Anti-FLT3 antibodies and compositions

Abstract

The present disclosure relates to anti-FLT3 antibodies and methods of their use in boosting immunity and in treating cancer in patients in need thereof.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority from U.S. Provisional Patent Application No. 63 / 009,578, filed on April 14, 2020. The disclosure of the priority application is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The electronic copy of the sequence listing was created on April 9, 2021, has the name 022675_WO047_SL.txt, and is 53,293 bytes in size.

[0003] Background of the Invention FMS - like tyrosine kinase 3 (FLT3), or CD135, is a class III receptor tyrosine kinase expressed on the surface of early hematopoietic progenitor cells that play an important role in the development of the immune system. Upon binding to the cytokine FLT3 ligand (FLT3L), FLT3 dimerizes and activates multiple signaling pathways that control cell differentiation, proliferation, and survival.

[0004] FLT3 has also been found to be expressed by dendritic cells (DCs), a class of specialized antigen - presenting cells. Upon contact with an antigen, dendritic cells internalize and process the antigen and present it to T cells in the context of an MHC class II complex, leading to T cell activation. FLT3 signaling plays a major role in dendritic cell differentiation and proliferation. Mice with deficiencies in FLT3 or FLT3 ligand (FLT3L) show reduced numbers of DCs, while mice treated with FLT3L show increased numbers of DCs. These observations indicate an important role for FLT3 in steady - state DC development.

[0005] Summary of the Invention The present disclosure provides anti-FTL3 antibodies that can stimulate the activity of dendritic cells. The antibodies can be used to enhance the immune response of a patient in need thereof, such as a patient having cancer or immunodeficiency. Also provided are pharmaceutical compositions comprising one or more of these antibodies, as well as the use of the antibodies and pharmaceutical compositions for the treatment of cancer. The antibodies and compositions described herein may be used in a method for treating cancer in a patient; may be used in the manufacture of a medicament for treating cancer in a patient; or may be for use in treating cancer in a patient. It is contemplated that, compared to currently available treatments (including antibody treatments) for such cancers, the antibodies and compositions described herein can provide an excellent clinical response, either alone or in combination with another cancer therapeutic.

[0006] In some embodiments, the present disclosure provides an anti-FLT3 antibody or an antigen-binding portion thereof that competes or cross-competes for binding to, or binds to, the same epitope of human FLT3 as antibody 17566, 17526, 17667, 17667-0, 17679, 17494, 17543, or 17497. In certain embodiments, the anti-FLT3 antibody or antigen-binding portion is defined by the six CDRs, the heavy and light chain variable domains, or the amino acid sequences of the heavy and light chains of said antibody.

[0007] In some embodiments, the present disclosure provides an anti-FLT3 antibody or an antigen-binding portion thereof: a) The heavy chain of said antibody comprises: i) Heavy chain complementarity determining regions (H-CDR)-1 to 3 each comprising the amino acid sequences of SEQ ID NOs: 5 to 7; ii) A heavy chain variable domain (VH) comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3; iii) A VH comprising the amino acid sequence of SEQ ID NO: 3; or iv) A heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 3 and 75; and b) The light chain of said antibody comprises: i) Light chain complementarity determining regions (L-CDR)-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 8 to 10; ii) A light chain variable domain (VL) containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4; iii) A VL containing the amino acid sequence of SEQ ID NO: 4; or iv) A light chain (LC) containing the amino acid sequences of SEQ ID NOs: 4 and 76.

[0008] In some embodiments, the present disclosure provides an anti-FLT3 antibody or an antigen-binding portion thereof: a) The heavy chain of said antibody comprises: i) H-CDR-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 15 to 17; ii) A VH containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13; iii) A VH containing the amino acid sequence of SEQ ID NO: 13; or iv) An HC containing the amino acid sequences of SEQ ID NOs: 13 and 75; and b) The light chain of said antibody comprises: i) L-CDR-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 18 to 20; ii) A VL containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14; iii) A VL containing the amino acid sequence of SEQ ID NO: 14; or iv) An LC containing the amino acid sequences of SEQ ID NOs: 14 and 76.

[0009] In some embodiments, the present disclosure provides an anti-FLT3 antibody or an antigen-binding portion thereof: a) The heavy chain of said antibody comprises: i) H-CDR-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 25 to 27; ii) A VH containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 23; iii) A VH containing the amino acid sequence of SEQ ID NO: 23; or iv) An HC containing the amino acid sequences of SEQ ID NOs: 23 and 75; and b) The light chain of said antibody comprises: i) L-CDR-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 28 to 30; ii) a VL containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 24; iii) a VL containing the amino acid sequence of SEQ ID NO: 24; or iv) an LC containing the amino acid sequences of SEQ ID NOs: 24 and 76.

[0010] In some embodiments, the present disclosure provides an anti-FLT3 antibody or an antigen-binding portion thereof: a) The heavy chain of said antibody comprises: i) H-CDR-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 35 to 37; ii) a VH containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 33; iii) a VH containing the amino acid sequence of SEQ ID NO: 33; or iv) an HC containing the amino acid sequences of SEQ ID NOs: 33 and 75; and b) The light chain of said antibody comprises: i) L-CDR-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 38 to 40; ii) a VL containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 34; iii) a VL containing the amino acid sequence of SEQ ID NO: 34; or iv) an LC containing the amino acid sequences of SEQ ID NOs: 34 and 76.

[0011] In some embodiments, the present disclosure provides an anti-FLT3 antibody or an antigen-binding portion thereof: a) The heavy chain of said antibody comprises: i) H-CDR-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 45 to 47; ii) a VH containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 43; iii) a VH containing the amino acid sequence of SEQ ID NO: 43; or iv) an HC containing the amino acid sequences of SEQ ID NOs: 43 and 75; and b) The light chain of said antibody comprises: i) L-CDR-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 48 to 50; ii) VL containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 44; iii) VL containing the amino acid sequence of SEQ ID NO: 44; or iv) LC containing the amino acid sequences of SEQ ID NOs: 44 and 76.

[0012] In some embodiments, the present disclosure provides an anti-FLT3 antibody or an antigen-binding portion thereof: a) The heavy chain of said antibody comprises: i) H-CDR-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 55 to 57; ii) VH containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53; iii) VH containing the amino acid sequence of SEQ ID NO: 53; or iv) HC containing the amino acid sequences of SEQ ID NOs: 53 and 75; and b) The light chain of said antibody comprises: i) L-CDR-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 58 to 60; ii) VL containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 54; iii) VL containing the amino acid sequence of SEQ ID NO: 54; or iv) LC containing the amino acid sequences of SEQ ID NOs: 54 and 76.

[0013] In some embodiments, the present disclosure provides an anti-FLT3 antibody or an antigen-binding portion thereof: a) The heavy chain of said antibody comprises: i) H-CDR-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 65 to 67; ii) VH containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 63; iii) VH containing the amino acid sequence of SEQ ID NO: 63; or iv) HC containing the amino acid sequences of SEQ ID NOs: 63 and 75; and b) The light chain of said antibody comprises: i) L-CDR-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 68 to 70; ii) VL containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 64; iii) VL containing the amino acid sequence of SEQ ID NO: 64; or iv) LC containing the amino acid sequences of SEQ ID NOs: 64 and 76.

[0014] In some embodiments, the present disclosure provides an anti-FLT3 antibody or an antigen-binding portion thereof: a) The heavy chain of said antibody comprises: i) H-CDR-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 25 to 27; ii) VH containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 73; iii) VH containing the amino acid sequence of SEQ ID NO: 73; or iv) HC containing the amino acid sequences of SEQ ID NOs: 73 and 75; and b) The light chain of said antibody comprises: i) L-CDR-1 to 3 each containing the amino acid sequences of SEQ ID NOs: 28 to 30; ii) VL containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 74; iii) VL containing the amino acid sequence of SEQ ID NO: 74; or iv) LC containing the amino acid sequences of SEQ ID NOs: 74 and 76.

[0015] The present disclosure also provides an isolated nucleic acid molecule, vector, and host cell comprising a nucleotide sequence encoding the heavy chain or an antigen-binding portion thereof, the light chain or an antigen-binding portion thereof, or both, of the anti-FLT3 antibody or antigen-binding portion described herein. Further, the present disclosure provides a method for producing the anti-FLT3 antibody or antigen-binding portion described herein by culturing said host cell, and a method for producing an antibody composition by mixing the antibody or antigen-binding portion described herein.

[0016] Other features, objects, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description shows embodiments and aspects of the present invention while being given by way of illustration only and not limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description.

Brief Description of the Drawings

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[0018] Detailed Description of the Invention The present disclosure provides novel agonist anti-human FLT3 antibodies that can be used to stimulate FLT3 activity in a patient, such as a cancer patient. Unless otherwise specified, as used herein, "FLT3" refers to human FLT3. The human FLT3 polypeptide sequence is available under UniProt accession number P36888 (FLT3_HUMAN) (SEQ ID NO: 77) as shown below: [Chemical formula]

[0019] The term "antibody" (Ab) or "immunoglobulin" (Ig), as used herein, refers to a tetramer comprising two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable domain (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable domain (VL) and a light chain constant region (CL). The VH and VL domains can be further subdivided into hypervariable regions called "complementary determining regions" (CDRs) that are interspersed among more conserved regions called "framework regions" (FRs). Each VH and VL is composed of three CDRs (H-CDRs in this specification indicate CDRs from heavy chains; and L-CDRs indicate CDRs from light chains) and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Amino acid numbering in the heavy or light chain, as well as the assignment of FR and CDR regions, can follow the definitions of IMGT® (EU numbering; Lefranc et al., Dev Comp Immunol (2003) 27(1):55-77); or Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J. Mol. Biol. (1987) 196:901-17; Chothia et al., Nature (1989) 342:878-83; MacCallum et al., J. Mol. Biol. (1996) 262:732-45; or Honegger and Pluckthun, J. Mol. Biol. (2001) 309(3):657-70.

[0020] The term "recombinant antibody" refers to an antibody expressed from a cell or cell line containing a nucleotide sequence encoding the antibody, wherein the nucleotide sequence is not naturally associated with the cell.

[0021] The terms "isolated protein", "isolated polypeptide", or "isolated antibody" refer, depending on their origin or derivation, to a protein, polypeptide, or antibody that is (1) not associated with the naturally associated components that accompany it in its native state, (2) free of other proteins from the same species, (3) expressed by cells from a different species, and / or (4) not produced in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cell line different from the cell from which it is naturally derived is "isolated" from its naturally associated components. A protein may also be isolated to substantially exclude the naturally associated components using protein purification techniques well known in the art.

[0022] The term "affinity" refers to a measure of the attractive force between an antigen and an antibody. The intrinsic attractive force of an antibody for an antigen is typically expressed as the binding affinity equilibrium constant (K D ) for a particular antibody-antigen interaction. An antibody is said to specifically bind an antigen when K D is ≤ 1 mM, for example, ≤ 1 μM, ≤ 100 nM, or ≤ 10 nM. The K D binding affinity constant can be measured, for example, by surface plasmon resonance (e.g., BIAcore (trademark)) using, for example, an IBIS MX96 SPR system from IBIS Technologies or a Carterra LSA SPR platform, or by biolayer interferometry using, for example, a ForteBio Octet (trademark) system.

[0023] As used herein, the term "epitope" refers to the portion (determinant) of an antigen that specifically binds to an antibody, or a related molecule such as a bispecific binding molecule. Epitope determinants are generally composed of chemically reactive surface groups of a molecule, such as amino acids or carbohydrates or sugar side chains, and generally have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope can be "linear" or "conformational". In a linear epitope, all of the points of interaction between a protein (e.g., an antigen) and an interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on a protein that are separated from each other in the primary amino acid sequence. Once the desired epitope on an antigen has been determined, it is possible to generate an antibody against that epitope using techniques well known in the art. For example, an antibody against a linear epitope can be generated, for example, by immunizing an animal with a peptide having the amino acid residues of the linear epitope. An antibody against a conformational epitope can be generated, for example, by immunizing an animal with a minidomain containing the relevant amino acid residues of the conformational epitope. An antibody against a specific epitope can also be generated, for example, by immunizing an animal with a target molecule (e.g., FLT3) of interest or a related portion thereof and then screening for binding to the epitope.

[0024] Whether an antibody binds to the same epitope as, or competes with, the anti-FLT3 antibodies of the present disclosure can be determined by methods known in the art, including but not limited to competitive assays, epitope binning, and alanine scanning. In some embodiments, the anti-FLT3 antibodies of the present disclosure are allowed to bind to FLT3 under saturating conditions, and then the ability of a test antibody to bind to FLT3 is measured. If the test antibody can bind to FLT3 simultaneously with a reference anti-FLT3 antibody, then the test antibody binds to a different epitope than the reference anti-FLT3 antibody. However, if the test antibody cannot bind to FLT3 simultaneously, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is proximal to the epitope bound by the anti-FLT3 antibodies of the present disclosure. This experiment can be performed using, for example, ELISA, RIA, BIACORE™, SPR, biolayer interferometry, or flow cytometry. To test whether an anti-FLT3 antibody cross-competes with another anti-FLT3 antibody, the competitive methods described above can be used in two directions, i.e., to determine whether a known antibody blocks the test antibody and vice versa. Such cross-competition experiments can be performed using, for example, an IBIS MX96 or Carterra LSA SPR instrument or an Octet™ system.

[0025] The term “human antibody” refers to an antibody in which the variable domain sequences and the constant region sequences are derived from human sequences. This term encompasses antibodies that are derived from human genes but that have been modified, for example, to reduce immunogenicity, increase affinity, and / or increase stability. Further, this term encompasses antibodies that are recombinantly produced in non-human cells, whereby glycosylation that is not typical of human cells can be imparted. This term also encompasses antibodies that are produced in transgenic non-human organisms (e.g., OmniRat® rats) that carry human antibody genes.

[0026] The term "antigen-binding portion" (or simply "antibody portion") of an antibody, as used herein, refers to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human FLT3, or a portion thereof). Certain fragments of a full-length antibody have been shown to be able to perform the antigen-binding function of the antibody. Examples of binding fragments included within the term "antigen-binding portion" are: (i) Fab fragment: a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab’)2 fragment: a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) Fd fragment consisting of the VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragment consisting of the VH domain; and (vi) isolated complementarity-determining regions (CDRs) capable of specifically binding to an antigen. Further, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be joined by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH domains pair to form a monovalent molecule (known as single-chain Fv (scFv)) using recombinant methods. Also within the disclosure are antigen-binding molecules that include VH and / or VL. In the case of VH, the molecule may also include one or more of the CH1, hinge, CH2, or CH3 regions. Such single-chain antibodies are also intended to be included within the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, etc., are also included. A diabody is a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain to create two antigen-binding sites.

