severe Combined Immunodeficiency (SCID).

Hematopoietic malignancy

Most hematopoietic malignancies contain functionally heterogeneous cells, of which only a subset (called cancer stem cells) are responsible for tumor maintenance. Cancer stem cells are therefore named because of their properties that suggest normal tissue stem cells, including self-renewal, prolonged survival and the ability to produce cells with more differentiated characteristics (Jones RJ and Armstrong SA, Biol Blood Marrow transfer. 2008. month 1; 14 (suppl 1): 12-16).

Depending on the degree of differentiation associated with carcinogenic assaults, transformation events in hematopoietic stem cells can produce several different malignancies, including, but not limited to, chronic myeloid leukemia, myelodysplastic syndrome, acute myeloid leukemia, and possibly even acute lymphocytic leukemia (Jones RJ and Armstrong SA, Biol Blood Marrow transplant. 2008. month 1; 14 (suppl 1): 12-16).

The cancer stem cell concept is based on the following idea: tumors of a particular tissue often appear to "try" to recapitulate the cellular heterogeneity found in the originating tissue, and thus stem cell-like cells are present in the tumor, resulting in different cell types. The basic test of the hypothesis is whether tumor cells can be isolated into cells with the ability to regenerate the tumor, and cells without the ability. This cellular hierarchy has recently been demonstrated in acute myeloid leukemia, where some AMLs possess cells with unique immunophenotypes that are capable of initiating leukemia in immunodeficient mice, while most cells are incapable of initiating leukemia progression. Furthermore, leukemia-initiating cells also give rise to cells that have lost tumor-initiating activity and thus recapitulate the cellular heterogeneity found in the original tumor (Lapidot T et al, Nature. 1994; 367: 645-.

Acute myelogenous leukemia

Acute Myeloid Leukemia (AML) is a clonal disorder characterized by a failure of differentiation in the myeloid lineage coupled with accumulation of immature progenitors in the bone marrow, resulting in hematopoietic failure (Pollyea DA et al, British Journal of Haematology (2011) 152(5): 523-542). There is extensive inter-patient heterogeneity in the appearance of leukemic blasts (leukemia blasts). The discovery of leukemia initiating cells in Acute Myeloid Leukemia (AML) begins with the following findings: most AML blasts do not proliferate and only a few are capable of forming new colonies (Testa U, Annals of Hematology (2011) 90(3): 245-. A common feature of all AML cases is the abnormal differentiation of the arrest leading to the accumulation of more than 20% of blasts in the bone marrow (Gilliland, DG and Tallman MS, Cancer Cell (2002) 1(5): 417-420).

More than 80% of myeloid leukemias are associated with at least one chromosomal rearrangement (Pandolfi PP, Oncogene (2001) 20(40): 5726-. These translocations often contain genes encoding transcription factors that have been shown to play an important role in hematopoietic lineage development. Thus, alterations in the transcription system appear to be a common mechanism for differentiation leading to stasis (Pandolfi PP, Oncogene (2001) 20(40): 5726-.

Clinical studies and experimental animal models suggest that at least two genetic alterations are required for the clinical manifestations of acute leukemia. According to the model proposed by Gilliland and Tallman (Cancer Cell (2002) 1(5): 417-. Class I mutations, such as mutations in the receptor tyrosine kinase genes FLT3 and KIT, RAS family members, and loss of function of neurofibromin 1, confer proliferative and/or survival advantages to hematopoietic progenitor cells, generally as a consequence of aberrant activation of signal transduction pathways. Class II mutations cause the cessation of differentiation by interfering with transcription factors or co-activators (Frankfurt O et al, Current Opinion in Oncology (2007) 19(6): 635-.

Although Leukemic Stem Cells (LSCs) appear to share many of the cell surface markers previously identified for HSCs such as CD34, CD38, HLA-DR and CD71, several groups have reported surface markers that are differentially expressed in both populations.

For example, CD90 or Thy-1 have been described as potentially specific for the LSC compartment. Thy-1 is down-regulated in normal hematopoiesis as most primitive stem cells progress toward the progenitor stage (Hope KJ et al, Archives of Medical Research (2003) 34(6): 507-514).

The interaction between CXCL12 (stromal cell derived factor-1 α) and its receptor CXCR4 on leukemic progenitor cells contributes to their homing to the bone marrow microenvironment. CXCR4 levels were significantly elevated in leukemic cells from patients with AML, and CXCR4 expression was associated with poor outcome (konompova MY and Jordan CT, Biology and Therapeutic Targeting (2011) 29(5): 591-599).

Constitutive activation of the nuclear factor kappa beta (NF-k β) pathway in primary human AML stem cells provides evidence that NF-k β plays a significant role in LSC and overall survival of AML cell types in general (Konoperva MY and Jordan CT, Biology and Therapeutic Targeting (2011) 29(5): 591-599).

FLT3, a member of the class III tyrosine kinase receptor family, is expressed in normal hematopoietic progenitor cells as well as in leukemic blasts, and it plays an important role in cell proliferation, differentiation and survival. Activation of the FLT3 receptor by FLT3 ligand results in receptor dimerization and phosphorylation as well as activation of downstream signaling pathways, including the Janus kinase (JAK) 2 signal transduction protein (JAK2), Signal Transducer and Activator of Transcription (STAT) 5, and mitogen-activated protein kinase (MAPK) pathways. It is believed that mutations in the FLT3 gene found in approximately 40% of patients with AML promote its autophosphorylation and constitutive activation, leading to ligand-independent proliferation (Frankfurt O et al, Current Opinion in Oncology (2007) 19(6): 635-.

Lymphoid malignancies

Self-renewal capacity in most tissues is lost as cells progress through their normal differentiation stages; for example, blood cells of myeloid lineage that exceed the level of hematopoietic stem cells no longer have self-renewal capacity. A significant exception to the self-renewing differentiation-related loss is the lymphoid system, where self-renewal capacity is maintained prior to the memory lymphocyte stage to maintain life-long immunological memory (Fearon DT et al, science 2001; 293: 248-. Somatic hypermutations serve as markers of the differentiation stage of B-cell malignant tumor origin. In general, the presence of somatic hypermutations identifies tumors as having originated from germinal centers or post-germinal B cells, while the absence of mutations identifies pre-germinal B cells. Unlike myeloid malignancies, but consistent with the maintained self-renewal capacity of the lineage, immunoglobulin (Ig) mutation patterns suggest that B cell malignancies can originate from cells throughout the entire B cell differentiation phase (Lapidot T et al, Nature 1994; 367: 645-.

Multiple Myeloma (MM) is commonly regarded as a malignant plasma cell disease with many clinical consequences of the disease caused by plasma cell mass (MM). However, normal plasma cells are terminally differentiated and lack self-renewal capacity, and it has been clear for more than 30 years that only a few cells from mouse and human MM are clonogenic. These rare clonogenic cells have been termed "tumor stem cells" (Park CH et al, J Natl Cancer Inst. 1971; 46: 411. sup. 422; Hamburger AW and Salmon SE, science 1977; 197: 461. sup. 463). MM plasma cells originate from a small population of self-renewing cancer stem cells similar to memory B cells. Not only do these clonal B cells circulate in most patients, they are also resistant to many standard anti-MM agents and thus appear to be responsible for most disease relapses (Matsui WH et al, Blood. 2004; 103: 2332-2336; Kukreja A et al, J Exp Med. 2006; 203: 1859-1865; Jones RJ and Armstrong SA, Biol Blood Marrow transfer. 2008.1 month; 14 (suppl 1): 12-16).

Reed-Sternberg (RS) cells, a marker for Hodgkin's Lymphoma (HL), are the only blood cells that occasionally express CD138 other than plasma cells (carbon A et al, blood 1998; 92: 2220-2228). HL Cell lines have been shown to include a small population of cells lacking the RS markers CD15 and CD30 present on the remaining cells, but expressing markers consistent with the memory B Cell phenotype (Newcom SR et al, Int J Cell cloning. 1988; 6: 417-. This small subset of phenotypic memory B cells had all clonogenic capacity within the HL cell line. Most HL patients, including those with early stage disease, carry circulating memory B cells with the same clonal Ig gene rearrangements as the RS cells of the patient (Jones RJ et al, Blood. 2006; 108: 470; Jones RJ and Armstrong SA, Biol Blood Marrow transfer. 2008.1 months; 14 (suppl 1): 12-16). These data suggest that these clonal memory B cells may represent HL stem cells.

Hematopoietic Stem Cells (HSCs) are used in bone marrow transplantation for the treatment of hematologic malignancies as well as non-malignant conditions (Warner et al, Oncogene (2004) 23(43): 7164-. Bone Marrow (BM) has been transplanted as an unfractionated Cell bank for many years before researchers find which cellular components are responsible for donor hematopoiesis and engraftment of the immune system in myeloresected patients (see, e.g., Sorgio Paulo Bydlowski and Felipe de Lara Janz (2012), Hematopoietic Stem Cell in enzyme granular Leukoid Development, Advances in Hematopoietic Stem Cell Research, Dr. Rosa Pelayo (eds.), ISBN: 978-.

The preparation or conditioning of patients for bone marrow/hematopoietic stem cell (BM/HSC) transplantation is a key element of the procedure. It plays two main roles: (1) it provides adequate immunosuppression of the patient and clears sufficient niche space in the bone marrow for the transplanted HSCs, which allows engrafting of the transplanted cells into the recipient; and (2) it often helps to eradicate the source of the malignancy.

Conditioning of patients has traditionally been achieved as follows: the maximum tolerated dose of the mixture of chemotherapeutic agents is administered with or without radiation. The components of the mixture are often selected to have non-overlapping toxicities. All preparations in use today are toxic and have serious side effects that can be life threatening. Among these are mucositis, nausea and vomiting, hair loss, diarrhea, rash, peripheral neuropathy, infertility, pulmonary toxicity and liver toxicity. Many of these side effects are particularly dangerous for elderly and ill patients, and often become a determining component in deciding whether a patient will receive a transplant.

Thus, there is a need to prepare or condition patients suitable for bone marrow/hematopoietic stem cell (BM/HSC) transplantation without these toxicities. The described invention provides compositions and methods for ablating hematopoietic stem/progenitor cells (HSC/HP) in a patient using bispecific antibodies that bind the human tyrosine kinase receptor FLT3/FLK2 receptor protein and the CD3 receptor protein expressed on T-cells.

Disclosure of Invention

According to one aspect, the invention provides a method of preparing or conditioning a patient in need thereof for hematopoietic cell transplantation, the method comprising: providing a recombinant single chain bispecific antibody that binds both human FLT3 and human CD3, and administering to the patient a therapeutic amount of a pharmaceutical composition comprising the bispecific antibody; wherein the therapeutic amount is effective to: reducing the level of a population of cells expressing one or more of CD45, CD3, FLT3, CD19, CD33 in peripheral blood by at least 90%, and reducing toxicity of a procedure used to prepare or condition the patient.

According to one embodiment, the amino acid sequence of the heavy chain of the antigen-binding portion of the bispecific antibody that binds FLT3 is SEQ ID No. 1 and the amino acid sequence of the light chain of the antigen-binding portion of the bispecific antibody that binds FLT3 is SEQ ID No. 2. According to another embodiment, the bispecific antibody comprises a monoclonal antibody reactive with a subunit of human CD 3. According to another embodiment, the bispecific antibody or antigen-binding portion thereof comprises an isotype selected from immunoglobulin g (igg), IgM, IgE, IgA, and IgD isotypes.

According to one embodiment, the effective amount comprises from 0.01 mg/kg to 10 mg/kg, better still from 0.05 mg/kg to 2mg/kg, better still from 0.1mg/kg to 0.5mg/kg, better still from 0.1mg/kg to 0.3mg/kg, better still from 0.1 mg/kg.

