Anti-CLL-1 antibodies and their uses

Abstract

Anti-CLL-1 antibodies and uses thereof are provided. According to one embodiment of the anti-CLL-1 antibody, the antibody can bind to CLL-1 with high binding affinity and induce T cell activation, and thus can be effectively used to prevent or treat cancers expressing CLL-1.

Description

[Technical field]

[0001] CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to Korean Provisional Patent Application No. 10-2021-0095142, filed on July 20, 2021, and Korean Provisional Patent Application No. 10-2021-0176638, filed on December 10, 2021, with the Korean Intellectual Property Office, the disclosures of which are incorporated by reference in their entireties into this application.

[0002] The present invention provides anti-CLL-1 antibodies and uses thereof. [Background technology]

[0003] Acute myeloid leukemia (AML) is the most common and fatal hematological malignancy in adult patients, with the majority of patients having a poor prognosis. AML remains difficult to treat due to the heterogeneity of the disease, the paucity of specific target antigens, and the presence of leukemic stem cells (LSCs) that mediate relapse after existing therapies.

[0004] CLEC12A (C-type lectin domain family 12 member A; also known as CLL-1 (C-type lectin-like molecule-1), CD371, DCAL2 (dendritic cell-associated lectin 2), MICL (myeloid inhibitory C-type lectin-like receptor), and KLRL1 (killer cell lectin-like receptor-1) is a myeloid differentiation antigen expressed in up to 90% of newly diagnosed and relapsed AML. It is a type II transmembrane glycoprotein that contains an extracellular C-terminal lectin domain, a transmembrane region, and an N-terminal cytoplasmic tail.

[0005] Monoclonal antibody (mAb)-based therapy has become an important therapeutic modality for cancer. Leukemia is highly suitable for such approaches because of the accessibility to malignant cells present in blood, bone marrow, spleen, and lymph nodes, and the well-defined immunophenotypes of diverse lineages and hematopoietic differentiation stages that can identify antigenic targets. Most research in acute myeloid leukemia (AML) has focused on CD33. However, responses to the unconjugated anti-CD33 mAb lintuzumab showed modest activity as a single agent in AML and failed to improve patient outcomes in two randomized trials in combination with existing chemotherapy.

[0006] There is a need in the art for safe and effective agents that target AML, including CLL-1, for the diagnosis and treatment of CLL-1-associated conditions, such as cancer. The present invention fulfills this need and also provides other advantages. Summary of the Invention [Problem to be solved by the invention]

[0007] One aspect of the present disclosure provides an isolated anti-CLL-1 antibody or antigen-binding fragment thereof.

[0008] Another aspect of the present disclosure provides an isolated nucleic acid encoding an anti-CLL-1 antibody.

[0009] Another aspect of the present disclosure provides a vector comprising the isolated nucleic acid.

[0010] Another aspect of the present disclosure provides a host cell comprising the vector.

[0011] Another aspect of the present disclosure provides a pharmaceutical composition comprising an anti-CLL-1 antibody.

[0012] Another embodiment of the present disclosure provides a method of treating or preventing cancer in a patient in need thereof, comprising administering to the patient an effective amount of an anti-CLL-1 antibody.

[0013] Another aspect of the present disclosure provides the use of an anti-CLL-1 antibody in the manufacture of a medicament for treating or preventing cancer.

[0014] Another aspect of the present disclosure provides the use of an anti-CLL-1 antibody for treating or preventing cancer. [Means for solving the problem]

[0015] One aspect of the present disclosure provides an isolated anti-CLL-1 antibody or antigen-binding fragment thereof, comprising: (a) a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3; (b) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 5, and 6; (c) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, and 11; (d) a VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, and 15; (e) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, and 18; and (f) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, and 22.

[0016] In one embodiment, the anti-CLL-1 antibody or fragment thereof may comprise a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:23 and 24.

[0017] In one embodiment, the anti-CLL-1 antibody or fragment thereof may comprise a light chain constant region comprising the amino acid sequence consisting of SEQ ID NO:55.

[0018] In one embodiment, the anti-CLL-1 antibody or fragment thereof may comprise a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:23 and 24, and a light chain constant region comprising an amino acid sequence consisting of SEQ ID NO:55.

[0019] In one embodiment, the anti-CLL-1 antibody or fragment thereof may comprise a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 74.

[0020] In one embodiment, the anti-CLL-1 antibody or fragment thereof may comprise a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44 and 75.

[0021] In one embodiment, the anti-CLL-1 antibody or fragment thereof can comprise a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 74, and a light chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, and 75.

[0022] In one embodiment, the anti-CLL-1 antibody or fragment thereof may comprise a heavy chain comprising the amino acid sequence consisting of SEQ ID NO:45.

[0023] In one embodiment, the anti-CLL-1 antibody or fragment thereof may comprise a light chain comprising the amino acid sequence consisting of SEQ ID NO:46.

[0024] In one embodiment, the anti-CLL-1 antibody or fragment thereof may comprise a heavy chain comprising the amino acid sequence consisting of SEQ ID NO:45 and a light chain comprising the amino acid sequence consisting of SEQ ID NO:46.

