specific antibodies to flt3 and uses thereof

专利摘要:
The present invention relates to antibodies that specifically bind to FLT3 (Tyrosine Kinase 3 of Type Fms). The invention further provides bispecific antibodies that bind to FLT3 and another antigen (for example, CD3). The invention further relates to antibodies encoding nucleic acids, and methods for obtaining such antibodies (monospecific and bispecific). The invention further relates to therapeutic methods for using these antibodies for the treatment of pathologies mediated by FLT3, including cancer such as Acute Myeloid Leukemia (AML).
公开号:BR112019025243A2
申请号:R112019025243-6
申请日:2018-05-31
公开日:2020-07-14
发明作者:Danielle Elizabeth DETTLING；Veena KRISHNAMOORTHY；Kristian Todd Poulsen；Cesar Adolfo Sommer；Yik Andy Yeung
申请人:Pfizer Inc.；
IPC主号:

专利说明:

[001] The present invention relates to antibodies, for example, life-size antibodies or antigen-binding fragments thereof, which specifically binds to Fms-like tyrosine kinase 3 (FLT3). The invention also relates to heteromultimeric antibodies (for example, bispecific antibodies) which comprise FLT3 antibody in a subdivision. Compositions comprising the FLT3 antibodies, methods for producing and purifying such antibodies, and their use in diagnostics and therapeutics are also provided. BACKGROUND
[002] [002] FIt3 (also known as CD135, FLK3, STK1), a target antigen well characterized by Acute Myeloid Leukemia (AML), is overexpressed in AML patient blasts compared to healthy cells, and is expressed in most cells of the patient (See, for example, Carow et al, Blood: 87 (3) (February 1996); and Birg et al., Blood: 80 (10) (November 1992)). In addition, Flt3 is the most frequently mutated gene in AML patients, and mutations that result in constitutive receptor activation are associated with poor prognosis (See, for example, Abu-Duhier et al. Br J Haematol., 111 (1) : 190-5 (Oct 2000), Yamamoto et al., Blood: 97 (8) (April 2001)).
[003] [003] The presence of an oncogenic conductor on the leukemic blast surface provides an opportunity for the development of targeted therapy. Small molecule FIt3 inhibitors have shown activity in clinical experiments; however, responses are generally transient, due to the acquisition of resistance. In addition, kinase inhibitors treat only a percentage of patients expressing the mutated form of FIt3, underscoring the urgent need for improved therapies.
[004] [004] Bispecific FIt3 antibody in the form of Bispecific Method involving T cell was also recently developed, since FIt3 has relatively low expression on tumor cells, compared to other tumor antigens. However, a limitation of many bispecific formats is the fact that they are of low molecular weight, of short half-life, thus requiring continuous infusion. Accordingly, there remains a need for antibodies (for example, monospecific or bispecific) for the treatment of cancer, such as AML with improved efficacy and safety profile, and suitable for use with human patients. summary
[005] [005] The invention described in this document is directed to antibodies (for example, monospecific or bispecific antibodies) that specifically bind to Fms-type Tyrosine Kinase 3 (FLT3). In particular, the inventors of the present invention have found that FLT3 antibodies as described here, in the bispecific life-size form have a longer half-life, minimized Fc interaction, and minimized nonspecific cytokine release in vivo through interaction with immune cells. . In addition, domain 4 targeting FLT3 antibodies to the FLT3 protein as described here, in the bi-specific life-size format is found to be more effective in AML cell depletion compared to other domains, including domains 1, 2, 3, and 5 in the bispecific format noticed.
[006] Consequently, in one aspect, the invention provides an isolated antibody that specifically binds to FLT3, wherein the antibody comprises (a) a heavy chain variable (VH) region comprising (i) a protein determining region one VH complementarity (CDR1) comprising the sequence shown in SEQ ID NO: 37, 38, 39, 43, 44, 45, 49, 50, 54, 55, 56, 60, 61, 62, 66, 67, 68, 72, 73, 74, 78, 79, 80, 84, 85, 86, 90, 91, 92, 96, 97, 98, 102, 103, 104, 108, 109, 110, 114, 115, 116, 120, 121, 122, 126, 127, 128, 132, 133, 134, 138, 139, 140, 246, or 247; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 40, 41, 46, 47, 51, 52, 57, 58, 63, 64, 69, 70, 75, 76, 81, 82, 87 , 88, 93, 94, 99, 100, 105, 106, 111, 112, 117, 118, 123, 124, 129, 130, 135, 136, 141, 142, 248, 249, 251, 252, 253, or 255; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 42, 48, 53, 59, 65, 71.77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 245, 250, or 254; and / or a light chain variable region (VL) comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 144, 147, 150, 153, 156, 159, 162, 165, 168, 171 , 174, 177, 180, 183, 186, 189, 192, 195, 257, 261, 263, 265, 268, 270, 273, or 275; (ii) a VL CDR 2 comprising the sequence shown in SEQ ID NO: 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190 , 193, 196, 259, 266, or 271; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 256, 258, 260, 262, 264, 267, 269, 272, or 274.
[007] In another aspect, an isolated antibody is provided which specifically binds to FLT3, wherein the antibody comprises: a VH region comprising a VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID No.: 2, 4,6,8,10,12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 205, 207, 209, 211, 213, 215, 217, 219, 221,223, 225, 227, 229, 231, or 233; and / or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31, 33, 35, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,
[008] [008] In some embodiments, an isolated antibody is provided which specifically binds to FLT3, wherein the antibody comprises: a VH region comprising the sequence shown in SEQ ID NO: 215, 229, or 231; and / or a VL region comprising the sequence shown in SEQ ID NO: 214, 228, or 230. In some embodiments, an isolated antibody is provided that specifically binds to FLT3, wherein the antibody comprises: a VH region comprising the sequence shown in SEQ ID NO: 229; and / or a VL region comprising the sequence shown in SEQ ID NO: 228.
[009] [009] In some embodiments, an antibody is provided that specifically binds to FLT3 and competes with an isolated antibody provided here that specifically binds to FLT3.
[0010] [0010] In another aspect, a bispecific antibody is provided in which the bispecific antibody is a life-size antibody, which comprises a first variable domain of the bispecific antibody specifically binding to a target antigen, and which comprises a second variable domain of bispecific antibody antibody capable of recruiting the activity of a human immune effector cell specifically by binding to an effector antigen located on the human immune effector cell, where the first antibody variable domain binds to FLT3 domain 4 comprising SEQ ID NO. : 279 or FLT3 domain 5 comprising SEQ ID NO: 280.
[0011] [0011] In another aspect, a bispecific antibody is provided in which the bispecific antibody is a life-sized antibody, comprising a first variable domain of the bispecific antibody specifically binding to a target antigen (e.g., FLT3), and which comprises a second antibody variable domain of the bispecific antibody capable of recruiting the activity of a human immune effector cell specifically binding to an effector antigen (e.g., Differentiation Cluster 3 (CD3)) located on the human immune effector cell. In some embodiments, the first antibody variable domain comprises a heavy chain variable region (VH) comprising a VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, or 233; and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15 , 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, or 232. In some embodiments, the first antibody variable domain comprises (a) a heavy chain variable region (VH) comprising (i) a VH complementarity determining region (CDR1) comprising the sequence shown in SEQ ID No. 37, 38, 39, 43, 44, 45, 49, 50, 54, 55, 56, 60, 61, 62, 66, 67, 68, 72.73,
[0012] [0012] In some embodiments, the second antibody variable domain comprises CD3 specific VH and / or VL regions. For example, the second antibody variable domain comprises a heavy chain variable region (VH) comprising a VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 282; and / or a light chain variable region (VL) comprising a VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 281.
[0013] [0013] In some embodiments, the first antibody variable domain comprises a heavy chain variable region (VH) comprising a VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 229; and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 228; and a second antibody variable domain that comprises a heavy chain variable region (VH) that comprises a VH CDR1, VH CDR 2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 282; and / or a light chain variable region (VL) comprising a VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 281.
[0014] [0014] In some embodiments, the second antibody variable domain comprises (a) a VH region comprising (i) a VH CDR1 comprising the sequence shown in SEQ ID NO: 285, 286, or 287; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 288 or 289; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 290; and / or a VL region comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 291; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 292; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 234.
[0015] [0015] In some embodiments, the antibodies described in this document comprise a constant region. In some embodiments, the antibodies described in this document are from the human IgG1, IgG2 or IgG2Aa, I9G3, or IgG4 subclass. In some embodiments, the antibodies described in this document comprise a glycosylated constant region In some embodiments, the antibodies described in this document comprise a constant region having reduced binding affinity to one or more human Fc gamma receptors.
[0016] [0016] In some embodiments, both: the first and the second antibody variable domain of the bispecific antibody comprises amino acid modifications at positions 223, 225, and 228 (for example, (C223E or C223R), (E225R), and (P228E or P228R)) in the hinge region and positions 409 or 368 (for example, K409R or L368E (European Union numbering scheme)) in the CH3 region of human I9G2 (SEQ ID NO: 290).
[0017] [0017] In some embodiments, both: the first and the second antibody variable domain of the bispecific antibody comprises amino acid modifications at position 265 (e.g., D265A) of human IgG2.
[0018] [0018] In some embodiments, both: the first and the second antibody variable domain of the bispecific antibody comprise amino acid modifications at one or more of positions 265 (for example, D265A), 330 (for example, A330S), and 331 ( for example, P331S) of human IgG2. In some embodiments, both: the first and the second antibody variable domain of the bispecific antibody comprise amino acid modifications at each of positions 265 (for example, D265A), 330 (for example, A330S), and 331 (for example, P331S ) of human IgG2.
[0019] [0019] In other embodiments, the invention provides pharmaceutical compositions that comprise any of the antibodies described herein.
[0020] [0020] The invention also provides cell lines that recombinantly produce any of the antibodies described here.
[0021] [0021] The invention also provides nucleic acids encoding any of the antibodies described here. The invention also provides nucleic acids encoding a heavy chain variable region and / or a light chain variable region of any of the antibodies described herein.
[0022] [0022] The invention also provides a host cell that comprises a nucleic acid or vector provided here. Also provided is a method of producing an antibody (for example, monospecific or bispecific) provided here, which comprises culturing a host cell provided here under conditions that result in the production of the antibody, and isolating the antibody from the host cell or culture.
[0023] [0023] The invention also provides kits that comprise an effective amount of any of the antibodies or antibody conjugates described herein.
[0024] [0024] An antibody or bispecific antibody provided here for use as a medicine is also provided.
[0025] The invention also provides methods of treating individuals in need thereof which comprises providing the isolated antibodies or bispecific antibodies described herein, and administering said antibodies to said individual.
[0026] [0026] Methods of treating a condition associated with malignant cells expressing FLT3 are also provided in an individual comprising administration to an individual in need of the same effective amount of the pharmaceutical composition comprising the antibodies as described herein. In some modalities, the condition is cancer. In some embodiments, the cancer is an FLT3-related cancer (for example, any cancer with FLT3 expression) selected from the group consisting of multiple myeloma, plasma cell malignancy, Hodgkin's lymphoma, predominant nodular lymphocyte Hodgkin's disease, Kahler and myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, capillary cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), lymphocytic leukemia acute (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, lymphoma - B lymphoblast precursor, myeloid leukemia, Waldenstrom's macroglobulienemia, lymphoma diffuse large B cell, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphatic tissue lymphoma, small cell lymphocytic lymphoma, cell lymphoma of the mantle, Burkitt's lymphoma, primary large mediastinal (thymic) lymphoma, lymphoplasmocytic lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma, large intravascular B cell lymphoma, primary effusion lymphoma , lymphomatoid granulomatosis, large B cell lymphoma rich in histocytes and T cells, primary central nervous system lymphoma, primary cutaneous diffuse large B cell lymphoma (leg type), EBV positive diffuse large B cell lymphoma of the elderly, diffuse large B cell associated with inflammation, large intravascular B cell lymphoma, positive ALK large B cell lymphoma, plasmoblastic lymphoma, large B cell lymphoma that arises in multicentric Castleman disease associated with HHVB8, B cell lymphoma not classified with intermediate characteristics between diffuse large B-cell lymphoma and Burkitt's lymphoma, unclassified B-cell lymphoma m intermediate characteristics between large diffuse B-cell lymphoma and classic Hodgkin's lymphoma, and other cancers related to hematopoietic cells.
[0027] In another aspect, the invention provides a method of inhibiting tumor growth or progression in an individual having malignant cells expressing FLT3, which comprises administering to the individual in need of an effective amount of the pharmaceutical composition comprising the antibodies bispecific isolates or antibodies, as described here.
[0028] [0028] In another aspect, the invention provides a method of inhibiting malignant cell metastasis expressing FLT3 in an individual,
[0029] [0029] In another aspect, the invention provides a method of inducing tumor regression in an individual who has malignant cells expressing FLT3, which comprises administering to the individual in need of the same effective amount of the pharmaceutical composition as the pharmaceutical composition comprising the isolated antibodies or bispecific antibodies, as described here.
[0030] [0030] In some embodiments, the methods as described herein, also comprise administering an effective amount of a second therapeutic agent. In some embodiments, the second therapeutic agent is a biotherapeutic agent, for example, an antibody.
[0031] [0031] In some embodiments, the second therapeutic agent is a cytokine, TNFa (Tumor Necrosis Factor alpha), a PAP inhibitor (phosphatidic acid phosphatase), an oncolytic virus, a kinase inhibitor, an IDO inhibitor (Indolamine -pyrrole 2,3-dioxigenase), a GLS1 glutaminase inhibitor, a T cell or T cell therapy (Chimeric Antigen Receptor), a TLR (Toll type receptor) agonist (for example, TLR3, TLR4, TLR5, TLR7, TLR9), or a tumor vaccine. In some embodiments, the cytokine is IL-15 (Interleukin-15). Brief Description of Figures / Drawings
[0032] [0032] Figure 1 shows that bispecific FLT3 / CD3 (subdivision of FLT3 is P5F7g, P5F791, P5F792, P5F793, or P5F794) induce cytotoxicity in AML Eol1 cell line.
[0033] [0033] Figure 2 shows that bispecific FLT3 / CD3 (subdivision of FLT3 is P1F1, P4A4, P4E5 or P5F7) with domain-binding epitopes
[0034] [0034] Figure 3 shows that bispecific FLT3 / CD3 (subdivision of FLT3 is 6B7 or P8B6) targeting domain 4 of the FLT3 protein improved tumor efficacy in an orthotopic graft in the presence of human T cells.
[0035] [0035] Figure 4A and Figure 4B demonstrate decreased ECs5o values for the bispecific FLT3 / CD3 antibody (P5F7) in the absence or presence of IL15, respectively.
[0036] [0036] Figures 5A, 5B, 5C, 5D, 5E, and 5PF demonstrate the death of two primary AML samples in the bone marrow aspirate ex vivo induced by increasing concentrations of bispecific FLT3 / CD3 (P5F7) in the presence of autologous T cells. A concentration-dependent increase in total activated T cells and T cells, as determined by the percentage of CD25 + cells, is described. Detailed Description
[0037] [0037] The invention described in this document provides antibodies (for example, monospecific or bispecific) that specifically binds to FLT3 (for example, human FLT3). The invention also provides polynucleotides encoding these antibodies, compositions that comprise these antibodies, and methods of making and using these antibodies. The invention also provides methods for treating a condition associated with FLT3-mediated pathologies in an individual, such as cancer. In particular, the inventors of the present invention have found that FLT3 antibodies as described here, in the bispecific life-size format have a longer half-life, minimized Fc interaction, and minimized in vivo release of nonspecific cytokine through interaction with cells immune. In addition, FLT3 antibodies targeting domain 4 of the FLT3 protein as described here, in bispecific life-size format are found to be more effective in AML cell depletion compared to other domains, including domains 1, 2, 3, and 5 in format bispecific. General Techniques
[0038] [0038] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunology, virology, generation and engineering of monoclonal antibody, which are within the technique. Such techniques are explained entirely in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrooket a /., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Human Press; Cell Biology: A Laboratory Notebook (JE. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.l. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et a / l., Eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et a /., Eds., 1994); Current Protocols in Immunology (J. E. Coliganet al., Eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A.Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical method (D. Catty., Ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical method (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).
[0039] [0039] An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in a variable region of the immunoglobulin molecule. As used here, the term encompasses not only intact monoclonal or polyclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab ”, F (ab ') 2, Fv), single chain (ScFv) and antibodies to domain (including, for example, shark and camelid antibodies), and fusion proteins that comprise an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. An antibody includes an antibody of any class, such as IgG, IgA or IgM (or a subclass thereof), and the antibody need not be of any particular class. Depending on the amino acid sequence of the antibody in the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can also be divided into subclasses (isotypes), for example, IgG1, IgG2, I9G3, I9gG4, IgA1 and IgA2. The heavy chain constant regions that correspond to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
[0040] [0040] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an intact antibody that retains the ability to specifically bind to an certain antigen (for example, FLT3). Antigen-binding functions of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen binding fragment" of an antibody include Fab; Fab '; F (ab ') :; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single antibody subdivision; a single domain antibody (dAb) fragment (Ward et al., Nature 341: 544-546, 1989), and an isolated complementarity determining region (CDR).