According to one embodiment, the patient in need thereof suffers from Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), chronic myeloid leukemia (CLL), CML, peripheral T-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, neuroblastoma, non-malignant genetic and acquired bone marrow disorders, multiple myeloma or SCID. According to another embodiment, the non-malignant genetic and acquired bone marrow disorder is selected from the group consisting of sickle cell anemia, β -thalassemia major, refractory Diamond-Blackfan anemia, myelodysplastic syndrome, idiopathic severe aplastic anemia, paroxysmal nocturnal hemoglobinuria, pure red cell aplasia, fanconi anemia, megakaryocyte deficiency, and congenital thrombocytopenia.

According to one embodiment, the composition further comprises an antineoplastic agent.

According to one embodiment, the bispecific antibody is a humanized antibody.

According to another aspect, the invention provides a method for making a recombinant single chain bispecific antibody that binds both human FLT3 and human CD3, the method comprising: the C-terminus of the Fab antigen-binding fragment of the Flt3 monoclonal antibody was linked to the CH2 domain of IgG1, and the single chain variable fragment (ScFv) of the monoclonal antibody reactive with the subunit of human CD3 (UCHT1) was linked to the CH2 domain of IgG 1.

According to another aspect, the invention provides a recombinant single chain bispecific antibody that binds both human FLT3 and human CD3, comprising: the C-terminus of the Fab antigen-binding fragment of the Flt3 monoclonal antibody linked to the CH2 domain of IgG1, and the single chain variable fragment (ScFv) of the monoclonal antibody reactive with the subunit of human CD3 (UCHT1) linked to the CH2 domain of IgG 1.

According to one embodiment, the amino acid sequence of the heavy chain binding domain of the Fab antigen binding fragment is SEQ ID NO 1 (H3113) and the amino acid sequence of the light chain binding domain of the Fab antigen binding fragment is SEQ ID NO 2 (L3133).

According to another aspect, the invention provides a monoclonal antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the light chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID No. 5, and the amino acid sequence of the heavy chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID No. 7.

According to another aspect, the invention provides a monoclonal antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the light chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID No. 9, and the amino acid sequence of the heavy chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID No. 11.

According to another aspect, the invention provides a monoclonal antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the light chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID No. 13, and the amino acid sequence of the heavy chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID No. 15.

According to another aspect, the invention provides a monoclonal antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the light chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID No. 17, and the amino acid sequence of the heavy chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID No. 19.

According to another aspect, the invention provides a monoclonal antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the light chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID No. 21, and the amino acid sequence of the heavy chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID No. 23.

According to another aspect, the invention provides a monoclonal antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the light chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID No. 25, and the amino acid sequence of the heavy chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID No. 27.

According to some embodiments, the antibody or fragment thereof has a half maximal Effective Concentration (EC) ₅₀ ) Is between 1 ng/mL (6.25 pM) and 2,000 ng/mL (12.5 nM). According to some embodiments, the antibody or antigen-binding fragment thereof has a half maximal Effective Concentration (EC) ₅₀ ) Is between 10 ng/mL (62.5 pM) and 200 ng/mL (1.25 nM). According to some embodiments, the FLT3 antibody that binds to a human FLT3/FLK2 receptor protein on a cell is effective to internalize the bound antibody or antigen-binding fragment by the cell.

Drawings

FIGS. 1A, 1B, 1C FIGS. 1A and 1B are the natural fluorescence of amino acids such as phenylalanine, tyrosine and tryptophan. FIG. 1C measurement of purity of the synthesized antibody.

Figure 2A, 2B, 2C, 2d. administration of bispecific antibody that binds FLT3/FLK2 expressed by HSC/HP and CD3 expressed by T-cells reduced the level of chimerism in the peripheral blood of humanized immunocompromised mice. FIG. 2A. example of flow cytometric analysis of peripheral blood of humanized NOG mice before (control; up-run) and 3 weeks after application of the CD3-FLT3 bispecific antibody. From left to right: analysis of the amount of human hCD45+ cells (percentage of total CD45+ cells), human hCD3+ cells (percentage of total hCD45+ cells; T-cells), human hCD19+ cells (percentage of total hCD45+ cells; B-cells), human hCD33+ cells (percentage of total hCD45+ cells; myeloid cells). Figure 2b. effect of bispecific antibody administration on chimerism levels in peripheral blood of humanized mice (n = 27). Figure 2c. effect of bispecific antibody administration on levels of T-cells (% hCD3+ cells of total hCD45+ cells), B-cells (% hCD19+ cells of total hCD45+ cells) and myeloid lineage (% hCD33+ cells of total hCD45+ cells) in peripheral blood (n = 27). Figure 2d. reduced effect of bispecific antibody application in humanized immunocompromised mice (marked with an asterisk in C) with reduced amount of human hCD3+ cells (n = 3).

Fig. 3A and 3b. screening of culture supernatants from clonally expanded hybridomas. Fig. 3a. fluorescence intensity histograms from flow cytometry analysis of supernatants of 9 positive hybridoma clones. The supernatants exhibited immunoreactivity to FLT3/FLK2 expressed by REH (human B-cell precursor leukemia cells established from peripheral blood of a 15 year old girl with first relapsed ALL) cells. Fig. 3b. table showing Median Fluorescence Intensity (MFI) of the histogram in fig. 3A. All 9 clones reacted with REH cells expressing the human FLT3/FLK2 receptor protein.

Fig. 4A and 4b. screening of purified monoclonal antibodies from expanded hybridomas fig. 4A. fluorescence intensity histograms obtained from flow cytometry analysis of purified monoclonal antibodies from 9 positive hybridoma clones. The supernatant showed immunoreactivity to the human FLT3/FLK2 receptor protein expressed by SP2/0 cells. The monoclonal antibody did not react with wild type SP2/0 cells that did not express the human FLT3/FL2 receptor protein. Fig. 4b. table showing Median Fluorescence Intensity (MFI) of the histogram in fig. 4A. All 9 clones reacted with SP2/0 cells expressing the human FLT3/FLK2 receptor protein and not with wild-type SP2/0 cells.

5A, 5B, 5C, 5D and 5E affinity of anti-human FLT3/FLK2 antibodies determined by an Effective Concentration (EC) curve using flow cytometry. Fig. 5a. antibody clone Ab 2-81. FIG. 5B antibody clone Ab1-23 DA. Figure 5c. antibody clone Ab3-16 HA. FIG. 5D antibody clone Ab 0-30A. FIG. 5E antibody clone Ab1-18 New.

FIG. 6 time course of internalization by anti-FLT 3/FLK2 monoclonal mouse anti-human CD135 antibody was tested for Mean Fluorescence Intensity (MFI) with a second Alexa Fluor 488 plotted against time for a live Reh cell population. Internalization assays were performed at 37 ℃ in parallel with control cells maintained on ice at 4 ℃ for 10, 30, 60, and 120 minutes. The percent change in MFI for each antibody (clones 123D, A281A, 330A and 316HA) was plotted versus time in triplicate at 4 ℃ and 37 ℃ for 2 hours, setting the MFI at 10 minutes to 100%.

Detailed Description

Glossary of terms

The term "activation" or "lymphocyte activation" refers to the stimulation of lymphocytes by specific antigens, non-specific mitogens or allogeneic cells, resulting in the synthesis of RNA, protein and DNA and the production of lymphokines; which is followed by proliferation and differentiation of various effector and memory cells. For example, a mature B cell can be activated by encountering an antigen that expresses an epitope recognized by its cell surface immunoglobulin Ig). The activation process can be a direct process, which relies on the cross-linking of membrane Ig molecules by antigens (cross-linking dependent B cell activation), or an indirect process, which occurs more efficiently in the context of intimate interactions with helper T cells ("cognate help process"). T-cell activation relies on the interaction of the TCR/CD3 complex with its cognate ligand (a peptide that binds in the groove of class I or class II MHC molecules). The molecular events driven by receptor engagement are complex. The earliest step appears to be the activation of tyrosine kinases, leading to tyrosine phosphorylation of a panel of substrates that control several signaling pathways. These include a group of adaptor proteins that link the TCR to the ras pathway, phospholipase C γ 1, whose tyrosine phosphorylation increases its catalytic activity and engages the inositol phospholipid metabolic pathway, leading to an increase in the intracellular free calcium concentration and activation of protein kinase C, and a series of other enzymes that control cell growth and differentiation. In addition to receptor engagement, full responsiveness of T cells requires co-stimulatory activity delivered by helper cells, e.g., engagement of CD80 and/or CD86 on Antigen Presenting Cells (APCs) to CD28 on T cells. The soluble product of activated B lymphocytes is immunoglobulin (antibody). The soluble product of activated T lymphocytes is lymphokine.

The term "administering" as used herein means administering or applying. The term "administration" and its various grammatical forms as used herein includes in vivo administration, as well as ex vivo administration directly to a tissue.

Antibody:

antibodies are serum proteins whose molecules have small regions of their surface that are complementary to small chemical groups on their targets. These complementary regions, which are at least 2 per antibody molecule, and in some types 10, 8, or in some species up to 12, can react with their corresponding complementary regions (epitopes or epitopes) on the antigen to link several molecules of multivalent antigens together to form a lattice.

The basic structural unit of a complete antibody molecule consists of 4 polypeptide chains: 2 identical light (L) chains (each containing about 220 amino acids) and 2 identical heavy (H) chains (each typically containing about 440 amino acids). The 2 heavy and 2 light chains are held together by a combination of non-covalent and covalent bonds (disulfide bonds). The molecule consists of 2 identical halves, each half having identical antigen binding sites consisting of the N-terminal region of the light chain and the N-terminal region of the heavy chain. Both the light and heavy chains typically cooperate to form an antigen-binding surface.

Human antibodies display two classes of light chains, kappa and lambda; a single immunoglobulin molecule is typically only one or the other. In normal serum, 60% of the molecules have been found to have kappa determinants and 30% lambda. Many other species have been found to exhibit two types of light chains, but in different proportions. For example, in mice and rats, λ chains account for several percent of the total; in dogs and cats, the kappa chain is very low; horses appear not to have any kappa chains; rabbits may have 5-40% λ, depending on strain and b-locus allotype; and chicken light chains are more homologous to λ than to κ.

In mammals, there are 5 antibody classes, IgA, IgD, IgE, IgG and IgM, each with its own heavy chain class- α (for IgA), δ (for IgD), epsilon (for IgE), γ (for IgG) and μ (for IgM). In addition, there are 4 IgG immunoglobulin subclasses (IgG1, IgG2, IgG3, IgG4) with γ 1, γ 2, γ 3, and γ 4 heavy chains, respectively. In its secreted form, IgM is a pentamer consisting of 5 4-chain units, providing it with a total of 10 antigen binding sites. Each pentamer contains one copy of the J chain, which is covalently inserted between 2 adjacent tail regions.

All 5 immunoglobulin classes differ from other serum proteins in that they exhibit a wide range of electrophoretic mobilities and are not homogeneous. The heterogeneity-individual IgG molecules, for example, differ from each other in net charge-is an intrinsic property of immunoglobulins.

An "antigenic determinant" or "epitope" is an antigenic site on a molecule. The sequential antigenic determinants/epitopes are substantially linear. In an ordered structure such as a helical polymer or protein, an antigenic determinant/epitope is essentially a limited region or patch within or on the surface of the structure that contains amino acid side chains from different parts of the molecule that are accessible to each other. These are conformational determinants.

The principle of complementarity, often compared to the fitting of a key in a lock, involves relatively weak binding forces (hydrophobic and hydrogen bonds, van der waals forces and ionic interactions) which can only effectively act when: two reactive molecules can be in close proximity to each other, and in fact, so close that the protruding component atoms or groups of atoms of one molecule can fit into complementary recesses or pockets of the other molecule. Antigen-antibody interactions exhibit a high degree of specificity, which is manifested at many levels. Reduced to the molecular level, "specificity" means that the antibody has complementarity to the binding site of the antigen, and also does not resemble antigenic determinants of unrelated antigens. Whenever the antigenic determinants of 2 different antigens have some structural similarity, some degree of coordination of one determinant into the binding site of some antibodies to another determinant may occur, and the phenomenon produces cross-reactivity. Cross-reactivity is of great importance in understanding the complementarity or specificity of antigen-antibody reactions. Immunological specificity or complementarity makes it possible to detect small amounts of impurities/contaminations in the antigen.