[0025] In one embodiment, the anti-CLL-1 antibody or fragment thereof can comprise the sequences of the heavy chain variable region, CDRH1, CDRH2, and CDRH3, and the light chain variable region, CDRL1, CDRL2, and CDRL3, which are any one of the following: (a) CDRH1, CDRH2, and CDRH3 are SEQ ID NOs: 1, 4, and 7, respectively, and CDRL1, CDRL2, and CDRL3 are SEQ ID NOs: 12, 16, and 19, respectively; (b) CDRH1, CDRH2, and CDRH3 are SEQ ID NOs: 2, 5, and 8, respectively, and CDRL1, CDRL2, and CDRL3 are SEQ ID NO: 13, respectively. , 17 and 20; (c) CDRH1, CDRH2 and CDRH3 are SEQ ID NOs: 3, 6, and 9, respectively, and CDRL1, CDRL2, and CDRL3 are SEQ ID NOs: 14, 18 and 21, respectively; (d) CDRH1, CDRH2 and CDRH3 are SEQ ID NOs: 1, 4, and 10, respectively, and CDRL1, CDRL2, and CDRL3 are SEQ ID NOs: 15, 16 and 22, respectively; and (e) CDRH1, CDRH2 and CDRH3 are SEQ ID NOs: 2, 5, and 11, respectively, and CDRL1, CDRL2, and CDRL3 are SEQ ID NOs: 13, 17 and 20, respectively.

[0026] In one embodiment, the anti-CLL-1 antibody or antigen-binding fragment thereof can be a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0027] In one embodiment, the anti-CLL-1 antibody or antigen-binding fragment thereof can be selected from the group consisting of whole IgG, Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv, scFv-Fc, scFab-Fc, diabody, minibody, scAb, dAb, half IgG, and combinations thereof.

[0028] In one embodiment, the anti-CLL-1 antibody or antigen-binding fragment thereof can be in the form of an IgG, preferably an IgG1.

[0029] In one embodiment, the anti-CLL-1 antibody or antigen-binding fragment thereof can be a Fab molecule.

[0030] In one embodiment, the anti-CLL-1 antibody or antigen-binding fragment thereof may have any one or more of the following properties: (a) binding to human CLL-1; (b) binding to cynomolgus monkey CLL-1; (c) binding to CLL-1 on the surface of human peripheral blood mononucleocyte cells (PBMCs); (d) binding to CLL-1 on the surface of cynomolgus monkey PBMCs; and (e) binding to CLL-1 on the surface of cancer cells.

[0031] In one embodiment, a cytotoxic agent can be conjugated to at least a portion of the anti-CLL-1 antibody or antigen-binding fragment.

[0032] In one embodiment, the cytotoxic agent can be attached to the anti-CLL-1 antibody or antigen-binding fragment via a linker.

[0033] In one embodiment, the linker is cleavable by a protease.

[0034] In one embodiment, the anti-CLL-1 antibody may be for use as a medicine.

[0035] In one embodiment, the anti-CLL-1 antibodies may be for use in the treatment or prevention of cancer.

[0036] Another embodiment of the present disclosure provides an immunoconjugate comprising the formula Ab-(LD)p, where (a) Ab is an anti-CLL-1 antibody as described above, (b) L is a linker, (c) D is a cytotoxic agent, and (d) p is in the range of 1 to 8.

[0037] Another aspect of the present disclosure provides an isolated nucleic acid encoding an anti-CLL-1 antibody.

[0038] Another aspect of the present disclosure provides a vector comprising the isolated nucleic acid.

[0039] Another aspect of the present disclosure provides a host cell comprising the vector.

[0040] Another aspect of the present disclosure provides a pharmaceutical composition comprising an anti-CLL-1 antibody.

[0041] Another embodiment of the present disclosure provides a method of treating or preventing cancer in a patient in need thereof, comprising administering to the patient an effective amount of an anti-CLL-1 antibody.

[0042] Another aspect of the present disclosure provides the use of an anti-CLL-1 antibody in the manufacture of a medicament for treating or preventing cancer.

[0043] Another aspect of the present disclosure provides the use of an anti-CLL-1 antibody for treating or preventing cancer.

[0044] In one embodiment, the pharmaceutical composition may be for treating or preventing cancer.

[0045] In one embodiment, the cancer can be a solid cancer or a blood cancer.

[0046] In one embodiment, the cancer is selected from the group consisting of leukemia, rectal cancer, endometrial cancer, nephroblastoma, basal cell carcinoma, nasopharyngeal carcinoma, bone tumors, esophageal cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular thyroid cancer, hepatocellular carcinoma, oral cancer, renal cell carcinoma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, mesenchymal tumors, soft tissue sarcoma, liposarcoma, gastrointestinal stromal tumor, malignant peripheral nerve sheath tumor (MPNST), Ewing's sarcoma, and / or rhabdomyosarcoma. sarcoma, leiomyosarcoma, mesenchymal chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, teratoma, neuroblastoma, medulloblastoma, glioma, benign skin tumor, Burkitt's lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma, neuroectodermal tumor, epithelial tumor, cutaneous T-cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), peripheral T cell lymphoma, pancreatic cancer, haematological malignancies, kidney cancer, tumor vasculature The cancer may be selected from the group consisting of human vasculature, breast cancer, kidney cancer, ovarian cancer, epithelial ovarian cancer, gastric cancer, liver cancer, lung cancer, colon cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer.

[0047] In one embodiment, the leukemia may be selected from the group consisting of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy-cell leukemia, myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML).