[0041] [0041] An antibody or polypeptide that "preferably binds" or "specifically binds" (used interchangeably here) to a target (e.g., FLT3 protein) is a term well known in the art, and methods for determining such specific binding or preferred are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more often, more quickly, with longer duration and / or greater affinity with a particular cell or substance than it does with alternative cells or substances . An antibody "specifically binds" or "preferably binds" to a target if it binds with greater affinity, avidity, more readily, and / or with a longer duration than it binds to other substances. For example, an antibody that specifically or preferably binds to an FLT3 epitope is an antibody that binds to this epitope with greater affinity, avidity, more easily, and / or with a longer duration than it binds to other FLT3 epitopes or epitopes not FLT3. It is also understood that according to the reading of this definition, for example, an antibody (or portion or epitope) that specifically or preferably binds to a first target may or may not specifically or preferably bind to a second target. As such, "specific link" or "preferred link" does not necessarily (although may include) exclusive link. Generally, but not necessarily, reference to link means preferred link.
[0042] [0042] An "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, alone or in combination. As known in the art, the variable regions of the heavy and light chain each consisting of four structure regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs in the other chain, contribute to the formation of the antibody antigen binding site. There are at least two techniques for determining CDRs: (1) a method based on cross-species sequence variability (ie, Kabat et al. Sequences of Proteins of Immunological Interest, (5th Edition, 1991, National Institutes of Health , Bethesda MD)); and (2) a method based on crystallographic studies of antigen-antibody complexes (Al-lazikaniet al., 1997, J. Molec. Biol. 273: 927-948). As used here, a CDR can refer to CDRs defined by either method or a combination of both methods.
[0043] [0043] A "CDR" of a variable domain are amino acid residues within a variable region that are identified according to the definitions of Kabat, Chothia, the accumulation of both: Kabat and Chothia, ADM, contact, and / or definitions conformational or any method of determining CDR well known in the art. Antibody CDRs can be identified as the hypervariable regions originally defined by Kabat et al. See, for example, Kabat et a /., 1992, Sequences of Proteins of Immunological Interest, 5a. Edition, Public Health Service, NIH, Washington D.C. The positions of CDRs can also be identified as the structural loop structures originally described by Chothia et al. See, for example, Chothia et a /., Nature 342: 877-883, 1989.
[0044] [0044] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in smaller quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. The "monoclonal" modifier indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used according to the present invention can be made by the hybridoma method first described by Kohler and Milstein, Nature 256: 495, 1975, or they can be made by the recombinant DNA methods as described in the Patent North American No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in McCafferty et al., Nature 348: 552-554, 1990, for example.
[0045] [0045] As used herein, "humanized" antibody refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab ', F (ab ') 2 or other subsequences of antigen-binding antibodies) containing minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a recipient complementarity determining region (CDR) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and ability. In some cases, human immunoglobulin Fv (FR) structure region residues are replaced by corresponding non-human residues. In addition, the humanized antibody may comprise residues that are not found in the recipient's antibody or in the imported CDR or framework sequences, but are included to further refine and optimize the antibody's performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a consensus sequence of human immunoglobulin. The optimally humanized antibody will also comprise at least a portion of an immunoglobulin (Fc) constant region or domain, typically a human immunoglobulin. Antibodies are preferred having modified Fc regions as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) that are altered with respect to the original antibody, which are also called one or more CDRs "derived from "one or more CDRs from the original antibody.
[0046] [0046] As used herein, "human antibody" means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or which were made using any of the techniques for making human antibodies known to those skilled in the art. or disclosed here. This definition of a human antibody includes antibodies that comprise at least one human heavy chain polypeptide or at least one human light chain polypeptide. Such an example is an antibody comprising human murine and light chain polypeptides. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et a., Nature Biotechnology, 14: 309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci. (USA) 95: 6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol,
[0047] [0047] The term "chimeric antibody" is intended to refer to antibodies in which variable region sequences are derived from one species and constant region sequences are derived from other species, such as an antibody in which region sequences variable are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
[0048] [0048] The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably here to refer to amino acid chains of any size. For example, the chain can be relatively short (for example, 10 to 100 amino acids), or longer. The chain can be linear or ramified, it can comprise modified amino acids, and / or it can be interrupted by non-amino acids. The terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation,
[0049] [0049] A "monovalent antibody" comprises an antigen-binding site per molecule (for example, IgG or Fab). In some cases, a monovalent antibody may have more than one antigen-binding site, but the binding sites are of different antigens.
[0050] [0050] A "monospecific antibody" comprises two identical antigen-binding sites per molecule (e.g., IgG) so that the two binding sites bind to identical epitopes on the antigen. In this way, they compete with each other for binding to an antigen molecule. Most antibodies found in nature are monospecific. In some cases, a monospecific antibody may also be a monovalent antibody (for example, Fab).
[0051] [0051] A "bivalent antibody" comprises two antigen-binding sites per molecule (for example, IgG). In some cases, the two binding sites have the same antigen specificities. However, divalent antibodies can be specific.
[0052] [0052] A "bispecific" or "dual-specific" is a hybrid antibody having two different antigen-binding sites. The two antigen-binding sites of a bispecific antibody bind to two different epitopes, which can reside on the same or different protein targets.
[0053] [0053] A "bifunctional" antibody is an antibody having identical antigen-binding sites (i.e., identical amino acid sequences) in the two subdivisions, but each binding site can recognize two different antigens.
[0054] [0054] A "heteromultimer", "heteromultimeric complex", or "heteromultimeric polypeptide" is a molecule comprising at least a first polypeptide and a second polypeptide, in which the second polypeptide differs in amino acid sequence from the first polypeptide by at least one amino acid residue. The heteromultimer can comprise a "heterodimer" formed by the first and second polypeptides or can form tertiary structures of a higher order where polypeptides in addition to the first and second polypeptides are present.
[0055] [0055] A "heterodimer," "heterodimeric protein," "heterodimeric complex," or "heteromultimeric polypeptide" is a molecule comprising a first polypeptide and a second polypeptide, in which the second polypeptide differs in amino acid sequence from the first polypeptide by at least one amino acid residue.
[0056] [0056] The "hinge region," "hinge sequence", and variations thereof, as used herein, include the meaning known in the art, which is illustrated in, for example, Janeway et al., ImmunoBiology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al., Protein Science (1997), 6: 407-415; Humphreys et al., J. Immunol. Methods (1997), 209: 193-202.
[0057] [0057] The "immunoglobulin-like hinge region," "immunoglobulin-like hinge sequence," and variations thereof, as used herein, refers to the hinge regions and hinge sequences of an immunoglobulin-like molecule or an antibody-type ( for example, immunoadhesins). In some embodiments, the immunoglobulin-like hinge region can be from or derived from any IgG1, IgG2, IgG3, or IgG4 subtype, or from IgA, IgE, IgD or IgM, including chimeric forms thereof, for example, a hinge region of chimeric Ig9G1 / 2.
[0058] [0058] The term "immune effector cell" or "effector cell" as used here refers to a cell within the natural repertoire of cells in the human immune system that can be activated to affect the viability of a target cell. The viability of a target cell can include cell survival, proliferation, and / or the ability to interact with other cells.
[0059] [0059] Antibodies of the invention can be produced using techniques well known in the art, for example, recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art (See, for example, Jayasena, SD, Clin. Chem., 45: 1628-50, 1999 and Fellouse, FA, et al, J. Mol. Biol., 373 (4): 924-40, 2007).
[0060] [0060] As known in the art, "polynucleotide," or "nucleic acid," as used interchangeably herein, refers to nucleotide strands of any size, and includes DNA and RNA. The nucleotides can be deoxoribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure can be granted before or after the strand is assembled. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can also be modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "coatings", replacement of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged bonds (eg, methyl phosphonates, phosphotriesters, phosphoamidates , carbamates,
[0061] [0061] As known in the art, an "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, alone or in combination.
[0062] [0062] As used herein, "substantially pure" refers to material that is at least 50% pure (i.e., free from contaminants), more preferably, at least 90% pure, more preferably, at least 95% pure, yet more preferably, at least 98% pure, and more preferably, at least 99% pure.
[0063] [0063] A "host cell" includes an individual cell or cell culture that can be or have a vector container for incorporating polynucleotide inserts. Host cells include progeny from a single host cell, and the progeny may not necessarily be completely identical (in morphology or in addition to genomic DNA) to the original progenitor cell due to natural, accidental or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide of this invention.
[0064] [0064] As known in the art, the term "Fc region" is used to define a C-termination region of an immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined to stretch from an amino acid residue in the Cys226 or Pro230 position to the carboxyl termination thereof. The waste numbering in the Fc region is that of the European Union index as in Kabat. Kabat et a /., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally comprises two constant regions, CH2 and cH3.
[0065] [0065] As used in the art, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. In addition, a preferred FcR is one that binds to an IgG antibody (a gamma receptor) and includes receptors of the subclasses FcyRI, FeyRII, and FeyRIII, including allelic variants and alternately joined forms of these receptors. FeyRIl receptors include FeyRIIA (an "activating receptor") and FCcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. FcRs are reviewed in Ravetch and Kinet, Ann. Rev. Immunol., 9: 457-92, 1991; Capelet al., Inmunomethods, 4: 25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126: 330-41, 1995. "FcR" also includes neonatal receptors, FcRn, which are responsible for the transfer of maternal IgGs to the fetus (Guyeret a /., J. Inmunol., 117: 587, 1976; and Kim et al., J. Inmunol., 24: 249, 1994).
[0066] [0066] The term "compete", as used here in connection with an antibody, means that a first antibody, or an antigen-binding fragment (or portion) thereof, binds to an epitope in a manner sufficiently similar to binding of a second antibody, or an antigen-binding portion thereof, so that the result of binding the first antibody to its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, may, however, not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody to its cognate or ligand epitope, whether to the same extent, to a greater or lesser extent, the antibodies are said to "cross-compete" with each other for binding their respective epitopes . Both: competing and cross-competing antibodies - are —globed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (for example, steric hindrance, conformational change, or attachment to a common epitope, or portion thereof), the skilled technician must appreciate, based on the teachings provided here, that such antibodies competing and / or cross-competing are included and may be useful for the methods disclosed here.
[0067] [0067] A "functional Fc region" has at least one effector function of a native sequence Fc region. Exemplary "effector functions" include C1qg binding; complement-dependent cytotoxicity; binding of Fc receptor; antibody-dependent cell-mediated cytotoxicity; phagocytosis; down regulation of cell surface receptors (eg B cell receptor), etc. Such effector functions generally require Fc region to be combined with a binding domain (for example an antibody variable domain) and can be evaluated using various assays known in the art to evaluate such antibody effector functions.
[0068] [0068] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequences of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification, yet retains at least one effector function of the native sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or the Fc region of a major polypeptide, for example, from about one to about ten amino acid substitutions, and preferably, from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the main polypeptide. The variant Fc region here will preferably have at least about 80% sequence identity with the native sequence Fc region and / or an Fc region of a major polypeptide, and more preferably, at least about 90% sequence identity. sequence, more preferably, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity.
[0069] [0069] The term "effector function" refers to the biological activities attributable to the Fc region of an antibody. Examples of antibody effector functions include, but are not limited to, antibody dependent cell mediated cytotoxicity (ADCC), Fc receptor binding, complement dependent cytotoxicity (CDC), phagocytosis, C1q binding, and receptor down regulation cell surface (eg B cell receptor; BCR). See, for example, U.S. Patent No. 6,737,056. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays known in the art to evaluate such antibody effector functions. An exemplary measure of effector function is by connecting Fcy3 and / or C1q.
[0070] [0070] As used herein "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (for example natural killer cells (NK) , neutrophils, and macrophages) recognize binding antibody in a target cell and subsequently cause lysis of the target cell. ADCC activity of a molecule of interest can be assessed using an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Alternatively, or in addition, ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model such as that disclosed in Clyneset a /., 1998, PNAS (USA), 95: 652-656.
[0071] [0071] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target in the presence of complement. The complement activation pathway is initiated by binding the first component of the complement system (C1lqg) to a molecule (for example, an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods, 202: 163 (1996), can be performed.
[0072] [0072] As used here, "treatment" is a method for obtaining desired beneficial or clinical results. For the purposes of this invention, desired beneficial or clinical results include, but are not limited to, one or more of the following: reduced proliferation of (or destruction) of neoplastic or cancer cells, inhibiting neoplastic cell metastasis, shrinkage or decrease in tumor size expressing FLT3, remission of a disease associated with FLT3 (for example, cancer), decreased symptoms resulting from a disease associated with FLT3 (for example, cancer),
[0073] [0073] "Improvement" means a decrease or improvement in one or more symptoms when compared to not administering an FLT3 antibody (monospecific or bispecific). "Improvement" also includes shortening or reducing the duration of a symptom.
[0074] [0074] As used herein, an "effective dosage" or "effective amount" of drug, compound, or pharmaceutical composition is an amount sufficient to affect any one or more desired or beneficial results. For prophylactic use, beneficial or desired results include elimination or reduced risk, decreased severity, or delayed disease onset, including biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during disease development. For therapeutic use, beneficial or desired results include clinical results, such as reducing the incidence or improving one or more symptoms of various diseases or conditions associated with FLT3 (such as, for example, multiple myeloma), lowering the dose of other necessary medications to treat the disease, improve the effect of another medication, and / or slow the progression of the disease associated with FLT3 in patients. An effective dosage can be administered in one or more administrations. For purposes of this invention, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to carry out prophylactic or therapeutic treatment directly or indirectly. As understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" can be considered in the context of administration of one or more therapeutic agents, and a single agent can be considered a single agent in an effective amount if, together with one or more other agents, a desirable result can be or is achieved.
[0075] [0075] An "individual (individual)" or an "individual (subject)" is a mammal, more preferably, a human. Mammals also include, but are not limited to, primates, horses, dogs, cats, mice and rats.
[0076] [0076] As used herein, "vector" means a construct, which is capable of releasing, and, preferably expressing, one or more gene (s) or sequence (s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA expression vectors or RNA encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
[0077] [0077] As used here, "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as an inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.
[0078] [0078] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that,
[0079] [0079] The term "acyl donor glutamine marker" or "glutamine marker" as used herein refers to a polypeptide or protein containing one or more Glnh residues that acts as an acceptor for transglutaminase amine. See, for example, WO02012059882 and WO2015015448.
[0080] [0080] The term "kKon" or "ka", as used here, refers to the rate constant for the association of an antibody with an antigen. Specifically, the rate constant (Kon / Ka € Kot / Ka) and equilibrium dissociation constants are measured using a whole antibody (i.e., divalent) and monomeric FLT3 proteins (for example, histidine-tagged FLT3 fusion protein).
[0081] [0081] The term "kof" or "ka", as used here, refers to the rate constant for dissociation of an antibody from the antibody / antigen complex.
[0082] [0082] The term "Kp", as used here, refers to the equilibrium dissociation constant of an antibody-antigen interaction.
[0083] [0083] Reference to "about" a value or parameter here includes (and describes) modalities that are directed to that value or parameter itself. For example, description referring to "about X" includes description of "X." Numeric ranges include the numbers defining the range. Generally speaking, the term "about" refers to the indicated values of the variable and all values of the variable that are within the experimental error of the indicated value (for example, within the 95% confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater. Where the term "about" is used within the context of a period of time (years, months, weeks, days, etc.), the term "about" means that a period of time plus or minus an amount of the next period of time subordinate (for example, about 1 year means 11 to 13 months; about 6 months means 6 months or so | week; about a week means 6 to 8 days; etc.), or within 10 percent of the value indicated, whichever is greater.
[0084] [0084] It is understood that whenever modalities are described in this document with the language "which comprises," otherwise similar, modalities described in terms of "consisting of" and / or "consisting essentially of" are also provided.
[0085] [0085] Where aspects or modalities of the invention are described in terms of a Markush group or other groupings of alternatives, the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the group main group, but also the main group absences one or more of the group members. The present invention also provides for the explicit exclusion of one or more of any of the group members in the claimed invention.
[0086] [0086] Unless otherwise defined, all technical and scientific terms used here have the same meaning as commonly by someone versed in the technique to which this invention belongs. In case of conflict, this specification, including definitions, will control. Throughout this specification and claims, the word "understand" or variations such as "understand" or "understand" should be understood to imply the inclusion of an established whole number or group of whole numbers, but not the exclusion of any another integer or group of integers. Unless otherwise required by context, singular terms must include pluralities and plural terms must include the singular.