"monoclonal antibodies" (mabs) can be generated as follows: mouse spleen cells from immunized donors are fused with a mouse myeloma cell line to produce established mouse hybridoma clones that are grown in selective media. "hybridoma cells" are immortalized hybrid cells derived from the in vitro fusion of antibody-secreting B cells with myeloma cells. "in vitro immunization" (which refers to the primary activation of antigen-specific B cells in culture) is another well-established means of producing mouse monoclonal antibodies.

Different libraries of immunoglobulin heavy (VH) and light (vk and V λ) chain V genes from peripheral blood lymphocytes can also be amplified by Polymerase Chain Reaction (PCR) amplification. By randomly combining the heavy and light chain V-genes using PCR, a gene can be made that encodes a single polypeptide chain in which the heavy and light chain variable domains are connected by a polypeptide spacer (single chain Fv or scFv). The combinatorial library can then be cloned for display on the surface of a filamentous bacteriophage via a small coat protein fused to the top of the bacteriophage.

The guided selection technique is based on shuffling of human immunoglobulin V genes with rodent immunoglobulin V genes. The method requires: (i) shuffling a human lambda light chain profile with the heavy chain variable region (VH) domain of a mouse monoclonal antibody reactive with an antigen of interest; (ii) selecting a semi-human Fab on said antigen; (iii) using the selected lambda light chain gene as the "docking domains" of the human heavy chain library in the second shuffling to isolate cloned Fab fragments with the human light chain gene; (v) transfecting a mouse myeloma cell by electroporation with a mammalian cell expression vector containing the gene; and (vi) expressing the V gene of Fab reactive with the antigen in a mouse myeloma as a whole IgG1, λ antibody molecule.

The term "antibody-dependent cellular cytotoxicity (ADCC)" as used herein is triggered when an antibody bound to the cell surface interacts with Fc receptors on Natural Killer (NK) cells. NK cells express the receptor Fc γ RIII (CD16), which recognizes the IgG1 and IgG3 subclasses. The killing mechanism is similar to that of cytotoxic T cells, involving the release of cytoplasmic granules containing perforin and granzyme (see below).

CD3 (TCR complex) is a protein complex consisting of 4 distinct chains. In mammals, the complex contains a CD3 γ chain, a CD3 δ chain, and 2 CD3 epsilon chains, which combine with the T Cell Receptor (TCR) and ζ -chains to generate an activation signal in T lymphocytes. The TCR, zeta-chain and CD3 molecules together comprise a TCR complex. The intracellular tail of the CD3 molecule contains a conserved motif called the immunoreceptor tyrosine-based activation motif (ITAM), which is essential for the signaling ability of the TCR. Upon phosphorylation of ITAMs, the CD3 chain can bind ZAP70 (zeta-related protein), a kinase involved in the signaling cascade of T cells.

The term "bind" and its various grammatical forms means a permanent attraction between chemical substances. Binding specificity includes binding to a specific partner and not binding to other molecules. Functionally important binding may occur in a range of affinities from low to high, and design elements may inhibit undesirable cross-interactions. Post-translational modifications may also alter the chemistry and structure of the interaction. "promiscuous binding" may involve a degree of structural plasticity that may result in different subpopulations of residues being important for binding to different partners. "relative binding specificity" is a characteristic whereby in a biochemical system, a molecule interacts differentially with its targets or partners, affecting them differently depending on the identity of the individual targets or partners.

The term "contact" and its various grammatical forms as used herein refers to a state or condition of contact or immediate or local proximity. Contacting the composition with a target object such as, but not limited to, an organ, tissue, or cell may occur by any mode of administration known to those of skill in the art.

The term "half maximal effective concentration" (EC) as used herein ₅₀ ) Means the antibody concentration that induces a response half way between baseline and maximum after the exposure time.

The term "hematopoietic cell transplantation" (HCT) is used herein to refer to blood and Bone Marrow Transplantation (BMT), including procedures for infusing cells (hematopoietic stem cells; also known as hematopoietic progenitor cells) to reconstitute the hematopoietic system of a patient.

The term "lymphocyte" refers to a small leukocyte formed in lymphoid tissues throughout the body, and accounts for about 22-28% of the total number of leukocytes in the circulating blood in normal adults, which plays an important role in defending the body against disease. Various lymphocytes are specialized in that they are typed to respond to a limited set of structurally related antigens. The stereotype, which is present before the first contact of the immune system with a given antigen, is expressed by the presence of receptors specific for determinants (epitopes) on the antigen on the surface membrane of lymphocytes. Each lymphocyte has a population of receptors, all of which have the same binding site. One pool or clone of lymphocytes differs from another by the structure of the binding region of its receptor and thus by the epitope it can recognize. Lymphocytes differ from one another not only in the specificity of their receptors, but also in their function.

Two major classes of lymphocytes were identified: b-lymphocytes (B-cells), which are precursors of antibody-secreting cells; and T-lymphocytes (T-cells).

B lymphocyte

B-lymphocytes are derived from hematopoietic cells of the bone marrow. Mature B-cells can be activated with an antigen that expresses an epitope recognized by its cell surface. The activation process may be direct, relying on antigen cross-linking to membrane Ig molecules (cross-linking dependent B-cell activation); or indirectly, via interaction with helper T-cells, in a process known as associative helper. In many physiological situations, receptor cross-linking stimulation and associated aids act synergistically to produce a more robust B-cell response (Paul, W.E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers, Philadelphia (1999)).

Cross-linking dependent B-cell activation requires that the antigen express multiple copies of an epitope complementary to the binding site of a cell surface receptor, since each B-cell expresses an Ig molecule with the same variable region. Such requirements are fulfilled by other antigens with repetitive epitopes, such as capsular polysaccharides of microorganisms or viral envelope proteins. Cross-linking dependent B-cell activation is The main protective immune response established against these microorganisms (Paul, W.E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers, Philadelphia (1999)).

Associative helper allows B-cells to establish a response to the antigen of the non-crosslinkable receptor and, at the same time, provide a costimulatory signal that rescues B cells from inactivation (when they are stimulated by a weak crosslinking event). Association aids rely on binding of the antigen by the membrane immunoglobulin (Ig) of the B-cell, endocytosis of the antigen and its fragmentation into peptides in the endosomal/lysosomal compartment of the cell. Some of the resulting peptides are loaded into the groove in a specialized collection of cell surface proteins called major histocompatibility complex class II (MHC) molecules. The resulting class II/peptide complexes are expressed on the cell surface and act as ligands for antigen-specific receptors of a group of T-cells designated CD4+ T-cells. CD4+ T-cells carry receptors on their surface that are specific for class II/peptide complexes of B-cells. B-cell activation is not only dependent on T-cell binding through its T-cell receptor (TCR), but the interaction also allows an activating ligand (CD40 ligand) on the T-cell to bind to its receptor on the B-cell (CD40), thereby signaling B-cell activation. In addition, T helper cells secrete several cytokines that regulate The growth and differentiation of stimulated B-cells by binding to cytokine receptors on B cells (Paul, W. E., "Chapter 1: The animal system: an introduction," Fundamental Immunology, 4 th edition, Paul, W. E. eds., Lippicott-Raven Publishers, Philadelphia (1999)).

During the cognate helper process of antibody production, CD40 ligand is transiently expressed on activated CD4+ T helper cells and it binds to CD40 on antigen-specific B cells, thereby transducing a second costimulatory signal. The latter signal is necessary for B cell growth and differentiation and for memory B cell production (by preventing apoptosis of germinal center B cells that have encountered antigen). Overexpression of CD40 ligand (overexpression) in B and T cells has been implicated in pathogenic autoantibody production in human SLE patients (Desai-Mehta, A. et al, "overexpression of CD40 ligand by B and T cells in human lung and its enzymes in pathological autoinduction," J. Clin. invest., 97(9): 2063-2073 (1996)).

T-lymphocytes

T-lymphocytes are derived from precursors in hematopoietic tissues, undergo differentiation in the thymus, and are then seeded into the surrounding lymphoid tissue and a recirculating pool of lymphocytes. T-lymphocytes or T-cells mediate a wide range of immunological functions. These include the ability to assist B cells in developing antibody-producing cells, the ability to increase monocyte/macrophage microbicidal action, the suppression of certain types of immune responses, direct killing of target cells, and the mobilization of inflammatory responses. These effects are dependent on The expression of their specific cell surface molecules and The secretion of cytokines (Paul, W. E., "Chapter 1: The animal system: an introduction," Fundamental Immunology, 4 th edition, Paul, W. E. eds., Lippicott-Raven Publishers, Philadelphia (1999)).

T cells differ from B cells in their antigen recognition mechanisms. Immunoglobulins (receptors for B cells) bind various epitopes on soluble molecules or on the surface of particles. B-cell receptors encounter epitopes expressed on the surface of natural molecules. Antibodies and B-cell receptors have evolved to bind and protect against microorganisms in extracellular fluids. In contrast, T cells recognize antigens on the surface of other cells and mediate their functions by interacting with and altering the behavior of these Antigen Presenting Cells (APCs). There are three major types of antigen presenting cells in the peripheral lymphoid organs that can activate T cells: dendritic cells, macrophages and B cells. The most potent of these are dendritic cells, whose sole function is to present foreign antigens to T cells. Immature dendritic cells are located in tissues throughout the body, including the skin, intestinal tract, and respiratory tract. When they encounter invading microorganisms at these sites, they endocytose the pathogens and their products and carry them to regional lymph nodes or gut-associated lymphoid organs via lymph. The encounter with the pathogen induces the dendritic cells to mature from antigen-capturing cells to Antigen Presenting Cells (APCs) that can activate T cells. APCs display three classes of protein molecules on their surface that play a role in activating T cells to become effector cells: (1) MHC proteins that present foreign antigens to T cell receptors; (2) a costimulatory protein that binds to a complementary receptor on the surface of a T cell; and (3) Cell-Cell adhesion molecules that enable T cells to bind to Antigen Presenting Cells (APC) for a length of time sufficient to become activated ("Chapter 24: The adaptive immune system," Molecular Biology of The Cell, Alberts, B.et al, Garland Science, NY, 2002).

T-cells are subdivided into two distinct classes based on the cell surface receptors they express. Most T cells express a T Cell Receptor (TCR) consisting of alpha and beta chains. Small T cell groups express receptors consisting of gamma and delta chains. There are two important sub-lineages in α/β T cells: those expressing the co-receptor molecule CD4 (CD4+ T cells); and those expressing CD8 (CD8+ T cells). These cells differ in how they recognize antigens and their effector and regulatory functions.

CD4+ T cells are the primary regulatory cells of the immune system. Their regulatory function depends on the expression of their cell surface molecules (such as CD40 ligand, which induces its expression when T cells are activated) and the bulk of the cytokines they secrete when activated.

T cells also mediate important effector functions, some of which are determined by the pattern of cytokines they secrete. The cytokine may be directly toxic to the target cell and may mobilize effective inflammatory mechanisms.

In addition, T cells, in particular CD8+ T cells, can develop cytotoxic T-lymphocytes (CTL) which are capable of effectively lysing target cells expressing The antigen recognized by The CTL (Paul, W. E., "Chapter 1: The animal system: an expression," Fundamental Immunology, 4 th edition, Paul, W. E. eds., Lippicott-Raven Publishers, Philadelphia (1999)).

T Cell Receptors (TCRs) recognize complexes composed of peptides derived from the proteolytic cleavage of antigens bound to the specialized groove of class II or class I MHC proteins. CD4+ T cells recognize only peptide/class II complexes, whereas CD8+ T cells recognize peptide/class I complexes (Paul, W. E., "Chapter 1: The immune system: an introduction," functional Immunology, 4 th edition, Paul, W. E. eds., Lippicott-Raven Publishers, Philadelphia (1999)).