[0048] In one embodiment, the cancer can be a CLL-1 expressing cancer. Effect of the Invention

[0049] According to one embodiment of the anti-CLL-1 antibody, it can bind to CLL-1 with high binding affinity and induce T cell activation, and therefore can be effectively used to prevent or treat cancers expressing CLL-1. [Brief description of the drawings]

[0050] [Figure 1] FIG. 1 is a graph showing the ligand binding activity of three chimeric antibodies according to one embodiment using huCLL1-His as the ligand. [Diagram 2] FIG. 2 is a graph showing the ligand binding activity of three chimeric antibodies according to one embodiment using hFc-huCLL1 as the ligand. [Diagram 3] FIG. 3 is a graph showing the ligand binding activity of three chimeric antibodies according to one embodiment using 150 ng / well of hFc-cynomolgus CLL as the ligand. [Figure 4] FIG. 4 is a graph showing the ligand binding activity of three chimeric antibodies according to one embodiment using 100 ng / well of hFc-cynomolgus CLL1 as the ligand. [Diagram 5]FIG. 5 is a graph showing the ligand binding activity of chimeric and humanized antibodies hu16C6 according to one embodiment. [Figure 6] FIG. 6 is a graph showing the ligand binding activity of a chimeric antibody and humanized antibody hu33C2 according to one embodiment. [Figure 7] FIG. 7 is a graph showing the ligand binding activity of various humanized antibody hu33C2, according to one embodiment. [Figure 8] FIG. 8 is a graph showing the cell binding activity of a chimeric antibody according to one embodiment in CLL1-negative cells and various CLL1-expressing cancer cells. [Figure 9] FIG. 9 is a graph showing the cell binding activity of a chimeric antibody according to one embodiment in HL60. [Figure 10] FIG. 10 is a graph showing the cell binding activity of a chimeric antibody according to one embodiment in U937. [Figure 11] FIG. 11 is a graph showing the cell binding activity of a chimeric antibody according to one embodiment in HEK293E overexpressing cynomolgus CLL-1. [Figure 12] FIG. 12 is a graph showing the cell binding activity of a chimeric antibody according to one embodiment and various humanized antibodies hu33C2. [Figure 13] FIG. 13 is a graph showing the cell binding activity of a chimeric antibody according to one embodiment and various humanized antibodies hu16C6. [Figure 14] FIG. 14 is a graph showing ADCC of ch84A2, hu16C6 and hu33C2 according to one embodiment. [Figure 15] FIG. 15 is a graph showing the cytostatic activity of an ADC according to an embodiment at EOL-1. [Figure 16] FIG. 16 is a graph showing the cytostatic activity of an ADC according to an embodiment in THP-1. [Figure 17] FIG. 17 is a graph showing the cell binding activity of a bispecific antibody according to one embodiment in CLL1-expressing cancer cells. [Figure 18]FIG. 18 is a graph showing the cell binding activity of a bispecific antibody according to one embodiment. [Figure 19] FIG. 19 is a graph showing T cell activation of a bispecific antibody according to one embodiment. [Figure 20] FIG. 20 is a graph showing T cell activation and cytolytic activity of a bispecific antibody according to one embodiment in HL-60. [Figure 21] FIG. 21 is a graph showing T cell activation and cytolytic activity of a bispecific antibody according to one embodiment in U937. [Figure 22] FIG. 22 is a graph showing antigen-dependent cytolytic activity of a bispecific antibody according to one embodiment. [Figure 23] FIG. 23 is a graph showing the in vivo efficacy of a bispecific antibody according to an embodiment in a U937 xenograft model. [Figure 24] FIG. 24 is an image showing the bioluminescence index (BLI) upon administration of a bispecific antibody according to one embodiment in an HL60-Lu orthotopic AML model. [Diagram 25] FIG. 25 is a graph showing quantitative analysis of BLI upon administration of a bispecific antibody according to one embodiment in an HL60-Lu orthotopic AML model (statistical analysis: Two-way ANOVA (Bonferroni's multiple comparisons test), *p<0.05, **p<0.01, ***p<0.001). [Figure 26] FIG. 26 is an image showing bioluminescence index (BLI) upon administration of a bispecific antibody according to one embodiment in an HL60-Lu orthotopic AML model. [Figure 27] FIG. 27 is a graph showing quantitative analysis of BLI upon administration of a bispecific antibody according to one embodiment in an HL60-Lu orthotopic AML model (***=P<0.005). [Figure 28]FIG. 28 is a graph showing the results of FACS measurement of intramedullary tumor cells after administration of a bispecific antibody according to one embodiment. [Figure 29] FIG. 29 is an image showing the results of IHC staining of intramedullary tumor cells after administration of a bispecific antibody according to one embodiment. [Diagram 30] FIG. 30 is a graph showing the cytolytic activity of a bispecific antibody according to one embodiment in AML blasts. [Diagram 31] FIG. 31 is a graph showing the T cell activation activity of a bispecific antibody according to one embodiment in AML blasts. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] <Definition>

[0052] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall also include the plural and vice versa.

[0053] The term "antibody" in this application is used in the broadest sense and includes a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0054] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, although possible variant antibodies, including naturally occurring mutations or those that arise during production of a monoclonal antibody preparation, are generally present in small amounts. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation acts against a single determinant on an antigen.

[0055] The term "monospecific" antibody as used herein means an antibody with one or more binding sites, each of which binds to the same epitope of the same antigen. The term "bispecific" means that the antibody can specifically bind to at least two distinct antigenic determinants, e.g., two binding sites formed by a pair of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL), respectively, bind to different antigens or different epitopes on the same antigen. Such bispecific antibodies are in the 1+1 format. Other bispecific antibody formats are in the 2+1 or 1+2 format (containing two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope), or in the 2+2 format (containing two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). Typically, bispecific antibodies contain two antigen binding sites, each of which is specific for a different antigenic determinant from the other.