[0027] Antibody portions, such as Fab and F(ab’) 2Fragments, etc. can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies. Further, antibodies, antibody portions, and immunoadhesin molecules can be obtained using standard recombinant DNA techniques, for example, as described herein.

[0028] The class (isotype) and subclass of the anti-FLT3 antibody can be determined by any method known in the art. Generally, the class and subclass of an antibody can be determined using an antibody that is specific for a particular class and subclass of antibody. Such antibodies are commercially available. The class and subclass can be determined by ELISA or Western blot as well as other techniques. Alternatively, the class and subclass can be determined by sequencing all or part of the constant region of the heavy and / or light chains of the antibody and comparing their amino acid sequences to the known amino acid sequences of the various classes and subclasses of immunoglobulins to determine the class and subclass of the antibody.

[0029] Unless otherwise indicated, all antibody amino acid residue numbers referred to in this disclosure are under the IMGT® numbering scheme (EU numbering).

[0030] anti-FLT3 antibody This disclosure provides antibodies directed against FLT3, and antigen-binding portions thereof. In certain embodiments, the antibodies disclosed herein are human antibodies generated from transgenic animals (e.g., rats) that can produce antibodies encoded by recombinant human antibody genes. In certain embodiments, the human antibodies may contain specific mutations (e.g., see the "simplex corrected" variant sequences in Table 1) in order to, for example, reverse changes by returning primer-derived mutations to the germline sequences.

[0031] In some embodiments, the anti-FLT3 antibodies of the present disclosure have an "LALA" mutation (L234A / L235A) in the Fc region. These mutations prevent the binding of the antibody to human FcγR (Fc gamma receptor). Such antibodies are advantageous because they have low levels of secondary effector function and therefore do not deplete effector T cells or target other non-malignant cells.

[0032] In some embodiments, the anti-FLT3 antibody or antigen-binding portion competes or cross-competes with, or binds to the same epitope of human FLT3 as, an antibody comprising: a) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 3 and 75 and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 4 and 76; b) an HC comprising the amino acid sequences of SEQ ID NOs: 13 and 75 and an LC comprising the amino acid sequences of SEQ ID NOs: 14 and 76; c) an HC comprising the amino acid sequences of SEQ ID NOs: 23 and 75 and an LC comprising the amino acid sequences of SEQ ID NOs: 24 and 76; d) an HC comprising the amino acid sequences of SEQ ID NOs: 33 and 75 and an LC comprising the amino acid sequences of SEQ ID NOs: 34 and 76; e) an HC comprising the amino acid sequences of SEQ ID NOs: 43 and 75 and an LC comprising the amino acid sequences of SEQ ID NOs: 44 and 76; f) an HC comprising the amino acid sequences of SEQ ID NOs: 53 and 75 and an LC comprising the amino acid sequences of SEQ ID NOs: 54 and 76; g) an HC comprising the amino acid sequences of SEQ ID NOs: 63 and 75 and an LC comprising the amino acid sequences of SEQ ID NOs: 64 and 76; or h) an HC comprising the amino acid sequences of SEQ ID NOs: 73 and 75 and an LC comprising the amino acid sequences of SEQ ID NOs: 74 and 76.

[0033] In some embodiments, the anti-FLT3 antibody or antigen-binding portion has a heavy chain CDR3 (H-CDR3) amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, or 67.

[0034] In some embodiments, the anti-FLT3 antibody or antigen-binding portion has heavy chain CDRs 1-3 (H-CDR1-3) each comprising the amino acid sequence of SEQ ID NOs: 5-7, 15-17, 25-27, 35-37, 45-47, 55-57, or 65-67.

[0035] In some embodiments, the anti-FLT3 antibody or antigen-binding portion has a heavy chain variable domain (VH) amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., at least 90% identical) to the amino acid sequence of SEQ ID NOs: 3, 13, 23, 33, 43, 53, 63, or 73.

[0036] In some embodiments, the anti-FLT3 antibody or antigen-binding portion has a VH comprising the amino acid sequence of SEQ ID NOs: 3, 13, 23, 33, 43, 53, 63, or 73.

[0037] In some embodiments, the anti-FLT3 antibody has a VH amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., at least 90% identical) to the amino acid sequence of SEQ ID NOs: 3, 13, 23, 33, 43, 53, 63, or 73; and a heavy chain constant region amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., at least 90% identical) to the amino acid sequence of SEQ ID NO: 75.

[0038] In some embodiments, the anti-FLT3 antibody comprises a VH amino acid sequence of SEQ ID NOs: 3, 13, 23, 33, 43, 53, 63, or 73 and a heavy chain constant region amino acid sequence of SEQ ID NO: 75.

[0039] In some embodiments, the anti-FLT3 antibody or antigen-binding portion has a light chain CDR3 (L-CDR3) amino acid sequence of SEQ ID NOs: 10, 20, 30, 40, 50, 60, or 70.

[0040] In some embodiments, the anti-FLT3 antibody or antigen-binding portion has light chain CDR1-3 (L-CDR1-3) each comprising the amino acid sequence of SEQ ID NO: 8-10, 18-20, 28-30, 38-40, 48-50, 58-60, or 68-70.

[0041] In some embodiments, the anti-FLT3 antibody or antigen-binding portion has a light chain variable domain (VL) amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., at least 90% identical) to the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, or 74.

[0042] In some embodiments, the anti-FLT3 antibody or antigen-binding portion has a VL comprising the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, or 74.

[0043] In some embodiments, the anti-FLT3 antibody has a VL amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., at least 90% identical) to the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, or 74; and a light chain constant region amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., at least 90% identical) to the amino acid sequence of SEQ ID NO: 76.

[0044] In some embodiments, the anti-FLT3 antibody comprises a VL amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, or 74 and a light chain constant region amino acid sequence of SEQ ID NO: 76.

[0045] In certain embodiments, the anti-FLT3 antibody comprises any one of the heavy chains described above and any one of the light chains described above.

[0046] In some embodiments, the anti-FLT3 antibody or antigen-binding portion of the present disclosure comprises the following H-CDR1-3 and L-CDR1-3 amino acid sequences: a) SEQ ID NOs: 5-10, respectively; b) SEQ ID NOs: 15-20, respectively; c) SEQ ID NOs: 25-30, respectively; d) SEQ ID NOs: 35-40, respectively; e) SEQ ID NOs: 45-50, respectively; f) SEQ ID NOs: 55-60, respectively; or g) SEQ ID NOs: 65-70, respectively.

[0047] In some embodiments, the anti-FLT3 antibody or antigen-binding portion of the present disclosure comprises VH and VL that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., at least 90% identical) to the following amino acid sequences: a) SEQ ID NOs: 3 and 4, respectively; b) SEQ ID NOs: 13 and 14, respectively; c) SEQ ID NOs: 23 and 24, respectively; d) SEQ ID NOs: 33 and 34, respectively; e) SEQ ID NOs: 43 and 44, respectively; f) SEQ ID NOs: 53 and 54, respectively; g) SEQ ID NOs: 63 and 64, respectively; or h) SEQ ID NOs: 73 and 74, respectively.

[0048] In some embodiments, the anti-FLT3 antibody or antigen-binding portion of the present disclosure comprises VH and VL that comprise the following amino acid sequences: a) SEQ ID NOs: 3 and 4, respectively; b) SEQ ID NOs: 13 and 14, respectively; c) SEQ ID NOs: 23 and 24, respectively; d) SEQ ID NOs: 33 and 34, respectively; e) SEQ ID NOs: 43 and 44, respectively; f) SEQ ID NOs: 53 and 54, respectively; g) SEQ ID NOs: 63 and 64, respectively; or h) SEQ ID NOs: 73 and 74, respectively.

[0049] In some embodiments, the anti-FLT3 antibodies of the present disclosure include the following: a) an HC comprising the amino acid sequences of SEQ ID NOs: 3 and 75 and an LC comprising the amino acid sequences of SEQ ID NOs: 4 and 76; b) an HC comprising the amino acid sequences of SEQ ID NOs: 13 and 75 and an LC comprising the amino acid sequences of SEQ ID NOs: 14 and 76; c) an HC comprising the amino acid sequences of SEQ ID NOs: 23 and 75 and an LC comprising the amino acid sequences of SEQ ID NOs: 24 and 76; d) an HC comprising the amino acid sequences of SEQ ID NOs: 33 and 75 and an LC comprising the amino acid sequences of SEQ ID NOs: 34 and 76; e) an HC comprising the amino acid sequences of SEQ ID NOs: 43 and 75 and an LC comprising the amino acid sequences of SEQ ID NOs: 44 and 76; f) an HC comprising the amino acid sequences of SEQ ID NOs: 53 and 75 and an LC comprising the amino acid sequences of SEQ ID NOs: 54 and 76; g) an HC comprising the amino acid sequences of SEQ ID NOs: 63 and 75, and an LC comprising the amino acid sequences of SEQ ID NOs: 64 and 76; or h) an HC comprising the amino acid sequences of SEQ ID NOs: 73 and 75, and an LC comprising the amino acid sequences of SEQ ID NOs: 74 and 76.

[0050] The present disclosure also provides an anti-FLT3 antibody or antigen-binding portion thereof that competes or cross-competes for binding with, or binds to the same epitope as, antibody 17566, 17526, 17667, 17667-0, 17679, 17494, 17543, or 17497.

[0051] In some embodiments, the anti-FLT3 antibody or antigen-binding portion of the present disclosure comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody 17566, 17526, 17667, 17667-0, 17679, 17494, 17543, or 17497.

[0052] In some embodiments, the anti-FLT3 antibody or antigen-binding portion of the present disclosure comprises VH and VL that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the VH and VL of antibody 17566, 17526, 17667, 17667-0, 17679, 17494, 17543, or 17497 (e.g., at least 90% identical).

[0053] In some embodiments, the anti-FLT3 antibody or antigen-binding portion of the present disclosure comprises VH and VL that are the VH and VL of antibody 17566, 17526, 17667, 17667-0, 17679, 17494, 17543, or 17497, respectively.

[0054] In some embodiments, the anti-FLT3 antibody of the present disclosure is antibody 17566, 17526, 17667, 17667-0, 17679, 17494, 17543, or 17497, or an antibody having the same amino acid sequence as said antibody.

[0055] The class of anti-FLT3 antibody obtained by the methods described herein may be changed or switched to another class or subclass. In some embodiments of the present disclosure, nucleic acid molecules encoding VL or VH are isolated using methods well known in the art such that they do not contain nucleic acid sequences encoding CL or CH, respectively. The nucleic acid molecules encoding VL or VH are then operably linked to nucleic acid sequences encoding CL or CH from different classes of immunoglobulin molecules. This can be accomplished using vectors or nucleic acid molecules containing the CL or CH sequences as described above. For example, an anti-FLT3 antibody that was originally IgM can be class switched to IgG. Further, class switching can be used to switch one IgG subclass to another, e.g., IgG 1 from IgG 2It may be converted to. The κ light chain constant region can be changed to, for example, the λ light chain constant region, or vice versa. An exemplary method for producing an antibody of the present disclosure with a desired Ig isotype includes isolating a nucleic acid molecule encoding the heavy chain of the anti-FLT3 antibody and a nucleic acid molecule encoding the light chain of the anti-FLT3 antibody, obtaining the variable domain of the heavy chain, ligating the coding sequence for the variable domain of the heavy chain with the coding sequence for the constant region of the heavy chain of the desired isotype, expressing the light and heavy chains encoded by the ligated sequences in a cell, and collecting the anti-FLT3 antibody with the desired isotype.

[0056] The anti-FLT3 antibodies of the present disclosure can be IgG, IgM, IgE, IgA, or IgD molecules, but typically are of the IgG isotype, for example, IgG subclass IgG 1 、IgG 2a or IgG 2b 、IgG 3 、or IgG 4 、or IgG 1 。In some embodiments, the antibody is of isotype subclass IgG

[0057] In some embodiments, the anti-FLT3 antibody can contain at least one mutation in the Fc region. Many different Fc mutations are known, and these mutations alter the effector function of the antibody. For example, in some embodiments, the anti-FLT3 antibody contains at least one mutation in the Fc region that reduces effector function, such as a mutation at one or more of positions 228, 233, 234, and 235, where the amino acid positions are numbered according to the IMGT® numbering scheme.

[0058] In some embodiments, for example, when the antibody is of the IgG 1 subclass, one or both of the amino acid residues at positions 234 and 235 can be mutated, for example, from Leu to Ala (L234A / L235A). These mutations are in IgG 1Reduces the effector function of the Fc region of the antibody. The amino acid positions are numbered according to the IMGT® numbering scheme.

[0059] In some embodiments, for example, if the antibody is of the IgG4 subclass, it may contain the mutation S228P, and this amino acid position is numbered according to the IMGT® numbering scheme. This mutation is known to reduce unwanted Fab arm exchange.

[0060] In some embodiments, the anti-FLT3 antibody or antigen-binding portion of the present disclosure is an agonist.

[0061] In some embodiments, the anti-FLT3 antibody or antigen-binding portion stimulates the proliferation of EOL-1 cells in vitro (e.g., at a concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μg / mL or less, such as 25 μg / mL or less).

[0062] In some embodiments, the anti-FLT3 antibody or antigen-binding portion stimulates the proliferation of OCI-AML5 cells in vitro (e.g., at a concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μg / mL or less, such as 25 μg / mL or less).

[0063] In some embodiments, the anti-FLT3 antibody or antigen-binding portion specifically binds to human FLT3 and cynomolgus FLT3 (e.g., expressed on CHO-S cells). In some embodiments, the anti-FLT3 antibody or antigen-binding portion specifically binds to human FLT3, cynomolgus FLT3, and mouse FLT3 (e.g., expressed on CHO-S cells).

[0064] In some embodiments, the anti-FLT3 antibody or antigen-binding portion does not block the binding of FLT3L to human FLT3 in vitro. In certain embodiments, the anti-FLT3 antibody or antigen-binding portion does not block the binding of FLT3L to immobilized FLT3 under saturated conditions.

[0065] In some embodiments, the anti-FLT3 antibody or antigen-binding portion does not block the binding of FLT3L-Fc to human, cynomolgus monkey, and / or mouse FLT3 protein presented on cells in vitro (e.g., at a concentration of at least 0.1, 0.5, 1, 5, 10, 30, 50, or 100 μg / mL, such as at a concentration of at least 30 μg / mL).

[0066] In some embodiments, the anti-FLT3 antibody or antigen-binding portion stimulates the proliferation of primary human CD34 + stem cells (e.g., at a concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μg / mL or less, such as at a concentration of 25 μg / mL or less).