[0087] [0087] Exemplary methods and materials are described here, although methods and materials similar or equivalent to those described in this document may also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and are not intended to be limiting. FLT3 Antibodies and Manufacturing Methods
[0088] [0088] The present invention provides an antibody that binds to FLT3 [for example, human FLT3 (for example, accession number: NP 004110 or SEQ ID NO: 235)] and characterized by any one or more of the following characteristics: ( a) treats, prevents, improves one or more symptoms of a condition associated with malignant cells expressing FLT3 in an individual (for example, cancer, such as access number AML); (b) inhibits tumor growth or progression in an individual (who has a malignant tumor expressing FLT3); (c) inhibits the metastasis of cancer cells (malignant) expressing FLT3 in an individual (who has one or more malignant cells expressing FLT3); (d) induces regression (e.g., long-term regression) of a tumor expressing FLT3; (e) exerts cytotoxic activity in malignant cells expressing FLT3; (f) blocks FLT3 interaction with others yet to be identified factors; and / or (g) induces a spectator effect that kills or inhibits growth of malignant cells expressing non-FLT3 in the vicinity.
[0089] [0089] In one aspect, an isolated antibody is provided which specifically binds to FLT3, wherein the antibody comprises (a) a heavy chain variable region (VH) comprising (i) a complementarity determining region (CDR1) ) of VH comprising the sequence shown at 37, 38, 39, 43, 44, 45, 49, 50, 54, 55, 56, 60, 61, 62, 66, 67, 68, 72, 73, 74, 78 , 79, 80, 84, 85, 86, 90, 91, 92, 96, 97, 98, 102, 103, 104, 108, 109, 110, 114, 115, 116, 120, 121, 122, 126, 127 , 128, 132, 133, 134, 138, 139, 140, 246, or 247; (ii) a VH CDR 2 comprising the sequence shown in SEQ ID NO: 40, 41, 46, 47, 51, 52, 57, 58, 63, 64, 69, 70, 75, 76, 81, 82 , 87, 88, 93, 94, 99, 100, 105, 106, 111, 112, 117, 118, 123, 124, 129, 130, 135, 136, 141, 142, 248, 249, 251, 252, 253 , or 255; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 42, 48, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 245, 250, or 254; and / or a light chain variable region (VL) comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 144, 147, 150, 153, 156, 159, 162, 165, 168, 171 , 174, 177, 180, 183, 186, 189, 192, 195, 257, 261, 263, 265, 268, 270, 273, or 275; (ii) a VL CDR 2 comprising the sequence shown in SEQ ID NO: 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190 , 193, 196, 259, 266, or 271; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 256, 258, 260, 262, 264, 267, 269, 272, or 274.
[0090] [0090] In another aspect, an isolated antibody is provided that specifically binds to FLT3, wherein the antibody comprises: a VH region comprising a VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID No.: 2, 4,6,8,10,12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 205, 207, 209, 211, 213, 215, 217, 219, 221,223, 225, 227, 229, 231, or 233; and / or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the sequence
[0091] [0091] In some embodiments, an antibody is provided that has any partial light chain sequence as listed in Table 1 and / or any partial heavy chain sequence as listed in Table 1. In Table 1, the underlined sequences are CDR sequences according to Kabat and bold according to Chothia, except for P4F6, P4C7, P3A1, PSA3, P9B5, P9F1, P1B4, P1B11, P7H3, P3E10, P1A5, P5F7, P4H11, P4H11, P4H11, P4H11, P4H11, P4H11 P12B6, P8B6, P14G2, and P7F9, the Chothia CDR sequence is underlined and the Kabat CDR sequence is in bold.
[0092] [0092] Portions of CDR binding to FLT3 antibody antigen domains (including CDRs Kabat, Chothia, and CDR contact regions) are also provided here. The determination of regions
[0093] [0093] In some embodiments, the present invention provides an antibody that binds to FLT3 and competes with the antibody as described in this document, including P4F6, P4C7, P3A, P5A3, P9B5, P9F1, P1B4, P1B11, P7H3, P3E10, P1A5 , P5F7, P4H11, P15F7, P12B6, P8B6, P14G2, P7F9, POS8BOGEE, PO4A04, PO1AOS5, PO8BO3, P5F7, P5F79g, P10A02g, P10A049, P10A05g, P10A07g, P10B5, P10F07g, P10B
[0094] [0094] In some embodiments, the invention also provides CDR portions of antibodies to FLT3 antibodies based on the CDR contact regions. Contact CDR regions are regions of an antibody that imbue the antibody with specificity for an antigen. In general, the CDR contact regions include the positions of the residues in the CDRs and Vernier zones that are restricted in order to maintain a suitable loop structure for the antibody to bind to a specific antigen. See, for example, Makabe et al., J. Biol. Chem., 283: 1156 to 1166, 2007. The determination of CDR contact regions is well within the technique.
[0095] [0095] The binding affinity (Kp) of the FLT3 antibody as described in this document, FLT3 (such as human FLT3 (for example, (SEQ ID NO: 201)) can be from about 0.001 to about 5000 nM. in some embodiments, the binding affinity is about any of 5000 nM, 4500 nM, 4000 nM, 3500 nM, 3000 nM, 2500 nM, 2000 NM, 1789 nM, 1583 nM, 1540 nM, 1500 nM, 1490 nM, 1064 nM, 1000 NM, 933 nM, 894 nM, 750 nM, 705 nM, 678 nM, 532 nM, 500 nM, 494 NM, 400 nM, 349 nM, 340 nM, 353 nM, 300 nM, 250 nM, 244 nM, 231 NM, 225 nM, 207 nM, 200 nM, 186 nM, 172 nM, 136 nM, 113 nM, 104 NM, 101 nM, 100 nM, 90 nM, 83 nM, 79 nM, 74 NM, 54 NM, 50 nM , 45 NM, 42 nM, 40 nM, 35 nM, 32 nM, 30 nM, 25 nM, 24 nM, 22 nM, 20 nM, 19 nM, 18 NM, 17 NM, 16 NM, 15 NM, 12 nM, 10 AM, 9 AM, 8 nM, 7.5 AM, 7 NM, 6.5 NM, 6 NM, 5.5 NM, 5 NM, 4 NM, 3 NM, 2 NM, 1 NM, 0.5 NM, 0 .3 NM, 0.1 nM, 0.01 nM, or 0.001 nM. In some embodiments, binding affinity is less than about any of 5000 nM,
[0096] [0096] Bispecific antibodies, monoclonal antibodies that have binding specificities for at least two different antigens, can be prepared using the antibodies described herein. Methods for making bispecifics are known in the art (See, for example, Suresh et al., Methods in Enzymology 121: 210, 1986). Traditionally, the recombinant production of bispecific antibodies was based on the coexpression of two pairs of heavy chain-immunoglobulin light chains, with the two heavy chains with different specificities (Millstein and Cuello, Nature 305, 537 to 539, 1983). Accordingly, in one aspect, a bispecific antibody is provided in which the bispecific antibody is a human-sized antibody, which comprises a first antibody variable domain of the bispecific antibody that specifically binds to a target antigen (e.g., FLT3) , and which comprises a second antibody variable domain of the bispecific antibody capable of recruiting the activity of a human immune effector cell specifically binding to an effector antigen located on the human immune effector cell.
[0097] The human immune effector cell can be any of a variety of immune effector cells known in the art. For example, the immune effector cell may be a member of the human lymphoid cell line, including, but not limited to, a T cell (for example, a cytotoxic T cell), a B cell, and a natural killer cell (NK). The immune effector cell can also be, for example, without limitation, a member of the human myeloid lineage, including, but not limited to, a monocyte, a neutrophilic granulocyte and a dendritic cell. Such immune effector cells can have a cytotoxic or apoptotic effect on a target cell or other desired effect after activation by binding an effector antigen.
[0098] [0098] The effector antigen is an antigen (for example, a protein or a polypeptide) expressed in the human immune effector cell. Examples of effector antigens that can be linked by the heterodimeric protein (for example, a heterodimeric antibody or a bispecific antibody) include, but are not limited to, human CD3 (or CD3 (Differentiation Cluster) complex), CD16, NKG2D, NKp46, CD2 , CD28, CD25, CD64, and CD8º9.
[0099] [0099] The target cell can be a native cell or foreign to humans. In a native target cell, the cell may have been transformed to be a malignant cell or pathologically modified (for example, a native target cell infected with a virus, plasmodium or bacterium). In a foreign target cell, the cell is an invading pathogen, like a bacterium, a plasmodium or a virus.
[00100] [00100] The target antigen is expressed in a target cell in a sick condition (for example, an inflammatory disease, a proliferative disease (for example, cancer), an immune disorder, a neurological disease, a neodegenerative disease, an autoimmune disease, an infectious disease (for example, a viral infection or a parasitic infection), an allergic reaction, a graft-versus-host disease, or a host-versus-graft disease. A target antigen is not an effector antigen. In some embodiments, the target antigen is FLT3.
[00101] [00101] In some embodiments, a bispecific antibody is provided in which the bispecific antibody is a life-size antibody, comprising a first variable domain of the bispecific antibody that specifically binds to a target antigen, and which comprises a second domain bispecific antibody antibody variable capable of recruiting the activity of a human immune effector cell specifically binding to an effector antigen located on the human immune effector cell, wherein the first variable domain of antibody bonds to domain 4 of FLT3 comprising SEQ ID No.: 279.
[00102] [00102] In some embodiments, a bispecific antibody is provided, wherein the bispecific antibody is a life-size antibody, which comprises a first bispecific antibody variable domain that specifically binds to a target antigen, and which comprises a second bispecific antibody antibody variable domain capable of recruiting the activity of a human immune effector cell specifically binding to an effector antigen located on the human immune effector cell, where the first antibody variable domain comprises a heavy chain variable region (VH) comprising a VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 , 32, 34, 36, 205, 207, 209, 211, 213, 215, 217,219,221,223, 225, 227, 229, 231, or 233; and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, or 232.
[00103] [00103] In some embodiments, a bispecific antibody is provided, wherein the bispecific antibody is a life-size antibody, which comprises a first variable domain of the bispecific antibody that specifically binds to a target antigen, and which comprises a second bispecific antibody variable domain capable of recruiting the activity of a human immune effector cell specifically binding to an effector antigen located on the human immune effector cell, where the first antibody variable domain comprises (a) a heavy chain variable region (VH) comprising (i) a VH one complementarity determining region (CDR1) comprising the sequence shown in SEQ ID NO: 37, 38, 39, 43, 44, 45, 49, 50, 54, 55, 56 , 60, 61, 62, 66, 67, 68, 72, 73, 74, 78, 79, 80, 84, 85, 86, 90, 91, 92, 96, 97, 98, 102, 103, 104, 108 , 109, 110, 114, 115, 116, 120, 121, 122, 126, 127, 128, 132, 133, 134, 138, 139, 140, 246, or 247; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 40, 41, 46, 47, 51, 52, 57, 58, 63, 64, 69, 70, 75, 76, 81, 82, 87, 88, 93, 94, 99, 100, 105, 106, 111, 112, 117, 118, 123, 124, 129, 130, 135, 136, 141, 142, 248, 249, 251, 252, 253, or 255 ; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 42, 48, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 245, 250, or 254; and / or a light chain variable region (VL) comprising (i) a CDR1 VL comprising the sequence shown in SEQ ID NO: 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 257, 261, 263, 265, 268, 270, 273, or 275; (ii) a CDR2 VL comprising the sequence shown in SEQ ID NO: 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 259, 266, or 271; and (iii) a CDR3 VL comprising the sequence shown in SEQ ID NO: 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194 , 197, 256, 258, 260, 262, 264, 267, 269, 272, or 274.
[00104] [00104] In some embodiments, the first antibody variable domain comprises (a) a heavy chain variable region (VH) comprising (i) a VH one complementarity determining region (CDR1) comprising the sequence shown in SEQ ID No. 102, 103, or 104; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 255 or 106; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 107; and / or (b) a light chain variable region (VL) comprising (i) a CDR1 VL comprising the sequence shown in SEQ ID NO: 177; (ii) a VR CDR 2 comprising the sequence shown in SEQ ID NO: 178; and (iii) a CDR3 VL comprising the sequence shown in SEQ ID NO: 179.
[00105] [00105] In some embodiments, the second antibody variable domain comprises a heavy chain variable region (VH) comprising a VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 282; and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 281.
[00106] [00106] In some embodiments, the first antibody variable domain comprises a heavy chain variable region (VH) comprising a VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 229; and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 228; and a second antibody variable domain comprising a heavy chain variable region (VH) comprising a VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 282; and / or a light chain variable region (VL) comprising a VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 281.
[00107] [00107] In some embodiments, the second antibody variable domain comprises (a) a heavy chain variable region (VH) comprising (i) a complementarity determining VH region (CDR1) comprising the sequence shown in SEQ ID No. 285, 286, or 287; (li) a VH CDR2 comprising the sequence shown in SEQ ID NO: 288 or 289; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 290; and / or (b) a light chain variable region (VL) comprising (i) a CDR1 VL comprising the sequence shown in SEQ ID NO: 291; (ii) a VR CDR 2 comprising the sequence shown in SEQ ID NO: 292; and (iii) a CDR3 VL comprising the sequence shown in SEQ ID NO: 234.
[00108] [00108] Table 3 shows the specific amino acid and nucleic acid sequences of the second variable domain of the antibody, which is specific for CD3. In Table 3, the underlined strings are CDR according to Kabat and in bold according to Chothia. Table 3 h2B4 H | DIVMTOSPDSLAVSLGERATINCKSSOS | EVQLVESGGGLVQPGGSLRLSCAASGFTFSD NPS VL | LENVRSRKNYLAWYQQKPGQPPKLLIS | YYMTWVROAPGKGLEWVAFIRNRARGYTSD OTK WASTRESGVPDRFSGSGSGTDFTLTIS | HNPSVKGRFTISRDNAKNSLYLQMNSLRAEDT SLOAEDVAVYYCKQSYDLFTFGSGTKL | AVYYCARDRPSYYVLDYWGQGTTVTVSS EIK (SEQ ID NO: 281) (SEQ ID NO: 282) h2B4 H | GACATTGTGATGACTCAATCCCCCGA | GAAGTCCAACTTGTCGAATCGGGAGGAGGC NPS VL | CTCCCTGGCTGTGTCCCTCGGCGAAC | CTTGTGCAACCOGGTGGATCCCTGAGGCTGE TK GCGCAACTATCAACTGTAAAAGCAGC | TCATGCGCGGCCTCGEGCTTCACCTTTTCC CAGTCCCTGTTCAACGTCCGGTCGAG | GATTACTACATGACCTGGGTCAGACAGGCC GAAGAACTACCTGGCCTGGTATCAGC | CCTGGAAMAGGGGTTGGAATGGGTGGCATTC AGAAACCTGGGCAGCCGCCGAAGCTT | ATCCGGAATAGAGCCCGCGGATACACTTCC CTGATCTCATGGGCCTCAACTCGGGA | GACCACAACCCCAGCGTGAAGGGGCEGTTC AAGCGGAGTGCCAGATAGATTCTCCG | ACCATTAGCCGCGACAACGCCAAGAACTCC GATCTGGCTCCGGAACCGACTTCACC | CTCTACCTCCAMATGAACAGCCTGCEGGCG CTGACGATTTCGAGCTTGCAAGCGGA | GAGGATACCGCTGTGTACTACTGCGCCCGC GGATGTGGCCGTGTACTACTGCAAGC | GACCGGCCGTCCTACTATGTGCTGGACTAC AGTCCTACGACCTCTTCACCTTTGGTT | TEGGGCCAGGGTACTACGGTCACCGTCTCC CGGGCACCAAGCTGGAGATCAAA TCA (SEQ ID NO: 284) (SEQ ID NO: 283)
[00109] [00109] Table 4 shows the examples of CDR sequences of the second antibody variable domain, which is specific for CD3.
[00110] [00110] In some embodiments, a bispecific antibody provided herein that contains a specific CD3 variable domain with an anti-CD3 sequence, as provided in North American Publication No. 20160297885, which is incorporated herein by reference for all purposes.
[00111] [00111] According to a method of preparing bispecific antibodies, variable domain of antibodies with the desired binding specificities (antibody-antigen combination sites), the immunoglobulin constant region sequences are fused. The fusion is preferably with an immunoglobulin Heavy Chain constant region, which comprises at least part of the hinge, CH2 and CH3 regions. It is preferable to have the first Heavy Chain constant region (CH1), containing the necessary site for Light Chain binding, present in at least one of the fusions. The DNAs encoding immunoglobulin Heavy Chain fusions and, if desired, the immunoglobulin Light Chain, are inserted into separate expression vectors, and are cotransfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in situations where unequal proportions of the three polypeptide chains used in construction provide the ideal yields. However, it is possible to insert the coding sequences for two or all three polypeptide chains in an expression vector when the expression of at least two polypeptide chains in equal proportions results in high yields or when the relationships have no particular meaning.