[0056] The term "valent" as used herein refers to the presence of a particular number of binding domains in an antibody or antibody fragment. Thus, the terms "monovalent", "bivalent", "tetravalent" and "hexavalent" refer to the presence of one, two, four and six binding domains in an antibody, respectively. A bispecific antibody according to the invention is at least "bivalent" and can be "trivalent" or "multivalent" (e.g., "tetravalent" or "hexavalent"). In a particular embodiment, an antibody of the invention has two or more binding sites and is bispecific. That is, an antibody may also be bispecific when more than one binding site is present (i.e., when the antibody is trivalent or multivalent).

[0057] The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a native antibody. "Native antibodies" refer to naturally occurring immunoglobulin molecules with diverse structures. For example, native IgG-class antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two light chains and two heavy chains disulfide-linked. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) called the variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3) called the heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) called the variable light domain or light chain variable domain, followed by a light chain constant domain (CL) called the light chain constant region. The heavy chain of an antibody can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0058] As mentioned above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL and VH domains of an antibody, or a subset of the complementarity determining regions (CDRs), combine to form the variable region that defines a three-dimensional antigen binding site. Such a quaternary antibody structure forms an antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three CDRs on each VH and VL chain (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In some cases, for example, when a given immunoglobulin molecule is derived from a camelid species or engineered based on camelid immunoglobulins, the complete immunoglobulin molecule can be composed of heavy chains alone without light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

[0059] The terms "CDR-H", "HCDR" and "CDRH" are used interchangeably herein to refer to the VH chain of a CDR (e.g., CDR-H1, HCDR1 and CDRH1 refer to the VH1 of a CDR). The terms "CDR-L", "LCDR" and "CDRL" are used interchangeably herein to refer to the VL chain of a CDR (e.g., CDR-L1, LCDR1 and CDRL1 refer to the VL1 of a CDR).

[0060] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen-binding domain are short non-contiguous amino acid sequences that are specifically positioned to form the antigen-binding domain as the antibody assumes a three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain, called the "framework" regions, show less intermolecular variability. The framework regions adopt a predominantly β-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. Thus, the framework regions act to form a scaffold that positions the CDRs in a correct orientation by interchain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. Such a complementary surface promotes non-covalent binding of the antibody to its cognate epitope. The amino acids which comprise the CDRs and framework regions for any given heavy or light chain variable region, respectively, can be readily identified by one of skill in the art since they have been precisely defined (see www.bioinf.org.uk: Dr. Andrew CR Martin's Group; "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196: 901-917 (1987)).

[0061] In the event that there are more than one definition for a term as used and / or accepted in the art, the definition of the term used herein is intended to include all of these meanings unless clearly stated to the contrary. As a specific example, the term "complementarity determining region" ("CDR") is used to describe the non-contiguous antigen binding sites found within all variable regions of heavy and light chain polypeptides. These specific regions are described in Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. MoI. Biol. 196: 901-917 (1987), the entire contents of which are incorporated herein by reference. The definitions of CDRs by Kabat and Chothia include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of the definitions to refer to the CDRs of an antibody or variants thereof is intended to fall within the scope of the term as defined and used herein. The appropriate amino acid residues which comprise the CDRs as defined by each of the above cited references are set forth in the table below (Table 1) for comparison. The exact residue numbers which comprise a particular CDR will vary depending on the sequence and size of the CDR. One of skill in the art can routinely determine whether a residue comprises a particular CDR given the variable region amino acid sequence of an antibody.

[0062] JPEG2024525916000002.jpg5281

[0063] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign the "Kabat numbering" system to any variable domain sequence without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system presented in Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0064] The antibodies disclosed herein can be from any animal source, including birds and mammals. Desirably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken.

[0065] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. As disclosed above, those skilled in the art will appreciate that the heavy chain constant region can be modified to vary the amino acid sequence from the naturally occurring immunoglobulin molecule.

[0066] The heavy chain constant regions of the antibodies disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide can comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived in part from an IgG3 molecule. As another example, the heavy chain constant region can comprise a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. As another example, the heavy chain portion can comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0067] As used herein, the term "light chain constant region" includes amino acid sequences derived from an antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.

[0068] "Light chain-heavy chain pair" refers to a collection of light and heavy chains that can form dimers through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0069] "Antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody, which includes a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv, scFv-Fc, scFab-Fc, diabody, minibody, scAb, dAb, half-IgG, or combinations thereof. The term "Fab" as used in Fab, Fab', F(ab')2, xFab, and scFab can include traditional Fab fragments and chimeric Fab-like domains as described in PCT / CN2018 / 106766 (Wuxibody), and can be derived from any mammal, including, but not limited to, human, mouse, rat, camelid, or rabbit. A functional portion of an antibody, such as one or more CDRs described herein, can be covalently linked to a second protein or small molecule compound, and can be used as a targeted therapeutic agent against a specific target. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or recombinant protein that acts like an antibody by binding to a specific antigen to form a complex.