[0067] In some embodiments, the anti-FLT3 antibody or antigen-binding portion stimulates the differentiation of primary human CD34 + stem cells (e.g., at a concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μg / mL or less, such as at a concentration of 25 μg / mL or less).

[0068] In some embodiments, the anti-FLT3 antibody or antigen-binding portion increases dendritic cell subpopulations, such as CD14 + cells, CD1c + cells, pDC cells, cDC cells, cDC1 cells, or cDC2 cells, or any combination thereof (e.g., at a concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μg / mL or less, such as at a concentration of 25 μg / mL or less).

[0069] In some embodiments, the anti-FLT3 antibody or antigen-binding portion induces the proliferation and / or mobilization of dendritic cells in immunocompetent mice, such as Balb / c mice (e.g., at a dose of 0.1, 1, or 10 mg / kg or less twice a week or less).

[0070] In some embodiments, the anti-FLT3 antibody or antigen-binding portion is human CD34 +Induce dendritic cell proliferation and / or mobilization in immunodeficient mice reconstituted with stem cells (e.g., twice weekly at a dose of 1 or 10 mg / kg or less). In certain embodiments, dendritic cell proliferation and / or mobilization is induced in the spleen, bone marrow, or both, of the mouse.

[0071] In some embodiments, the anti-FLT3 antibody or antigen-binding portion binds to human FLT3 with a K of about 20, 15, 10, 9, 8, 7, 6, 5, 4, or 3 nM or less (e.g., 9 nM or less). D Bind to human FLT3.

[0072] In some embodiments, the anti-FLT3 antibody or antigen-binding portion binds to domain 1 (D1) of the extracellular domain (ECD) of human FLT3. In certain embodiments, the antibody or portion binds to residues at the C-terminus of D1 (e.g., on the inner surface of D1 for FLT3 ligand). In certain embodiments, the antibody or portion binds to residues on the outer surface of D1 for FLT3 ligand. In certain embodiments, the distance between two epitopes on each D1 in the FLT3 ligand / receptor complex is 90 - 120 Å.

[0073] In some embodiments, the anti-FLT3 antibody or antigen-binding portion binds to a linear epitope comprising residues 78 - 87, 78 - 97, or 138 - 147 of the human FLT3 amino acid sequence. In some embodiments, the anti-FLT3 antibody or antigen-binding portion binds to an epitope comprising residue A79 in combination with residues A80 and / or V81 of the human FLT3 amino acid sequence. In certain embodiments, the anti-FLT3 antibody or antigen-binding portion binds to an epitope comprising the following residues of the human FLT3 amino acid sequence: a) A79, A80, V81, T157, R161; b) A79, A80, V81, I89, T90, R161; c) A79, V81; d) A79, V81, V83, A87, I89, V125, T157; or e) N100, L104 - V106, H109 - S111, E140, L142, N151, T153. The antibody or antigen-binding portion can bind to an epitope as shown, for example, for antibodies 17566, 17526, 17667, 17667-0, 17679, 17494, 17543, or 17497 in Table 8. In certain embodiments, the antibody or binding portion does not bind to the same epitope as antibody IMC-EB10.

[0074] Also contemplated herein are anti-FLT3 antibodies or antigen-binding portions described herein with any combination of the above characteristics.

[0075] In some embodiments, the anti-FLT3 antibodies or antigen-binding portions described herein have at least one of the following characteristics (e.g., all of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11): a) Stimulates the proliferation of EOL-1 cells in vitro; b) Stimulates the proliferation of OCI-AML5 cells in vitro; c) Binds to human FLT3 with a K D of 20 nM or less; d) Specifically binds to cynomolgus FLT3; e) Specifically binds to mouse FLT3; f) Does not block FLT3 ligand binding to human FLT3 in vitro; g) Does not block the binding of FLT3L-Fc to human, cynomolgus, or mouse FLT3 protein presented on cells in vitro; h) Stimulates the proliferation of primary human CD34 + stem cells; i) Stimulates the differentiation of primary human CD34 + stem cells; j) Induces dendritic cell mobilization in vivo in Balb / c mice; and k) Induces dendritic cell mobilization in vivo in immunodeficient mice reconstituted with human CD34 + stem cells. For example, in certain embodiments, the anti-FLT3 antibodies or antigen-binding portions described herein can have characteristics a) and c)-h); a), c), d), and f)-i); a), c), d), and f)-h); a), c), and f)-h); a)-c), h), i), and k); a)-c), h), and i); or a)-c) and j).

[0076] In some embodiments, the anti-FLT3 antibodies or antigen-binding portions described herein can increase the proliferation and / or activation of dendritic cells in a patient. In some embodiments, the anti-FLT3 antibodies or antigen-binding portions described herein enhance the ability of dendritic cells to take up tumor antigens.

[0077] In some embodiments, the anti-FLT3 antibodies or antigen-binding portions described herein can inhibit tumor growth in vivo and / or induce tumor growth regression. In some embodiments, the anti-FLT3 antibodies or antigen-binding portions described herein can delay or reverse metastasis in cancer patients. In some embodiments, the anti-FLT3 antibodies or antigen-binding portions described herein can extend the survival of cancer patients. Any combination of the above characteristics is also contemplated.

[0078] In certain embodiments, the antibodies or antigen-binding portions thereof of the present disclosure can be part of larger immunoadhesin molecules formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesin molecules include the use of streptavidin core regions to create tetrameric scFv molecules (Kipriyanov et al., Human Antibodies and Hybridomas (1995) 6:93-101), as well as the use of cysteine residues, marker peptides, and C-terminal polyhistidine tags to create bivalent biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol. (1994) 31:1047-58). Other examples include cases where one or more CDRs from an antibody are incorporated into a molecule either covalently or non-covalently to create an immunoadhesin that specifically binds to the antigen of interest. In such embodiments, the CDRs can be covalently linked to another polypeptide chain that can be incorporated as part of a larger polypeptide chain, non-covalently incorporated, or non-covalently incorporated.

[0079] In another embodiment, a fusion antibody or immunoadhesin comprising all or a portion of an anti-FLT3 antibody of the present disclosure linked to another polypeptide may be made. In certain embodiments, only the variable domain of the anti-FLT3 antibody is linked to the polypeptide. In certain embodiments, the VH domain of the anti-FLT3 antibody is linked to a first polypeptide while the VL domain of the anti-FLT3 antibody is linked to a second polypeptide that associates with the first polypeptide in a manner such that the VH and VL domains can interact with each other to form an antigen-binding site. In some embodiments, the VH domain is separated from the VL domain by a linker (e.g., single-chain antibody) such that the VH and VL domains can interact with each other. The VH-linker-VL antibody is then linked to the polypeptide of interest. Also, fusion antibodies in which two (or more) single-chain antibodies are linked to each other can be made. This is useful when one wishes to make a bivalent or multivalent antibody on a single polypeptide chain or when one wishes to make a bispecific antibody.

[0080] To make a single-chain antibody (scFv), DNA fragments encoding VH and VL are operably linked, for example, to another fragment encoding a flexible linker encoding the amino acid sequence (Gly 4 -Ser) 3 (SEQ ID NO: 78) such that the VL and VH domains are linked by the flexible linker and the VH and VL sequences can be expressed as a continuous single-chain protein. See, for example, Bird et al., Science (1988) 242:423-6; Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85:5879-83; and McCafferty et al., Nature (1990) 348:552-4. A single-chain antibody can be monovalent if only one VH and VL are used, bivalent if two VH and VL are used, or multivalent if more than two VH and VL are used. For example, bispecific or multivalent antibodies that specifically bind to human FLT3 and another molecule may be generated.

[0081] In other embodiments, other modified antibodies may be prepared using nucleic acid molecules encoding anti-FLT3 antibodies. For example, "camelid antibodies" (Ill et al., Protein Eng. (1997) 10:949-57), "minibodies" (Martin et al., EMBO J. (1994) 13:5303-9), "diabodies" (Holliger et al., Proc. Natl. Acad. Sci. USA (1993) 90:6444-8), or "Janusins" (Traunecker et al., EMBO J. (1991) 10:3655-9 and Traunecker et al., Int. J. Cancer (Suppl.) (1992) 7:51-2) may be prepared using standard molecular biology techniques according to the teachings herein.

[0082] The anti-FLT3 antibodies or antigen-binding portions of the present disclosure can be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, the antibody or portion thereof is derivatized such that FLT3 binding is not detrimentally affected by the derivatization or labeling. Accordingly, the antibodies and antibody portions of the present disclosure are intended to include both the intact and modified forms of the human anti-FLT3 antibodies described herein. For example, the antibodies or antibody portions of the present disclosure can be (chemically coupled, genetically fused, non-covalently bound, or otherwise) functionally linked to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a pharmaceutical agent, and / or a protein or peptide (e.g., streptavidin core region or polyhistidine tag, etc.) that can mediate the association of the antibody or antibody portion with another molecule.

[0083] One type of derivatized antibody is produced by cross-linking two or more antibodies (e.g., of the same or different types, for making bispecific antibodies). Suitable cross-linkers are heterobifunctional and have two distinct reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or are homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available, for example, from Pierce Chemical Company (Rockford, Illinois).

[0084] The anti-FLT3 antibody or antigen-binding portion can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups can be useful, for example, for improving the biological characteristics of the antibody, such as increasing serum half-life.

[0085] Antibodies or antigen-binding portions according to the present disclosure can also be labeled. As used herein, the term "label" or "labeled" refers to the incorporation of another molecule within the antibody. In some embodiments, the label is a detectable marker, for example, the incorporation of a radiolabeled amino acid, or the addition of a biotinyl moiety to a polypeptide that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity that can be detected by an optical or colorimetric method). In some embodiments, the label or marker can be a therapeutic agent, such as a drug conjugate or a toxin. Various methods for labeling polypeptides and glycoproteins are known in the art and these may be used. Examples of labels for polypeptides include, but are not limited to: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope tags), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogs or homologs. In some embodiments, the label is attached by spacer arms of various lengths to reduce potential steric hindrance.

[0086] In some embodiments, an antibody or antigen-binding portion according to the present disclosure can be conjugated to a cytotoxic agent to form an immunoconjugate. In some embodiments, an antibody or antigen-binding portion according to the present disclosure can be conjugated to a radioisotope.

[0087] In certain embodiments, the antibodies of the present disclosure can exist in a neutral form (including zwitterionic forms), or as positively or negatively charged species. In some embodiments, the antibody can complex with a counterion to form a pharmaceutically acceptable salt.

[0088] Anti-FLT3 antibody composition The present disclosure also provides combination therapies (e.g., compositions) comprising one, two, three, four, or more of the anti-FLT3 antibodies or antigen-binding portions thereof described herein. In certain embodiments, the combination therapy (e.g., composition) comprises two of the anti-FLT3 antibodies or antigen-binding portions. The combination therapy can take the form of, for example, a method of treatment using the antibodies or antigen-binding portions described above, or a pharmaceutical composition comprising the antibodies or antigen-binding portions described above.

[0089] In some embodiments, the present disclosure provides a composition comprising a first anti-FLT3 antibody or antigen-binding portion thereof and a second anti-FLT3 antibody or antigen-binding portion thereof, wherein the first and second antibodies are: - Antibodies 17566 and 17526, respectively; - Antibodies 17566 and 17667 (or 17667-0), respectively; - Antibodies 17566 and 17679, respectively; - Antibodies 17566 and 17494, respectively; - Antibodies 17566 and 17543, respectively; - Antibodies 17566 and 17497, respectively; - Antibodies 17526 and 17667 (or 17667-0), respectively; - Antibodies 17526 and 17679, respectively; - Antibodies 17526 and 17494, respectively; - Antibodies 17526 and 17543, respectively; - Antibodies 17526 and 17497, respectively; - Antibodies 17667 (or 17667-0) and 17679, respectively; - Antibodies 17667 (or 17667-0) and 17494, respectively; - Antibodies 17667 (or 17667-0) and 17543, respectively; - Antibodies 17667 (or 17667-0) and 17497, respectively; - Antibodies 17679 and 17494, respectively; - Antibodies 17679 and 17543, respectively; - Antibodies 17679 and 17497, respectively; - Antibodies 17494 and 17543, respectively; - Antibodies 17494 and 17497, respectively; - Antibodies 17543 and 17497, respectively; or - Antibodies 17667 and 17667-0, respectively.

[0090] In some embodiments, the composition comprises an antibody or an antigen-binding portion thereof that binds to the same epitope as, or competes with, the first and second antibodies.

[0091] In some embodiments, the composition comprises an antibody or an antigen-binding portion thereof that comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of the first antibody, and an antibody or an antigen-binding portion thereof that comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of the second antibody.

[0092] In some embodiments, the composition comprises an antibody or an antigen-binding portion thereof comprising VH and VL with amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, respectively, to the VH and VL amino acid sequences of the first antibody (e.g., at least 90% identical), and an antibody or an antigen-binding portion thereof comprising VH and VL with amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, respectively, to the VH and VL amino acid sequences of the second antibody (e.g., at least 90% identical).

[0093] In some embodiments, the composition comprises an antibody or an antigen-binding portion thereof comprising the VH and VL amino acid sequences of the first antibody, and an antibody or an antigen-binding portion thereof comprising the VH and VL amino acid sequences of the second antibody.

[0094] In some embodiments, the composition comprises an antibody or an antigen-binding portion thereof comprising the HC and LC amino acid sequences of the first antibody, and an antibody or an antigen-binding portion thereof comprising the HC and LC amino acid sequences of the second antibody.

[0095] In certain embodiments, the composition can comprise one, two, or more antibodies or antigen-binding portions thereof selected from the group consisting of: a) an antibody comprising H-CDR1-3 each comprising the amino acid sequences of SEQ ID NOs: 5-7, 15-17, 25-27, 35-37, 45-47, 55-57, or 65-67; b) an antibody in which the VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NOs: 3, 13, 23, 33, 43, 53, 63, or 73; c) an antibody comprising the amino acid sequence of SEQ ID NOs: 3, 13, 23, 33, 43, 53, 63, or 73 in the VH; d) an antibody in which the HC comprises the amino acid sequences of SEQ ID NOs: 3 and 75, 13 and 75, 23 and 75, 33 and 75, 43 and 75, 53 and 75, 63 and 75, or 73 and 75; e) An antibody comprising L-CDR1-3 each containing an amino acid sequence of SEQ ID NO: 8-10, 18-20, 28-30, 38-40, 48-50, 58-60, or 68-70; f) An antibody in which VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, or 74; g) An antibody in which VL contains the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, or 74; h) An antibody in which LC contains the amino acid sequence of SEQ ID NO: 4 and 76, 14 and 76, 24 and 76, 34 and 76, 44 and 76, 54 and 76, 64 and 76, or 74 and 76; i) An antibody in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequence of SEQ ID NO: 5-10, 15-20, 25-30, 35-40, 45-50, 55-60, or 65-70; j) An antibody comprising VH and VL each containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3 and 4, 13 and 14, 23 and 24, 33 and 34, 43 and 44, 53 and 54, 63 and 64, or 73 and 74; k) An antibody comprising VH and VL each containing the amino acid sequence of SEQ ID NO: 3 and 4, 13 and 14, 23 and 24, 33 and 34, 43 and 44, 53 and 54, 63 and 64, or 73 and 74; l) An antibody comprising HC and LC each containing the amino acid sequence of 3 and 75, and 4 and 76; 13 and 75, and 14 and 76; 23 and 75, and 24 and 76; 33 and 75, and 34 and 76; 43 and 75, and 44 and 76; 53 and 75, and 54 and 76; 63 and 75, and 64 and 76; or 73 and 75, and 74 and 76.