[00112] [00112] In another method, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one subdivision and a pair of hybrid immunoglobulin heavy chain (providing a second binding specificity) in the other subdivision. This asymmetric structure, with an immunoglobulin light chain in only half of the bispecific molecule, facilitates the separation of the desired bispecific compound from unwanted combinations of immunoglobulin chains. This method is described in PCT Publication No. WO 94/04690.
[00113] [00113] In another method, bispecific antibodies are composed of amino acid modifications in the first hinge region of a subdivision, and the substituted / replaced amino acid in the first hinge region has a charge opposite to the corresponding amino acid in the second hinge region of another subdivision. This method is described in International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545).
[00114] [00114] In another method, the formation of a desired heteromultimeric or heterodimeric protein (eg, bispecific antibody) is enhanced by altering or creating an interface between a first immunoglobulin-like Fc region and a second one (for example, a hinge region and / or a CH3 region). In this method, specific antibodies can be composed of a CH3 region, where the CH3 region comprises a first CH3 polypeptide and a second CH3 polypeptide that interact together to form a CH3 interface, where one or more amino acids within the CH3 interface destabilizes the formation of the homodimer and is not electrostatically unfavorable to the formation of the homodimer. This method is described in International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545).
[00115] [00115] In another method, bispecific antibodies can be generated using a peptide marker containing modified glutamine for the antibody targeting an epitope (for example, FLT3) in a subdivision and another peptide marker (for example, a peptide marker containing Lys or an endogenous reactive Lys) modified to a second antibody directed to a second epitope in another subdivision in the presence of transglutaminase. This method is described in International Patent Application No. PCT / IB2011 / 054899 (WO2012 / 059882).
[00116] [00116] In some embodiments, the heterodimeric protein (e.g., bispecific antibody) as described herein, comprises a human-sized antibody, wherein a first bispecific antibody antibody variable domain that specifically binds to a target antigen ( for example, FLT3), and which comprises a second antibody variable domain of the bispecific antibody capable of recruiting the activity of a human immune effector cell specifically binding to an effector antigen (eg CD3) located on the human immune effector cell, in whereas the first and second antibody variable domains of the heterodimeric protein comprise amino acid modifications at positions 223, 225, and 228 (for example, (C223E or C223R), (E225E or E225R), and (P228E or
[00117] [00117] In some embodiments, the first and second variable domains of heterodimeric protein vectors comprise amino acid modifications at positions 221 and 228 (for example, (D221R or D221E) and (P228R or P228E)) in the joint region and position 409 or 368 (for example, K409R or L368E (EU numbering scheme)) in the CH3 region of human IgG1 (SEQ ID NO: 237).
[00118] [00118] In some embodiments, the first and second variable domains of heterodimeric protein vectors comprise amino acid modifications at positions 228 (for example, (P228E or P228R)) in the joint region and at position 409 or 368 (for example, R409 or L368E (EU numbering scheme)) in the CH3 region of human IgG4 (SEQ ID NO: 238).
[00119] [00119] Antibodies useful in the present invention can include antibodies - monoclonal, polyclonal antibodies, antibody fragments (e.g., Fab, Fab ', F (ab') 2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies , heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion (e.g., a domain antibody), humanized antibodies, and any other modified configuration of the immunoglobulin molecule comprising a recognition site antigen of the required specificity, including antibody glycosylation variants, antibody amino acid sequence variants and covalently modified antibodies. The antibodies can be murine, rats, humans or any other source (including chimeric or humanized).
[00120] [00120] In some embodiments, the monospecific antibody FLT3 or the bispecific antibody FLT3 (for example, FLT3-CD3) as described in this document, is a monoclonal antibody. For example, the monospecific antibody FLT3 is a human monoclonal antibody. In another example, the FLT3 subdivision of the bispecific FLT3-CD3 antibody is a human monoclonal antibody, and the CD3 subdivision of the bispecific FLT3-CD3 antibody is a humanized monoclonal antibody.
[00121] [00121] In some embodiments, the antibody comprises a modified constant region, such as, without limitation, a constant region that has an increased potential to elicit an immune response. For example, the constant region can be modified to have increased affinity for an Fc gamma receptor, such as, for example, FeyRI, FeyRIIA or Fevylll.
[00122] [00122] In some embodiments, the antibody comprises a region - constant - modified, as an immunologically inert constant region, that is, with a reduced potential to provoke an immune response. In some embodiments, the constant region is modified as described in Eur. J. Immunol., 29: 2613 to 2624, 1999; PCT Publication No. PCT / GB99 / 01441; and / or United Kingdom Patent Application No. 98099518. The Fc can be human IgG1, human IgG2, human IgG3 or human IgG4. The Fc can be human IgG2 containing the A330P331 to S330S331 (IgG2Aa) mutation, where the amino acid residues are numbered with reference to the wild-type IgG2 sequence. Eur. J. Immunol., 29: 2613 - 2624, 1999. In some embodiments, the antibody comprises an IgG constant region, which comprises the following mutations (Armor et al, Molecular Immunology 40 585 - 593, 2003): E233F234L235 a P233V234A235 (IgG4Ac), where the reference number refers to wild type IgG4. In yet another embodiment, the Fc is human I1g9G4 E233F234L235 to P233V234A235 with the G236 (I9G4Ab) deletion. In another embodiment, the Fc is any human IgG4 Fc (I9G4, IGgG4Ab or IgG4Ac) containing stabilization mutation of the S228 to P228 joint (Aalberse et al., Immunology 105, 9 - 19, 2002). In another embodiment, the Fc may be aglycosylated Fc.
[00123] [00123] In some embodiments, the constant region is aglycosylated by mutating the oligosaccharide-binding residue (such as Asn297) and / or flanking residues that are part of the glycosylation recognition sequence in the constant region. In some situations, the constant region is aglycosylated for N-linked glycosylation enzymatically. The constant region can be aglycosylated for N-linked glycosylation enzymatically or by expression in a host cell deficient in glycosylation.
[00124] [00124] In some embodiments, the constant region has a modified constant region that removes or reduces the binding to the Fc gamma receptor. For example, the Fc can be human IgG2 containing the D265 mutation, where the amino acid residues are numbered with reference to the wild type IgG2 sequence (SEQ ID NO: 236). Consequently, in some embodiments, the constant region has a modified constant region having the sequence shown in SEQ ID NO: 239: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLOSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKV DKTVERKCRVRCPRCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCV VVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQODWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD
[00125] [00125] In some embodiments, the constant region has a modified constant region having the sequence shown in SEQ ID NO: 241:
[00126] [00126] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSNFGTQTYTCNVD HKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMIS RTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKT
[00127] [00127] The amino acid of the human kappa constant region is shown in SEQ ID NO: 243:
[00128] [00128] GTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
[00129] [00129] “One way to determine the binding affinity of FLT3 is by measuring the binding affinity of the divalent antibody to the monomeric FLT3 protein. The affinity of an FLT3 antibody can be determined by surface plasmon resonance (Biacore '3000'Y (SPR) surface plasmon resonance system (SPR), Biacore'Y, INC, Piscataway NJ) equipped with pre-immobilized anti-mouse Fc or Anti-human Fc using HBS-EP running buffer (0.01M HEPES, pH 7.4, 0.15 NaCl, 3 MM EDTA, 0.005% v / v surfactant P20) The 8-histidine-labeled human FLT3 extracellular domain monomeric can be diluted in the HBS-EP buffer to a concentration below 0.5 µg / mL and injected through the individual chip channels using varying contact times, to achieve two antigen density ranges, 50 to 200 response units ( RU) for detailed kinetic studies or 800 to 1,000 RU for analysis assays Regeneration studies have shown that 25 mM NaOH in 25% v / v ethanol effectively removes the bound FLT3 protein while maintaining an activated FLT3 activity on the chip for more than 200 injections, typically serial dilutions (covering concentrations rations of 0.1 to 10x estimated Kp) of FLT3 samples labeled with purified 8-histidine are injected for 1 min at 100 μl / minute and dissociation times of up to two hours are allowed. The concentrations of FLT3 proteins are determined by absorbance at 280 nm based on the sequence-specific extinction coefficient of the FLT3 protein labeled with 8-histidine. The kinetic association rates (Kon or ka) and the dissociation rates (Kkor or ka) are obtained simultaneously, adjusting the data globally to a Langmuir 1: 1 connection model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99 to 110) using the BlAevaluation program. The values of the equilibrium dissociation constant (Ko) are calculated as koff / kon. This protocol is suitable for use in determining the binding affinity of an antibody to any monomeric FLT3, including human FLT3, FLT3 from another mammal (such as mouse FLT3, rat FLT3, or primate FLT3), as well as different forms of FLT3 (eg glycosylated FLT3). The binding affinity of an antibody is usually measured at 25ºC, but it can also be measured at 37ºC.
[00130] [00130] Antibodies, as described in this document, can be made by any method known in the art. For the production of hybridoma cell lines, the host animal's immunization route and schedule are generally in accordance with conventional and established techniques for stimulation and antibody production, as described in this document below. General techniques for producing human and mouse antibodies are known in the art and / or are described in this document.
[00131] [00131] It is contemplated that any individual mammal,
[00132] [00132] Hybridomas can be prepared from immortalized lymphocytes and myeloma cells, using the general somatic cell hybridization technique of Kohler, B. and Milstein, C., Nature 256: 495-497, 1975 or as modified by Buck, DW, et al., In Vitro, 18: 377-381,
[00133] [00133] Hybridomas that can be used as a source of antibodies cover all derivatives, cells derived from parental hybridomas that produce monoclonal antibodies specific for FLT3, or portions thereof.
[00134] [00134] Hybridomas that produce these antibodies can be cultured in vitro or in vivo using known procedures. Monoclonals can be isolated from the culture medium or body fluids, by conventional immunoglobulin purification procedures, such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography and ultrafiltration, if desired. The unwanted activity, if present, can be removed, for example, by running the preparation on adsorbents made from the immunogen attached to a solid phase and eluting or releasing the desired antibodies from the immunogen. Immunization of a host animal with cells that express human FLT3, a human FLT3 protein, or a fragment containing the target amino acid sequence conjugated to an immunogenic protein in the species to be immunized, for example, keyhole limpet hemoglobin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCI2 = or RIÍN = C = NR, where R and R 'are different alkyl groups, can produce a population of antibodies (for example, monoclonal antibodies).
[00135] [00135] If desired, the antibody (monoclonal or polyclonal) of interest can be sequenced and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in vector in a host cell and the host cell can be expanded and frozen for future use. The production of recombinant monoclonal antibodies in cell culture can be performed by cloning B cell antibody genes by means known in the art. See, for example, Tiller et al., J. Immunol. Methods 329, 112, 2008; United States Patent No. 7,314,622.
[00136] [00136] In an alternative, the polynucleotide sequence can be used for genetic manipulation to "humanize" the antibody or to improve the affinity or other characteristics of the antibody. For example, the constant region can be modified to more closely resemble human constant regions to prevent the immune response if the antibody is used in clinical trials and treatments in humans. It may be desirable to genetically manipulate the antibody sequence to obtain greater affinity for FLT3 and greater effectiveness in inhibiting FLT3.
[00137] [00137] There are four general steps to humanize a monoclonal antibody. They are: (1) determining the predicted amino acid sequence for the nucleotide and the light and heavy variable domains of the starting antibody (2) designing the humanized antibody, that is, deciding which region of the antibody structure to use during the humanization process (3) the humanization of real methodologies / techniques and (4) the transfection and expression of the humanized antibody. See, for example, US Patent Nos. 4,816,567; 5,807,715; 5,866,692;
[00138] [00138] A number of "humanized" antibody molecules comprising a binding to an antigen site derived from a non-human immunoglobulin have been described, including chimeric assays with modified rodent or rodent V regions and their associated CDRs fused to human constant regions . See, for example, Winter et al. Nature 349: 293 to 299, 1991, Lobuglio et al. Proc. Nat. Acad. Sci. USA 86: 4220 to 4224, 1989, Shaw et al. J Immunol. 138: 4534 to 4538, 1987, and Brown et al. Cancer Res. 47: 3577 to 3583, 1987. Other references describe CDRs of rodents grafted into a human support structure (FR) region prior to fusion with an appropriate human antibody constant region. See, for example,
[00139] [00139] The general principles related to humanized antibodies discussed above are also applicable to the customization of filters for use, for example, in dogs, cats, primates, horses and cattle. In addition, one or more aspects of the humanization of an antibody described herein can be combined, for example, CDR graft, structure mutation and CDR mutation.
[00140] [00140] In one variation, fully human antibodies can be obtained using commercially available mice that have been “modified to express specific human immunoglobulin proteins. Transgenic animals that are designed to produce a more desirable immune response (eg, fully human antibodies) or more robust can also be used to generate humanized or human antibodies. Examples of this technology are Xenomouse '“by Abgenix, Inc. (Fremont, CA) and HuMAb- Mouseº and TC Mouse" by Medarex, Inc. (Princeton, NJ).
[00141] [00141] Alternatively, controls can be done recombinantly and expressed using any method known in the art. Alternatively, accessories can be recombinant by phage display technology. See, for example, US Patent Nos. 5,565,332, 5,580,717, 5,733,743, and 6,265,150; and Winter et al, Annu. Rev. Immunol. 12: 433 to 455, 1994. Alternatively, phage display technology (McCafferty et al., Nature 348: 552 to 553, 1990) can be used to produce in vitro fragments of human antibodies and antibodies from gene repertoires immunoglobulin variable domain (V) from unimmunized donors. According to this technique, the genes of the antibody V domain are cloned into the structure into a gene of the main or minor coat protein of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. . Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the antibody's functional properties also result in the selection of the gene encoding the antibody that exhibits these properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for review see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3: 564 to 571, 1993. Various sources of V gene segments can be used for phage display. Clackson et al., Nature 352: 624 to 628, 1991, isolated a diverse array of antioxazolone from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies from a diverse set of antigens
[00142] [00142] Antibodies can be made recombinantly by first isolating antibodies and antibody-producing cells from host animals, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in host cells (e.g., CHO cells). Another method that can be used is to express the sequence of antibodies in plants (for example, tobacco) or transgenic milk. “Methods for expressing recombinantly in plants or milk have been disclosed. See, for example, Peeters, et al. Vaccine 19: 2756, 2001; Lonberg, N. and D. Huszar Int. Rev. Immunol 13:65, 1995; and Pollock, et al., J. Immunol Methods 231: 147, 1999. Methods for producing derivatives of antibodies, e.g., humanized, single chain, etc. are known in the art.
[00143] [00143] Immunoassay classification techniques and flow cytometry as well as fluorescence-activated cell classification (FACS) can also be employed for isolated antibodies that are specific to FLT3, or tumor antigens of interest.
[00144] [00144] Antibodies, as described in this document, can be linked to many different vehicles. Vehicles can be active and / or inert. Examples of well-known vehicles include polypropylene, polystyrene, polyethylene, dextran, nylon, amylases, glass, natural and modified celluloses, polyacrylamides, agarose and magnetite. The nature of the vehicle can be soluble or insoluble for the purposes of the invention. Those skilled in the art will know of other vehicles suitable for antibody binding, capable of ascertaining this,
[00145] [00145] The DNA encoding the monoclonal antibody is easily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes capable of binding specifically to the genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of this DNA. Once isolated, DNA can be placed into expression vectors (such as the expression vectors disclosed in PCT Publication No. WO 87/04462), which are then transfected into host cells such as E. coli cells, simian COS cells, cells Chinese hamster ovary (CHO), or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of activated monoclonal in recombinant host cells. See, for example, PCT Publication No. WO 87/04462. DNA can also be modified, for example, by replacing the coding sequence with human heavy and light chain constant regions instead of homologous murine sequences, Morrison et al., Proc. Nat. Acad. Sci. 81: 6851, 1984, or by covalently joining the immunoglobulin coding sequence, all or part of the coding sequence for a non-immunoglobulin polypeptide. In this way, "chimeric" or "hybrids" are prepared which have the binding specificity of a monoclonal antibody here.
[00146] [00146] FLT3 antibodies as described in this document can be identified or characterized by methods known in the art, in which the reduction of FLT3 expression levels is detected and / or measured. In some embodiments, an FLT3 antibody is identified by incubating a candidate agent with FLT3 and monitoring the binding and / or resulting reduction in FLT3 expression levels. The binding assay can be performed with purified FLT3 polypeptide (s), or with cells that express naturally, or transfected to express FLT3 polypeptide (s). In one embodiment, the binding assay is a competitive binding assay, in which the ability of a candidate antibody to compete with a FLT3 antibody known for binding to FLT3 is assessed. The assay can be performed in several formats, including the ELISA format.
[00147] [00147] After initial identification, an activity of a candidate FLT3 antibody can be confirmed and refined by bioassays, known to test the target biological activities. Alternatively, bioassays can be used to analyze candidates directly. Some of the methods for identifying and characterizing antibodies are described in detail in the Examples.