[0070] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, as well as production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0071] Papain digestion of an intact antibody produces two identical antigen-binding fragments, called "Fab" fragments, containing the heavy and light chain variable domains, the constant domain of the light chain, and the first constant domain (CH1) of the heavy chain. Thus, as used herein, the term "Fab fragment" refers to an antibody fragment containing the VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments in that they contain several additional residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. Pepsin treatment produces an F(ab')2 fragment with two antigen-binding sites (two Fab fragments) and a portion of the Fc region. In the present application, the "F(ab')2 fragment" includes two light chains and two heavy chains including a variable region, a CH1, and a part of a constant region between the CH1 and CH2 domains, thereby forming an intrachain disulfide bond between the two heavy chains. Thus, the F(ab')2 fragment is composed of two Fab' fragments, and the two Fab' fragments are bound to each other by disulfide bonds between them.

[0072] The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which the variable or constant regions of the heavy and light chains have been exchanged. Two different chain compositions of crossover Fab molecules are possible and are included in the bispecific antibodies of the present invention, on the one hand, the variable regions of the Fab heavy and light chains are exchanged, i.e. the cross-Fab molecule contains a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1), and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). Such a cross-Fab molecule is also called CrossFab. (VLVH)On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the crossover Fab molecule contains a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1). Such a crossover Fab molecule is also called CrossFab. (CLCH1) It is called.

[0073] A "single chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, said antibody domains and said linker being arranged N-terminally towards the C-terminus in one of the following orders: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL, said linker being a polypeptide having at least 30 amino acids, preferably 32-50 amino acids. The single chain Fab fragment is stabilized by a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (eg, position 44 in the variable heavy chain and position 100 in the variable light chain according to the Kabat numbering).

[0074] A "crossover single chain Fab fragment" or "x-scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, said antibody domains and said linker having, from N-terminus to C-terminus, one of the following orders: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL, said VH and VL together forming an antigen binding domain that specifically binds to an antigen, said linker being a polypeptide having at least 30 amino acids. These x-scFab molecules can also be further stabilized by the creation of interchain disulfide bonds by the insertion of cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to the Kabat numbering).

[0075] An "Fv region" is an antibody that contains the variable regions of the heavy and light chains, but does not contain the constant region. An scFv is an Fv linked by a flexible linker. An scFv-Fc is an Fc linked to an scFv. A minibody is an scFv linked to a CH3. A diabody contains two molecules of an scFv. A "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some embodiments, the regions are linked with a short linker peptide having 10 to about 25 amino acids. The linker may be glycine-rich for flexibility or serine- or threonine-rich for solubility, and may link the N-terminus of the VH to the C-terminus of the VL, or vice versa. Such proteins retain the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of a linker. ScFv molecules are well known in the art and are described, for example, in US Patent No. 5,892,019.

[0076] A "short-chain antibody (scAb)" is a single polypeptide chain that contains one heavy chain variable region or a light chain constant region, in which the heavy and light chain variable regions are linked by a flexible linker. See, e.g., U.S. Patent No. 5,260,203, which is incorporated herein by reference.

[0077] A "domain antibody (dAb)" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In one embodiment, two or more VH regions are covalently linked by a peptide linker to form a bivalent domain antibody. The two VH regions of such a bivalent domain antibody can target the same or different antigens.

[0078] The term "full length IgG" according to the present invention is defined to include essentially the entire IgG, but it does not necessarily have all the functions of an intact IgG. For the avoidance of doubt, full length IgG includes two heavy chains and two light chains. Each chain includes a constant (C) and a variable (V) region, which are divided into domains designated CH1, CH2, CH3, VH, and CL, VL. IgG antibodies bind to antigens through the variable region domains contained in the Fab portion and can interact with cells and molecules of the immune system after binding through the constant domains and mostly through the Fc portion. The terms "variable region domain", "variable region", "variable domain", "VH / VL pair", "VH / VL", "Fab portion", "Fab arm", "Fab" or "arm" are used interchangeably herein. Full length antibodies according to the present invention include IgG molecules in which mutations that provide desired characteristics may be present. Such mutations should not result in the deletion of a substantial portion of any region. However, IgG molecules in which one or more amino acid residues have been deleted without essentially altering the binding properties of the resulting IgG molecule are included in the term "full-length IgG." For example, such IgG molecules may desirably have one or more deletions of 1-10 amino acid residues in the non-CDR regions, where the deletion of amino acids is not essential to the binding specificity of the IgG.

[0079] Full-length IgG antibodies are preferred because of their advantageous half-life and because they must remain as close as possible to the fully self (human) molecule for immunogenicity. According to the invention, bispecific IgG antibodies are used. In one preferred embodiment, bispecific full-length IgG1 antibodies are used. IgG1 is preferred because of its long circulating half-life in humans. To prevent any immunogenicity in humans, the bispecific IgG antibodies according to the invention are preferably human IgG1. The term "bispecific" (bs) means that one arm of the antibody binds to a first antigen while the second arm binds to a second antigen, where the first and second antigens are not the same. According to the invention, the first and second antigens are in fact two different molecules located on two different cell types. The term "one arm [of an antibody]" preferably means one Fab portion of a full-length IgG antibody. Bispecific antibodies, which recruit and activate endogenous immune cells to mediate cytotoxicity, are an emerging class of next-generation antibody therapeutics. This can be achieved by combining antigen binding specificities for target cells (i.e., tumor cells) and effector cells (i.e., T cells, NK cells, and macrophages) in one molecule (Cui et al. JBC 2012 (287) 28206-28214; Kontermann, MABS 2012 (4) 182-197; Chames and Baty, MABS 2009 (1) 539-547; Moore et al. Blood 2011 (117) 4542-4551; Loffler et al. 2000 Blood 95:2098; Zeidler et al. 1999 J. Immunol. 163:1246). According to the present invention, bispecific antibodies are provided in which one arm binds to a CLL-1 antigen on abnormal (tumor) cells, whereas a second arm binds to an antigen on immune effector cells.