The ligand (i.e., peptide/MHC protein complex) of the TCR is produced within an Antigen Presenting Cell (APC). In general, MHC class II molecules bind peptides derived from proteins that have been taken up by APCs through the process of endocytosis. These peptide-loaded class II molecules are then expressed on the cell surface where they can be bound by CD4+ T cells with TCRs that are capable of recognizing the expressed cell surface complex. Thus, CD4+ T cells are specialized to react with antigens derived from extracellular sources (Paul, W. E., "Chapter 1: The animal system: an introduction," Fundamental Immunology, 4 th edition, Paul, W. E. eds., Lippicott-Raven Publishers, Philadelphia (1999)).

In contrast, class I MHC molecules are loaded primarily with peptides derived from internally synthesized proteins, such as viral proteins. These peptides are produced from cytoplasmic proteins by proteolysis of proteosomes and transported into the rough endoplasmic reticulum. Such peptides (typically 9 amino acids in length) bind into MHC class I molecules and reach the cell surface where they can be recognized by CD8+ T cells expressing the appropriate receptor. This gives The T cell system, in particular CD8+ T cells, The ability to detect cells expressing proteins which are different from or produced in much larger amounts than those of cells of other parts of The organism (e.g. viral antigens) or mutant antigens such as active oncogene products, even though these proteins in their intact form are neither expressed nor secreted on The cell surface (Paul, w. e., "Chapter 1: The animal system: an introduction," Fundamental Immunology, 4 th edition, Paul, w. e. eds., lippiccott-Raven Publishers, philiadelphia (1999)).

T cells can also be classified based on their function as: helper T cells; t cells involved in inducing cellular immunity; an inhibitory T cell; and cytotoxic T cells.

Helper T cell

Helper T cells are T cells that stimulate B cells to generate antibody responses to proteins and other T cell-dependent antigens. T cell-dependent antigens are immunogens in which individual epitopes are present only once or a limited number of times, making them unable or inefficient to cross-link the membrane immunoglobulin (Ig) of B cells. B cells bind antigen through their membrane Ig, and the complex undergoes endocytosis. Within the endosomal and lysosomal compartments, the antigen is fragmented into peptides by proteolytic enzymes, and one or more of the produced peptides are loaded into MHC class II molecules, which are transported through the vesicular compartment. The resulting peptide/MHC class II complex is then exported to the B-cell surface membrane. T cells with receptors specific for peptide/class II molecule complexes recognize The complexes on The B-cell surface (Paul, W.E., "Chapter 1: The immune system: an introduction," functional Immunology, 4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers, Philadelphia (1999)).

B-cell activation depends on both T-cell binding through its TCR and the interaction of T-cell CD40 ligand (CD40L) with CD40 on B-cells. T cells do not constitutively express CD 40L. In contrast, CD40L expression was induced as a result of interaction with APCs that express both the cognate antigen recognized by the TCR of the T cell and CD80 or CD 86. CD80/CD86 is typically expressed by activated B cells rather than resting, so that helper interactions involving activated B cells and T cells can lead to efficient antibody production. However, in many cases, the initial induction of CD40L on T cells is dependent on their recognition of antigens on the surface of APCs (such as dendritic cells) that constitutively express CD 80/86. Such activated helper T cells can then efficiently interact with and help B cells. Crosslinking of membrane Ig on B cells, even if inefficient, may cooperate with CD40L/CD40 interactions to produce robust B-cell activation. Later events in B-cell responses, including proliferation, Ig secretion and class switching (of The Ig class expressed) are dependent on or enhanced by The action of T-cell-derived cytokines (Paul, W. E., "Chapter 1: The immune system: an expression," Fundamental immunity, 4 th edition, Paul, W. E. eds., Lippicott-Raven Publishers, Philadelphia (1999)).

CD4+ T cells tend to differentiate into cells that secrete primarily the cytokines IL-4, IL-5, IL-6, and IL-10 (TH2 cells), or into cells that produce primarily IL-2, IFN- γ, and lymphotoxins (TH1 cells). TH2 cells very efficiently aid B-cells in developing antibody producing cells, while TH1 cells are potent inducers of cellular immune responses, involving an enhancement of the microbicidal activity of monocytes and macrophages and a consequent increased efficiency of lysis of microorganisms in the intracellular capsule compartment. Although CD4+ T cells, which have the phenotype of TH2 cells (i.e., IL-4, IL-5, IL-6, and IL-10), are potent helper cells, TH1 cells also have the ability to become helper cells. (Paul, W.E., "Chapter 1: The animal system: an introduction," Fundamental Immunology, 4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers, Philadelphia (1999)).

T cells involved in the induction of cellular immunity

T cells may also act to enhance the ability of monocytes and macrophages to destroy intracellular microorganisms. Specifically, interferon- γ (IFN- □) produced by helper T cells enhances several mechanisms by which mononuclear phagocytes destroy intracellular bacteria and parasitism, including the production of nitric oxide and the induction of Tumor Necrosis Factor (TNF). TH1 cells are effective in enhancing microbicidal action because they produce IFN- γ. In contrast, the two major cytokines IL-4 and IL-10 produced by TH2 cells block these activities. (Paul, W.E., "Chapter 1: The animal system: an introduction," Fundamental Immunology, 4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers, Philadelphia (1999)).

Suppressor or regulatory T (Treg) cells

A controlled balance between initiation and downregulation of immune responses is important for maintaining immune homeostasis. Apoptosis and T cell anergy (a tolerance mechanism, in which T cells are intrinsically functionally inactivated upon encountering antigen (Scrwartz, R. H., "T cell interference," Annu. Rev. immunity, 21: 305-334 (2003)) are both important mechanisms contributing to the down-Regulation of the immune response.A third mechanism is provided by the inhibitory or regulatory activity inhibition of activated T cells by CD4+ T (Treg) cells (reviewed in Kroneberg, M. et al, "Regulation of immunity by self-reactive T cells," Nature 435: 598-604 (2005)). the CD4+ Treg (CD4+ CD25+) constitutively expressing the IL-2 receptor alpha (IL-2R □) chain is an anergy and inhibitory subset of naturally occurring T cells (Taams, L.S. et al, "Human/environmental + CD25+ CD25+) which are inactive and inhibitory, "Eur. J. Immunol., 31: 1122-1131 (2001)). Exclusion of CD4+ CD25+ tregs resulted in the development of systemic autoimmune disease in mice. In addition, the metastasis of these tregs prevents the development of autoimmune diseases. Human CD4+ CD25+ tregs (similar to their murine counterparts) are produced in the thymus and are characterized by the ability to suppress proliferation of responding T cells (responder T cells) through cell-cell contact-dependent mechanisms, the inability to produce IL-2, and an in vitro anergic phenotype. Human CD4+ CD25+ T cells can be divided into suppressive (high CD 25) and non-suppressive (low CD 25) cells based on CD25 expression levels. FOXP3, a member of the forkhead family of transcription factors, has been shown to be expressed in murine and human CD4+ CD25+ Tregs and appears to be the dominant gene controlling the development of CD4+ CD25+ Tregs (Battaglia, M. et al, "Rapamycin proteins expansion of functional CD4+ CD25+ Foxp3+ relative T cells of bone height basic subjects and type 1 diagnostic markers," J. Immunol., 177: 8338-8347 (200)).

Cytotoxic T Lymphocytes (CTL)

CD8+ T cells that recognize peptides from proteins produced within target cells have cytotoxic properties because they cause lysis of the target cells. The mechanism of CTL-induced lysis involves the production of perforin, a molecule that can insert into the membrane of the target cell and facilitate lysis of the cell, by CTLs. A series of enzymes produced by activated CTLs (called granzymes) enhance perforin-mediated lysis. Many active CTLs also express large numbers of fas ligands on their surface. The interaction of fas ligand on the surface of CTLs with fas on the surface of target cells initiates apoptosis in the target cells, leading to the death of these cells. CTL-mediated lysis appears to be the major mechanism of destruction of virus-infected cells.

Sensitization

The term "naive cells" (also referred to as daughter, naive or inexperienced cells) as used herein refers to T cells and B cells that have produced antigen receptors with specific specificities (TCR for T cells and BCR for B cells), but have never encountered the antigen. The term "priming" as used herein refers to the process whereby T cells and B cell precursors encounter antigens for which they are specific.

For example, antigen-specific T cell precursors must be primed before helper T and B cells can interact to produce specific antibodies. Sensitization involves several steps: antigen uptake, processing and cell surface expression of antigen presenting cells in association with MHC class II molecules, recycling and antigen-specific trapping of helper T cell precursors in lymphoid tissues, and T cell proliferation and differentiation. Janeway, CA, Jr., "The priming of helper T cells, Semin. Immunol. 1(1): 13-20 (1989). Helper T cells express CD4, but not all CD 4T cells are helper cells. The above is presented. The signals required for clonal expansion of helper T cells differ from those required for other CD 4T cells. Antigen presenting cells critical for helper T cell priming appear to be macrophages; and the key second signal for helper T cell growth is the macrophage product interleukin 1 (IL-1). The above is presented. If the primed T-cells and/or B-cells receive the second costimulatory signal, they become activated T-cells or B-cells.

The term "transplantation" as used herein refers to the removal of a cell, tissue or organ from one part or individual and transfer to another.

According to one aspect, the invention provides recombinant bispecific antibodies that bind both human Flt3 and human CD 3. According to some embodiments, the Flt3 antibody binds to the Flt3/FLK2 receptor protein. According to some embodiments, the FLT3/FLK2 receptor protein is a mammalian protein. According to some embodiments, the FLT3/FLK2 receptor protein is a human protein. According to some embodiments, the FLT3/FLK2 receptor protein is native. According to some embodiments, the FLT3/FLK2 receptor protein is in a modified form. According to some embodiments, the FLT3/FLK2 receptor protein is in a denatured form. According to some embodiments, the FLT3/FLK2 receptor protein is in an unmodified form. According to some embodiments, the Flt3 antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, an antibody fragment, and a synthetic antibody mimetic. According to some embodiments, the Flt3 antibody is a monoclonal antibody. According to some embodiments, the FLt3 monoclonal antibody is selected from the group consisting of a synthetic antibody and an artificially engineered antibody. According to some embodiments, the synthetic antibody is a recombinant antibody. According to some embodiments, the recombinant antibody is a single chain variable fragment (scFv) antibody. According to some embodiments, the single chain antibody comprises the C-terminus of a Fab fragment of the Flt3 antibody linked to the CH2 domain of IgG 1. According to some embodiments, the CH2 domain of IgG1 is linked to a single chain variable fragment (ScFv) of an antibody reactive with a subunit of human CD 3. According to some embodiments, the single chain variable fragment is a monoclonal antibody. According to some embodiments, the subunit of human CD3 is UCHT 1. According to some embodiments, the artificially engineered antibody is a chimeric antibody. According to some embodiments, the artificially engineered antibody is a humanized antibody.

According to some embodiments, the Flt3 antibody that binds Flt3 effectively blocks the binding of Flt3 ligand to the Flt3/FLK2 receptor protein. According to some embodiments, the Flt3 antibody that binds Flt3 on a cell is effective for internalizing the bound antibody by the cell.