[0096] In some embodiments, the anti-FLT3 antibody compositions described herein can inhibit tumor growth and / or induce tumor growth regression in vivo. In some embodiments, the anti-FLT3 antibody compositions described herein can slow or reverse metastasis in cancer patients. In some embodiments, the anti-FLT3 antibody compositions described herein can extend the survival of cancer patients.

[0097] The present disclosure also provides a method for producing the anti-FLT3 antibody compositions described herein, the method comprising providing a first anti-FLT3 antibody or antigen-binding portion and a second anti-FLT3 antibody or antigen-binding portion, and mixing the two antibodies or portions.

[0098] Bispecific binding molecule The present disclosure also provides a bispecific binding molecule having the binding specificity of the anti-FLT3 antibodies described herein (e.g., an antigen-binding portion, e.g., including six CDRs or VH and VL, etc.). In some embodiments, the bispecific binding molecule additionally has the binding specificity of another, distinct anti-FLT3 antibody (e.g., another anti-FLT3 antibody described herein) or a different protein, such as an antibody that targets a cancer antigen or another cell surface molecule whose activity mediates a disease state, such as cancer, etc. Such bispecific binding molecules are known in the art, and examples of different types of bispecific binding molecules are given elsewhere in this specification.

[0099] Nucleic acid molecules and vectors The present disclosure also provides nucleic acid molecules and sequences encoding the anti-FLT3 antibodies or antigen-binding portions thereof described herein. In some embodiments, different nucleic acid molecules encode the heavy and light chain amino acid sequences of the anti-FLT3 antibody or antigen-binding portion. In other embodiments, the same nucleic acid molecule encodes the heavy and light chain amino acid sequences of the anti-FLT3 antibody or antigen-binding portion.

[0100] References to nucleotide sequences include, unless otherwise specified, their complements. Thus, references to nucleic acids having a particular sequence are to be understood to include the complementary strand thereof, with its complementary sequence. As used herein, the term "polynucleotide" means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. The term includes single-stranded and double-stranded forms.

[0101] In some embodiments, the disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain or an antigen-binding portion thereof of an anti-FLT3 antibody or an antigen-binding portion thereof described herein, or a nucleotide sequence encoding a light chain or an antigen-binding portion thereof, or both.

[0102] The present disclosure also provides nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical (e.g., at least 90% identical) to one or more nucleotide sequences recited herein, such as nucleotide sequences selected from the group consisting of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 51, 52, 61, 62, 71, and 72, or nucleotide sequences encoding amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 4, 13, 14, 23, 24, 33, 34, 43, 44, 53, 54, 63, 64, 73, and 74. The term "percent sequence identity" in the context of nucleic acid sequences refers to residues in two sequences that are the same when aligned for maximum correspondence. The length of the sequence identity comparison can span at least about 9 nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48, or more nucleotides. There are numerous different algorithms known in the art that can be used to measure nucleotide sequence identity. For example, polynucleotide sequences can be compared using programs in the Wisconsin Package Version 10.0, Genetics Computer Group (GCG) (Madison, Wis.), such as FASTA, Gap, or Bestfit. FASTA includes, for example, programs FASTA2 and FASTA3, and provides an alignment of the best overlapping regions between a query sequence and a search sequence and percent sequence identity (e.g., Pearson, Methods Enzymol. (1990) 183:63-98; Pearson, Methods Mol. Biol. (2000) 132:185-219; Pearson, Methods Enzymol. (1996) 266:227-58; and Pearson, J. Mol. Biol. (1998) 276:71-84; incorporated herein by reference).Unless otherwise specified, default parameters for a particular program or algorithm are used. For example, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (word size 6 and NOPAM coefficient for the scoring matrix), or using Gap with its default parameters, as provided in GCG version 6.1 (incorporated herein by reference).

[0103] In some embodiments, the present disclosure provides nucleic acid molecules comprising nucleotide sequences selected from the group consisting of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 51, 52, 61, 62, 71, and 72. In certain embodiments, the nucleic acid molecule comprises the nucleotide sequences of SEQ ID NOs: 1 and 2, 11 and 12, 21 and 22, 31 and 32, 41 and 42, 51 and 52, 61 and 62, or 71 and 72.

[0104] In any of the above embodiments, the nucleic acid molecule can be isolated. As used herein, a nucleic acid molecule referred to as "isolated" or "purified" is (1) separated from the nucleic acids of their source genomic DNA or cellular RNA, and / or (2) a nucleic acid that does not occur naturally.

[0105] In further embodiments, the present disclosure provides a vector suitable for expressing one or both strands of the antibody or antigen-binding portion thereof described herein. The term "vector," as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded piece of DNA to which additional DNA segments can be ligated. Further, certain vectors are capable of directing the expression of operably linked genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0106] The present disclosure provides a vector comprising a nucleic acid molecule encoding a heavy chain, a light chain, or both a heavy chain and a light chain of an anti-FLT3 antibody or an antigen-binding portion thereof described herein. In certain embodiments, the vector of the present disclosure comprises the nucleic acid molecule described herein. The present disclosure further provides a vector comprising a nucleic acid molecule encoding a fusion protein, a modified antibody, an antibody fragment, and a probe thereof. The vector may further comprise an expression control sequence.

[0107] As used herein, the term "expression control sequence" means a polynucleotide sequence necessary to effect the expression and processing of a coding sequence to which it is ligated. Expression control sequences include suitable transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing signals and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences differs depending on the host organism; in prokaryotes, such control sequences generally include a promoter, ribosome binding site, and transcription termination sequence; in eukaryotes, such control sequences generally include a promoter and transcription termination sequence. The term "control sequence" is intended to include at a minimum all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0108] In some embodiments, the nucleic acid molecule described herein comprises a nucleotide sequence encoding a VH domain from the anti-FLT3 antibody or antigen-binding portion described herein, ligated in-frame to a nucleotide sequence encoding a heavy chain constant region from any source. Similarly, the nucleic acid molecule described herein can comprise a nucleotide sequence encoding a VL domain from the anti-FLT3 antibody or antigen-binding portion described herein, ligated in-frame to a nucleotide sequence encoding a light chain constant region from any source.

[0109] In further embodiments of the present disclosure, the nucleic acid molecule encoding VH and / or VL can be "converted" into a full-length antibody gene. In some embodiments, the nucleic acid molecule encoding the VH or VL domain is converted into a full-length antibody gene by insertion into an expression vector that already encodes the heavy chain constant (CH) region or the light chain constant (CL) region, respectively, such that the VH segment is operably linked to the CH segment within the vector and / or the VL segment is operably linked to the CL segment within the vector. In another embodiment, the nucleic acid molecule encoding the VH and / or VL domain is converted into a full-length antibody gene by ligating, e.g., ligating, the nucleic acid molecule encoding the VH and / or VL domain to the nucleic acid molecule encoding the CH and / or CL region using standard molecular biology techniques. The nucleic acid molecules encoding the full-length heavy chain and / or light chain can then be expressed from the cells into which they are introduced, and the anti-FLT3 antibody can be isolated.

[0110] In some embodiments, the resulting framework region is mutated such that it has the amino acid sequence of the corresponding germline gene. Mutations can be made, for example, in the framework region or the constant region to increase the half-life of the anti-FLT3 antibody. See, e.g., PCT Publication WO00 / 09560. Mutations in the framework region or constant region can also be made to modify the immunogenicity of the antibody and / or to provide sites for covalent or non-covalent attachment to another molecule. According to the present disclosure, the antibody can have mutations in any one or more of the CDRs or framework regions of the variable domain or in the constant region.

[0111] Host Cells and Methods for Producing Antibodies and Antibody Compositions The present disclosure also provides antibody compositions as described herein and methods for producing antibodies and antigen-binding portions thereof. In some embodiments, the present disclosure relates to a method for producing an anti-FLT3 antibody or antigen-binding portion as described herein, the method comprising providing a host cell (e.g., a recombinant host cell) comprising a nucleotide sequence encoding the heavy chain or antigen-binding portion thereof of the anti-FLT3 antibody or antigen-binding portion as described herein, and a nucleotide sequence encoding the light chain or antigen-binding portion thereof; culturing the host cell under conditions appropriate for expression of the antibody or antigen-binding portion; and isolating the resulting antibody or antigen-binding portion. An antibody or antigen-binding portion produced by such expression in such a recombinant host cell is referred to herein as a "recombinant" antibody or antigen-binding portion. The present disclosure also provides progeny cells of such host cells and antibodies or antigen-binding portions produced thereby.

[0112] The term "recombinant host cell" (or simply "host cell") as used herein means a cell into which a recombinant expression vector has been introduced. By definition, a recombinant host cell does not occur naturally. The present disclosure provides, for example, host cells that can contain a vector as described herein. The present disclosure also provides, for example, host cells that contain a nucleotide sequence encoding the heavy chain or antigen-binding portion thereof of an anti-FLT3 antibody or antigen-binding portion as described herein, a nucleotide sequence encoding the light chain or antigen-binding portion thereof, or both. It should be understood that "recombinant host cell" and "host cell" mean not only a particular subject cell but also the progeny of such a cell. Such progeny may not actually be identical to the parent cell due to, for example, mutations or environmental influences that may occur in subsequent generations, but are still included within the scope of the term "host cell" as used herein.

[0113] Anti-FLT3 antibodies, nucleic acid molecules encoding antigen-binding portions thereof, and vectors containing these nucleic acid molecules can be used for transfection of suitable mammalian, plant, bacterial, or yeast host cells. Transformation can be by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Also, nucleic acid molecules can be introduced into mammalian cells by viral vectors.

[0114] Antibodies expressed by different cell lines or in transgenic animals are likely to have different glycosylation patterns. However, all antibodies encoded by the nucleic acid molecules provided herein or containing the amino acid sequences provided herein are part of the present disclosure, regardless of the glycosylation state of the antibody, and more generally, regardless of the presence or absence of post-translational modifications.

[0115] Pharmaceutical composition Another embodiment of the present disclosure is a pharmaceutical composition comprising, as an active ingredient (or as the only active ingredient), an anti-FLT3 antibody or an antigen-binding portion thereof of the present disclosure, an antibody composition, or a bispecific binding molecule. The pharmaceutical composition may additionally contain a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is intended for remission, prevention, and / or treatment of cancer, such as the cancers described herein. In certain embodiments, the cancer is in a tissue, such as skin, lung, intestine, colon, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bone, bladder, breast, stomach, uterus, cervix, and pancreas.

[0116] The pharmaceutical compositions of the present disclosure can include one or more anti-FLT3 antibodies, antigen-binding portions, antibody compositions, or bispecific binding molecules of the present disclosure, such as one or two anti-FLT3 antibodies, antigen-binding portions, or bispecific binding molecules. In some embodiments, the composition includes a single anti-FLT3 antibody or antigen-binding portion thereof of the present disclosure. In another embodiment, the composition includes two different anti-FLT3 antibodies or antigen-binding portions thereof of the present disclosure.

[0117] In some embodiments, the pharmaceutical composition can include at least one anti-FLT3 antibody or antigen-binding portion thereof of the present disclosure, such as one anti-FLT3 antibody or portion, and one or more additional antibodies that target one or more related cell surface receptors, such as one or more cancer-related receptors.

[0118] In some embodiments, the pharmaceutical composition can include at least one anti-FLT3 antibody or antigen-binding portion thereof of the present disclosure, such as one anti-FLT3 antibody or portion, and one or more additional agents selected, for example, from immunostimulants, vaccines, chemotherapeutic agents, anti-neoplastic agents, anti-angiogenic agents, and tyrosine kinase inhibitors.

[0119] Generally, the antibodies, antigen-binding portions, and bispecific binding molecules of the present disclosure are suitable for administration as formulations in association with one or more pharmaceutically acceptable excipients, as described, for example, below.

[0120] The term "excipient" is used herein to describe any component other than the compounds of the present disclosure. The choice of excipient can depend heavily 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. As used herein, "pharmaceutically acceptable excipients" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying 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, as well as combinations thereof. In many cases, it will be preferable to include in the composition an isotonic agent such as sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride. 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 enhance the shelf life or effectiveness of the antibody.

[0121] The pharmaceutical compositions of the present disclosure and 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 (Good Manufacturing Practice) conditions.

[0122] The pharmaceutical compositions of the present disclosure can be prepared, packaged, or sold in bulk as a single unit dose or as multiple single unit doses. As used herein, a "unit dose" is a separate amount of a pharmaceutical composition that contains a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient that can be administered to a subject or a convenient fraction of such a dose, for example, one half or one third of such a dose.

[0123] Formulations of suitable pharmaceutical compositions for parenteral administration typically include an 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 administration or for continuous administration. Injectable formulations can be prepared, packaged, or sold in unit dosage form, for example, in ampoules containing a preservative or in multi-dose containers. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, etc. Such formulations can further include one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In some embodiments of formulations for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granule) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations can also include aqueous solutions containing excipients, such as salts, carbohydrates, and buffering agents (preferably up to pH 3-9), although for some applications they may be more suitably formulated as sterile non-aqueous solutions or in a dry form for use in combination with a suitable vehicle, such as sterile pyrogen-free water. Exemplary parenteral administration forms include sterile aqueous solutions, such as solutions or suspensions in aqueous propylene glycol or dextrose solutions. Such administration forms can be suitably buffered if desired. Other parenterally administrable formulations that are useful include formulations containing the active ingredient in microcrystalline form or in liposomal formulations.

[0124] Therapeutic Use of the Antibodies and Compositions of the Present Disclosure In some embodiments, the anti-FLT3 antibodies and antigen-binding portions thereof, anti-FLT3 antibody compositions, and bispecific binding molecules of the present disclosure are used, for example, to enhance or activate the immune system in a patient (e.g., a mammal such as a human) in need thereof by stimulating FLT3 activity. In certain embodiments, the patient is immunosuppressed. In certain embodiments, a physician can enhance the anti-cancer activity of the patient's own immune system by administering an anti-FLT3 antibody or antigen-binding portion thereof, composition, or bispecific binding molecule described herein. For example, a physician can enhance anti-tumor activity in a patient by administering an anti-FLT3 antibody or antigen-binding portion, antibody composition, or bispecific binding molecule of the present disclosure alone or in combination with other therapeutic agents (sequentially or simultaneously).