[0080] As used herein, the term "antigen binding domain" or "antigen-binding site" refers to a portion of an antibody or antibody fragment that specifically binds to an antigenic determinant. More specifically, the term "antigen binding domain" refers to a portion of an antibody that specifically binds to a part or all of an antigen and includes a region complementary thereto. When an antigen is large, an antibody or antibody fragment can only bind to a specific portion of the antigen, and such a portion is called an epitope. An antigen binding domain can be provided, for example, by one or more variable domains (also called variable regions). Desirably, an antigen binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one embodiment, an antigen binding domain can bind to an antigen and block or partially block its function. Antigen binding domains that specifically bind to CLL-1 or CD3 include antibodies and fragments thereof as further defined herein. Antigen binding domains may also comprise scaffold antigen binding proteins, such as binding domains based on designed repeat proteins or designed repeat domains (see, for example, WO2002 / 020565).

[0081] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site (e.g., a conformational structure made up of a contiguous stretch of amino acids or a distinct region of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen conjugate. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, in serum-free blood, and / or in the extracellular matrix (ECM). Proteins useful as antigens herein may be any naturally occurring protein from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. In a particular embodiment, the antigen is a human protein. When referring to a particular protein herein, these terms include the "full-length," unprocessed protein as well as all forms of the protein that are processed and produced by the cell. These terms also include naturally occurring variants of the proteins, such as splice variants or allelic variants.

[0082] "Specific binding" means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of an antibody or antibody fragment to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as Surface Plasmon Resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329(2000)), and traditional binding analysis (Heeley, Endocr Res 28, 217-229(2002)).

[0083] "Affinity" or "binding affinity" refers to the strength of the total set of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally measured by the dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (koff and kon, respectively). Thus, equivalent affinities can include rate constants that are different from each other, as long as the ratio of the rate constants remains equal. Affinity can be measured by common methods known in the art, including the methods described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0084] As used herein, the term "high affinity" of an antibody refers to an antibody that has a high affinity for a target antigen. D to 10 -9 M or less, more specifically 10 -10 The term "low affinity" of an antibody means that the antibody has a K D to 10 -8 This means that it has M or more.

[0085] An "affinity matured" antibody refers to an antibody that has one or more modifications in one or more hypervariable regions (HVRs) that improve the affinity of the antibody for an antigen, compared to a parent antibody that does not contain such modifications.

[0086] The terms "bispecific antibody that specifically binds CLL-1 and CD3," "bispecific antibody specific for CLL-1 and CD3," or "anti-CLL-1 / anti-CD3 antibody" are used interchangeably herein and refer to a bispecific antibody that is capable of binding to CLL-1 and CD3 with sufficient affinity that the antibody targeted to CLL-1 and CD3 is useful as a diagnostic and / or therapeutic agent.

[0087] The term "C-type lectin-like molecule-1 (CLL-1)", also known as MICL or CLEC12A, is a type II transmembrane glycoprotein and a member of a large family of C-type lectin-like receptors involved in immune regulation. CLL-1 was previously identified in bone marrow-derived cells. The intracellular domain of CLL-1 contains an immunotyrosine-based inhibition motif (ITIM) and a YXXM motif. Phosphorylation of the ITIM-containing receptor in various cells leads to inhibition of activation pathways by recruitment of protein tyrosine phosphatases SHP-1, SHP-2, and SHIP. The YXXM motif harbors a potential SH2 domain binding site for the p85 subunit of PI-3 kinase, 13, which in conjunction with the cell activation pathway provides a potential dual role for CLL-1 as an inhibitory and activating molecule for bone marrow cells. Indeed, binding of CLL-1 to SHP-1 and SHP-2 was experimentally demonstrated in transfected bone marrow-derived cell lines.

[0088] CLL-1 has a restricted expression pattern in hematopoietic cells. It is specifically found in most AML blasts, as well as bone marrow cells derived from peripheral blood and bone marrow. Recent studies have also shown that CLL-1 is present in most leukemic stem cells in the CD34+ / CD38- compartment in AML, but is absent from CD34+ / CD38- cells in normal and regenerating bone marrow controls, which helps to distinguish leukemic stem cells from normal cells. (See, e.g., Zhao et al., Haematologica 95:71-78(2010); Bakker et al., Cancer Res.64:8443-8450(2004)). Nucleoside and protein sequences of CLL-1 for many species are known. For example, the human sequence can be found at Genbank accession number AF247788.1 and Uniprot accession number Q5QGZ9.

[0089] The terms "CLL-1 antibody," "antibody that binds CLL-1," and "antibody comprising an antigen binding domain that binds CLL-1" refer to an antibody that can bind to CLL-1, particularly a CLL-1 polypeptide expressed on a cell surface, with sufficient affinity that the antibody targeting CLL-1 is useful as a diagnostic and / or therapeutic agent. In one embodiment, the extent of binding of the anti-CLL-1 antibody to unrelated, non-CLL-1 proteins is less than about 10% of the binding of the antibody to CLL-1, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS) or surface plasmon resonance analysis using a biosensor system such as a Biacore system. In certain embodiments, an antigen binding protein that binds human CLL-1 has a binding affinity K for binding to human PD1 that is less than about 10% of the binding affinity K for binding to human PD1. D Values ​​of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 The term "anti-CLL-1 antibody" also includes bispecific antibodies capable of binding to CLL-1 and to a different antigen.