According to some embodiments, the Flt3 antibody has a half-maximal Effective Concentration (EC) between about 1 ng/mL (6.25 pM) and about 2,000 ng/mL (12.5nM) ₅₀ ). According to some embodiments, the Flt3 antibody has an affinity at aboutHalf maximal Effective Concentration (EC) between 10 ng/mL (62.5 pM) and about 200 ng/mL (1.25nM) ₅₀ ). According to some embodiments, the bispecific antibody that binds both human Flt3 and human CD3 is effective to deplete one or more of hematopoietic stem cells (HPCs), early hematopoietic progenitor cells (HPs), and cancer cells. According to some embodiments, one or more of the HPCs, and cancer cells express FLT 3. According to some embodiments, the subject in need thereof is a patient eligible to receive, will receive, or is receiving a BM/HPCPC transplant. Examples of cancer cells include, but are not limited to, blast cells of Acute Myeloid Leukemia (AML), Acute Lymphocytic Leukemia (ALL), blast cells of blast crisis stage of chronic myeloid leukemia (BC-CML) and Chronic Lymphocytic Leukemia (CLL). According to some embodiments, the bispecific antibody is effective to condition a patient undergoing a Bone Marrow (BM)/Hematopoietic Stem Cell (HSC) transplant. According to some embodiments, the HSC/HP transplantation is used to treat hematological malignancies or hyperproliferative disorders (hyperproliferative disorders), such as Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML), peripheral T-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, neuroblastoma, non-malignant genetic and acquired myeloid disorders (e.g., sickle cell anemia, beta-thalassemia major, refractory Diamond-blaken anemia, myelodysplastic syndrome, idiopathic severe aplastic anemia, paroxysmal nocturnal hemoglobinuria, monocytic aplasia, kanney anemia, megakaryocytic deficiency or congenital thrombocytopenia), Multiple myeloma and Severe Combined Immunodeficiency (SCID).

According to another aspect, a method for making a recombinant single chain bispecific antibody that binds both human FLT3 and human CD3 comprises linking the C-terminus of the Fab fragment of the FLT3 monoclonal antibody to the CH2 domain of IgG1, and linking a single chain variable region fragment (ScFv) of a monoclonal antibody reactive with a subunit of human CD3 (UCHT1) to the CH2 domain of IgG 1.

According to another aspect, the invention provides a method of depleting hematopoietic stem/progenitor cells (HSC/HP) in a patient in need thereof. According to some embodiments, the method comprises administering to the patient a bispecific antibody that specifically binds HSC/HP and T-cells. Specifically, the bispecific antibody binds human FLT3 expressed by HSC/HP and human CD3 expressed by T cells. Simultaneous binding of the antibodies redirects T-cells to specifically eliminate HSC/HP from the patient.

The method also provides for the administration of an effective amount of a specific antibody to the patient. The effective amount is 0.01 mg/kg to 10 mg/kg, preferably 0.05 mg/kg to 2mg/kg, more preferably 0.1mg/kg to 0.5mg/kg, more preferably 0.1mg/kg to 0.3mg/kg, more preferably 0.1 mg/kg.

According to some embodiments, the bispecific antibody that binds primate and human CD3 is a humanized antibody.

According to some embodiments, the bispecific antibody or antigen-binding portion thereof comprises the amino acid sequence of the FLT3 antibody.

According to some embodiments, the bispecific antibody, or antigen-binding portion thereof, comprises the amino acid sequence of a CD3 antibody.

According to some embodiments, the bispecific antibody or antigen-binding portion thereof comprises an isotype selected from immunoglobulin g (igg), IgM, IgE, IgA, or IgD isotypes.

According to another aspect, the invention also provides a method of eliminating HSC/HP in a patient in need thereof, wherein the HSC/HP expresses FLT 3. The method comprises selecting a patient in need of HSC/HP elimination and administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a bispecific antibody that specifically binds human FLT3 expressed by HSC/HP and human CD3 expressed by T-cells, wherein the bispecific antibody redirects the T-cells to kill the HSC/HP in the patient.

Patients in need of HSC/HP elimination are patients suffering from Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML), peripheral T-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, hodgkin's lymphoma, non-hematologic malignancies such as neuroblastoma, non-malignant genetic and acquired bone marrow disorders (e.g., sickle cell anemia, beta-thalassemia major, refractory Diamond-Blackfan anemia, myelodysplastic syndrome, idiopathic severe aplastic anemia, paroxysmal nocturnal hemoglobinuria, monocytic aplasia, convinical anemia, megakaryocytic deficiency or congenital thrombocytopenia), multiple myeloma, Severe Combined Immunodeficiency (SCID) and treatment of hematopoietic stem cells transplantation using Bone Marrow (BM)/Hematopoietic Stem Cells (HSC) Other conditions of treatment.

The pharmaceutical composition comprises the antibody and a pharmaceutically acceptable carrier, diluent or excipient. The carrier is selected from, for example, one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, and the like, and combinations thereof. The pharmaceutically acceptable carrier may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers which enhance the shelf-life or effectiveness of the binding protein. The pharmaceutical compositions may be formulated so as to provide rapid, sustained or delayed release of the active ingredient after administration (Mishra, m.k. (2016), as is well known in the art.Handbook of encapsulation and controlled release. Boca Raton, CRC Press, Taylor &Francis Group, CRC Press is Taylor&Publisher name of Francis Group, Informata, incorporated herein by reference in its entirety).

The pharmaceutical composition may further comprise another component such as a T-cell or an antineoplastic agent. Antineoplastic agents to be administered with the antibodies include any agent that destroys or damages a tumor or malignant cells.

The antineoplastic agent is selected from suitable anticancer agents known to those skilled in the art and includes anthracyclines (e.g., daunorubicin and doxorubicin), auristatins (auristatins), Methotrexate (MTX), vindesine, neocarzinostain, cisplatin, chlorambucil, cytarabine, 5-floxuridine, melphalan, ricin and calicheamicin, including combination chemotherapies such as with doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD), beacop or enhanced doses of beacop (escalated beacop) (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone) and Stanford V (doxorubicin, vinblastine, nitrogen mustard, vincristine, bleomycin, etoposide and prednisone). The antineoplastic agent may also be an immunotherapy (e.g., the anti-CD 20 antibody rituximab (rituximab)), an immunotoxin (e.g., Brentuximab vedotin (SGN-35) is an immunotoxin consisting of a CD-30-directed antibody linked to the anti-tubulin agent monomethyl auristatin e (mmae)), an adoptive immunotherapy (cytotoxic T lymphocytes), programmed death 1 (PD-1) blockade (e.g., nivolumab, pembrolizumab)).

According to another aspect, the invention further provides a method of testing for a bispecific antibody that redirects T-cells to kill HSC/HP in an in vivo animal model, wherein the animal model is an immunocompromised humanized mouse with a chimeric mouse-human hematopoietic system, wherein the humanized mouse is produced by: transplanting human HSC/HP or transplanting human post-natal hematopoietic endothelial cells into the severely myelosuppressed (myeloablated) immunocompromised mice.

Bispecific antibodies of the invention have been synthesized according to the methods described in Durben et al (Molecular Therapy, vol.23, No. 4, month 4 2015), which is incorporated herein by reference in its entirety.

The FLT3 antibody sequence used is described in U.S. Pat. No. 9,023,996 to Grosse-Hovest et al, also incorporated herein by reference in its entirety.

It is to be understood that while the invention has been described in conjunction with the preferred embodiments thereof, those skilled in the art will recognize that other embodiments may be made without departing from the spirit of the invention.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may also be included in the invention, and are independently included in the smaller ranges, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the described invention, exemplary methods and materials have been described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric.

In order to better illustrate the invention, the following examples are given.

Example 1: description of antibody Synthesis

Background information

Fabsc is a recombinant bispecific antibody format. The Fabsc versions of bispecific antibodies targeting FLT3 (using the 4G8 clone) and CD3 (using the UCHT1 antibody sequence, also known as huxCD3v1) are as follows: the C-terminus of the Fab fragment of Flt3 mAb will be linked to the CH2 domain of IgG1, followed by a ScFv of UCHT 1.

The sequences of the 4G8 clone and UCHT1 were obtained from patent nos. US 9,023,996B 2 and 6,054,297, respectively, which are incorporated herein by reference in their entirety.

Extent of the experiment

Gene synthesis based on the forms of 4G8 and UCHT1 variable heavy and light chain sequences described in the background.

Molecular construction of IgG expression vectors.

Transient production of 0.1 elevated mass (premium) in HEK293 cells.

Custom purification (kappa as electrode and protein l (protein l) -column and elution at pH 2.3).

Protein aggregation analysis by SE-HPLC.

Target Deliverables (Target delivery)

All purified proteins from 0.1 liter production.

The study report includes: certificate analysis, CE-SDS analysis, SE-HPLC analysis report.

Depending on the yields obtained after expression and purification, the customer will decide whether the antibodies produced are used in the following analytical steps:

assay purity, monomer content, and aggregation (0.1 mg) by SE-HPLC.

The association and dissociation using different concentrations of antigen and calculation of kD were performed by ForeBio Octet QKe (0.2 mg).

Results

The Fabsc antibody was cloned into a high expression mammalian vector system and small scale (0.1 liter) high quality transient production was achieved in HEK293 cells. The protein was purified by protein L purification and 20.17 mg of protein was obtained. The yield is reported and the customer confirms that SE-HPLC should be performed. The antibody was 92% non-aggregated monomer as determined by SE-HPLC.

Vector construction and transient production

Molecular construction of expression vectors

The DNA Studio gene was synthesized and the programmed sequences were cloned into one of the high expressing mammalian vectors. The completed construct was sequence confirmed before proceeding to transfection.

TABLE A

Small-scale transient transfection

HEK293 cells were seeded in shake flasks 1 day before transfection and cultured using serum-free chemically defined media. The DNA expression constructs were transiently transfected into 0.1 liter suspension HEK293 cells using standard procedures for transient transfection. After 20 hours, cells were sampled for viability and viable cell count, and titers were measured (Octet QKe, ForteBio). Cultures were harvested on day 5 and additional readings were made.

Protein L affinity purification

The conditioned medium of Fabsc was harvested and clarified from the transient transfection production process by centrifugation and filtration. The supernatant was run on a protein L column and eluted with low pH buffer. Filtration was performed using a 0.2 μm membrane filter before aliquots. After purification and filtration, protein concentration was calculated from OD280 and extinction coefficient. See table 1 for a summary description of yields and aliquots. CE-SDS analysis (LabChip GXII, Perkin Elmer) was performed and electrophoretograms were plotted and shown in FIGS. 1A and 1B.

SE-HPLC analysis

5 μ L of purified antibody was injected into a MAbPac SEC-1, 5 μm, 4 x 300 mm column at a flow rate of 0.2 mL/min for 25 minutes. The protein eluted at the expected time, 92% in its non-aggregated form. The chromatogram and the illustration of SE-HPLC can be observed in FIG. 1C.

See table 1 for a summary description of aggregation levels.

。

TABLE 1 Final yields and aliquots

Brief description of the project

The Fabsc antibody was cloned into the high expression mammalian vector system of LakePharma and small scale (0.1 liter) high quality transient production was accomplished in HEK293 cells. The protein was purified by protein L purification and yielded 20.17 mg of protein and delivered 19.07 mg. The antibody was 92% non-aggregated as determined by SE-HPLC. See table 1 for a summary description of yields and aliquots.

Results of protein purification

Summary of the procedures and descriptions

Protein L affinity chromatography

Sterile filtration of 0.2 μm

Name of protein	Fabsc
		Batch number	4622-848799
Extinction coefficient (for concentration calculation)	1.67 mg/ml - 1 cm -1
		Protein concentration	5.45 mg/ml
Volume of	0.50 ml
		Total protein	2.72 mg
Endotoxin	Not measured
		Physical state	Liquid, method for producing the same and use thereof
Buffer solution	230 mM HEPES, 115 mM NaCl, 58 mM naOAc, pH 7.0

TABLE 2

Test of	SE-HPLC
		SR #	3916
Sample ID	Fabsc (PP4622)
		Date	2015-12-09
Scientists	SW

TABLE 3

Method

Peak numbering	Time (min)	Peak size (kDa)	Peak area%	Peak ID
						1	11.2	~230	5.8	Aggregate and method of making same
2	12.7	~100	91.8	Monomer
					3	13.9	~40	2.4	Fragments
4
					5
6

TABLE 4

Column	MabPac SEC-1, 5µm, 4x 300 mm
		Mobile phase	50 mM sodium phosphate, 300 mM NaCl, pH 6.2
Equal degree	0-25 min
		Flow rate (mL/min)	0.2
Injection volume (mu L)	5

TABLE 5

Example 2: preparation or conditioning of patients for bone marrow/hematopoietic stem cell (BM/HSC) transplantation.