[0125] In certain embodiments, the antibodies or antigen-binding portions thereof, compositions, and bispecific binding molecules of the present disclosure are for use in the treatment of cancer, such as FLT3-positive cancer. The cancer can be in one or more tissues, such as the skin, lung, intestine, colon, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bone, bladder, breast, stomach, uterus, cervix, and pancreas, among others. In some embodiments, the antibodies or antigen-binding portions thereof, compositions, or bispecific binding molecules of the present disclosure are for use in treating tumors with low immune cell infiltration (e.g., dendritic cell infiltration).

[0126] In some embodiments, the cancers treated by the anti-FLT3 antibodies, antigen-binding portions, compositions, and bispecific binding molecules of the present disclosure include, for example, melanoma (e.g., cutaneous, mucosal, or ocular melanoma; progressive or metastatic melanoma), cutaneous basal cell carcinoma, glioblastoma, glioma, gliosarcoma, astrocytoma, meningioma, neuroblastoma, adrenocortical carcinoma, head and neck squamous cell carcinoma, oral cancer, salivary gland cancer, nasopharyngeal cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer, and squamous cell lung cancer), esophageal cancer, gastroesophageal junction cancer, gastric cancer, gastrointestinal cancer, primary peritoneal cancer, liver cancer, hepatocellular carcinoma, biliary tract cancer, colon cancer, rectal cancer, colorectal cancer, ovarian cancer, fallopian tube cancer, bladder cancer, upper urinary tract cancer, urothelial cancer, renal cell carcinoma, kidney cancer, genitourinary cancer, cervical cancer, prostate cancer, fibrosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, histiocytoma, pancreatic cancer, endometrial cancer, appendiceal cancer, advanced Merkel cell carcinoma, multiple myeloma, sarcoma, choriocarcinoma, erythroleukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, mast cell leukemia, small lymphocyte lymphoma, Burkitt lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, monocytic lymphoma, HTLV-associated T-cell leukemia / lymphoma, mesothelioma, and solid tumors. The cancer can be, for example, in an early, intermediate, late, locally advanced, or metastatic stage, but can be recurrent or refractory to other therapeutic agents (e.g., other anti-FLT3 therapeutic agents or checkpoint inhibitors), or there may be no available standard treatment.

[0127] In some embodiments, the conditions treated by the anti-FLT3 antibodies, antigen-binding portions, compositions, and bispecific binding molecules of the present disclosure include, for example, melanoma (e.g., cutaneous, mucosal, or ocular melanoma), glioma, glioblastoma multiforme, head and neck squamous cell carcinoma, breast cancer, non-small cell lung cancer, colorectal cancer, renal cell carcinoma, kidney cancer, lymphoma (e.g., B-cell lymphoma or non-Hodgkin lymphoma), leukemia (e.g., acute myeloid leukemia), multiple myeloma, plasmacytoma tumors, and myelodysplastic and / or myeloproliferative disorders.

[0128] In some embodiments, the antibodies or antigen-binding portions thereof, compositions, or bispecific binding molecules of the present disclosure are for use in the treatment of immune disorders.

[0129] In some embodiments, antibodies or antigen-binding portions, compositions, or bispecific binding molecules may be used to treat patients who are immunocompromised (e.g., due to chemotherapy or radiation therapy) or at risk of becoming immunocompromised. In some embodiments, antibodies or antigen-binding portions, compositions, or bispecific binding molecules may be used to expand stem cells in patients after stem cell transplantation.

[0130] In some embodiments, antibodies or antigen-binding portions, compositions, or bispecific binding molecules are for use in treating viral and / or parasitic infections, e.g., when the pathogen inhibits the host immune response. Pathogens can be, for example, HIV, hepatitis (A, B, or C), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), adenovirus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T-cell lymphotropic virus (HTLV), human cytomegalovirus (HCMV), dengue virus, molluscum contagiosum virus, poliovirus, rabies virus, John Cunningham (JC) virus, arbovirus encephalitis virus, simian immunodeficiency virus (SIV), influenza, herpes, giardia, malaria, leishmania, Staphylococcus aureus, Mycobacterium tuberculosis, or Pseudomonas aeruginosa.

[0131] "Treat," "treating," and "treatment" refer to a method of reducing or arresting at least one of a biological disorder and / or its attendant symptoms. As used herein, "reducing" a disease, disorder, or condition means decreasing the severity and / or frequency of symptoms of the disease, disorder, or condition. Further, references to "treatment" herein include references to curative, palliative, and prophylactic treatment.

[0132] "Therapeutically effective amount" refers to the amount of a therapeutic agent administered that reduces to some extent one or more of the symptoms of the disorder being treated. A therapeutically effective amount of an anti-cancer therapeutic agent can, for example, result in a delay in tumor growth, a reduction in tumor size, an increase in survival rate, the elimination of cancer cells, a slow or decreased disease progression, a reversal of metastasis, or other clinical endpoints desired by a healthcare provider.

[0133] The anti-FLT3 antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules described herein can be administered alone or in combination with one or more other drugs or antibodies (or as any combination thereof). Accordingly, the pharmaceutical compositions, methods, and uses described herein include embodiments of combination (co-administration) with other active agents, as detailed below.

[0134] As used herein, the terms "co-administered," "co-administering," and "in combination with" refer to the anti-FLT3 antibodies and antigen-binding portions thereof, antibody compositions, and bispecific binding molecules of the present disclosure in combination with one or more other therapeutic agents and are intended to mean, refer to, and include the following: a) co-administration of such a combination of an antibody / antigen-binding portion / antibody composition / bispecific binding molecule of the present disclosure and a therapeutic agent to a patient in need of treatment, where such components are formulated together in a single dosage form that releases such components to the patient substantially simultaneously, b) substantially simultaneous administration of such a combination of an antibody / antigen-binding portion / antibody composition / bispecific binding molecule of the present disclosure and a therapeutic agent to a patient in need of treatment, where such components are formulated separately in separate dosage forms that are substantially simultaneously ingested by the patient, where such components are released to the patient substantially simultaneously, c) Administering to a patient in need of treatment such a combination of an antibody / antigen-binding portion / antibody composition / bispecific binding molecule of the present disclosure and a therapeutic agent in a sequential manner, wherein such components are formulated separately in distinct dosage forms that are each taken by the patient at successive times with a significant time interval between each administration, whereby the components are released to the patient at substantially different times; and d) Administering to a patient in need of treatment such a combination of an antibody / antigen-binding portion / antibody composition / bispecific binding molecule of the present disclosure and a therapeutic agent in a sequential manner, wherein such components are formulated together in a single dosage form that releases the components in a controlled manner, whereby they are released to the patient simultaneously, sequentially, and / or repeatedly, at the same and / or different times, and each portion can be administered by either the same or different routes.

[0135] The anti-FLT3 antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule of the present disclosure may be administered without an additional therapeutic treatment, i.e., as a monotherapy. Alternatively, treatment with the anti-FLT3 antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule of the present disclosure may include at least one additional therapeutic treatment (combination therapy), e.g., another immunostimulatory agent, an anti-cancer agent (e.g., a chemotherapeutic agent, an anti-neoplastic agent, an anti-angiogenic agent, or a tyrosine kinase inhibitor), or a vaccine (e.g., a tumor vaccine).

[0136] In some embodiments, the antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule may be co-administered or formulated with another medicament / drug for the treatment of cancer. The additional therapeutic treatment may include, for example, an immunostimulant, a vaccine, a chemotherapeutic agent, an anti-neoplastic agent, or an anti-angiogenic agent, a tyrosine kinase inhibitor, and / or radiation therapy. In some embodiments, the additional therapeutic treatment may include different anti-cancer antibodies.

[0137] The pharmaceutical articles comprising the anti-FLT3 antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule described herein, and at least one other agent (e.g., chemotherapeutic agent, anti-cancer agent, or anti-angiogenic agent) can be used as a combination treatment for simultaneous, separate, or sequential administration in cancer treatment. The other agent can be any agent appropriate for the treatment of the particular cancer in question, such as, for example, alkylating agents such as platinum derivatives such as cisplatin, carboplatin, and / or oxaliplatin; plant alkaloids such as paclitaxel, docetaxel, and / or irinotecan; antitumor antibiotics such as doxorubicin (adriamycin), daunorubicin, epirubicin, idarubicin, mitoxantrone, dactinomycin, bleomycin, actinomycin, luteomycin, and / or mitomycin; topoisomerase inhibitors such as topotecan; antimetabolites such as fluorouracil and / or other fluoropyrimidines; FOLFOX; osimertinib; cyclophosphamide; anthracycline; dacarbazine; gemcitabine; or any combination thereof. In some embodiments, responsiveness to the other agent is re-established by the anti-FLT3 antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule described herein.

[0138] The anti-FLT3 antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present disclosure may also be used in combination with other anti-cancer therapies, such as vaccines, cytokines, enzyme inhibitors, immunostimulatory compounds, and T cell therapies. In the case of vaccines, it can be, for example, a protein, peptide, or DNA vaccine containing one or more antigens relevant to the cancer being treated, or a vaccine containing dendritic cells together with the antigen. Suitable cytokines include, for example, IL-2, IFN-gamma, and GM-CSF. An example of the type of enzyme inhibitor having anti-cancer activity is an indoleamine-2,3-dioxygenase (IDO) inhibitor, such as 1-methyl-D-tryptophan (1-D-MT). Also contemplated is adoptive T cell therapy, which refers to various immunotherapy techniques that involve growing or engineering the patient's own T cells to recognize and attack their tumors.

[0139] It is also contemplated that the anti-FLT3 antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present disclosure can be used in adjuvant therapy in the context of tyrosine kinase inhibitors. These are synthetic, mainly quinazoline-derived low molecular weight molecules that interact with the intracellular tyrosine kinase domain of the receptor and inhibit ligand-induced receptor phosphorylation, for example, by competing for the intracellular Mg-ATP binding site.

[0140] In some embodiments, an antibody or antigen-binding portion thereof, an antibody composition, or a bispecific binding molecule can be used in combination with an agent (including, but not limited to, agents that modulate the expression or activity of A2AR, A1AR, A2BR, A3AR, ADA, ALP, AXL, BTLA, B7-H3, B7-H4, CTLA-4, CD116, CD123, CD27, CD28, CD39, CD40, CD47, CD55, CD73, CD122, CD137, CD160, CGEN-15049, CHK1, CHK2, CTLA-3, CEACAM (e.g., CEACAM-1 and / or CEACAM-5, etc.), EGFR, FLT3, FLT3L, GAL9, GITR, HVEM, LAG-3, LILRB1, LY108, LAIR1, ICOS, IDO, IL2R, IL4R, KIR, LAIR1, MET, NKG2A, PAP, PD-1 / PD-L1 / PD-L2, OX40, STING, TIGIT, TIM-3, TGFR-beta, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TNFR2, VEGF, VEGFR, VISTA, LILRB2, CMTM6, and / or 2B4) that mediates the activation of the immune system. In some embodiments, the agent enhances the activity, differentiation, proliferation, or mobilization of dendritic cells. In certain embodiments, the agent is a small molecule inhibitor. In certain embodiments, the agent is an antibody or antigen-binding fragment thereof that binds to one of the above molecules. The anti-FLT3 antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule of the present disclosure may also be used in combination with cytokines (e.g., IL-1, IL-2, IL-12, IL-15, or IL-21), EGFR inhibitors, VEGF inhibitors, etc.

[0141] In certain embodiments, an antibody or antigen-binding portion thereof, an antibody composition, or a bispecific binding molecule can be used in combination with an agent that modulates the expression or activity of FLT3L, CD40, AXL, TLR, or PD-1.

[0142] The present disclosure also contemplates the use of the sequences of the anti-FLT3 antibodies or antigen-binding portions described herein (e.g., the six CDRs or the VH and VL sequences) in the preparation of chimeric antigen receptors, which may be for use in CAR-T technology.

[0143] The antibodies and antigen-binding portions thereof, antibody compositions, and bispecific binding molecules of the present disclosure may be used in the methods of treatment described herein, may be for use in the treatment described herein, and / or may be for use in the manufacture of a medicament for the treatment described herein.

[0144] Dosage and Route of Administration The antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present disclosure may be administered in an effective amount for the treatment of the condition in question, i.e., in a dosage and for a period necessary to achieve the desired result. The therapeutically effective amount may vary depending on factors such as the particular condition being treated, the age, sex, weight, and antibody of the patient, and whether the antibody is administered as a single treatment or in combination with one or more additional anti-cancer treatments.

[0145] The dosing regimen may be adjusted to provide the optimal desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dosage may be proportionally decreased or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate the parenteral compositions in unit dosage form. A unit dosage form, as used herein, refers to physically discrete units suitable as unitary dosages for the patient / subject to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present disclosure are generally determined by and directly dependent on (a) the particular characteristics of the therapeutic agent and the specific therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds for the treatment of hypersensitivity in individuals.

[0146] Thus, those skilled in the art will understand that, based on the disclosure provided herein, the dosage and administration regimen will be adjusted according to methods well known in the therapeutic art. That is, the maximum tolerated dose can be readily established, and the effective amount that provides a detectable therapeutic benefit to the patient can be determined, as well as the time requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while specific dosages and administration regimens are exemplified herein, these examples in no way limit the dosages and administration regimens that can be provided to a patient in the practice of the present disclosure.

[0147] It should be noted that the dosage values can vary with the type and severity of the condition being alleviated and can include single or multiple dosages. For any particular subject, a specific administration regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the composition, and further understand that the dosage ranges shown herein are exemplary only and are not intended to limit the scope or practice of the specific compositions. Furthermore, the administration regimen using the compositions of the present disclosure can be based on a variety of factors (including the type of disease, the patient's age, weight, gender, medical condition, severity of the condition, route of administration, and the specific antibody used). Thus, the administration regimen can vary widely but can be routinely determined using standard methods. For example, the dosage can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which can include clinical effects such as toxic effects and / or clinical test values. Thus, the present disclosure encompasses dose escalation within the patient as determined by those skilled in the art. The determination of suitable dosages and regimens is well known in the relevant art and will be understood to be encompassed by those skilled in the art once the teachings disclosed herein are provided.