[0090] The terms "CLL-1" and "CLL1" are used interchangeably in this application.

[0091] The term "immune effector cell" or "effector cell" as used herein refers to a cell in the natural repertoire of mammalian immune system cells that can affect the viability of target cells when activated. Immune effector cells include lymphoid lineage cells, such as natural killer (NK) cells, T cells including cytotoxic T cells, or B cells, but also myeloid lineage cells, such as monocytes or macrophages, dendritic cells, and neutrophil granulocytes, can be considered as immune effector cells. Thus, the effector cells are preferably NK cells, T cells, B cells, monocytes, macrophages, dendritic cells, or neutrophil granulocytes. According to the present invention, recruiting effector cells to abnormal cells means that immune effector cells are located close to abnormal target cells so that the effector cells can directly kill or indirectly initiate the death of the recruited abnormal cells. To avoid non-specific interactions, it is preferred that the bispecific antibodies of the present invention specifically recognize antigens on immune effector cells that are at least overexpressed by these immune effector cells compared to other cells of the body. Target antigens present on immune effector cells can include CD3, CD16, CD25, CD28, CD64, CD89, NKG2D, and NKp46. Preferably, the antigen on immune effector cells is CD3 expressed on T cells, or a functional equivalent thereof (the functional equivalent can be a CD3-like molecule that is similarly distributed on T cells and has a similar function (related to type, not necessarily related to amount)). Also, as used herein, the term "CD3" includes functional equivalents of CD3. The most preferred antigen for immune effector cells is the CD3ε chain. This antigen has been shown to be highly effective in recruiting T cells to abnormal cells. Thus, the bispecific IgG antibodies of the present invention preferably include one arm that specifically recognizes CD3ε.

[0092] The term "CD3" refers to any native CD3 from any vertebrate source, including mammals, such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. The term includes all forms of CD3 that occur in cells through processing, as well as "full-length" unprocessed CD3. The term also includes naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, the CD3 is human CD3, specifically the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in UniProt (www.uniprot.org) accession no. P07766 (version 189), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. The amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3ε is shown in NCBI GenBank no. BAB71849.1.

[0093] The terms "anti-CD3 antibody," "antibody that binds CD3," and "antibody comprising an antigen-binding domain that binds CD3" refer to an antibody that can bind CD3 with sufficient affinity that the antibody targeted to CD3 is useful as a diagnostic and / or therapeutic agent. In one embodiment, the extent of binding of an anti-CD3 antibody to an unrelated, non-CD3 protein is less than about 10% of the binding of the antibody to CD3, e.g., as measured by radioimmunoassay (RIA). In certain embodiments, an antibody that binds CD3 has a dissociation constant (K D ) is ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10-8 M or less, e.g., 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 In certain embodiments, the anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3 from different species. Furthermore, the term "anti-CD3 antibody" includes bispecific antibodies that can bind to CD3 and a different antigen.

[0094] The term "mouse" antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from mouse germline immunoglobulin sequences. Murine antibodies of the present disclosure can include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0095] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remaining heavy and / or light chains are derived from a different source or species.

[0096] The "class" of an antibody refers to the type of constant domain or constant region possessed by the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the various classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0097] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will contain substantially all of at least one, and usually two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody and all or substantially all of the FRs correspond to the FRs of a human antibody.

[0098] A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g. a non-human antibody, refers to an antibody that has been humanized. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been further modified or altered from that of the original antibody to generate properties according to the invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0099] A "human" antibody is an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0100] The term "Fc domain" or "Fc region" as used herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain can vary slightly, the human IgG heavy chain Fc region generally extends from Cys226 or Pro230 to the carboxy-terminus of the heavy chain. However, antibodies produced by host cells can undergo post-translational truncation of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain can comprise a full-length heavy chain or a truncated variant of the full-length heavy chain (also referred to herein as a "truncated variant heavy chain"). This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (K447) may or may not be present. The amino acid sequence of a heavy chain comprising an Fc domain (or a subunit of an Fc domain as defined herein) is depicted herein without a C-terminal glycine-lysine dipeptide unless otherwise indicated. In one embodiment of the invention, a heavy chain comprising a subunit of an Fc domain as disclosed herein, comprised in an antibody or bispecific antibody according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to Kabat EU index). In one embodiment of the invention, a heavy chain comprising a subunit of an Fc domain as disclosed herein, comprised in an antibody or bispecific antibody according to the invention, comprises an additional C-terminal glycine residue (G446, numbering according to Kabat EU index). Compositions of the invention, such as pharmaceutical compositions described herein, comprise populations of antibodies or bispecific antibodies of the invention. The populations of antibodies or bispecific antibodies may comprise molecules with full-length heavy chains and molecules with truncated variant heavy chains.The antibody or bispecific antibody population can be composed of a mixture of molecules with full-length heavy chains and molecules with truncated mutant heavy chains, where at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the antibodies or bispecific antibodies have truncated mutant heavy chains. In one embodiment of the invention, a composition comprising an antibody or bispecific antibody population of the invention comprises an antibody or bispecific antibody comprising a heavy chain with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to Kabat EU index) and a subunit of the Fc domain disclosed herein. In one embodiment of the invention, a composition comprising an antibody or bispecific antibody population of the invention comprises an antibody or bispecific antibody comprising a heavy chain with an additional C-terminal glycine residue (G446, numbering according to Kabat EU index) and a subunit of the Fc domain disclosed herein. In one embodiment of the invention, such compositions comprise: a molecule comprising a heavy chain having an Fc domain subunit as disclosed herein; a molecule comprising a heavy chain having an Fc domain subunit as disclosed herein and an additional C-terminal glycine residue (G446, numbering according to the Kabat EU index); and a population of antibodies or bispecific antibodies comprising a molecule comprising a heavy chain having an Fc domain subunit as disclosed herein and an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the Kabat EU index). Unless otherwise specified in the present application, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also called EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991 (see also supra). As used herein, the term "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide having a C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association.For example, the subunits of the IgGFc domain include the IgGCH2 and IgGCH3 constant domains.