[00148] [00148] FLT3 antibodies can be characterized using methods well known in the art. For example, one method is to identify the epitope to which it attaches, or "epitope mapping". There are many methods known in the art to map and characterize the location of epitopes in proteins, including resolving the crystalline structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and assays based on synthetic peptides, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodys, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. In an additional example, epitope mapping can be used to determining the sequence to which an antibody binds Epitope mapping is commercially available from several sources, for example, Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitope can be a linear epitope, that is, contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that cannot be contained in a single stretch.
[00149] [00149] - Yet another method that can be used to characterize an FLT3 antibody is to use competition assays with other antibodies known to bind to the same antigen, that is, several fragments in FLT3, to determine whether the FLT3 antibody binds to the same epitope like other antibodies. Competition trials are well known to those skilled in the art.
[00150] [00150] An expression vector can be used to target the expression of an FLT3 antibody. Someone skilled in the art is familiar with the administration of expression vectors to obtain expression of an exogenous protein in vivo. See, for example, US Patent Nos. 6,436,908, 6,413,942, and 6,376,471. Administration of expression vectors includes local or systemic administration, including injection, oral administration, particulate gun or catheterized administration, and topical administration. In another modality, the expression vector is administered directly to the sympathetic trunk or ganglion, inside a coronary artery, atrium, ventricle or pericardium.
[00151] [00151] Targeted delivery of therapeutic compositions containing an expression vector or subgenomic polynucleotides can also be used. Receptor-mediated DNA release techniques are described in, for example, Findeis et al, Trends Biotechnol., 1993, 11: 202; Chiou et al., Gene Therapeutics: Methods and Applications Of Direct Gene Transfer, J.A. Wolff, ed., 1994; Wu et al., J. Biol. Chem., 263: 621, 1988; Wu et al., J. Biol. Chem., 269: 542, 1994; Zenke et al., Proc. Natl. Acad. Sci. USA, 87: 3655, 1990; and Wu et al., J.
[00152] [00152] Virus-based vectors for the release of a polynucleotide and desired expression in a desired cell are well known in the art. Exemplary virus-based vehicles include, but are not limited to, recombinant retroviruses (See, for example, PCT Publication No. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; United States Patents No. 5,219,740 and 4,777,127; GB Pat.
[00153] [00153] Non-viral vehicles and delivery methods can also be employed, including, but not limited to, polycationic condensed DNA linked or not linked only to dead adenovirus (See, for example, Curiel, Humiel. Gene Ther ,, 3: 147, 1992); Ligand-linked DNA (See, for example, Wu, J. Biol. Chem., 264: 16985, 1989); cells from eukaryotic cell release vehicles (See, for example, U.S. Patent No. 5,814,482; PCT Publication No. WO 95/07994; WO 96/17072, WO 95/30763; and WO 97/42338) and neutralization nucleic charge or fusion with cell membranes. Naked DNA can also be used. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and United States Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in United States Patent No. 5,422,120; PCT Publication No. WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional methods are described in Philip, Mol. Cell Biol., 14: 2411, 1994 and in Woffendin, Proc. Natl. Acad. Sci., 91: 1581, 1994.
[00154] [00154] In some embodiments, the invention encompasses compositions, including pharmaceutical compositions, which comprise antibodies described herein or made by the methods and with the characteristics described herein. As used herein, the compositions comprise one or more vectors that bind to FLT3, and / or one or more polynucleotides that comprise sequences that encode one or more of these phrases. Such compositions may further comprise — suitable excipients, such as pharmaceutically acceptable excipients, including buffers, which are well known in the art.
[00155] [00155] The invention also provides methods for making any of these items. The antibodies of this invention can be made by procedures known in the art. Polypeptides can be produced by proteolytic or other degradation of antibodies, by recombinant methods (i.e., fusion or single polypeptides) as described above or by chemical synthesis. Antibody polypeptides, especially the shortest polypeptides up to about 50 amino acids, are conveniently produced by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available. For example, an antibody can be produced by an automated polypeptide synthesizer using the solid phase method. See also, US Patent No.
[00156] [00156] Alternatively, controls can be done recombinantly using procedures that are well known in the art. In one embodiment, a polynucleotide comprises a sequence that encodes the variable regions of the Heavy Chain and / or Light Chain of antibody P4F6, P4C7, P3A ', PSA3, P9B5, P9F1, P1BA4, P1B11, P7H3, P3E10, P1A5, P5F7, P4H11, P15F7, P12B6, P8B6, P14G2, P7F9, POS8BOGEE, PO4A04, PO1AOS5, PO8BO3, P5F7, P5F79, P10A02g, P10A049g, P10A05g, P10A07g, P10B03g, P10B4g, P10B5 The sequence encoding the antibody of interest can be maintained in a vector in a host cell and the host cell can be expanded and frozen for future use. Vectors (including expression vectors) and host cells are described here.
[00157] [00157] Heteroconjugate antibodies, which comprise two covalently joined antibodies, are also within the scope of the invention. Such antibodies have been used to target immune cells to unwanted cells (North Patent
[00158] [00158] Chimers or hybrids can also be prepared in vitro using methods known from synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
[00159] [00159] In recombinant humanized vectors, the Fcy portion can be modified to avoid interaction with the Fcy receptor and the immune and complementary systems. Techniques for the preparation of such antibodies are described in WO 99/58572. For example, the constant region can be modified to look more like human constant regions to prevent the immune response if the antibody is used in clinical trials and treatments in humans. See, for example, US Patent No. 5,997,867 and
[00160] [00160] The invention encompasses modifications to the antibodies and polypeptides of the invention, including variants shown in Table 5, including functionally equivalent equivalents that do not significantly affect its properties and variants that have increased or decreased activity and / or affinity. For example, the amino acid sequence can be mutated to obtain an antibody with the desired binding affinity for FLT3. Modification of polypeptides is a routine practice in the art and need not be described in detail here.
[00161] [00161] Amino acid sequence inserts include amino- and / or carboxyl- terminal fusions of length ranging from one residue to polypeptides containing one hundred or more residues, as well as intrassequent insertions of single or multiple amino acid residues. Examples of terminal inserts include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other variants of insertion of the antibody molecule include fusion to the N or C terminal of the antibody of an enzyme or a polypeptide that increases the half-life of the antibody in the bloodstream.
[00162] [00162] The substitution variants have at least one amino acid residue in the removed antibody molecule and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include hypervariable regions, but changes in RF are also contemplated. Conservative substitutions are shown in Table 5, under the heading "conservative substitutions". If such substitutions will result in a change in biological activity, more substantial changes, called "exemplary substitutions", can be introduced in Table 5, or as described below in reference to the amino acid classes and the selected products. In some embodiments, the antibody substitution variants provided here have no more than 15, 14, 13, 12, 11, 10,9,8,7,6,5, 4, 3, 2, or 1 conservative substitution in the VH or VL region compared to the parental reference antibody. In some situations, substitutions are not within a VH or VL region CDR.
[00163] [00163] “Substantial modifications in the biological properties of the antibody are carried out by selecting substitutions that differ significantly in their effects on maintaining (a) the structure of the polypeptide backbone in the substitution area, for example, as a leaf or helical conformation, (bj) the charge or hydrophobicity of the molecule at the target site, or (c) most of the side chain. Naturally occurring amino acid residues are divided into groups based on common side chain properties:
[00164] [00164] Non-polar: Norleucine, Met, Ala, Val, Leu, lle;
[00165] [00165] —Polars without charge: Cys, Ser, Thr, Asn, Gin;
[00166] [00166] Acid (negative charge): Asp, Glu;
[00167] [00167] Basic (positive charge): Lys, Arg;
[00168] [00168] Residues that influence the orientation of the chain: Gly, Pro; and
[00169] [00169] Aromatic: Trp, Tyr, Phe, His.
[00170] [00170] Non-conservative substitutions are made by exchanging a member of one of these classes for another class.
[00171] [00171] Any cysteine residue not involved in maintaining the proper conformation of the antibody can also be replaced, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. On the other hand, cysteine bonds can be added to the antibody to improve its stability, particularly when the antibody is an antibody fragment, such as an Fv fragment.
[00172] [00172] Amino acid modifications can range from altering or modifying one or more amino acids to the complete redesign of a region, such as a variable region. Changes in a variable region can alter the affinity and / or binding specificity. In some embodiments, no more than one to five conservative amino acid substitutions are made within a CDR domain. In other embodiments, no more than one to three conservative amino acid substitutions are made within a CDR domain. In other classes, the CDR domain is CDR H3 and / or CDR L3.
[00173] [00173] The modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation and phosphorylation. Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, Chem. Immunol. 65: 111 to 128, 1997; Wright and Morrison, TIbTECH 15:26 to 32, 1997) The oligosaccharide side chains of immunoglobulins affect the protein function (Boyd et al., Mol. Immunol. 32: 1311 to 1318, 1996; Wittwe and Howard, Biochem. 29: 4175 to 4180, 1990) and the intramolecular interaction between portions of the glycoprotein, which can affect conformation, presented a three-dimensional surface of the glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, Current Opin. Biotech. 7: 409 to 416, 1996). Oligosaccharides can also serve to target a given glycoprotein to certain molecules based on specific recognition structures. Antibody glycosylation has also been reported to affect antibody-dependent cell cytotoxicity (ADCC). In particular, it was reported that CHO cells with expression regulated by B (1,4) N-acetylglucosaminyltransferase Ill (GnTIII), a catalytic formation of GICNAc glycosyltransferase in bisector, improved ADCC activity (Umana et al. , Mature Biotech. 17: 176 to 180, 1999).
[00174] [00174] Glycosylation of antibodies is typically N-ligand or O-ligand. N-linker refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine and asparagine-X-cysteine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linker glycosylation refers to the binding of one of the N-acetylgalactosamine, galactose or xylose sugars to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.
[00175] [00175] The addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence so that it contains one or more of the tripeptide sequences described above (for N-linker glycosylation sites). The change can also be made by adding or replacing one or more serine or threonine residues to the original antibody sequence (for O-linker glycosylation sites).
[00176] [00176] The glycosylation pattern of the vehicle can also be changed without changing the underlying nucleotide sequence. Glycosylation depends largely on the host cell used to express the antibody. Since the cell type used for the expression of recombinant glycoproteins, for example, antibodies, as a therapeutic potential is rarely the native cell, variations in the pattern of glycosylation of antibodies can be expected (See, for example, Hse et al., J. Chem 272: 9062 to 9070, 1997).
[00177] [00177] In addition to the choice of host cells, factors that affect glycosylation during recombinant block production include growth mode, media formulation, culture density, oxygenation, pH, purification schemes and the like. Several methods have been proposed to alter the glycosylation pattern achieved in a particular host organism, including the introduction or overexpression of certain enzymes involved in the production of oligosaccharides (U.S. Pat.
[00178] [00178] Other methods of modification include the use of technical coupling techniques, including, but not limited to, enzymatic media, oxidative substitution and chelation. Modifications can be used, for example, to attach markers for immunoassay. The modified polypeptides are made using procedures established in the art and can be analyzed using standard assays known in the art, some of which are described below and in the Examples.
[00179] [00179] Other antibody modifications include antibodies that have been modified as described in PCT Publication No. WO 99/58572. These antibodies comprise, in addition to a binding domain directed to the target molecule, an effector domain with an amino acid sequence substantially homologous to all or part of a constant region of a human heavy chain immunoglobulin. These antibodies are able to bind to the target molecule without triggering significant complement-dependent lysis or destroying the cell-mediated target. In some situations, the effector domain is able to bind FcRn and / or FeyRilb. These are typically based on chimeric domains derived from two or more Cx2 heavy chain domains of human immunoglobulin. Antibodies modified in this way are particularly suitable for use in chronic antibody therapy, to avoid inflammatory reactions and other adverse reactions to conventional antibody therapy.
[00180] [00180] The invention includes matured affinity modalities.
[00181] [00181] The following methods can be used to adjust the affinity of an antibody and to characterize a CDR. One way to characterize a CDR of an antibody and / or to alter (as it improves) the binding affinity of a polypeptide, such as an antibody, is called "library scan mutagenesis". Generally, library scan mutagenesis works as follows. One or more amino acid positions in the CDR are replaced by two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20) amino acids using methods recognized in the art. This generates small libraries of clones (in some modalities, one for each amino acid position analyzed), each with a complexity of two or more members (if two or more amino acids are substituted in all positions). Generally, the library also includes a clone that comprises the native (unsubstituted) amino acid. A small number of clones, for example, about 20 to 80 clones (depending on the complexity of the library), from each library, are analyzed for affinity binding to the target polypeptide (or other binding target), and candidates with increased, decrease, equal, or no link is identified. Methods for determining binding affinity are well known in the art. Binding affinity can be determined using Biacore'Y surface plasmon resonance analysis, which detects differences in binding affinity about two times or more. Biacore "" is particularly useful when the initial antibody already binds to a relatively high affinity, for example, a Kp of about 10 nM or less. Analysis using Biacore surface plasmon resonance "" is described in the Examples in this document.
[00182] [00182] The binding affinity can be determined using the Kinexa Biocensor, scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescence extinction, fluorescence transfer, and / or yeast display. The binding affinity can also be analyzed using a suitable bioassay.
[00183] [00183] In some embodiments, every amino acid position in a CDR is replaced (on occasion, one at a time) with all 20 natural amino acids using mutagenesis methods recognized in the art (some of which are described in this document). This generates small libraries of clones (sometimes, one for each amino acid position analyzed), each with a complexity of 20 members (if all 20 amino acids are replaced in all positions).
[00184] [00184] In some modalities, the library to be analyzed comprises substitutions in two or more positions, which can be in the same CDR or in two or more CDRs. Thus, the library can comprise substitutions in two or more positions on a CDR. The library can comprise substitution in two or more positions on two or more CDRs. The library can comprise substitution in 3, 4, 5 or more positions, positions mentioned in two, three, four, five or six CDRs. The replacement can be prepared using low redundancy codons. See, for example, Table 2 of Balint et al., Gene 137 (1): 109 to 18, 1993.
[00185] [00185] The CDR can be CDRH3 and / or CDRL3. The CDR can be one or more CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and / or CDRH3. The CDR can be a Kabat CDR, a Chothia CDR or an extended CDR.
[00186] [00186] Candidates with improved binding can be sequenced, thereby identifying a CDR substitution mutant that results in improved affinity (also called "improved" substitution). Candidates who bind can also be sequenced, thereby identifying a CDR replacement that retains the binding.
[00187] [00187] Multiple rounds of analysis can be performed. For example, candidates (each comprising an amino acid substitution at one or more positions on one or more CDRs) with enhanced binding are also useful for the design of a second containing at least the original and substituted amino acid at each improved CDR position ( that is, position of amino acids in the CDR at which a substitution mutant showed improved binding). The preparation, analysis or selection of this library is described below.
[00188] [00188] Library scan mutagenesis also provides a means to characterize a CDR, in that the frequency of clones with improved binding, the same binding, reduced binding or without binding also provides information related to the importance of each amino acid position for the stability of the antibody-antigen complex. For example, if a CDR position retains binding when changed to all 20 amino acids, that position is identified as a position that is unlikely to be necessary for antigen binding. On the other hand, if a position of the CDR retains the bond only in a small percentage of substitutions, that position will be identified as an important position for the function of the CDR. Thus, library scanning mutagenesis methods generate information about positions in CDRs that can be changed for many different amino acids (including all 20 amino acids), and positions in CDRs that cannot be changed or that can only be changed for some amino acids .
[00189] [00189] Candidates with enhanced affinity can be combined into a second library, which includes the enhanced amino acid, the original amino acid in that position, and may also include additional substitutions in that position, depending on the complexity of the desired library, or is allowed using the method of desired analysis or selection. In addition, if desired, the adjacent amino acid position can be randomized to at least two or more amino acids. The randomization of adjacent amino acids may allow for additional conformational flexibility in the mutant CDR, which may, in turn, allow or facilitate the introduction of a greater number of improved mutations. The library may also include substitution in positions that did not show improved affinity in the first cycle of analysis.
[00190] [00190] The second library is analyzed or selected for library members with altered and / or enhanced binding affinity using any method known in the art, including analysis using Biacore'Y surface plasmon resonance analysis and selection using any method known in the art for selection, including phage display, yeast display and ribosome display.
[00191] [00191] The invention also encompasses fusion proteins that comprise one or more fragments or regions of the chapters of this invention. In one embodiment, a fusion polypeptide is provided that comprises at least 10 contiguous amino acids from the Light Chain variable region shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31, 33, 35, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, or 232, and / or at least 10 amino acids from the heavy chain variable region shown in SEQ ID NO: 2, 4,6,8,10,
[00192] [00192] A fusion polypeptide can be created by methods known in the art, for example, synthetically or recombinantly. Typically, the fusion proteins of this invention are produced by preparing a polynucleotide that encodes them using recombinant methods described herein, although they can also be prepared by other methods known in the art, including, for example, chemical synthesis.