The preparation or conditioning of patients for bone marrow/hematopoietic stem cell (BM/HSC) transplantation is a key element of the procedure. It plays two main roles: (1) it provides adequate immunosuppression for the patient and clears sufficient niche space in the bone marrow for the transplanted HSCs. This allows for engraftment of the transplanted cells into the recipient; and (2) it often helps to eradicate the source of the malignancy.

Conditioning of patients has traditionally been achieved as follows: the maximum tolerated dose of the mixture of chemotherapeutic agents is administered with or without radiation. The components of the mixture are often selected to have non-overlapping toxicities. All preparations in use today are toxic and have serious side effects that can be life threatening. Among these side effects are mucositis, nausea and vomiting, hair loss, diarrhea, rash, peripheral neuropathy, infertility, lung and liver toxicity. Many of these side effects are particularly dangerous for elderly and ill patients, and often become a determining component in deciding whether a patient will receive a transplant.

To eliminate the use of chemotherapeutic agents for conditioning patients undergoing BM/HSC transplantation, we developed methods to selectively eliminate hematopoietic stem/progenitor cells (HSC/HP) using redirected T-cell killing. The method is based on the use of bispecific antibodies that bind to a target on the surface of HSC/HP (FLT3), and also bind to a target on the surface of T-cells (CD3), thereby recruiting T-cells to HSC/HP.

As evidence of the principle that the developed approach can be used to eliminate HSC/HP, we tested a bispecific (FLT3xCD3) antibody designed to kill leukemic blasts in primary peripheral blood mononuclear cells from Acute Myeloid Leukemia (AML) patients (Durben, Schmiedel et al.2015).

Female immunocompromised NOG (NOD. Cg-Prkdcscid Il2rgtm1Sug/JicTac) mice (4-6 weeks old) were used for transplantation of human CD34+ HSC/HP from umbilical Cord Blood (CB). The mononuclear cell fraction of CB was separated by gradient density centrifugation using Ficoll-Paque (GE Healthcare Life Sciences). Briefly, CB treated with anticoagulant was mixed with Phosphate Buffered Saline (PBS) in a 1:1 ratio and overlaid (35ml mixture) on a ficoll layer (10ml) in a 50ml conical centrifuge tube. The tube was then rotated at a speed of 400 x g. The monocyte-lymphocyte layer was carefully removed and the cells from the layer were washed 2 times with PBS.

CD34+ HSC/HP was isolated by negative selection with platelet depletion (Stemcell Technologies). Unwanted cells are targeted for removal with tetrameric antibody complexes that recognize CD2, CD3, CD11b, CD11c, CD14, CD16, CD19, CD24, CD56, CD61, CD66b, glycophorin a, and dextran-coated magnetic particles. The labeled cells were separated using an EasySep chamber magnet without the use of a column.

CD34+ HSC/HP were resuspended in PBS at 10,000-50,000 cells/200 μ l for transplantation into severely myelosuppressed NOG mice.

Mice were severely myelosuppressed 24 hours prior to transplantation with busulfan (10 mg/kg) via intraperitoneal injection. Transplantation of CD34 by tail vein injection of 200 μ l cell suspension (n =52) ⁺ HSC/HP. Eighteen (18) weeks post-transplantation, against human CD45 ⁺ The presence of cells peripheral blood of the transplanted mice was tested. Select a CD of 40% or more (human CD 45) ⁺ The percentage of cells ≧ 40% of total CD45+ cells) for further experiments (n = 27; fig. 1A, fig. 1B). The level of chimerism was tested in peripheral blood and calculated as follows:

。

it is also directed against human B-cells (hCD 19) ⁺ ) Human T-cells (hCD 3) ⁺ ) And human cells belonging to the myeloid lineage (hCD 33) ⁺ ) The peripheral blood of the selected mice was tested. Most mice showed robust development of all three lineages (fig. 1A, fig. 1B). Some smallMurine (n =3) is CD3 ⁺ Defective in cell development (FIG. 1B asterisk, FIG. 1D). These T-cell deficient mice were used as internal controls within the experiment.

Protein sequence of the insert

Signal peptide

Variable weight

The weight can be reduced.

DNA sequence of the insert

Example 3: production and characterization of monoclonal antibodies against Flt3/FLK2 human receptor protein

Cells from the murine myeloma cell line SP2/0 were transduced with lentiviruses expressing the complete coding sequence of the human FT3/FLK2 receptor protein and a selection marker for puromycin resistance. Transduced cells were selected in vitro in the presence of puromycin. Cells (SP2/0-Hu-FLT3) selected and validated for expression of human FLT3/FLK2 protein cells were used as antigens.

8-week-old Balb/c mice were immunized 3 times with 107 SP2/0-Hu-FLT3 cells by intraperitoneal injection every 5 days to generate antibodies specific for the FLT3/FLK2 protein. Development of antibodies was tested by screening sera of immunized mice for binding to FLT3/FLK2 antigen using flow cytometry.

Approximately 3 weeks after the first immunization, spleens of immunized mice were collected and used to isolate splenocytes. Isolated splenocytes were fused with SP2/0 cells and selected for the hybrid phenotype (hybridomas). Hybridomas were cultured in vitro and supernatants from hybridoma cultures were screened by flow cytometry for the presence of anti-FLT 3/FLK2 antibodies (fig. 3A and 3B). 9 hybridoma clones showed production of anti-FLT 3/FLK2 antibody. These hybridoma clones were expanded for isolation of monoclonal antibodies. Isolated monoclonal anti-FLT 3/FLK2 antibodies were purified and tested for their selectivity (FIGS. 4A and 4B).

Example 4: characterization of the specificity of monoclonal antibodies to the human FLT3/FLK2 receptor protein

The specificity of the monoclonal antibodies was determined by evaluating their affinity for the FLT3/FLK2 antigen. To determine the affinity of anti-human FLT3/FLK2 antibodies, Effective Concentration (EC) curves were established using flow cytometry. 9 monoclonal antibody clones were used to stain human REH cells endogenously expressing human FLT3/FLK 2. The concentration of the clones ranged from 1 ng/ml (6.25 pM) up to 10,000 ng/ml (62.5 nM). 5 ECs were selected with a range of about 70 ng/ml (437.5 pM) up to 1566 ng/ml (9.79 nM) ₅₀ The clones of (FIGS. 5A, 5B, 5C, 5D and 5E) were used for sequencing. Sequencing of the clones revealed that clones 1-23DA and 1-18 have identical amino acid sequences. The cloned sequences are shown below.

MHC1692-1-23DA sequence:

amino acid sequence in FASTA format (MHC16992LC.2 \ M13F) -light chain (SEQ ID NO: 5)

Nucleotide sequence in FASTA format (MHC16992LC.2 \ M13F) -light chain (SEQ ID NO: 6)

The amino acid sequence of the FASTA format (MHC 16992HC.1 \ M13F) -heavy chain (SEQ ID NO:7)

nucleotide sequence in FASTA format (MHC16992HC.1 \ M13F) -heavy chain (SEQ ID NO: 8)

MHC 1693-16 HA sequence:

amino acid sequence in FASTA format (MHC169383 LC.1\ M13F) -light chain (SEQ ID NO: 9)

Nucleotide sequence in FASTA format (MHC169383 LC.1\ M13F) -light chain (SEQ ID NO: 10)

Amino acid sequence in FASTA format (MHC169iphC.3 \ M13F) -heavy chain (SEQ ID NO: 11)

Nucleotide sequence in FASTA format (MHC169iphC.3 \ M13F) -heavy chain (SEQ ID NO: 12)

MHC1695-3-3OA sequence:

amino acid sequence in FASTA format (MHC1695LC.8\ M13F) -light chain (SEQ ID NO: 13)

Nucleotide sequence in FASTA format (MHC1695LC.8\ M13F) -light chain (SEQ ID NO: 14)

Amino acid sequence of FASTA Format (MHC1695HC.3\ M13F) -heavy chain (SEQ ID NO: 15)

Nucleotide sequence in FASTA format (MHC1695HC.3\ M13F) -heavy chain (SEQ ID NO: 16)

MHC1696-2-8IA sequence:

amino acid sequence in FASTA format (MHC1696LC.3\ M13F) -light chain (SEQ ID NO: 17)

Nucleotide sequence in FASTA format (MHC1696LC.3\ M13F) -light chain (SEQ ID NO: 18)

Amino acid sequence of FASTA Format (MHC1696HC.2\ M13F) -heavy chain (SEQ ID NO: 19)

Nucleotide sequence in FASTA format (MHC1696HC.2\ M13F) -heavy chain (SEQ ID NO: 20)

MHC2279-1B11.E7 sequence:

amino acid sequence in FASTA format (MHC2279LC.5\ M13F) -light chain (SEQ ID NO: 21)

Nucleotide sequence in the format of FASTA (MHC2279LC.5\ M13F) -light chain (SEQ ID NO: 22)

Amino acid sequence of FASTA Format (MHC2279HC.1\ M13F) -heavy chain (SEQ ID NO: 23)

Nucleotide sequence in the format of FASTA (MHC2279HC.1\ M13F) -heavy chain (SEQ ID NO: 24)

MHC1694-1-18BA sequence:

amino acid sequence in FASTA format (MHC1694LC.2\ M13F) -light chain (SEQ ID NO: 25)

Nucleotide sequence in FASTA format (MHC1694LC.2\ M13F) -light chain (SEQ ID NO: 26)

Amino acid sequence in FASTA format (MHC1694HC.1\ M13F) -heavy chain (SEQ ID NO: 27)

Nucleotide sequence in FASTA format (MHC1694HC.1\ M13F) -heavy chain (SEQ ID NO: 28)

TABLE 6

Example 5: characterization of the internalization of monoclonal antibodies against the human FLT3/FLK2 receptor protein

Internalization of monoclonal antibodies directed against FLT3/FLK2 (example 3) was quantified by an internalization assay.

Briefly, 2X (4 μ g/ml) working stock of antibodies was prepared on ice in staining buffer (1X Phosphate Buffered Saline (PBS) containing 2% calf serum (BCS)) for antibodies 281A, 330A, 316HA and 123 DA. Stock solutions of 4 μ g/ml anti-human CD135 (FLT3/FLK2) antibody (BioLegend #313302, clone BV10A4H2) and 4 μ g/ml isotype control (BioLegend #400102, clone MOPC-21) were prepared as positive and negative CD135 staining controls, respectively. Reh cells (human cell line expressing CD 135) were washed and washed at 2x10 ⁶ The concentration of individual cells/ml was resuspended in staining buffer. The primary antibody was added 1:1 with equal volume of cells to a final concentration of 2 μ g/ml. Cells were stained in a 15 mL centrifuge tube for washing. Then, the cells were incubated on ice for 30 minutes, and then washed 3 times in 5ml PBS to remove unbound primary antibody. Stained cells were resuspended in complete medium (RPMI 1640 with glutamine and 2% BCS) and plated in parallel 96-well plates at 100 μ l/well, using a separate plate at each time point in triplicate wells. Transfer one set of plates to a 37 ℃ incubator with 5% CO ₂ And the second set of plates was maintained at 4 ℃. Incubation times were 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours and 4 hours. After incubation, the plates were washed in 1x PBS. Cells were then stained on ice in the dark with anti-mouse IgG Alexa 488 secondary antibody at a 1:800 dilution for 30 minutes (Jackson Immuno # 115545164). Triplicate control wells containing unstained cells and cells stained with secondary antibody only were also prepared. After incubation with the secondary antibody, cells were washed for the last time in 1x PBS containing 2% BCS and stained with 7AAD immediately before FACS.