[0148] An effective amount for tumor treatment may be measured by its ability to stabilize disease progression and / or relieve symptoms in a patient and, preferably, its ability to reverse disease progression, for example, by reducing tumor size. The ability of an antibody, antigen-binding portion, antibody composition, or bispecific binding molecule of the present disclosure to inhibit cancer can be evaluated by in vitro assays (e.g., as described in the Examples) and in suitable animal models that predict efficacy in human tumors. An appropriate dosing regimen is selected to provide an optimal therapeutic response in each particular situation and may be administered, for example, as a single bolus or by continuous infusion, with possible adjustment of the dosage indicated by the exigencies of each case.

[0149] The antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule of the present disclosure may be administered by any method for administering a peptide, protein, or antibody that is accepted in the art and is typically suitable for parenteral administration. As used herein, "parenteral administration" includes any route of administration characterized by physical rupture of the tissue of a subject and administration through the rupture in the tissue, and thus generally results in direct administration into the bloodstream, muscle, or viscera. Parenteral administration thus includes administration by injection, by application through a surgical incision, by application through a tissue-penetrating non-surgical wound, etc., but is not limited thereto. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intrasinus, intravenous, intraarterial, intrathecal, intraurethral, intracranial, intratumoral, and intrasynovial injection or infusion. Certain embodiments include intravenous and subcutaneous routes.

[0150] Diagnostic Uses and Compositions The antibodies and antigen-binding portions of the present disclosure are also useful in diagnostic processes (e.g., in vitro or ex vivo). For example, the antibodies and antigen-binding portions can be used to detect and / or measure the level of FLT3 in a sample from a patient (e.g., a tissue sample, or a body fluid sample such as an inflammatory exudate, blood, serum, intestinal fluid, saliva, or urine). Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assay, radioimmunoassay, and immunohistology. The present disclosure further encompasses kits (e.g., diagnostic kits) comprising the antibodies and antigen-binding portions described herein.

[0151] Manufactured articles and kits The present disclosure also provides a kit comprising a manufactured article, e.g., a pharmaceutical composition of an anti-FLT3 antibody or an antigen-binding portion thereof, a composition, or a bispecific binding molecule described herein, optionally one or more additional biologically active molecules (e.g., another therapeutic agent), in one or more containers (e.g., disposable or reusable containers), and instructions for use. The antibody or antigen-binding portion, composition, or bispecific binding molecule, and any additional biologically active molecules can be separately packaged in a suitable package, e.g., a vial or an ampoule made of non-reactive glass or plastic. In certain embodiments, the vial or ampoule holds a concentrated stock (e.g., 2-fold, 5-fold, 10-fold or more) of the antibody or antigen-binding portion, composition, or bispecific binding molecule, and optionally the biologically active molecule. In certain embodiments, the manufactured article, e.g., the kit, includes a medical device (e.g., a syringe and a needle) for administering the antibody or antigen-binding portion, composition, or bispecific binding molecule and / or the biologically active molecule; and / or a suitable diluent (e.g., sterile water and physiological saline). The present disclosure also includes a method for manufacturing the said article.

[0152] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control.

[0153] Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, pharmaceuticals and pharmaceutical chemistry, and protein and nucleic acid chemistry, as well as hybridization, described herein are well known and commonly used in the art. Enzyme reactions and purification techniques are performed according to the manufacturer's specifications, as commonly accomplished in the art or as described herein.

[0154] Further, unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise", or variations such as "has", "having", "comprises", or "comprising", etc., are meant to imply the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0155] All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not amount to an admission that any of these documents forms part of the common general knowledge in the art.

[0156] To better understand the present disclosure, the following examples are described. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present disclosure in any way.

[0157] Example

[0158] Example 1. Cloning of anti-FLT3 antibody from rat B cells Materials and methods Antibodies against human FLT3 were isolated from the antibody repertoire derived from OmniRat® rats (Osborn et al., J Immunol. 190(4):1481-90 (2013)) (a transgenic rat strain from Ligand Pharmaceuticals Inc. that produces antibodies with fully human idiotypes). Cloning of rat-derived antibody genes from single cell sorted antibody secreting B cells (ASCs) was performed using Symplex™ antibody discovery technology (Meijer et al., J Mol Biol 358(3):764-72 (2006)).

[0159] IgG 1 An antibody repertoire construct encoding a fully human immunoglobulin in the -LALA format (see below) was transfected into HEK293 cells. The cell supernatants were screened for binding to FLT3 expressed on the surface of CHO cells using flow cytometry in a high-throughput format. FLT3-reactive clones were analyzed by DNA sequencing and the DNA sequences encoding the antibodies were extracted. The selected antibody clones were expressed and functionally tested as described below.

[0160] The missense mutations at the amino termini of the heavy and light chains introduced by the use of degenerate primers in the Symplex™ cloning of the cDNA fragments encoding the antibodies were corrected by reverting them to the germline sequences. Table 1 shows the nucleotide sequences of the heavy and light chain variable domains of the germline antibodies designated 17566, 17526, 17667, 17679, 17494, 17543, and 17497. The correction process included the correction of the amino terminal sequences to the germline and the optimization of codon usage. Targets for matching the human germline sequences were identified by BLAST homology searches for the heavy and light chain variable regions. Table 1 also includes the heavy and light chain variable domain sequences for the uncorrected version of antibody 17667 (17667.0).

[0161] The protein sequences of the variable domains, constant regions, and complementarity-determining regions (CDRs) of antibodies 17566, 17526, 17667, 17667-0, 17679, 17494, 17543, and 17497 are shown in Tables 2, 3, and 4, respectively.

[0162] Results Table 1 shows the nucleotide sequences encoding the variable domains of antibodies 17566, 17526, 17667, 17667-0, 17679, 17494, 17543, and 17497. [Table 1] TIFF0007679399000003.tif254169 TIFF0007679399000004.tif59169

[0163] Table 2 shows the deduced amino acid sequences of antibodies 17566, 17526, 17667, 17667-0, 17679, 17494, 17543, and 17497. The CDRs are shown in bold / underlined. [Table 2]

[0164] Table 3 shows the amino acid sequences of the heavy and light chain constant regions (CH and CL, respectively). "IgG 1 -LALA" refers to the presence of the "LALA" mutation in the heavy chain (numbered according to the IMGT® numbering scheme) that is known to reduce the effector function of the Fc region of the IgG 1 antibody (Hezareh et al., J Virol. (2001) 75(24):12161-8; Hessell et al., Nature (2007) 449(7158):101-4).

Table 3

[0165] Table 4 shows the heavy and light chain CDR amino acid sequences of antibodies 17566, 17526, 17667, 17667-0, 17679, 17494, 17543, and 17497, where the CDRs are defined according to the IMGT® system.

Table 4

[0166] Table 5 shows the sequence number information for antibodies 17566, 17526, 17667, 17667-0, 17679, 17494, 17543, and 174978. Unless otherwise stated, the sequences are amino acid sequences.

Table 5

[0167] Example 2. In Vitro Functional Screening of Anti-FLT3 Antibodies in the EOL-1 Proliferation Assay This example describes the in vitro functional evaluation of a panel of anti-FLT3 monoclonal antibodies for the purpose of identifying lead candidates with agonist activity. The antibodies were evaluated for their ability to stimulate the proliferation of the FLT3-expressing cancer cell line EOL-1.

[0168] Materials and Methods A panel of anti-FLT3 antibodies was evaluated in vitro for their ability to induce the proliferation of the FLT3-expressing cancer cell line EOL-1. EOL-1 cells were seeded in RPMI 1640 Glutamax medium supplemented with 0.5% FBS and 1% P / S and incubated with the indicated antibodies at a final concentration of 25 μg / mL for 5 days. Cell proliferation was quantified using the WST-1 cell proliferation reagent (Roche) according to the manufacturer's instructions. Results The proliferation of EOL-1 cells after treatment with anti-FLT3 antibodies is shown in Figure 1. The ability to induce the proliferation of EOL-1 cells varied widely among the antibodies tested. Some antibodies showed no effect in this assay, while other antibodies had stimulatory capacities as indicated by their ability to induce the proliferation of EOL-1 cells.

[0169] Example 3. Cloning of Anti-FLT3 Reference Antibody Analogs Materials and Methods The amino acid sequences encoding the heavy and light chain variable domains of the antibody analogs in Table 6 were obtained from the patent applications listing them. The protein sequences were reverse-translated into DNA sequences using human codon usage. The corresponding DNA sequences were gene synthesized and cloned into expression vectors containing the human heavy or light chain constant regions, resulting in the expression of full-length antibody chains. The human antibody isotypes selected for expression are listed in the antibody format column. The resulting expression plasmids were transfected into CHO cells using a standard protein expression system. The corresponding antibody supernatants were purified using standard protein A purification column chromatography. [Table 6]

[0170] Example 4. Direct Binding of Anti-FLT3 Antibodies to CHO-S Cells Transfected with Human, Cynomolgus Monkey, or Mouse FLT3 Protein This example demonstrates the binding of anti-FLT3 antibodies to human, mouse, and cynomolgus monkey FLT3 proteins transiently expressed on cells.

[0171] Materials and Methods The binding of seven anti-FLT3 antibodies to human, cynomolgus monkey, or mouse FLT3 proteins expressed on CHO-S cells was evaluated and compared to that of the IMC-EB10 analog.

[0172] Anti-FLT3 antibodies were incubated with hamster CHO-S cell lines transiently expressing human, cynomolgus monkey, or mouse FLT3 at 4°C for 30 minutes. The cells were washed twice and then incubated with an AF647-conjugated secondary anti-human IgG (H+L) antibody for an additional 20 minutes. After the washing steps, antibody binding was detected using a high-throughput flow cytometer, the iQue Screener PLUS (Sartorius), and the GeoMean of the AF647 signal in each well was measured. All concentrations were assayed in triplicate, and a 12-point titration curve was generated for each antibody. Results The binding curves of the antibodies to human, cynomolgus monkey, or mouse FLT3 expressed on the cells are shown in Figure 2. The antibodies assayed bind to the human, cynomolgus monkey, or mouse FLT3 proteins presented on the cells with different potencies and efficacies.

[0173] Example 5. Blockade of FLT3 Ligand Binding to Human FLT3 by Seven Anti-FLT3 Antibodies Materials and Methods Binding of anti-FLT3 antibodies to recombinant human FLT3 ECD HIS-tagged fusion receptor (Sino Biological) and their blocking / non-blocking of FLT3L were measured by Biolayer Interference (BLI) on an OCTET QK384 instrument (ForteBio). HIS-tagged FLT3 was immobilized on a pre-equilibrated Anti-Penta-HIS (HIS1K) Biosensor (ForteBio) for 600 seconds, followed by 600 seconds of association with 500 nM anti-FLT3 antibody and then 300 seconds of association with 100 nM FLT3L and 500 nM MAB. Total FLT3L binding response to immobilized FLT3 was measured in parallel. Data were analyzed in the ForteBio Data Analysis (8.2) program.

[0174] Results The blocking profiles of antibodies 17543, 17494, 17679, 17667, 17526, 17566, 17497, and control mAb are shown in Figure 3. None of the anti-FLT3 mAbs blocked the binding of ligand to FLT3 under saturation conditions, except for the IMC-EB10 analog that completely blocked the binding of ligand to FLT3.

[0175] Example 6. Blocking of FLT3 Ligand Fc Binding to CHO-S Cells Transfected with Human, Cynomolgus Monkey, or Mouse FLT3 Protein by Seven Anti-FLT3 Antibodies This example describes the blocking of FLT3 ligand Fc protein binding to human, cynomolgus monkey, or mouse FLT3 protein expressed on CHO-S cells by anti-FLT3 antibodies.

[0176] Materials and Methods Binding of seven anti-FLT3 antibodies to human, cynomolgus monkey, or mouse FLT3 protein expressed on CHO-S cells was evaluated and compared to that of the IMC-EB10 analog in the presence of FLT3 ligand-Fc protein.

[0177] Anti-FLT3 antibodies were incubated with hamster CHO-S cell lines transiently expressing human, cynomolgus monkey, or mouse FLT3 at 4°C for 30 minutes. The cells were washed twice and then incubated with AF647-conjugated FLT3 ligand Fc for an additional 20 minutes. After the washing steps, residual binding of the AF647-conjugated FLT3 ligand Fc protein was detected using a high-throughput flow cytometer, the iQue Screener PLUS (Sartorius), and the GeoMean of the AF647 signal in each well was measured. All concentrations were assayed in triplicate and a 12-point titration curve was generated for each antibody.

[0178] Results The blocking curves of the antibodies to human, cynomolgus monkey, or mouse FLT3 expressed on the cells are shown in Figure 4. Binding of the AF647-conjugated FLT3L-Fc protein to human, cynomolgus monkey, or mouse FLT3 protein presented to the cells was not blocked at any of the concentrations tested by the antibodies assayed. Binding of the AF647-conjugated FLT3 ligand Fc protein to human, cynomolgus monkey, or mouse FLT3 protein presented to the cells was partially blocked by the IMC-EB10 analog antibody.

[0179] Example 7. In Vitro Functional Activity of Anti-FLT3 Antibodies in the EOL-1 Proliferation Assay In this example, the in vitro functional evaluation of seven anti-FLT3 monoclonal antibodies is described for the purpose of demonstrating dose-dependent agonist activity. The antibodies were evaluated for their ability to stimulate the proliferation of the FLT3-expressing cancer cell line, EOL-1. FLT3 ligand was included for comparison.

[0180] Materials and Methods Seven anti-FLT3 antibodies were further evaluated in vitro for their ability to induce the proliferation of the FLT3-expressing cancer cell line EOL-1. EOL-1 cells were seeded in RPMI 1640 Glutamax medium supplemented with 0.5% FBS and 1% P / S and incubated for 5 days with a 2-fold titration of the indicated antibodies starting at 25 μg / mL. A 2-fold titration of FLT3 ligand starting at 1 μg / mL was included for comparison. Cell proliferation was quantified using the WST-1 cell proliferation reagent (Roche) according to the manufacturer's instructions.

[0181] Results The proliferation of EOL-1 cells after treatment with anti-FLT3 antibodies is shown in Figure 5. All seven antibodies tested demonstrated a dose-dependent stimulatory ability, as shown by their ability to induce the proliferation of EOL-1 cells.

[0182] Example 8. In Vitro Functional Activity of Anti-FLT3 Antibodies in Different IgG Formats in a Cell Proliferation Assay This example demonstrates the in vitro functional evaluation of anti-FLT3 monoclonal antibodies in either the IgG 1 -LALA or IgG 2 format for the purpose of demonstrating dose-dependent agonist activity. The antibodies were evaluated for their ability to stimulate the proliferation of the FLT3-expressing cancer cell lines EOL-1 and OCI-AML5.