[00193] [00193] This invention also provides compositions that comprise conjugated antibodies (e.g., bound) to an agent that facilitates coupling to a solid support (such as biotin or avidin). For simplicity, reference will generally be made to antibodies with the understanding that these methods apply to any of the FLT3 antibodies described here. Conjugation generally refers to the binding of these components as described in this document. The connection (which generally fixes these components in close association at least for administration) can be obtained in several ways. For example, a direct reaction between an agent and an antibody is possible when each has a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, in which one may be able to react with a group containing carbornyl, such as an anhydride or an acid halide, or the other with an alkyl group containing a good leaving group (for example, a halide).
[00194] [00194] The invention also provides isolated polynucleotides that encode the vectors of the invention, vectors and host cells that comprise the polynucleotide.
[00195] [00195] Consequently, the invention provides polynucleotides (or compositions, including pharmaceutical compositions), which comprise polynucleotides that encode any of the following: P4F6, P4C7, P3A, PS5A3, P9B5, P9F1, P1B4, P1B11, P7H3, P3E10, P3E10, P3E10 , P5F7, P4H11, P15F7, P12B6, P8B6, P14G2, P7F9, POSBOGEE, PO4A0A, PO1AO5, PO8BO3, P5F7, P5F7g9, P1IOAO02g9, P1IOAO4g9, P10F05g, P10F07, P10F07, P10F07, with the ability to bind to FLT3.
[00196] [00196] In another aspect, the invention provides polynucleotides that encode any of the antibodies (including antibody fragments) and polypeptides described herein, such as antibodies and polypeptides that have impaired effective function. Polynucleotides can be produced and expressed by procedures known in the art.
[00197] [00197] In another aspect, the invention provides compositions (such as pharmaceutical compositions) that comprise any of the polynucleotides of the invention. In some combinations, the composition comprises an expression vector that comprises a polynucleotide that encodes any of those described herein.
[00198] [00198] The vectors of expression and administration of polynucleotide compositions are further described here.
[00199] [00199] In another aspect, the invention provides a method for producing any of the polynucleotides described herein.
[00200] [00200] Polynucleotides complementary to any of these sequences are also covered by the present invention. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA molecules (genomics, cCcDNA or synthetic) or RNA. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in an individual way, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may be present within a polynucleotide of the present invention and a polynucleotide may, but need not, be linked to other molecules and / or support materials.
[00201] [00201] —Pollucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes an antibody or a portion thereof) or may comprise a variant of such an sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and / or insertions so that the immunoreactivity of the encoded polypeptide is not decreased with respect to a native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assessed as described in this document. Variants preferably exhibit at least about 70% identity, more preferably, at least about 80% identity, even more preferably, at least about 90% identity, and most preferably, at least about 95% identity for a polynucleotide sequence that encodes a native antibody or a portion thereof.
[00202] [00202] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences in a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used here, refers to a segment of at least about 20 contiguous positions, generally 30 to about 75, or 40 to about 50, where a sequence can be compared to a sequence of reference of the same number of contiguous positions after the two sequences are ideally aligned.
[00203] [00203] Optimal alignment of sequences for comparison can be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc. Madison, WI), using standard parameters. This program incorporates several alignment schemes described in the following references: Dayhoff, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Supl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., 1989, CABIOS 5: 151-153; Myers, EW. and Muller W., 1988, CABIOS 4: 11-17; Robinson, E.D., 1971, Comb. Theor. 11: 105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4: 406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., 1983, Proc. Natl. Acad. Sci. USA 80: 726-730.
[00204] [00204] Preferably, the "percent sequence identity" is determined by comparing two sequences ideally aligned in a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (ie, intervals) of 20 percent or less, usually 5 to 15 percent, or 12 percent, when compared to reference sequences (which do not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in which identical nucleic acid bases or amino acid residues occur in both sequences to generate the number of corresponding positions, dividing the number of corresponding positions by the total number of positions in the sequence. reference (ie the window size) and multiplying the results by 100 to produce the percent sequence identity.
[00205] [00205] Variants may also, or alternatively, be substantially homologous to a native gene, or a portion or complement thereof. Such polynucleotide variants are able to hybridize under moderately stringent conditions to a naturally occurring DNA sequence, encoding a native antibody (or a complementary sequence).
[00206] [00206] Suitable "moderately stringent conditions" include prewash in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50ºC to 65ºC, 5 X SSC, overnight; followed by washing twice at 65ºC for 20 minutes with each of 2X, 0.5X and 0.2XK SSC containing 0.1% SDS.
[00207] [00207] As used here, "highly stringent conditions" or "high stringency conditions" are those that: (1) employ low ionic intensity and high temperature for washing, for example, 0.015 M sodium chloride / sodium citrate at 0.0015 M / 0.1% sodium dodecyl sulfate at 50ºC; (2) employ during denaturation a denaturing agent, such as formamide, for example 50% (v / v) formamide
[00208] [00208] It will be appreciated by those skilled in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described in this document. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that vary due to differences in use of the codon are specifically contemplated by the present invention. In addition, alleles of the genes comprising the polynucleotide sequences provided herein fall within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and / or substitutions of nucleotides. The resulting mMRNA and protein may, however, not need, have an altered structure or function. Alleles can be identified using standard techniques (such as hybridization, amplification and / or database sequence comparison).
[00209] [00209] The polynucleotides of this invention can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of synthesizing chemical polynucleotides are well known in the art and need not be described in detail here. Someone skilled in the art can use the sequences provided here and a commercial DNA synthesizer to produce a desired DNA sequence.
[00210] [00210] To prepare polynucleotides using - recombinant methods, a polynucleotide comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a host cell suitable for replication and amplification, as also described herein. Polynucleotides can be inserted into host cells by any method known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as an unintegrated vector (such as a plasmid) or integrated into the host cell's genome. The polynucleotide thus amplified can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et a /., 1989.
[00211] [00211] —Alternatively, PCR allows the reproduction of DNA sequences. PCR technology is well known in the art and is described in US Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.
[00212] [00212] RNA can be obtained using the DNA isolated in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those skilled in the art, as mentioned in Sambrook et al., 1989, supra, for example.
[00213] [00213] Suitable cloning vectors can be constructed according to standard techniques, or can be selected from the large number of cloning vectors available in the technique. Although the selected cloning vector may vary depending on the host cell intended for use, useful cloning vectors will generally have the ability to self-replicate, may have a single target for a particular restriction endonuclease, and / or may carry genes for a marker that can be used in the selection of clones that contain the vector. - Suitable examples include plasmids and bacterial viruses, for example, pUC18, pUC19, Bluescript (for example, pBS SK +) and their derivatives, mp18, mp119, pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and transport vectors , such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors, such as BioRad, Strategene and Invitrogen.
[00214] [00214] Expression vectors are generally replicable polynucleotide constructs that contain a polynucleotide according to the invention. It is implied that an expression vector must be replicable in host cells either as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including - adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector (s) described in PCT Publication No. WO 87/04462. Vector components can generally include, but are not limited to, one or more of the following: a signal sequence; a source of replication; one or more gene markers; suitable transcriptional control elements (such as promoters, enhancers and terminators). For expression (i.e., translation), one or more elements of translational control are also generally required, such as ribosome binding sites, translation initiation sites, and interruption codons.
[00215] [00215] The vectors containing the polynucleotides of interest can be introduced into the host cell by any of several appropriate methods, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microproject bombardment; lipofection; and infection (for example, where the vector is an infectious agent, such as the vaccinia virus). The choice of introducing vectors or polynucleotides will often depend on aspects of the host cell.
[00216] [00216] The invention also provides host cells comprising any of the polynucleotides described herein. Any host cells capable of overexpressing heterologous DNA can be used to isolate the genes encoding the antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa and CHO cells. See also PCT Publication No. WO 87/04462. Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeasts (such as S. cerevisae, S. pombe; or K. lactis). Preferably, the host cells express the cDNAs at a level of about 5 times higher, more preferably, 10 times higher, even more preferably, 20 times higher than that of the corresponding endogenous antibody or protein of interest, if present, host cells. The screening of host cells for a specific binding to FLT3 is carried out by an immunoassay or FACS. A cell that overexpresses the antibody or protein of interest can be identified. Methods of Using FLT3 Antibodies
[00217] [00217] The antibodies of the present invention are useful in a variety of applications, including, but not limited to, therapeutic treatment methods and diagnostic treatment methods.
[00218] [00218] Antibodies (for example, monospecific and bispecific) obtained by the methods described above can be used as a medicine. In some modalities, this medication can be used to treat cancer. In some modalities, cancer is a cancer of hematopoietic origin, such as lymphoma or leukemia. In some embodiments, the cancer is malignant myeloma malignant plasma cell neoplasm, Hodgkin's lymphoma, predominant nodular lymphocyte, predominant Hodgkin's lymphoma, Kahler's disease and myelomatosis, plasma cell leukemia, plasmacytoma, B cell leukemia, prolinocytic leukemia hair cells, non-Hodgkin's B-cell lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma, lymphoma Burkitt's disease, marginal zone lymphoma, lining cell lymphoma, large cell lymphoma, B lymphocyte precursor lymphoma, myeloid leukemia, Waldenstrom macroglobulienemia, diffuse large B cell lymphoma, follicular lymphoma, marginal zone lymphoma, tissue lymphoma mucosa-associated lymphatic system, small lymphocytic cell lymphoma, lining cell lymphoma, Burkitt's lymphoma, large B-cell lymphoma primary thymic (thymic) lymphoplasmoctatic lymphoma, Waldenstrom macroglobulinemia, nodal marginal B cell lymphoma, splenic marginal zone lymphoma, large B cell intravascular lymphoma, primary effusion lymphoma, lymphoid granulomatosis, large B cell lymphoma rich in cells / histiocytes, primary central nervous system lymphoma, primary large-cell diffuse cutaneous lymphoma (leg type), diffuse EBV-positive large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma associated with inflammation, B-cell lymphoma large intravascular, large B cell lymphoma positive ALK, plasma-plasma lymphoma, large B cell lymphoma arising in multicenter Castleman disease associated with HHVB8, B-cell lymphoma not classified with intermediate characteristics between diffuse large B cell lymphoma and lymphoma Burkitt, B-cell lymphoma not classified with intermediate characteristics between li diffuse large B cell lymphoma and classic Hodgkin's lymphoma or other cancers related to hematopoietic cells. In a preferred embodiment, the cancer is AML. In a preferred embodiment, the cancer is ALL.
[00219] [00219] In some embodiments, a method is provided to inhibit tumor growth or progression in an individual who has malignant cells expressing FLT3, comprising administering to the individual in need of an effective amount of a composition comprising the FLT3 antibodies (for example , bispecific antibodies FLT3-CD3) as described herein. In other embodiments, a method is provided to inhibit metastasis of cells that express FLT3 in an individual, comprising administering to the individual in need an effective amount of a composition comprising FLT3 antibodies (e.g., bispecific FLT3-CD3 antibodies), as herein described. In other embodiments, a method is provided to induce tumor regression in malignant cells in an individual, comprising administering to the individual in need of an effective amount of a composition comprising FLT3 antibodies (e.g., bispecific FLT3-CD3 antibodies), such as described here.
[00220] [00220] In some embodiments, the antibody (for example, bispecific FLT3-CD3 antibody) according to the invention can be used in the manufacture of a drug for treating cancer in a patient who needs it.
[00221] [00221] In some embodiments, treatment may be in combination with one or more therapies against a cancer selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, targeted therapy, vaccine therapy, dendritic cell therapy, therapy genetics, hormonal therapy, surgical resection, laser light therapy and radiation therapy.
[00222] [00222] In some embodiments, the cytokine used in cytokine therapy is interleukin (IL) - 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. In some embodiments, the cytokine is IL-15, IL-12 or IL-
[00223] [00223] In some embodiments, the FLT3 antibodies (for example, bispecific FLT3-CD3 antibodies) of the present invention are administered to a patient in conjunction with (for example, before, simultaneously or after) treatment with a therapeutic agent, for example, an antibody, including but not limited to, an anti-CTLA-4 antibody, an anti-4-1BB antibody (for example, PF-04518600), an anti-PD-1 antibody (for example, nivolumab, pembrolizumab or PF- 06801591), an anti-PD-L1 antibody (for example, avelumab, atezolizumab or durvalumab), an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an IL-8 antibody , an anti-HVEM antibody, an anti-BTLA antibody, an anti-CD40 antibody, an anti-CD40L antibody, an anti-CD47 antibody, an anti-CSF1IR antibody, an anti-cCSF1 antibody, an anti-MARCO antibody, an antibody anti-CXCRA4, an anti-VEGFR1 antibody, an anti-VEGFR2 antibody, an anti-TNFR1 antibody, an anti-MCSF antibody (for example lo, PD-0360324), an anti-TNFR2 antibody, a bispecific anti-CD3 antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-Her2 antibody, an anti-EGFR antibody, an anti-ICOS antibody, an anti-CD22 antibody, anti-CD 52 antibody, anti-CCR4 antibody, anti-CCR8 antibody, anti-CD200R antibody, anti-VISGA antibody, anti-CCR2 antibody, anti-LILRb2 antibody, anti-CXCRA antibody, anti antibody -CD206, anti-CD163 antibody, anti-KLRG1 antibody, an anti-B7-H4 antibody, an anti-B7-H3 antibody or an anti-GITR antibody.
[00224] [00224] In some embodiments, the FLT3 antibodies (for example, bispecific FLT3-CD3 antibodies) of the present invention are administered to a patient in conjunction with (for example, before, simultaneously or after) treatment with a CCR2 antagonist (for example , INC-8761), an antiviral agent, cidofovir and interleukin-2, treatment with cytarabine (also known as ARA-C) or nataliziimab for patients with MS or treatment with efaliztimab for patients with psoriasis or other treatments for patients with PML. In some embodiments, the FLT3 antibodies (for example, FLT3-CD3 bispecific antibodies) of the present invention can be used in combination with chemotherapy, radiation, immunosuppressive agents (such as cyclosporine, azatioprinay methotrexate, mycotenolate and FK506) or other immunoablative agents such as CAMPATH , cytotoxin, fludaribine, cyclosporine, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines and / or irradiation. These drugs inhibit calcium-dependent calcineurin phosphatase (cyclosporine and FK506) or inhibit p70S6 kinase which is important for growth factor-induced signaling (rapamycin). In other embodiments, the FLT3 antibodies (for example, bispecific FLT3-CD3 antibodies) of the present invention can be used in combination with kinase inhibitors, including, but not limited to, mTOR inhibitors, midostaurine, lestaurtinib, sorafenib,
[00225] [00225] In some embodiments, the FLT3 antibodies (for example, bispecific FLT3-CD3 antibodies) of the present invention can also be used in combination with epigenetic modulators, proteasome inhibitors, immunomodulatory agents (for example, lenalidomide), Hedgehog inhibitors, TNFa (tumor necrosis factor alpha), PAP inhibitors (phosphatidic acid phosphatase), oncolytic viruses, IDO inhibitors (indoleamine-pyrrole 2,3-dioxigenase), GLS1 glutaminase inhibitors, tumor vaccines, TLR agonists toll) (for example, TLR3, TLR4, TLR5, TLR5, TLR5, or TLR9) or isocitrate dehydrogenase (HDI) inhibitors.
[00226] [00226] In an additional embodiment, the FLT3 antibodies (for example, bispecific FLT3-CD3 antibodies) of the present invention are administered to a patient in conjunction with (for example, before, simultaneously or after) the bone marrow transplant, CART cells (Chimeric T Antigen Receptor), T cell ablative therapy using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide or antibodies such as OKT3 or alemtuzumab.
[00227] [00227] The administration of the antibodies (e.g., monospecific or bispecific) according to the invention can be carried out in any convenient manner, including by inhalation, injection, ingestion, transfusion, implantation or aerosol transplantation. The compositions described herein can be administered to a patient by subcutaneous, intradermal, intratumoral, intracranial, intranodal, intramedullary, intramuscular, by intravenous or intralymphatic or intraperitoneal injection. In one embodiment, the antibody compositions of the invention are preferably administered by intravenous injection.
[00228] [00228] In some embodiments, administration of antibodies
[00229] [00229] In some embodiments, the anti-FLT3 antibodies provided here can be used for diagnostic purposes, such as in assays to identify the FLT3 protein in samples (for example, in immunohistochemical assays) or in patients.
[00230] [00230] In one aspect, the present invention provides a pharmaceutical composition comprising an antibody (for example, monospecific or bispecific) of the invention or portion thereof as described above in a pharmaceutically acceptable carrier. In certain embodiments, the polypeptides of the invention may be present in a neutral form (including ionic forms of zwitter) or as a positively or negatively charged species. In some embodiments, polypeptides can be complexed with a counterion to form a "pharmaceutically acceptable salt", which refers to a complex comprising one or more polypeptides and one or more counterions, in which the counterions are derived from organic and inorganic acids and pharmaceutically acceptable bases.