Stained cells were analyzed by flow cytometry on a Beckman Coulter Cytoflex at a sample flow rate of 60 μ l/min. 10,000 events were captured for each well and the FCS file was evaluated using FloJo software version 10. Mean Fluorescence Intensity (MFI) of Alexa 488 was calculated for the live cell population and the change in MFI for each antibody was plotted versus time at 4 ℃ and 37 ℃.

As shown in fig. 6, all clones showed internalization, with clones 330A and 123DA showing the most rapid internalization (fig. 6). Without being limited by theory, it is hypothesized that the internalization properties of the anti-FLT 3/FLK2 antibodies (clones 330A, 123DA, 316HA, and 281A) make them effective as carriers (e.g., antibody-drug-conjugate-ADC) to deliver drugs/toxins into targeted cells.

While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Sequence listing

<110> HEMOGENYX LLC

<120> method for depleting hematopoietic stem/progenitor cells (HSC/HP) in a patient using bispecific antibodies

<130> 128557-00119

<140>

<141>

<150> US 16/506,764

<151> 2019-07-09

<150> US 16/091,139

<151> 2018-10-04

<150> PCT/US2017/025951

<151> 2017-04-04

<150> US 62/317,906

<151> 2016-04-04

<160> 28

<170> PatentIn version 3.5

<210> 1

<211> 613

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 1

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Leu Lys Leu Ser Cys Lys Ser Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Met His Trp Val Arg Gln Arg Pro Gly His Gly Leu

50 55 60

Glu Trp Ile Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Asn

85 90 95

Thr Ala Tyr Met His Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly

115 120 125

Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Pro Val Ala Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu

275 280 285

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Ala Lys Gly Gly Gly Gly Ala Gly Gly Gly Gly

355 360 365

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

370 375 380

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

385 390 395 400

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

405 410 415

Ala Leu Ile Asn Pro Tyr Lys Gly Val Thr Thr Tyr Ala Asp Ser Val

420 425 430

Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr

435 440 445

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

450 455 460

Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp

465 470 475 480

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

485 490 495

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

500 505 510

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

515 520 525

Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys

530 535 540

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu

545 550 555 560

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr

565 570 575

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

580 585 590

Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys

595 600 605

Val Glu Ile Lys Arg

610

<210> 2

<211> 234

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 2

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

Val Thr Pro Gly Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Ile Ser Asn Asn Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro

50 55 60

Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn

85 90 95

Ser Val Glu Thr Glu Asp Phe Gly Val Tyr Phe Cys Gln Gln Ser Asn

100 105 110

Thr Trp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 3

<211> 1842

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 3

atggaatgga gctgggtctt tctcttcttc ctgtcagtaa cgactggtgt ccactcccag 60

gtgcagctgc agcagcctgg tgccgagctc gtgaaacctg gcgcctccct gaagctgtcc 120

tgcaagtcct ccggctacac cttcaccagc tactggatgc actgggtgcg acagaggcct 180

ggccacggac tggaatggat cggcgagatc gacccctccg actcctacaa ggactacaac 240

cagaagttca aggacaaggc caccctgacc gtggacagat cctccaacac cgcctacatg 300

cacctgtcct ccctgacctc cgacgactcc gccgtgtact actgcgccag agccatcaca 360

accaccccct tcgatttctg gggccagggc accacactga cagtgtcctc cgcttccacc 420

aagggcccct ccgtgtttcc tctggcccct tccagcaagt ccacctctgg cggaacagcc 480

gctctgggct gcctcgtgaa ggactacttc cccgagcctg tgaccgtgtc ctggaactct 540

ggcgctctga catccggcgt gcacaccttc cctgctgtgc tgcagtctag cggcctgtac 600

tccctgtcca gcgtcgtgac cgtgccttcc agctctctgg gcacccagac ctacatctgc 660

aacgtgaacc acaagccttc caacaccaag gtggacaaga aggtggaacc caagtcctgc 720

gacaagaccc acaccagccc tccaagccct gctcctcctg tggctggccc tagcgtgttc 780

ctgttccctc caaagcccaa ggataccctg atgatctccc ggacccccga agtgacctgc 840

gtggtcgtgg gagtgtctca cgaggaccct gaagtgaagt tcaattggta cgtggacggc 900

gtggaagtgc acaacgccaa gaccaagcct agagaggaac agtaccagtc cacctaccgg 960

gtggtgtccg tgctgaccgt gctgcaccag gattggctga acggcaaaga gtacaagtgc 1020

aaggtgtcca acaagcagct gcccagcccc atcgaaaaga ccatctccaa ggctaagggc 1080

ggaggcggag ctggtggtgg cggagaagtg cagctggtgg aatctggcgg cggactggtg 1140

cagcctggcg gatctctgag actgtcttgt gccgccagcg gctactcttt caccggctat 1200

accatgaatt gggtgcgcca ggcccctgga aagggcctgg aatgggtggc cctgatcaac 1260

ccctacaagg gcgtgaccac ctacgccgac tccgtgaagg gccggttcac catctccgtg 1320

gacaagtcca agaataccgc ttacctgcag atgaactccc tgcgggccga ggacaccgct 1380

gtgtattact gtgctagatc cggctactac ggcgacagcg attggtactt cgacgtgtgg 1440

ggacagggaa ccctcgtgac tgtgtcatca ggcggcggtg gttctggcgg agggggatct 1500

gggggcggtg gatccgatat ccagatgacc cagtccccca gctccctgtc tgcctctgtg 1560

ggcgacagag tgaccatcac ctgtcgggcc tctcaggaca tccggaacta cctgaactgg 1620

tatcagcaga agcccggcaa ggcccccaag ctgctgatct actacacctc ccggctggaa 1680

agcggcgtgc cctccagatt ctccggctct ggctctggaa ccgactatac cctgaccatc 1740

tctagcctgc agcccgagga cttcgccacc tactactgcc agcagggcaa caccctgccc 1800

tggacctttg gccagggaac aaaggtggaa atcaagcggt ga 1842

<210> 4

<211> 705

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 4

atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60

gacatcgtgc tgacccagtc tcccgccacc ctgtctgtga cccctggcga ctctgtgtcc 120

ctgtcctgca gagcctccca gtccatctcc aacaacctgc actggtatca gcagaagtcc 180

cacgagagcc ctcggctgct gattaagtac gccagccagt ctatctccgg catcccctcc 240

agattctccg gctctggctc tggcaccgac ttcaccctgt ccatcaactc cgtggaaacc 300

gaggacttcg gcgtgtactt ctgccagcag tccaacacct ggccctacac ctttggcgga 360

ggcaccaagc tggaaatcaa gcggaccgtg gccgccccca gcgtgttcat cttccctccc 420

agcgacgagc agctgaagtc tggcaccgcc agcgtggtgt gcctgctgaa caacttctac 480

ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 540

gagagcgtga ccgagcagga ctccaaggac agcacctaca gcctgagcag caccctgacc 600

ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggga 660

ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gctaa 705

<210> 5

<211> 107

<212> PRT

<213> mouse (Mus musculus)

<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu His Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Arg Leu Glu Ser

65 70 75 80

Glu Asp Val Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Arg

100 105

<210> 6

<211> 321

<212> DNA

<213> mouse (Mus musculus)

<400> 6

gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggaaattagt ggttacttaa gctggcttca gcagaaacca 120

gatggaacta ttaaacgcct gatctacgcc gcatccactt tacattctgg tgtcccaaaa 180

aggttcagtg gcagtaggtc tgggtcagat tactctctca ccatcagcag gcttgagtct 240

gaagatgttg cagactatta ctgtctacaa tatgctagtt atccattcac gttcggctcg 300

gggacaaagt tggaaataag a 321

<210> 7

<211> 123

<212> PRT

<213> mouse (Mus musculus)

<400> 7

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Thr Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Leu His Ile Leu Trp Asn Asp Ser Lys Tyr Tyr Asn Pro Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Tyr Asn Lys Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Val Tyr Tyr Ser Thr Tyr Val Gly Tyr Phe Asp Val

100 105 110

Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 8

<211> 369

<212> DNA

<213> mouse (Mus musculus)

<400> 8

caggttactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg 60

acttgttctt tctctgggtt ttctctgagc acttctacta tgggtgtagg ctggattcgt 120

cagccttcag ggaagggtct ggagtggctg ttacacattt tgtggaatga tagtaagtat 180

tataacccag ccctgaagag ccggctcaca atctccaagg atacctacaa caagcaggta 240

ttcctcaaga tcgccaatgt ggacactgca gatactgcca catactactg tgctcgaata 300

gtttactact ctacctacgt cgggtacttc gatgtctggg gcgcagggac cacggtcacc 360

gtctcctca 369

<210> 9

<211> 111

<212> PRT

<213> mouse (Mus musculus)

<400> 9

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Val Ser Asn Gln Gly Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 10

<211> 333

<212> DNA

<213> mouse (Mus musculus)

<400> 10

gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60

atctcctgca gagccagcga aagtgttgat aattatggca ttagttttat gaactggttc 120

caacagaaac caggacagtc acccaaactc ctcatctatg ctgtatccaa ccaaggatcc 180

ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat 240

cctatggagg aggatgatac tgcaatgtat ttctgtcagc aaagtaagga ggttccgtgg 300

acgttcggtg gaggcaccaa gctggaaatc aaa 333

<210> 11

<211> 116

<212> PRT

<213> mouse (Mus musculus)

<400> 11

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Leu Val Lys Leu Ser Cys Lys Gly Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Glu Asn Asp Ile Thr Met Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Gly Asn Phe Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ala

115

<210> 12

<211> 348

<212> DNA

<213> mouse (Mus musculus)

<400> 12

gaggttcagc tgcagcagtc tggggctgag cttgtgaggc caggggcctt agtcaagttg 60

tcctgcaaag gttctggctt caacattaaa gactactata tacactgggt gaagcagagg 120

cctgaacagg gcctggagtg gattggaagg attgatcctg agaatgatat tactatgtat 180

gacccgaagt tccagggcaa ggccagtata acagcagaca catcctccaa cacagcctac 240

ctgcagctca gcagcctgac atctgaggac actgccgtct attactgtgc tagaaatggt 300

aatttctttg cttactgggg ccaagggact ctggtcactg tctctgca 348

<210> 13

<211> 107

<212> PRT

<213> mouse (Mus musculus)

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asn Ser Gly Val Pro Arg Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 14

<211> 321

<212> DNA

<213> mouse (Mus musculus)

<400> 14

gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggaaattagt ggttacttaa gctggcttca gcagaaacca 120

gatggaacta ttaaacgcct gatctacgcc gcatccactt taaattctgg tgtcccaaga 180

aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240

gaagattttg cagactatta ctgtctacaa tatgctagtt atccattcac gttcggctcg 300

gggacaaagt tggaaataaa a 321

<210> 15

<211> 124

<212> PRT

<213> mouse (Mus musculus)

<400> 15

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

His Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Leu His Ile Leu Trp Asn Asp Ser Val Tyr Tyr Asn Pro Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Tyr Asn Lys Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Val Tyr Tyr Gly Ile Ser Tyr Val Gly Tyr Phe Asp

100 105 110

Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 16

<211> 372

<212> DNA

<213> mouse (Mus musculus)

<400> 16

caggttactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg 60

acttgttctt tctctgggtt ttcactgagc acttctcaca tgggtgtagg ctggattcgt 120

cagccttcag ggaagggtct ggagtggctg ttacacattt tgtggaatga tagtgtgtac 180

tataacccag ccctgaagag ccggctcaca atctccaagg atacctacaa caagcaggta 240

ttcctcaaga tcgccaatgt ggacactgca gatactgcca catactactg tgctcgaata 300

gtttactacg gtattagtta cgtcgggtac ttcgatgtct ggggcgcagg gaccacggtc 360

accgtctcct ca 372

<210> 17

<211> 107

<212> PRT

<213> mouse (Mus musculus)

<400> 17

Asp Thr Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Gly Phe Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Asn Ser Val Glu Thr