[0183] Materials and Methods EOL-1 and OCI-AML5 cells were seeded in RPMI 1640 Glutamax medium supplemented with 0.5% FBS and 1% P / S and incubated for 5 days with a 2-fold titration of the indicated antibodies starting at 25 μg / mL. Cell proliferation was quantified using the WST-1 cell proliferation reagent (Roche) according to the manufacturer's instructions. Results The proliferation of EOL-1 cells and OCI-AML5 cells after treatment with anti-FLT3 antibodies is shown in Figures 6A and 6B, respectively. The IgG 1 -LALA and IgG 2Both isotypes demonstrated dose - dependent stimulatory capacity, as shown by their ability to induce the proliferation of both the EOL - 1 cell line and the OCI - AML5 cell line.

[0184] Example 9. Primary Human CD34 + Effect of Anti - FLT3 Antibodies on the Proliferation of Stem Cells In this example, primary human CD34 + stem cells were used to verify the agonistic activity. Seven anti - FLT3 monoclonal antibodies, as well as IgG 1 -LALA or IgG 2 formats of the selected antibodies were described for in vitro functional evaluation. The antibodies were evaluated for their ability to stimulate the proliferation of primary human CD34 + stem cells. FLT3 ligand was included for comparison.

[0185] Materials and Methods Primary human bone marrow - derived CD34 + stem cells were obtained from the American Type Culture Collection (ATCC). The CD34 + stem cells were seeded in hematopoietic progenitor cell (HPC) growth medium DXF (PromoCell) supplemented with 50 ng / mL thrombopoietin (TPO) and 25 ng / mL IL - 3, and incubated with the indicated antibodies (25 μg / mL) or FLT3 ligand (250 ng / mL) for 7 days. Cell proliferation was quantified using the WST - 1 cell proliferation reagent (Roche) according to the manufacturer's instructions.

[0186] Results Proliferation of primary human CD34 + stem cells after treatment with anti - FLT3 antibodies is shown in Figure 7. It is clear that all 7 tested anti - FLT3 antibodies induced the proliferation of primary human CD34 + stem cells, and their stimulatory ability was confirmed (left panel). IgG 1 -LALA and IgG 2 formats of the selected antibodies also did not stimulate the proliferation of primary human CD34 +Stem cell proliferation was induced (right panel). FLT3 ligand was included as a positive control.

[0187] Example 10. Human primary CD34 + Effect of anti-FLT3 antibody on stem cell differentiation This example describes primary human CD34 + stem cells for the purpose of verifying agonist activity, IgG 1 -LALA and IgG 2 format to describe the in vitro functional evaluation of two anti-FLT3 monoclonal antibodies. The antibodies were evaluated for their ability to induce differentiation of primary human CD34 + stem cells. FLT3 ligand was included for comparison.

[0188] Materials and methods Primary human bone marrow-derived CD34 + stem cells were obtained from the American Type Culture Collection (ATCC). CD34 +Stem cells were seeded in IMDM medium containing 10% FBS, 1% PenStrep, 10 mM HEPES, 20 μM 2-mercaptoethanol, 20 ng / mL IL-3, 20 ng / mL GM-CSF, and 20 ng / mL IL-4. Subsequently, anti-FLT3 antibody (25 μg / mL) or FLT3 ligand (250 ng / mL) was added to the culture, and the cells were incubated for 2 weeks. Fresh medium containing the supplements and anti-FLT3 antibody or FLT3 ligand was added twice during the 2-week culture. Cells were collected and the expression of CD14 and CD1c was analyzed by flow cytometry. Briefly, cells were washed twice with PBS and stained with Human BD Fc Block and Zombie Aqua Fixable Viability dye (dead cell marker) for 20 minutes at 4°C. Subsequently, cells were washed and stained with antibodies against cell surface markers (anti-CD14-FITC, anti-CD1c-PE-CF594, anti-CD11c-BV421, anti-CD123-PE, anti-CD141-PerCPCy5.5) for 30 minutes at 4°C in the dark. After two final washes, cells were analyzed using a BD FACSCelesta flow cytometer and FacsDiva software. Data analysis was performed using GraphPad Prism 5.0.

[0189] Results The agonistic activities of two anti-FLT3 antibodies were evaluated through their ability to induce the differentiation of primary human CD34 + stem cells. Both of the anti-FLT3 antibodies tested were able to induce the differentiation of primary human CD34 + (Figure 8A) and CD1c + (Figure 8B) cells, as well as the frequencies of dendritic cell subsets (pDC, cDC1, and cDC2) (Figure 8C), as indicated by their ability to increase the frequencies compared to the untreated control. FLT3 ligand was included as a positive control. + stem cells.

[0190] Example 11. In Vivo Functional Activity of Anti-FLT3 Antibody in Balb / c Mice This example describes the in vivo functional evaluation of anti-FLT3 monoclonal antibodies in the IgG 1 -LALA or IgG 2 format for their ability to induce dendritic cell proliferation and mobilization in immunocompetent mice. FLT3 ligand was included for comparison.

[0191] Materials and Methods Forty female Balb / c mice were divided into eight treatment groups of five mice each. Treatments were initiated on day 0 and ended on day 13. Mice received vehicle; anti-FLT3 antibody at 0.1 mg / kg, 1 mg / kg, or 10 mg / kg by intraperitoneal injection twice a week; or 10 μg of FLT3L by intraperitoneal injection five times a week. At the end, spleens were harvested from all mice and analyzed by flow cytometry. Cells were stained with anti-CD3-FITC antibody, anti-CD370-PE antibody, anti-CD8-PerCP-Cy5.5 antibody, anti-CD11b-PE-Cy7 antibody, anti-I-A / I-E-APC-Cy antibody, anti-CD11c-BV421 antibody, anti-Ly6C-FITC antibody, anti-CD3-PerCP-Cy5.5 antibody, and anti-CD45R-APC antibody. Zombie Aqua was used for identification of live / dead cells. Cells were analyzed using a BD FACSVerse flow cytometer and FacsDiva software. Data analysis was performed using GraphPad Prism 5.0.

[0192] Results IgG 1 -LALA or IgG 2 The in vivo agonist activity of anti-FLT3 antibodies in the IgG1-LALA and IgG2 formats was evaluated for their ability to induce dendritic cell (DC) mobilization in immunocompetent mice at three different doses. The most efficient dose of anti-FLT3 antibodies in the IgG1-LALA and IgG2 formats for inducing mobilization of the majority of DC subpopulations was 10 mg / kg, as demonstrated by the fold increase in cell numbers compared to vehicle (Figures 9A and 9B). FLT3 ligand was included as a positive control.

[0193] Example 12. In vivo functional activity of anti-FLT3 antibody in CD34 humanized mice This example describes the in vivo functional evaluation of an anti-FLT3 monoclonal antibody for the purpose of verifying its ability to induce dendritic cell mobilization in immunodeficient mice reconstituted with human CD34 + hematopoietic stem cells ("CD34 humanized mice"). FLT3 ligand was included for comparison.

[0194] Materials and methods Thirty-six female NOD / Shi-SCID / IL-2Rγ null (NCG) mice were humanized using hematopoietic stem cells (CD34 + ) isolated from human umbilical cord blood. Only mice with a humanization rate (hCD45 / total CD45) above 25% were used for the study. Mice were randomized into six treatment groups with six animals in each group based on humanization rate and CD34 donor. Treatments were initiated on day 0 and ended on day 11. Mice received twice-weekly intraperitoneal injections of vehicle, 1 or 10 mg / kg of anti-FLT3 antibody, or five-times-weekly intraperitoneal injections of 10 μg of FLT3L. At the end, spleens and bone marrows were collected from all mice and analyzed by flow cytometry. Cells were stained with anti-CD1c-BV421 antibody, anti-CD11c-BV510 antibody, anti-CD14-BV650 antibody, anti-CD123-FITC antibody, anti-CD3-PerCPVio700 antibody, anti-CD20-PerCPVio700 antibody, anti-CD56-PerCPVio700 antibody, anti-CD301-PE antibody, anti-CD141-PE-Vio615 antibody, anti-hCD45-PE-Vio770 antibody, anti-CD370-APC antibody, and anti-HLA-DR-APC-Cy7 antibody. Fixable Yellow was used for live / dead cell discrimination. Cells were analyzed using an Attune NxT flow cytometer and FacsDiva software. Data analysis was performed using GraphPad Prism 5.0.

[0195] Results IgG 1 -LALA or IgG 2The in vivo agonist activity of the anti-FLT3 antibody in the format was evaluated with respect to its ability to induce DC mobilization in CD34 humanized mice at two different doses. In both formats at both doses, the antibody was able to induce mobilization of the majority of subsets of DCs in the spleen (Figures 10A and 10B) and bone marrow (Figures 10C and 10D), as demonstrated by the fold increase compared to the vehicle. FLT3 ligand was included as a positive control.

[0196] Example 13. Human primary CD34 + Effect of anti-FLT3 antibody and FLT3 ligand on gene expression in stem cells This example demonstrates that in vitro stimulation of human primary CD34 + stem cells with an agonist anti-FLT3 antibody induces changes in gene expression similar to those of FLT3 ligand and provides evidence of a similar signaling pathway.

[0197] Materials and methods Primary human bone marrow-derived CD34 + stem cells were obtained from the American Type Culture Collection (ATCC). CD34 +Stem cells were seeded in IMDM medium containing 10% FBS, 1% PenStrep, 10 mM HEPES, 20 μM 2-mercaptoethanol, 20 ng / ml IL-3, 20 ng / ml GM-CSF, and 20 ng / ml IL-4. Subsequently, anti-FLT3 antibody (25 μg / mL) or FLT3 ligand (250 ng / ml) was added to the cultures, and the cells were incubated for 2 weeks. Fresh medium containing the supplements and anti-FLT3 antibody or FLT3 ligand was added twice during the 2-week culture. Cells were harvested, and RNA was extracted using the RNeasy Micro kit (Qiagen) according to the manufacturer's instructions. 100 ng of RNA was used as input for gene expression analysis on the nCounter SPRINT Profiler. Gene expression was analyzed using the nCounter Myeloid Innate Immunity Panel (XT_PGX_huV2_Myeloid, NanoString Technologies), and nSolver Analysis software was used for data quality control, normalization, and gene expression difference analysis. Spearman's rank correlation was used to analyze the relatedness between the genes tested.

[0198] Results As shown in Figure 11, IgG 1 -LALA (left panel) and IgG 2 (right panel) In both formats, the tested antibodies induced changes in gene expression similar to FLT3 ligand. The strong correlation between the observed changes in gene expression induced by the agonist anti-FLT3 antibody and FLT3 ligand indicates that they stimulate human primary CD34 + stem cells in a similar manner.

[0199] Example 14. Binding Kinetics of Anti-FLT3 Fab Fragment to Human FLT3 This example evaluates the binding of an anti-FLT3 Fab fragment to human FLT3 domain 1 as measured by surface plasmon resonance (SPR).

[0200] Materials and Methods The cDNA encoding human FLT3 domain 1 (UniProt accession number P36888) was synthesized and cloned into a vector containing the CMV promoter and the human IgFc sequence (residues P101 - K330), resulting in a fusion of IgFc to the C - terminus and transiently expressed in the ExpiCHO™ expression system. After collection, the supernatant was tested for binding to anti - FLT3 Fab by surface plasmon resonance (SPR) using Carterra LSA. The HC200M (Carterra) chip was functionalized with goat anti - human IgFc (Southern Biotech) using amine coupling. The chip was activated with freshly prepared 0.4M EDC, 0.1M sulfo - NHS, and 0.1M MES, pH 5.5 (1:1:1 v / v / v) for 5 minutes, conjugated with 75 μg / mL anti - human IgFc in 10 mM sodium acetate, pH 4.5 for 10 minutes, and quenched of excess reactive esters by injection of 1M ethanolamine, pH 8.5 for 3 minutes. The instrument was primed with running buffer (PBS pH 7.4, 0.01% Tween - 20, 0.5 mg / ml BSA). After priming and washing, the FLT3 fusion protein in the culture supernatant was captured as replicates on individual spots of the chip for 12 minutes. Fab analyte was prepared in running buffer each. Kinetic analysis was performed by applying a kinetic titration series of monomeric Fab at increasing concentrations. Fab association was performed for 5 minutes and antigen dissociation was recorded for 5 minutes. After each cycle of Fab injection, the surface was regenerated with 0.45% H 3 PO 4 , 2×20 seconds and washed in running buffer for 5 minutes. The binding response was processed and analyzed using Carterra's KIT software tool. The processed data was fit to a simple Langmuir 1:1 binding model for calculation of the on - rate (k on or k a ), off - rate (k off or k d ), and affinity (K D ) constants.

[0201] Results The binding kinetics of the Fab fragments of antibodies 17566, 17526, 17667, 17543, and 17497 to human FLT3 domain 1 are shown in Table 7 below. Data are presented as mean ± SEM, n = 4. [[Table 7]]

[0202] Example 15. Epitope binning of anti-FLT3 antibodies This example describes the grouping of anti-FLT3 antibodies into epitope bins based on the competition patterns of pairs measured by surface plasmon resonance (SPR). Antibodies belonging to different epitope bins recognize different epitopes on the FLT3 ECD.

[0203] Materials and methods Studies of paired antibody competition were performed by SPR using an IBIS-MX96 instrument (IBIS, Netherlands). Anti-FLT3 antibodies were diluted to 3 μg / mL in PBS and spotted onto G-a-hu-IgG Fc SensEye® by capture over 15 minutes using a Continuous Flow Microspotter, followed by blocking of residual binding sites with Herceptin (trastuzumab) and crosslinking with the SensEye FixIt kit (IBIS, Netherlands). After sensor preparation, antibody competition analysis was performed using a classical sandwich assay. Recombinant FLT3-his ECD antigen (Sino Biological Inc) was diluted in PBS, 0.05% Tween 20, 200 nM Herceptin running buffer and injected at a concentration of 100 nM and captured by a conjugate array of anti-FLT3 antibodies. Next, individual injections of each FLT3 antibody diluted to 100 nM in running buffer were performed to establish the antibody competition pattern. Recombinant FLT3 ligand (100 nM) was included as an analyte to characterize ligand-blocking antibodies. Data were analyzed by Epitope Binning 2.0 (Wasatch, USA).

[0204] Results Figure 12 shows the node plots of anti-FLT3 mAbs 17667, 17566, 17526, and 17543, IMC-EB10 analogs, and FLT3 ligand (FLT3L) derived from epitope binning analysis. The anti-FLT3 antibodies were assigned to three separate groups, or bins. Bin 1 (light gray) contains antibodies 17526, 17543, 17566, and 17667, all of which cross-block each other, indicating that these antibodies bind to similar epitopes on the FLT3 extracellular domain. Bin 2 (gray) consists of the IMC-EB10 analog, which blocks FLT3L in accordance with published data (U.S. Patent Publication 2011 / 0091470). Bin 3 (white) consists of only antibody 17497, indicating that this antibody recognizes a different epitope in the assay than the other antibodies.