[00231] [00231] The antibody (for example, monospecific or bispecific) or portions thereof, can be administered alone or in combination with one or more other polypeptides of the invention or in combination with one or more other drugs (or as any combination thereof) . The pharmaceutical compositions, methods and uses of the invention also encompass modalities of combinations (co-administration) with other active agents, as detailed below.
[00232] [00232] When used herein, the terms "co-administration", "co-administered" and "in combination with", referring to the antibodies of the invention and one or more other therapeutic agents, are intended to mean and refer to and include the following: (i) simultaneous administration of such a combination of an antibody described herein and therapeutic agent (s) to a patient in need of treatment, when such components are formulated together in a single dosage form that releases said components substantially at the same time. said patient; (ii) substantially simultaneous administration of such a combination of an antibody described herein and therapeutic agent (s) to a patient in need of treatment, when these components are formulated separately from each other in separate dosage forms, which are taken substantially at the same time by said patient, after which said components are released substantially at the same time to said patient; (iii) sequential administration of such a combination of an antibody described herein and a therapeutic agent (s) to a patient in need of treatment, when such components are formulated separately from each other in separate dosage forms, which are taken at times consecutive by said patient with a significant time interval between each administration, after which said components are released at substantially different times for said patient; and (iv) sequential administration of such a combination of an antibody described herein and therapeutic agent (s) to a patient in need of treatment, when such components are formulated together in a single dosage form that releases said components from a controlled way, when they are simultaneously, consecutively and / or overlapping, released in the same and / or at different times for the referred patient, in which each part can be administered by the same or by a different route.
[00233] [00233] Generally, the antibody (for example, monospecific or bispecific) described herein or portions thereof is suitable to be administered as a formulation in combination with one or more pharmaceutically acceptable excipient (s). The term 'excipient' is used here to describe any ingredient other than the compound (s) of the invention. The choice of excipient (s) will depend to a large extent on factors such as the particular mode of administration, the effect of the excipient on solubility and stability and the nature of the dosage form. When used herein, "pharmaceutically acceptable excipient" includes any and all solvents, dispersion medium, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which increase the useful life or effectiveness of the antibody.
[00234] [00234] The pharmaceutical compositions of the present invention and the methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19th edition (Mack Publishing Company, 1995). The pharmaceutical compositions are preferably manufactured under GMP conditions.
[00235] [00235] A pharmaceutical composition of the invention can be prepared, packaged or sold in bulk, as a single unit dose or as a plurality of single unit doses. When used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to an individual or a convenient fraction of such dosage, such as, for example, half or a third of that dose. Any method for administering peptides, proteins or antibodies accepted in the art can be suitably employed for the heterodimeric proteins and their portions described herein.
[00236] [00236] The pharmaceutical compositions of the invention are typically suitable for parenteral administration. When used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical violation of an individual's tissue and administration of the pharmaceutical composition through rupture in the tissue, generally resulting in direct administration into the bloodstream, muscle or in an internal organ. Parenteral administration thus includes, but is not limited to, the administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a non-surgical wound that penetrates tissues and the like. In particular, parenteral administration is considered to include, but is not limited to, injection - subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial or infusions; renal dialysis infusion techniques. Preferred embodiments include intravenous and subcutaneous routes.
[00237] [00237] Formulations of a pharmaceutical composition suitable for parenteral administration generally comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations can be prepared, packaged or sold in a form suitable for bolus or continuous administration. Injectable formulations can be prepared, packaged or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to suspensions, solutions, emulsions in oily or aqueous vehicles, pastes and the like.
[00238] [00238] Dosage regimens can be adjusted to provide the desired optimal response. For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the requirements of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in unit dosage form to facilitate dosage administration and uniformity. The dosage unit form, when used here, refers to physically discrete units suitable as unitary dosages for the patients / individuals to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the unit dosage forms of the invention is generally dictated and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the specific therapeutic or prophylactic effect to be achieved; and (b)
[00239] [00239] Thus, the person skilled in the art would appreciate, based on the description provided here, that the dose and dosage regimen is adjusted according to methods well known in the therapeutic techniques. That is, the maximum tolerable dose can be easily established and the effective amount that provides a detectable therapeutic benefit to the patient can also be determined, as well as the time requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Therefore, while certain dosage and administration regimens are exemplified herein, these examples in no way limit the dosage and administration regimen that can be provided to a patient in the practice of the present invention.
[00240] [00240] It should be noted that dosage values may vary with the type and severity of the condition to be relieved and may include single or multiple doses. It should also be understood that, for any particular individual, the specific dosage regimens must be adjusted over time, according to the individual need and the professional judgment of the person who administers or supervises the administration of the compositions, and that the intervals dosage forms set out herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. In addition, the dosage regimen with the compositions of this invention can be based on a variety of factors, including the type of disease, age, weight, sex, medical condition of the patient, severity of the condition, route of administration, and the particular antibody employee. Thus, the dosage regimen can vary widely, but it can be determined routinely using standard methods. For example, doses can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which can include clinical effects, such as toxic effects and / or laboratory values. Thus, the present invention encompasses intrapatient dose escalation as determined by the person skilled in the art. The determination of appropriate dosages and regimens is well known in the relevant art and should be understood as covered by the person skilled in the art, once the teachings described herein are provided.
[00241] [00241] Generally, for administration of the antibodies described here (monospecific or bispecific), the candidate dosage can be administered daily, every week, every two weeks, every three weeks, every four weeks, every five weeks, to every six weeks, every seven weeks, every eight weeks, every ten weeks, every twelve weeks or more than every twelve weeks. For repeated administrations for several days or more, depending on the condition, treatment is continued until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved, for example, to reduce symptoms associated with cancer. The progress of this therapy is easily monitored by conventional techniques and trials. The dosage regimen (including the monospecific or bispecific anti-FLT antibody used) may vary over time.
[00242] [00242] In some embodiments, the candidate dosage is administered daily with a dosage ranging from about 1 vg / kg to 30 pug / kg to 300 po / kg to 3 mg / kg, to 30 mg / kg, to 100 mg / kg kg or more, depending on the factors mentioned above. For example, daily dosage of about 0.01 mg / kg, about 0.03 mg / kg, about 0.1 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about of 2.5 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg and about 25 mg / kg can be used.
[00243] [00243] In some embodiments, the candidate dosage is administered every week with a dosage ranging from 1 µg / Kkg to 30 µg / kg to 300 µg / kg to 3 mg / kg, to 30 mg / kg, to 100 mg / kg kg or more, depending on the factors mentioned above. For example, a weekly dose of about 0.01 mg / kg, about 0.03 mg / kg, about 0.1 mg / kg, about 0.3 mg / kg, about 0.5 mg / kg kg, about 1 mg / kg, about 1 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 25 mg / kg and about mg / kg can be used.
[00244] [00244] In some embodiments, the candidate dosage is administered every two weeks, with the dosage ranging from 1 µg / kg to 30 µg / kg to 300 µg / kg to 300 mg / kg to 3 mg / kg to 30 mg / kg, to 100 mg / kg or more, depending on the factors mentioned above. For example, a biweekly dose of about 0.1 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 25 mg / kg and about 30 mg / kg can be used.
[00245] [00245] In some embodiments, the candidate dosage is administered every three weeks, with dosage ranging from about 1 µg / kg to 30 µg / kg to 300 µg / kg to 300 mg / kg to 3 mg / kg, 30 mg / kg, to 100 mg / kg or more, depending on the factors mentioned above. For example, a three-weekly dosage of about 0.1 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg and about 50 mg / kg can be used.
[00246] [00246] In some modalities, the candidate dosage is administered every month or every four weeks, with the dosage varying from 1 ug / kg to 30 ug / kg to 300 upg / kg to 3 mg / kg to 3 mg / kg , at 30 mg / kg, at 100 mg / kg or more, depending on the factors mentioned above. For example, a monthly dosage of about 0.1 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2.5 mg / kg,
[00247] [00247] In other modalities, the candidate dosage is administered daily with the dosage ranging from about 0.01 mg to about 1200 mg or more, depending on the factors mentioned above. For example, daily dosage of about 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg or about 1200 mg can be used.
[00248] [00248] In other modalities, the candidate dosage is administered every week with the dosage ranging from about 0.01 mg to about 2000 mg or more, depending on the factors mentioned above. For example, weekly dosage of about 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg can be used.
[00249] [00249] In other modalities, the candidate dosage is administered every two weeks with the dosage ranging from about 0.01 mg to about 2000 mg or more, depending on the factors mentioned above. For example, biweekly dosage of about 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg or about 2000 mg can be used.
[00250] [00250] In other modalities, the candidate dosage is administered every three weeks with the dosage ranging from about 0.01 mg to about 2500 mg or more, depending on the factors mentioned above. For example, three-weekly dosage of about 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg or about 2500 mg can be used.
[00251] [00251] In other modalities, the candidate dosage is administered every four weeks or month, with the dosage ranging from about 0.01 mg to about 3000 mg or more, depending on the factors mentioned above. For example, monthly dosage of about 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, about 2500, about 2600 mg, about 2700 mg, about 2800 mg, about 2900 mg or about 3000 mg can be used. Kits
[00252] [00252] The invention also provides kits for use in instantaneous methods. Kits of the invention include one or more containers comprising the antibody (for example, monospecific or bispecific) as described herein and instructions for use according to any of the methods of the invention described herein. Generally, these instructions comprise a description of the administration of the antibody protein for the therapeutic treatments described above.
[00253] [00253] Instructions related to the use of the antibody (for example, monospecific or bispecific), as described herein, generally include information on dosage, dosing schedule and route of administration for the intended treatment. The containers can be unit doses, bulk packages (for example, multi-dose packages) or doses of subunits. The instructions provided in the kits of the invention are generally instructions written on a packaging label or insert (for example, a sheet of paper included in the Kit), but machine-readable instructions (for example, instructions carried on a magnetic or optical storage disk) ) are also acceptable.
[00254] [00254] The prevention kits are in suitable packaging. Proper packaging includes, but is not limited to, vials, bottles, flasks, flexible packaging (for example, sealed Mylar or plastic bags) and the like. Packages are also considered for use in combination with a specific device, such as an inhaler, a nasal delivery device (for example, an atomizer) or an infusion device, such as a mini-pump. A kit can have a sterile access port (for example, the container can be a bag of intravenous solution or a vial with a stopper that can be pierced by a hypodermic injection needle). The container may also have a sterile access port (for example, the container may be a bag of intravenous solution or a vial with a stopper that can be pierced by a hypodermic injection needle). At least one active agent in the composition is a bispecific antibody. The container can further comprise a second pharmaceutically active agent.
[00255] [00255] Kits can optionally provide additional components, such as buffers and interpretive information. The kit usually comprises a container and a label or packaging insert or associated with the container.
[00256] [00256] The representative materials of the present invention were deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA, on June 1, 2017. The vector PS5F7g2-VL with accession number ATCC PTA -124230 is a polynucleotide encoding the P5F792 light chain variable region and the vector PSF7g2-VH with ATCC accession number PTA-124229 is a polynucleotide encoding the P5F792 heavy chain variable region. The deposits were made in accordance with the provisions of the Budapest Treaty on international recognition of the deposit of microorganisms for the purposes of patent procedures and regulations (Treaty of Budapest). This ensures the maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty and subject to an agreement between Pfizer, Inc. and ATCC, which guarantees permanent and unrestricted availability of the deposit culture progeny to the public by issuing the relevant US patent. or when opening any US or foreign patent application to the public, whichever comes first, and ensures progeny availability for one determined by the US Commissioner of Patents and Trademarks to be entitled to this under 35 USC $ 122 and Commissioner rules under the terms thereof (including 37 CFR 8 1.14 with particular reference to 886 OG 638).
[00257] [00257] The assignee of the present application has agreed that if a culture of the deposited materials dies or is lost or destroyed when cultivated under suitable conditions, the materials will be immediately replaced by notification by another of them. The availability of the deposited material should not be interpreted as a license to practice the invention in violation of the rights granted under the authority of any government, in accordance with its patent laws.
[00258] [00258] Reference is also made to material described here and deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA, on June 1, 2017. Vector PE310-VL with ATCC accession number PTA-124228 is a polynucleotide that encodes the variable region of the PE310 light chain, and the P3E10-VH vector with the ATCC accession number. PTA-124227 is a polynucleotide that encodes the variable region of the PE310 heavy chain. Deposits were also made in accordance with the provisions of the Budapest Treaty, in accordance with its terms and conditions, as summarized above.
[00259] [00259] The following examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way. In fact, several modifications of the invention, in addition to those shown and described in this document, will become evident to those skilled in the art from the previous description and fall within the scope of the attached claims. Examples Example 1: Determination of the kinetics and affinity of human FLT3 / FLT3 antibody interactions at 37 ºC
[00260] [00260] This example determines the kinetics and affinity of various anti-FLT3 antibodies at 37ºC. All experiments were performed on a Biacore T200 surface Plasmid resonance biosensor (GE Lifesciences, Piscataway NJ).
[00261] [00261] The preparation of the sensor chip was carried out at 25ºC with a buffer of 10 mM HEPES, 150 mM NaCl, 0.05% (v / v) of Tween-20, pH 7.4. A human anti-Fc sensor chip was produced by activating all flow cells of a Biacore CM4 sensor chip with a 1: 1 (v / v) mixture of 400 mM EDC and 100 mM NHS for 7 minutes, at a flow rate of 10 ul / min. An anti-human Fc reagent (anti-goat human IgG Fc, Southern Biotech Catalog tt 2081-01) was diluted to 30 upg / mL in 10 mM sodium acetate pH 4.5 and injected into all flow cells for 7 minutes at 20 ul / min. All flow cells were blocked with 100 mM ethylenediamine in 150 mM borate buffer pH 8.5 for 7 minutes at 10 uL / min.
[00262] [00262] The experiments were performed at 37ºC using a fluent buffer of 10 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween-20, Tween-20, pH 7.4, 1 mg / ml of BSA. FLT3 antibodies were captured from undiluted supernatants in the flow cells downstream (flow cells 2, 3 and 4) at a flow rate of 10 uL / min for 1 minute. Different antibodies were captured in each flow cell. Flow cell 1 was used as a reference surface. After the capture of FLT3 antibodies, the analyzed (buffer, hFLT3) was injected at 30 ul / min in all flow cells for two minutes. After the injection of the analyte, dissociation was monitored for 10 minutes, followed by the regeneration of all flow cells with three 1-minute injections of 75 mM phosphoric acid. For each captured FLT3 antibody, the following analyte injections were performed: buffer, 11 nM hFLT3, 33 NM hFLT3, 100 nM hFLT3 and 300 NM hFLT3. Buffer cycles were collected for each FLT3 antibody captured for double reference purposes (double reference as described in Myszka, D.G. Improving biosensor analysis. J. Mol. Recognit. 12, 279-284 (1999)). The double reference sensor diagrams have been globally adapted to a simple Langmuir 1: 1 with mass transport connection model.
[00263] [00263] The kinetic and affinity parameters for the tested anti-FLT3 antibodies are shown in Tables 6. Table 6 The e and Tee PO
[00264] [00264] This example illustrates the in vitro cytotoxicity of anti-FIt3 / CD3 bispecific hlgG2AA D265A in FIt3 positive cells.
[00265] [00265] Human anti-FIt3 antibodies (P5F7p, P5F792, P5F792, P5F793, PSF794) and human anti-CD3 (h2B4-VH-hnps VL-TK ("H2B4")) were expressed as human E26EA-designed IgG2dA in EEEE in RRR in the other subdivision for bispecific exchange in positions 223, 225 and 228 (for example, (C223E or C223R), (E225E or E225R) and (P228E or P228R)) in the joint region and in position 409 or 368 (for example, K409R or L368E (EU numbering scheme)) in the CH3 region of human IgG2 (SEQ ID NO: 236). The bispecific FLT3 / CD3 antibody also has the D to A mutation at position 265 (EU numbering scheme).
[00266] [00266] CD3 + T cells from human PBMC were selected negatively using the human Pan T Cell Isolation kit (Miltenyi, San Diego CA). The cells expressing the target (Eol1) and CD3 + T cells were seeded in transparent U-base plates at 20,000 and 100,000 cells / well, respectively. The cells were treated with bispecific antibody serially diluted 8 times. Depletion of AML cells was determined by flow cytometry analysis 24 hours after treatment. Cell depletion was measured in contrast to the control of treated cells, in this case H2B4 only in Figure 1. EC50 was calculated using the Prism software. Cytotoxicity was observed in this Eol1 cell line, as shown in Figure 1. Example 3: Bispecific FIt3-CD3 IgG induces tumor ablation in the AML orthopedic xenograft model
[00267] [00267] This example illustrates tumor regression and inhibition in an Eol1 xenograft orthotopic model.