65 70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Thr Asn Ser Trp Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 18

<211> 321

<212> DNA

<213> mouse (Mus musculus)

<400> 18

gatactgtgc taactcaatc tccagccacc ctgtctgtga ctccaggaga tagcgtcagt 60

ctttcctgca gggccagcca aagtattagc aacaacctac actggtatca acaaaaatca 120

catgagtctc caaggcttct catcaagtat ggtttccagt ccatctctgg gatcccctcc 180

aggttcagtg gcagtggatc agggacagat ttcactctca gaatcaacag tgtggagact 240

gaagattttg gaatgtattt ctgtcaacag actaacagct ggccgctcac gttcggtgct 300

gggaccaagc tggagctgaa a 321

<210> 19

<211> 115

<212> PRT

<213> mouse (Mus musculus)

<400> 19

Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ile Asp Tyr

20 25 30

Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Gly Gly Thr Ser Asn Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe

65 70 75 80

Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Thr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser

115

<210> 20

<211> 345

<212> DNA

<213> mouse (Mus musculus)

<400> 20

gagatccagc tgcagcagtc tggacctgaa ctggtgaagc ctggggcttc agtgaaggta 60

tcctgcaagg cttctggtta ctcattcatt gactacaaca tgtactgggt gaagcagagc 120

catggaaaga gccttgagtg gattggatat attaatcctt acaatggtgg tactagcaac 180

aaccagaagt tcaaggacaa ggccacattg actgttgaca agtcctccag cacagccttc 240

atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagaggtact 300

acgggtgact actggggcca aggcaccact ctcacagtct cctca 345

<210> 21

<211> 111

<212> PRT

<213> mouse (Mus musculus)

<400> 21

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 22

<211> 333

<212> DNA

<213> mouse (Mus musculus)

<400> 22

gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60

atctcctgca gagccagcga aagtgttgat aattatggca ttagttttat gaactggttc 120

caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa ccaaggatcc 180

ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat 240

cctatggagg aggatgatac tgcaatgtat ttctgtcagc aaagtaagga ggttccgtgg 300

acgttcggtg gaggcaccaa gctggaaatc aaa 333

<210> 23

<211> 119

<212> PRT

<213> mouse (Mus musculus)

<400> 23

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Met Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ala Tyr Tyr Ser Lys Arg Asp Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 24

<211> 357

<212> DNA

<213> mouse (Mus musculus)

<400> 24

caggtccaac tgcagcagcc tggggctgag cttgtgatgc ctggggcttc agtgaagctg 60

tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120

cctggacaag gccttgagtg gatcggagag attgatcctt ctgatagtta tactaactac 180

aatcaaaagt tcaagggcaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagatcagcc 300

tactatagta aaagggatga ctactggggc caaggcacca ctctcacagt ctcctca 357

<210> 25

<211> 107

<212> PRT

<213> mouse (Mus musculus)

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu His Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Arg Leu Glu Ser

65 70 75 80

Glu Asp Val Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Arg

100 105

<210> 26

<211> 321

<212> DNA

<213> mouse (Mus musculus)

<400> 26

gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggaaattagt ggttacttaa gctggcttca gcagaaacca 120

gatggaacta ttaaacgcct gatctacgcc gcatccactt tacattctgg tgtcccaaaa 180

aggttcagtg gcagtaggtc tgggtcagat tactctctca ccatcagcag gcttgagtct 240

gaagatgttg cagactatta ctgtctacaa tatgctagtt atccattcac gttcggctcg 300

gggacaaagt tggaaataag a 321

<210> 27

<211> 123

<212> PRT

<213> mouse (Mus musculus)

<400> 27

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Thr Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Leu His Ile Leu Trp Asn Asp Ser Lys Tyr Tyr Asn Pro Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Tyr Asn Lys Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Val Tyr Tyr Ser Thr Tyr Val Gly Tyr Phe Asp Val

100 105 110

Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 28

<211> 369

<212> DNA

<213> mouse (Mus musculus)

<400> 28

caggttactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg 60

acttgttctt tctctgggtt ttctctgagc acttctacta tgggtgtagg ctggattcgt 120

cagccttcag ggaagggtct ggagtggctg ttacacattt tgtggaatga tagtaagtat 180

tataacccag ccctgaagag ccggctcaca atctccaagg atacctacaa caagcaggta 240

ttcctcaaga tcgccaatgt ggacactgca gatactgcca catactactg tgctcgaata 300

gtttactact ctacctacgt cgggtacttc gatgtctggg gcgcagggac cacggtcacc 360

gtctcctca 369

Claims (25)

1. A monoclonal antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the light chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is selected from the group consisting of SEQ ID NO 5, SEQ ID NO 9, SEQ ID NO 13, SEQ ID NO 17, SEQ ID NO 21 and SEQ ID NO 25, and the amino acid sequence of the heavy chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is selected from the group consisting of SEQ ID NO 7, SEQ ID NO 11, SEQ ID NO 15, SEQ ID NO 19, SEQ ID NO 23 and SEQ ID NO 27.

2. The monoclonal antibody, or antigen-binding fragment thereof, of claim 1, wherein the amino acid sequence of the light chain of the antigen-binding portion of the antibody, or fragment thereof, that binds human FLT3/FLK2 receptor protein is SEQ ID No. 5, and the amino acid sequence of the heavy chain of the antigen-binding portion of the antibody, or fragment thereof, that binds human FLT3/FLK2 receptor protein is SEQ ID No. 7.

3. The monoclonal antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof has a half maximal Effective Concentration (EC) ₅₀ ) Is between 1 ng/mL (6.25 pM) and 2,000 ng/mL (12.5 nM).

4. The monoclonal antibody or antigen-binding fragment thereof of claim 3, wherein the antibody or antigen-binding fragment thereof has a half maximal Effective Concentration (EC) ₅₀ ) Is between 10 ng/mL (62.5 pM) and 200 ng/mL (1.25 nM).

5. The monoclonal antibody or antigen-binding fragment thereof of claim 2, wherein the FLT3 antibody that binds the human FLT3/FLK2 receptor protein on a cell is effective to internalize the bound antibody or antigen-binding fragment by the cell.

6. The monoclonal antibody, or antigen-binding fragment thereof, of claim 1, wherein the amino acid sequence of the light chain of the antigen-binding portion of the antibody, or fragment thereof, that binds human FLT3/FLK2 receptor protein is SEQ ID No. 9, and the amino acid sequence of the heavy chain of the antigen-binding portion of the antibody, or fragment thereof, that binds human FLT3/FLK2 receptor protein is SEQ ID No. 11.

7. The monoclonal antibody or antigen-binding fragment thereof of claim 6, wherein the antibody or fragment thereof has a half maximal Effective Concentration (EC) ₅₀ ) Is between 1 ng/mL (6.25 pM) and 2,000 ng/mL (12.5 nM).

8. The monoclonal antibody or antigen-binding fragment thereof of claim 7, wherein the antibody or antigen-binding fragment thereof has a half maximal Effective Concentration (EC) ₅₀ ) Is between 10 ng/mL (62.5 pM) and 200 ng/mL (1.25 nM).

9. The monoclonal antibody or antigen-binding fragment thereof of claim 6, wherein the FLT3 antibody that binds the human FLT3/FLK2 receptor protein on a cell is effective to internalize the bound antibody or antigen-binding fragment by the cell.

10. The monoclonal antibody, or antigen-binding fragment thereof, of claim 1, wherein the amino acid sequence of the light chain of the antigen-binding portion of the antibody, or fragment thereof, that binds human FLT3/FLK2 receptor protein is SEQ ID No. 13, and the amino acid sequence of the heavy chain of the antigen-binding portion of the antibody, or fragment thereof, that binds human FLT3/FLK2 receptor protein is SEQ ID No. 15.

11. The monoclonal antibody or antigen-binding fragment thereof of claim 10, wherein the antibody or antigen-binding fragment thereof has a half maximal Effective Concentration (EC) ₅₀ ) Is between 1 ng/mL (6.25 pM) and 2,000 ng/mL (12.5 nM).

12. The monoclonal antibody or antigen-binding fragment thereof of claim 11, wherein the antibody or antigen-binding fragment thereof has a half maximal Effective Concentration (EC) ₅₀ ) Is between 10 ng/mL (62.5 pM) and 200 ng/mL (1.25 nM).

13. The monoclonal antibody or antigen-binding fragment thereof of claim 11, wherein the FLT3 antibody that binds the human FLT3/FLK2 receptor protein on a cell is effective for internalizing the bound antibody or antigen-binding fragment by the cell.

14. The monoclonal antibody, or antigen-binding fragment thereof, of claim 1, wherein the amino acid sequence of the light chain of the antigen-binding portion of the antibody, or fragment thereof, that binds human FLT3/FLK2 receptor protein is SEQ ID No. 17, and the amino acid sequence of the heavy chain of the antigen-binding portion of the antibody, or fragment thereof, that binds human FLT3/FLK2 receptor protein is SEQ ID No. 19.

15. The monoclonal antibody or antigen-binding fragment thereof of claim 14, wherein the antibody or antigen-binding fragment thereof has a half maximal Effective Concentration (EC) ₅₀ ) Is between 1 ng/mL (6.25 pM) and 2,000 ng/mL (12.5 nM).

16. The monoclonal antibody or antigen-binding fragment thereof of claim 15, wherein the antibody or antigen-binding fragment thereof has a half maximal Effective Concentration (EC) ₅₀ ) Is between 10 ng/mL (62.5 pM) and 200 ng/mL (1.25 nM).

17. The monoclonal antibody or antigen-binding fragment thereof of claim 14, wherein the FLT3 antibody that binds the human FLT3/FLK2 receptor protein on a cell is effective for internalizing the bound antibody or antigen-binding fragment by the cell.

18. A monoclonal antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the light chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID NO: 21 and the amino acid sequence of the heavy chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID NO: 23, wherein said monoclonal antibody or antigen-binding fragment thereof does not compete with FLT3 ligand (FLT 3L) when binding FLT3/FLK 2.

19. The monoclonal antibody or antigen thereof according to claim 18Binding fragment, wherein the antibody or fragment thereof has a half maximal Effective Concentration (EC) ₅₀ ) Is between 1 ng/mL (6.25 pM) and 2,000 ng/mL (12.5 nM).

20. The monoclonal antibody or antigen-binding fragment thereof of claim 19, wherein the antibody or antigen-binding fragment thereof has a half maximal Effective Concentration (EC) ₅₀ ) Is between 10 ng/mL (62.5 pM) and 200 ng/mL (1.25 nM).

21. The monoclonal antibody or antigen-binding fragment thereof of claim 18, wherein the FLT3 antibody that binds the human FLT3/FLK2 receptor protein on a cell is effective for internalizing the bound antibody or antigen-binding fragment by the cell.

22. A monoclonal antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the light chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID NO: 25 and the amino acid sequence of the heavy chain of the antigen-binding portion of the antibody or fragment thereof that binds human FLT3/FLK2 receptor protein is SEQ ID NO: 27, wherein said monoclonal antibody or antigen-binding fragment thereof does not compete with FLT3 ligand (FLT 3L) when binding FLT3/FLK 2.

23. The monoclonal antibody or antigen-binding fragment thereof of claim 22, wherein the antibody or fragment thereof has a half maximal Effective Concentration (EC) ₅₀ ) Is between 1 ng/mL (6.25 pM) and 2,000 ng/mL (12.5 nM).

24. The monoclonal antibody or antigen-binding fragment thereof of claim 23, wherein the antibody or antigen-binding fragment thereof has a half maximal Effective Concentration (EC) ₅₀ ) Is between 10 ng/mL (62.5 pM) and 200 ng/mL (1.25 nM).

25. The monoclonal antibody or antigen-binding fragment thereof of claim 22, wherein the FLT3 antibody that binds the human FLT3/FLK2 receptor protein on a cell is effective for internalizing the bound antibody or antigen-binding fragment by the cell.

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