[0205] In conclusion, with the exception of the IMC-EB10 analog, none of the tested antibodies blocked FLT3L. Antibodies 17526, 17543, 17566, and 17667 bind to overlapping epitopes, while 17497 binds to a different epitope.

[0206] Example 16. Epitope Mapping of Anti-FLT3 Antibodies by Mutagenesis and Surface Plasmon Resonance This example illustrates how the epitopes recognized by monoclonal anti-FLT3 antibodies 17566, 17526, 17667, 17543, and 17497 are distributed on the FLT3 extracellular domain (ECD). Linear and conformational epitopes were characterized by mutagenesis approaches and surface plasmon resonance (SPR).

[0207] Materials and Methods The protein sequences of human and rat (Rattus norvegicus) FLT3 were downloaded from UniProt (accession numbers P36888 and A0A0G2JW59, respectively) and aligned. To map linear epitopes, an Fc fusion protein of domain 1 of human FLT3 ECD was generated and had 10 amino acids sequentially replaced by the corresponding rat FLT3 sequences in overlapping segments by five amino acids. Conformational epitopes were characterized by alanine-scan mutagenesis of FLT3 domain 1.

[0208] The cDNA encoding human FLT3 domain 1 was synthesized and cloned into a vector containing the CMV promoter and the human IgFc sequence (residues P101-K330), resulting in a fusion of IgFc to the C-terminus. Wild-type (wt) and mutant human FLT3 domain 1 Fc fusion constructs were generated by standard gene synthesis techniques and the proteins were transiently expressed in the ExpiCHO™ expression system. After collection, the supernatants were tested for binding to anti-FLT3 Fab by surface plasmon resonance (SPR) using Carterra LSA. The HC200M (Carterra) chip was functionalized with goat anti-human IgFc (Southern Biotech) using amine coupling. The chip was activated with freshly prepared 0.4M EDC, 0.1M sulfo-NHS, and 0.1M MES, pH 5.5 (1:1:1 v / v / v) for 5 minutes and conjugated with 75 μg / mL anti-human IgFc in 10 mM sodium acetate, pH 4.5 for 10 minutes. Excess reactive esters were quenched by injection of 1M ethanolamine, pH 8.5 for 3 minutes. The instrument was primed with running buffer (PBS pH 7.4, 0.01% Tween-20, 0.5 mg / ml BSA). After priming and washing, the FLT3 fusion proteins in the culture supernatants were captured as duplicates on individual spots of the chip for 12 minutes. The Fab analyte was prepared in running buffer for each. Kinetic analysis was performed by applying a kinetic titration series of monomeric Fab at increasing concentrations. Fab association was performed for 5 minutes and antigen dissociation was recorded for 5 minutes. After each cycle of Fab injection, the surface was regenerated with 0.45% H 3 PO 4 , 2 x 20 seconds and washed in running buffer for 5 minutes. The binding responses were processed and analyzed using Carterra's KIT software tool. The processed data were used to determine the on-rate (k on or k a ), the off-rate (k off or k d ), and the affinity (K D)For the calculation of constants, it was fitted to a simple Langmuir 1:1 binding model. Mutations that generate a common inert protein for all Fab fragments were de-selected. To identify amino acids that cause a significant loss of binding, epitopes were defined using a cutoff of at least a 5-fold decrease in binding affinity and / or a z-score greater than 3 compared to wild-type human FLT3.

[0209] Results The linear and conformational epitopes of anti-FLT3 antibodies 17566, 17526, 17667, 17543, and 17497 are shown in Table 8.

Table 8

[0210] The extracellular domain of FLT3 consists of five immunoglobulin (Ig)-like domains (D1-D5). All of the anti-FLT3 antibodies in this example bound to FLT3 D1. As shown in Table 8, antibodies 17566, 17526, 17667, and 17543 bound to similar epitopes, while the mouse cross-reactive antibody 17497 bound to a different epitope predicted by epitope binning (Example 15).

[0211] The epitope was mapped onto the FLT3 ligand-receptor complex (PDB entry: 3QS9, Figure 13). The crystal structure consists of two receptor molecules that bind bivalently to the FLT3 ligand (FLT3L) with a binding interface at the tip of FLT3 D3. This complex forms an open-ring-like structure in which the N-terminal D1 is highly flexible and has at least two different orientations around the linker region between D1 and D2 and does not interact with the rest of the protein complex (Verstrate et al., Blood (2011) 1:60-68).

[0212] The common section in the epitopes of 17566, 17526, 17667, and 17543 was positioned at the C-terminus of D1 immediately before the start of D2. This shared section was positioned on the inner surface of D1, while the epitope of 17497 was positioned on the outer surface of D1 with respect to FLT3L. The distance between the epitopes on each D1 in the ligand-receptor complex was about 90 Å to 120 Å for each of the epitopes (Figure 13), which is within the optimal distance for IgG molecule binding to the two epitopes (Zhang et al., Nature Communication (2020) 11:3114 and Zhang et al., Scientific Reports (2015) 5:9803). Considering that D1 is highly flexible and can adapt in different directions (Verstrate et al., supra), antibodies binding to these epitopes are likely to dimerize FLT3 and activate receptor signaling independent of the FLT3 ligand.

[0213] In conclusion, epitope mapping analysis showed that the epitopes of antibodies 17566, 17526, 17667, and 17543 share residues on the inner surface of FLT3 D1, while antibody 17497 binds to a different epitope on the outer surface of D1. The epitopes positioned on FLT3 D1 appear to be optimal for agonist anti-FLT3 antibodies and receptor activation.

Claims

1. An anti-FLT3 antibody or an antigen-binding portion thereof, comprising: a) SEQ ID NOs: 5 to 10, respectively; b) SEQ ID NOs: 15-20, respectively; c) SEQ ID NOs: 25-30, respectively; d) SEQ ID NOs: 35-40, respectively; e) SEQ ID NOs: 45-50, respectively; f) SEQ ID NOs: 55-60, respectively; or g) SEQ ID NOs: 65 to 70, respectively The H-CDR1-3 and L-CDR1-3 amino acid sequences of An anti-FLT3 antibody or an antigen-binding portion thereof.

2. 2. The anti-FLT3 antibody or antigen-binding portion of claim 1, wherein the antibody comprises: a) SEQ ID NOs: 3 and 4, respectively; b) SEQ ID NOs: 13 and 14, respectively; c) SEQ ID NOs: 23 and 24, respectively; d) SEQ ID NOs: 33 and 34, respectively; e) SEQ ID NOs: 43 and 44, respectively; f) SEQ ID NOs: 53 and 54, respectively; g) SEQ ID NOs: 63 and 64, respectively; or h) SEQ ID NOs: 73 and 74, respectively a heavy chain variable domain amino acid sequence and a light chain variable domain amino acid sequence that are at least 90% identical to the amino acid sequence of Anti-FLT3 antibodies or antigen-binding portions.

3. 2. The anti-FLT3 antibody or antigen-binding portion of claim 1, wherein the antibody comprises: a) SEQ ID NOs: 3 and 4, respectively; b) SEQ ID NOs: 13 and 14, respectively; c) SEQ ID NOs: 23 and 24, respectively; d) SEQ ID NOs: 33 and 34, respectively; e) SEQ ID NOs: 43 and 44, respectively; f) SEQ ID NOs: 53 and 54, respectively; g) SEQ ID NOs: 63 and 64, respectively; or h) SEQ ID NOs: 73 and 74, respectively a heavy chain variable domain and a light chain variable domain comprising the amino acid sequence Anti-FLT3 antibodies or antigen-binding portions.

4. The anti-FLT3 antibody according to any one of claims 1 to 3, which is an IgG.

5. IgG １ The anti-FLT3 antibody of claim 4,

6. The anti-FLT3 antibody of any one of claims 1 to 5, comprising at least one mutation in the Fc region.

7. IgG １ and comprising a mutation at one or more of heavy chain amino acid positions 234 and 235, numbered according to the IMGT® numbering scheme.

8. The anti-FLT3 antibody of claim 7 , wherein one or both of the amino acid residues at positions 234 and 235 are mutated from Leu to Ala.

9. a) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 3 and 75 and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 4 and 76; b) a HC comprising the amino acid sequences of SEQ ID NOs: 13 and 75 and a LC comprising the amino acid sequences of SEQ ID NOs: 14 and 76; c) a HC comprising the amino acid sequences of SEQ ID NOs: 23 and 75 and a LC comprising the amino acid sequences of SEQ ID NOs: 24 and 76; d) a HC comprising the amino acid sequences of SEQ ID NOs: 33 and 75 and a LC comprising the amino acid sequences of SEQ ID NOs: 34 and 76; e) a HC comprising the amino acid sequences of SEQ ID NOs: 43 and 75 and a LC comprising the amino acid sequences of SEQ ID NOs: 44 and 76; f) a HC comprising the amino acid sequences of SEQ ID NOs: 53 and 75 and a LC comprising the amino acid sequences of SEQ ID NOs: 54 and 76; g) a HC comprising the amino acid sequences of SEQ ID NOs: 63 and 75 and a LC comprising the amino acid sequences of SEQ ID NOs: 64 and 76; or h) a HC comprising the amino acid sequences of SEQ ID NOs: 73 and 75 and a LC comprising the amino acid sequences of SEQ ID NOs: 74 and 76 An anti-FLT3 antibody comprising:

10. 10. The anti-FLT3 antibody or antigen-binding portion of any one of claims 1 to 9, wherein the antibody or antigen-binding portion is: a) stimulates proliferation of EOL-1 cells in vitro; b) stimulates proliferation of OCI-AML5 cells in vitro; c) specifically binds to cynomolgus monkey FLT3; d) specifically binds to mouse FLT3; e) does not block FLT3 ligand binding to human FLT3 in vitro; f) does not block binding of FLT3L-Fc to cell-presented human, cynomolgus monkey, or mouse FLT3 protein in vitro; g) Primary human CD34 ＋ Stimulates stem cell proliferation; h) Primary human CD34 ＋ Stimulates stem cell differentiation; i) induce dendritic cell mobilization in Balb / c mice in vivo; and j) Human CD34 ＋ Inducing dendritic cell recruitment in vivo in stem cell-reconstituted immunodeficient mice having at least one characteristic selected from the following: Anti-FLT3 antibodies or antigen-binding portions.

11. 11. The anti-FLT3 antibody or antigen-binding portion of claim 10, having at least 2, 3, 4, 5, 6, 7, 8, 9, or all of the above properties.

12. A pharmaceutical composition comprising the anti-FLT3 antibody or antigen-binding portion of any one of claims 1 to 11.

13. 13. The pharmaceutical composition of claim 12, further comprising an immunostimulant, a vaccine, a chemotherapeutic agent, an anti-neoplastic agent, an anti-angiogenic agent, or a tyrosine kinase inhibitor.

14. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain or an antigen-binding portion thereof and a nucleotide sequence encoding the light chain or an antigen-binding portion thereof of an anti-FLT3 antibody or antigen-binding portion thereof of any one of claims 1 to 11.

15. 15. The isolated nucleic acid molecule of claim 14, comprising the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 51, 52, 61, 62, 71, and 72.

16. 16. A vector comprising the isolated nucleic acid molecule of claim 14 or 15, further comprising an expression control sequence.

17. A host cell comprising a nucleotide sequence encoding the heavy chain or an antigen-binding portion thereof and a nucleotide sequence encoding the light chain or an antigen-binding portion thereof of the anti-FLT3 antibody of any one of claims 1 to 11.

18. A method for producing an anti-FLT3 antibody or antigen-binding portion thereof, comprising providing a host cell as described in claim 17, culturing the host cell under conditions suitable for expression of the antibody or portion, and isolating the resulting antibody or portion.

19. A bispecific binding molecule comprising the antigen-binding domains of one or two distinct anti-FLT3 antibodies according to any one of claims 1 to 11.

20. An anti-FLT3 antibody or antigen-binding portion thereof according to any one of claims 1 to 11, a pharmaceutical composition according to claim 12 or 13, or a bispecific binding molecule according to claim 19 for use in a diagnostic process.

21. 20. An anti-FLT3 antibody or antigen-binding portion thereof according to any one of claims 1 to 11, a pharmaceutical composition according to claim 12 or 13, or a bispecific binding molecule according to claim 19, for use in enhancing immune activity in a patient in need thereof.

22. 20. An anti-FLT3 antibody or antigen-binding portion thereof according to any one of claims 1 to 11, a pharmaceutical composition according to claim 12 or 13, or a bispecific binding molecule according to claim 19, for use in treating cancer in a patient.

23. The anti-FLT3 antibody, antigen-binding portion, pharmaceutical composition, or bispecific binding molecule of claim 22, wherein the patient has cancer of the skin, lung, intestine, colon, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bone, bladder, breast, stomach, uterus, cervix, or pancreas.

24. 23. The anti-FLT3 antibody, antigen-binding portion, pharmaceutical composition, or bispecific binding molecule of claim 22, wherein the patient has melanoma, glioma, breast cancer, non-small cell lung cancer, colorectal cancer, renal cell carcinoma, kidney cancer, lymphoma, leukemia, multiple myeloma, plasma cell neoplasm, myelodysplastic disorder, or myeloproliferative disorder.

25. An anti-FLT3 antibody, antigen-binding portion, pharmaceutical composition, or bispecific binding molecule described in any one of claims 21 to 24 for use in combination with an immunostimulant, a vaccine, a chemotherapeutic agent, an anti-neoplastic agent, an anti-angiogenic agent, a tyrosine kinase inhibitor, or radiation therapy.

26. 20. An anti-FLT3 antibody or antigen-binding portion thereof according to any one of claims 1 to 11, a pharmaceutical composition according to claim 12 or 13, or a bispecific binding molecule according to claim 19, for use in treating an immune disorder in a patient in need thereof.

27. According to the application defined in any one of claims 21 to 26, a) enhancing immune activity in a patient; b) treating cancer in a patient; or c) Treating immune disorders in patients 20. Use of an anti-FLT3 antibody or antigen-binding portion thereof according to any one of claims 1 to 11, a pharmaceutical composition according to claim 12 or 13, or a bispecific binding molecule according to claim 19, for the manufacture of a medicament for

28. According to the application defined in any one of claims 21 to 26, a) enhancing immune activity in a patient; b) treating cancer in a patient; or c) Treating immune disorders in patients An anti-FLT3 antibody or antigen-binding portion thereof according to any one of claims 1 to 11, a pharmaceutical composition according to claim 12 or 13, or a bispecific binding molecule according to claim 19, for use in

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