[00268] [00268] An in vivo efficacy study of bispecific FIt3 with Eol1, expressing luciferase and GFP, orthotopic model, was carried out. Three hundred thousand Eol1 LucGFP cells were injected intravenously through the tail vein in 6- to 8-week-old female Nod / Scid / IL2Rg - / - (NSG) animals. The intraperitoneal injection of D-luciferin (Regis Technologies, Morton Grove, IL) (200 uL per animal at 15 mg / mL), followed by anesthesia with isofluorane and subsequent bioluminescence imaging of the entire body (BLI) allowed the monitoring of the load of the tumor. The bioluminescent signals emitted by the interaction between luciferase expressed by tumor cells and luciferin were captured by image using an IVIS Spectrum CT (Perkin Elmer, MA) and quantified as total flow (photons / s) using Living Image
[00269] [00269] This example illustrates the improved tumor activity of antibodies targeting domain 4 of FIt3 compared to antibodies targeting domain 5.
[00270] [00270] The Eol1 orthotopic xenograft model was performed as described in Example 3. In this case, a bispecific 6B7 directed to domain 4 or a specific P8b6 directed to domain 5 was dosed in a single dose of 10 upk. Stumpy represents CD3 that binds only the bispecific control antibody. At the tested dose, P8B6 had no antitumor activity, while 6B7 was an ablative tumor. Example 5: EC50 values for the bispecific of FIt3 are significantly reduced in the presence of IL15 in a long-term in vitro death assay
[00271] [00271] This example illustrates the in vitro cytotoxicity of the bispecific Anti-FIt3 / CD3 P5F7 antibody in FIt3i positive cells, in combination with IL-15.
[00272] [00272] —The previously frozen human Pan T olymphocytes were thawed and recovered in RPMI-1640 medium supplemented with 10% serum (Hyclone), Pen Strep 1% (Corning) and 15 units per mL of human IL-2 (eBioscience) per one day. Human T lymphocytes recovered for 1 day were collected and resuspended in 1 x 10 th cells / mlL in complete RPMI medium. FIt3 expressing EOL1 cells were seeded into 50,000 cells in 100 µl in a 96-well U-bottom plate. Fifty thousand (50,000) viable human CD3 + lymphocytes were added to tumor cells seeded in 1 µl of medium per well. Serial dilutions of 5 points and 5 times of the bispecific antibody FIt3 / CD3 P5F7 were prepared (dose range 1x10 * nM to 8 x 10º nM final concentration). The cytotoxicity assay was started by adding diluted bispecific antibody to the plates and incubating at 37 ºC for 2 days, 5 days or 7 days. To test the effect of I1L15 on the anti-tumor efficacy of T cells redirected to bispecific FIt3 / CD3 antibodies, cell cultures received 10 ng / ml of IL15 or vehicle control. The depletion of AML cells was determined by analysis of luciferase at the respective times. EC50 values were determined by a nonlinear regression graph of specific percentage cytotoxicity versus the Log10 concentration of bispecific FIt3 / CD3 P5F7 using the GraphPad Prism 7.0 software (GraphPad Software). Figure 4A and Figure 4B illustrate the improved anti-tumor activity of bispecific FIt3 / CD3 in the absence or presence of I | L15 in a long-term death trial, respectively. Example 6: Autologous T cells present in bone marrow aspirates from AML patients are effective in killing AML blasts in the presence of bispecific FIt3 / CD3 P5F7
[00273] [00273] This example illustrates that the bispecific anti-FIt3 / CD3 P5F7 antibody effectively redirects autologous T cells to eliminate AML blasts ex vivo.
[00274] [00274] To determine cytotoxic activity using patient T cells and AML cells, fresh bone marrow aspirates were purchased from Fred Hutchinson Cancer Research Center (Seattle, WA). The number of target cells (AML blasts) and effector cells (T cells) was determined by staining each sample with PerCP-cy5.5 anti-human CD3 (BioLegend, San Diego, CA), anti-CD8 antibody
[00275] [00275] After 4 days of incubation, cell viability of AML patients, T cell proliferation and activation were assessed by counting the number of CD33 + CD45dim AML blasts, the number of CD4 + CD8 + cells and the percentage of cells CD25 + CD4 + or CD25 + CDB8 +, respectively, in an LSRII flow cytometry instrument using the FACS Diva software (BD Biosciences). The results demonstrate effective death of primary AML cells induced by increasing concentrations of bispecific FLT3 / CD3 (PSF7) in the presence of autologous T cells (see, Figures 5A, 5B, 5C, 5D, 5E and 5P).
[00276] [00276] Although the teachings described have been described with reference to various applications, methods, kits and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings here and the invention claimed below. The previous examples are provided to better illustrate the teachings described and are not intended to limit the scope of the teachings presented here. Although the present teachings have been described in terms of these exemplary modalities, the person skilled in the art will easily understand that numerous variations and modifications of these exemplary modalities are possible without undue experimentation. All of these variations and modifications are within the scope of current teachings.
[00277] [00277] All references cited herein, including patents, patent applications, papers, textbooks and the like, and the references cited, to the extent that they are not yet, are incorporated by reference in their entirety. In the event that one or more of the embedded literature and similar materials differ or contradict this application, including, but not limited to defined terms, use of terms, described or similar techniques, that application controls.
[00278] [00278] The description and previous examples detail certain specific embodiments of the invention and describe the best mode considered by the inventors. It should be appreciated, however, that no matter how detailed the above may appear in the text, the invention can be practiced in several ways and the invention must be interpreted in accordance with the attached claims and any equivalents thereof.

权利要求:
Claims (28)
[1]
1. Isolated antibody, which specifically binds to Fms-type tyrosine kinase 3 (FLT3), the antibody characterized by the fact that it comprises (a) a heavy chain variable region (VH) comprising (i) a complementarity determining region of VH (CDR1) comprising the sequence shown in SEQ ID NO: 37, 38, 39, 43, 44, 45, 49, 50, 54 55, 56, 60, 61, 62, 66, 67, 68, 72, 73, 74, 78, 79, 80, 84, 85, 86, 90, 91, 92, 96, 97, 98, 102, 103, 104, 108, 109, 110, 114, 115, 116, 120, 121, 122, 126, 127, 128, 132, 133, 134, 138, 139, 140, 246 or 247; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 40, 41, 46, 47, 51, 52, 57, 58, 63, 64, 69, 70, 75, 76, 81, 82, 87, 88, 93, 94, 99, 100, 105, 106, 111, 112, 117, 118, 123, 124, 129, 130, 135, 136, 141, 142, 248, 249, 251, 252, 253 or 255; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 42, 48, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 245, 250 or 254; and / or (b) a light chain variable region (VL) comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 257, 261, 263, 265, 268, 270, 273 or 275; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 259, 266 or 271; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194 , 197, 256, 258, 260, 262, 264, 267, 269, 272 or 274.
[2]
2. Isolated antibody that specifically binds to Fms-type tyrosine kinase 3 (FLT3), the antibody characterized by the fact that it comprises:
a VH region comprising a VH CDR1, VH CDR2 and VH CDR3 of the VH sequence shown in SEQ ID NO: 2,4,6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26 , 28, 30, 32, 34, 36, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231 or 233; and / or a VL region comprising VL CDR1, VL CDR2 and VL CDR3 of the VL sequence shown in SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23 , 25, 27, 29, 31, 33, 35, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230 or 232.
[3]
3. Isolated antibody, characterized by the fact that it specifically binds to FLT3 and competes with the antibody according to claim 1.
[4]
4. Bispecific antibody in which the bispecific antibody is a life-size antibody, characterized by the fact that it comprises a first variable domain of the bispecific antibody that specifically binds to a target antigen, and comprises a second variable domain of the antibody antibody bispecific capable of recruiting the activity of a human immune effector cell by specific binding to an effector antigen located on the human immune effector cell, where the first variable domain of the antibody binds to domain 4 of FLT3 comprising SEQ ID NO: 279.
[5]
5. Bispecific antibody, where the bispecific antibody is a natural-sized antibody, characterized by the fact that it comprises a first variable domain of the bispecific antibody that specifically binds to a target antigen, and comprises a second variable domain of the antibody of the bispecific antibody capable of recruiting the activity of a human immune effector cell by specific binding to an effector antigen located on the human immune effector cell, wherein the first variable domain of the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1 , VH CDR2 and VH CDR3 of the VH sequence shown in SEQ ID NO: 2, 4, 6.8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 , 36, 205, 207, 209, 211, 213, 215, 217, 219, 221,223,225, 227, 229, 231 or 233; and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 of the VL sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 , 19, 21, 23, 25, 27, 29, 31, 33, 35, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230 or 232.
[6]
6. Bispecific antibody, where the bispecific antibody is a natural-sized antibody, characterized by the fact that it comprises a first variable domain of the bispecific antibody that specifically binds to a target antigen, and comprises a second variable domain of the antibody of the bispecific antibody capable of recruiting the activity of a human immune effector cell by specific binding to an effector antigen located on the human immune effector cell, wherein the first variable domain of the antibody comprises a heavy chain variable region (VH) comprising (i) a VH complementarity determining region (CDR1) comprising the sequence shown in SEQ ID NO: 37, 38, 39, 43, 44, 45, 49, 50, 54, 55, 56, 60, 61, 62, 66, 67 , 68, 72, 73, 74, 78, 79, 80, 84, 85, 86, 90, 91, 92, 96, 97, 98, 102, 103, 104, 108, 109, 110, 114, 115, 116 , 120, 121, 122, 126, 127, 128, 132, 133, 134, 138, 139, 140, 246 or 247; (li) a VH CDR2 comprising the sequence shown in SEQ ID NO: 40, 41, 46, 47, 51, 52, 57, 58, 63, 64, 69, 70, 75, 76, 81, 82, 87, 88, 93, 94, 99, 100, 105, 106, 111, 112, 117, 118, 123, 124, 129, 130, 135, 136, 141, 142, 248, 249, 251, 252, 253 or 255; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 42, 48, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 245, 250 or 254; and / or b. a light chain variable region (VL)
comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 144, 147, 150, 153, 156, 159, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189 , 192, 195, 257, 261, 263, 265, 268, 270, 273 or 275; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 259, 266 or 271; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194 , 197, 256, 258, 260, 262, 264, 267, 269, 272 or 274.
[7]
7. Bispecific antibody according to claim 6, characterized by the fact that the second variable domain of the antibody specifically binds to the CD3 effector antigen.
[8]
Bispecific antibody according to claim 7, characterized in that the second variable domain of the antibody comprises a. a heavy chain variable region (VH) comprising (i) a complementary VH determining region (CDR1) comprising the sequence shown in SEQ ID NO: 285, 286 or 287; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 288 or 289; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 290; and / or b. a light chain variable region (VL) comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 291; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 292; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 234.
[9]
Bispecific antibody according to claim 4 or 5, characterized in that the first and second variable domains of heterodimeric protein antibodies comprise amino acid modifications at positions 223, 225 and 228 in the joint region and at positions 409 or 368 (EU numbering scheme) in the CH3 region of a human IgG2 (SEQ ID NO: 290).
[10]
Bispecific antibody according to claim 9, characterized by the fact that it further comprises a modification of amino acids in one or more of the positions 265, 330 and 331 of human IgG2.
[11]
11. Nucleic acid, characterized by the fact that it encodes the antibody as defined in any one of claims 1 to 10.
[12]
12. Vector, characterized by the fact that it comprises the nucleic acid as defined in claim 11.
[13]
13. Host cell, characterized by the fact that it comprises the nucleic acid as defined in claim 11.
[14]
14. Use of an antibody, as defined in any one of claims 1 to 10, characterized in that it is for preparing a drug or kit.
[15]
15. Use of an antibody, according to claim 14, characterized by the fact that the drug or kit is for use in the treatment of a FLT3-related cancer that you select from the group consisting of multiple myeloma, malignant plasma cell neoplasm, Hodgkin's lymphoma, predominant nodular lymphocyte Hodkin's lymphoma, Kahler's disease and Myelomatosis, plasma cell leukemia, plasmacytoma, B cell prolinocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (NHL) AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, lining cell lymphoma, large cell lymphoma, limphoblastic B lymphoma precursor, myeloid leukemia, Waldenstrom macroglobulienemia, diffuse large B cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoma of the associated lymphatic tissue mucosa, small cell lymphocytic lymphoma, lining cell lymphoma, Burkitt's lymphoma, primary mediastinal (thymic) B-cell lymphoma, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, B-cell lymphoma of the marginal nodal zone, marginal zone lymphoma splenic, intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, large B cell lymphoma rich in T cells / histiocytes, primary central nervous system lymphoma, primary cutaneous diffuse B cell lymphoma (leg type), EBV-positive diffuse large B cells in the elderly, inflammation-associated diffuse large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in multicentric Castleman disease associated with HHVB8, unclassified B cell lymphoma with intermediate characteristics between d lymphoma and diffuse large B cells and Burkitt's lymphoma, unclassified B-cell lymphoma with intermediate characteristics between diffuse large B-cell lymphoma and classic Hodgkin's lymphoma and other hematopoietic cell-related cancer.
[16]
16. Use of an antibody as defined in any one of claims 1 to 10, characterized in that it is for preparing a drug or kit to be administered to treat an individual in need thereof.
[17]
17. Pharmaceutical composition, characterized in that it comprises the antibody as defined in any one of claims 1 to 10.
[18]
18. Use of the antibody as defined in any of claims 1 to 10, characterized in that it is for preparing a pharmaceutical composition as defined in claim 17, to treat a condition associated with malignant cells that express FLT3 in an individual.
[19]
19. Use, according to claim 18, characterized by the fact that the condition is a cancer.
[20]
20. Use according to claim 19, characterized by the fact that cancer is a FLT3-related cancer selected from the group consisting of multiple myeloma, malignant plasma cell neoplasm, Hodgkin's lymphoma, predominant nodular lymphoma of Hodgkin's lymphocytes, Kahler's disease and myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), leukemia acute lymphocytic (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, lining cell lymphoma, large cell lymphoma, precursor B lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's lymphoma, diffuse large B cells, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphatic tissue lymphoma, small cell lymphocytic lymphoma, lymphoma coating cells, Burkitt's lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, marginal nodal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, lymphoma effusion, lymphomatoid granulomatosis, large B cell lymphoma rich in T cells / histiocytes, primary central nervous system lymphoma, primary B diffuse cutaneous primary (leg type) lymphoma, large B cell lymphoma diffuse positive for EBV of the elderly , diffuse inflammation-associated large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasma lymphoma, large B-cell lymphoma arising in multicentric Castleman disease associated with HHVB8, non-B cell lymphoma classified with intermediate characteristics between diffuse large B-cell lymphoma and Burkitt's lymphoma, lymphoma B-cell unclassified with intermediate characteristics between diffuse large B-cell lymphoma and classic Hodgkin's lymphoma, and other hematopoietic cell-related cancer.
[21]
21. Use of a pharmaceutical composition as defined in claim 17, characterized in that it is for preparing a medicament or kit to inhibit tumor growth or progression in an individual who has malignant cells that express FLT3.
[22]
22. Use of a pharmaceutical composition as defined in claim 17, characterized in that it is for preparing a drug or kit to inhibit metastasis of malignant cells that express FLT3 in an individual.
[23]
23. Use of a pharmaceutical composition as defined in claim 17, characterized in that it is for preparing a medicament or kit to induce tumor regression in an individual who has FLT3-expressing malignant cells.
[24]
24. Use according to any one of claims 18 to 23, characterized in that the use further comprises administering an effective amount of a second therapeutic agent.
[25]
25. Use according to claim 24, characterized by the fact that the second therapeutic agent is a cytokine, TNFa (Tumor Necrosis alpha factor), a PAP inhibitor (phosphatidic acid phosphatase), an oncolytic virus, a kinase inhibitor, an IDO inhibitor (indoleamine-pyrrole 2.3-dioxigenase), a glutaminase GLS1 inhibitor, a T cell therapy of CAR (Chimeric Antigen Receptor) or T cell, a TLR Agonist (Fold Type Receptor) of Bell) or a tumor vaccine.
[26]
26. Use according to claim 25, characterized by the fact that the cytokine is I1L-15.
[27]
27. Use according to claim 25, characterized by the fact that the kinase inhibitor is midostaurin, lestaurtinib, sorafenib, sunitinib, quizartinib, ponatinib, crenolanib, palbocyclib or gilteritinib.
[28]
28. Method of producing an antibody, characterized in that it comprises the culture of the host cell as defined in claim 13 under conditions that result in the production of the antibody, and the isolation of the antibody from the host cell or culture.

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法律状态:
2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762514574P| true| 2017-06-02|2017-06-02|

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US62/514,574|2017-06-02|

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US201862660908P| true| 2018-04-20|2018-04-20|

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US62/660,908|2018-04-20|

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PCT/IB2018/053908|WO2018220584A1|2017-06-02|2018-05-31|Antibodies specific for flt3 and their uses|

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