[0101] A "modification that promotes binding of the first and second subunits of the Fc domain" refers to a peptide backbone manipulation or post-translational modification of the Fc domain subunit that reduces or prevents binding of the same polypeptide to form homodimers with a polypeptide comprising the Fc domain subunit. Modifications that promote binding as used herein include individual modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are to be bound, in particular, where the modifications are complementary to each other and therefore promote binding of the two Fc domain subunits. For example, a modification that promotes binding can alter the structure or charge of one or both of the Fc domain subunits to make binding sterically or electrostatically favorable, respectively. Thus, (hetero)dimers can occur between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may not be identical in that additional components are fused to each subunit (e.g., antigen-binding moieties). In some embodiments, modifications that promote binding include amino acid mutations, particularly amino acid substitutions, in the Fc domain. In one particular embodiment, the binding enhancing modification comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.

[0102] The term "peptide linker" refers to a peptide that includes one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are well known in the art or are described herein. A suitable non-immunogenic linker peptide is, for example, (G4S) n , (SG4) n or G4 (SG4) na peptide linker, where "n" is generally a number between 1 and 10, usually between 2 and 4, specifically 2, i.e. the peptide is selected from the group consisting of GGGGS (SEQ ID NO:58), GGGGSGGGGS (SEQ ID NO:59), SGGGGSGGGG (SEQ ID NO:60) and GGGGSGGGGSGGGG (SEQ ID NO:61) and comprises the sequences GSPGSSSSGS (SEQ ID NO:62), GGGGSGGGGSGGGGS (SEQ ID NO:63), GSGSGSGS (SEQ ID NO:64), GSGSGNGS (SEQ ID NO:65), GGSGSGSG (SEQ ID NO:66), GGSGSG (SEQ ID NO:67), GGSG (SEQ ID NO:68), GGSGNGSG (SEQ ID NO:69), GGNGSGSG (SEQ ID NO:70) and GGNGSG (SEQ ID NO:71).

[0103] The term "effector functions" refers to biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immunoconjugate-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0104] As used herein, the terms "engineer, engineered, engineer" are intended to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes not only modifications of the amino acid sequence, glycosylation patterns, or side groups of individual amino acids, but also combinations of such approaches.

[0105] The term "amino acid mutation" as used herein is meant to include substitution, deletion, insertion and modification of amino acids. Any combination of substitution, deletion, insertion and modification can be used to arrive at the final structure, provided that the final structure has the desired properties, e.g., decreased binding to Fc receptors or increased binding to other peptides. Deletion and insertion of amino acid sequences include deletion of amino and / or carboxy termini and insertion of amino acids. Particular amino acid mutations are amino acid substitutions. For example, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid that has different structural and / or chemical properties, are particularly desirable in order to alter the binding properties of the Fc region. Amino acid substitutions include replacement of non-naturally occurring amino acids or naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is believed that methods other than genetic recombination, such as methods of changing the side chain group of an amino acid by chemical modification, can also be useful. Various designations can be used herein to indicate the same amino acid mutation. For example, a proline to glycine substitution at position 329 of the Fc domain can be represented as 329G, G329, G329, P329G, or Pro329Gly.

[0106] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the ratio of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence, after aligning the sequences and, if necessary, incorporating gaps to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the art, for example, using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes of this application, amino acid sequence identity (%) values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c or later versions, with the BLOSUM50 comparison matrix. The FASTA program package is described in WWR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; WR Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et. al. (1997) Genomics 46:24-36, and is publicly available at http: / / fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml.Alternatively, sequences can be compared using the public server accessible at http: / / fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch(globalprotein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2) so that a global alignment is performed rather than a local alignment. The percent amino acid identity (%) is provided in the output alignment header.

[0107] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent.

[0108] As used herein, the term "polypeptide" is intended to include the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule that includes monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any one of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with well-known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides can be derived from natural or biological sources or produced by recombinant technology, but are not necessarily translated from a specified nucleic acid sequence. It can be produced in any manner, including chemical synthesis. The term "polypeptide" also includes variants of polypeptides and derivatives of polypeptides. Furthermore, "polypeptide fragment" refers to a polypeptide having an amino-terminal amino acid sequence deletion, a carboxy-terminal amino acid sequence deletion, and / or an internal deletion compared to the full-length protein. Such a fragment can also contain modified amino acids compared to the full-length protein. In one embodiment, the fragment can be about 5-900 amino acids in length, for example at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or more amino acids in length. For purposes of the present invention, useful polypeptide fragments include immunologically functional fragments of antibodies that contain an antigen-binding domain.In the case of CLL-1 or CD3 binding antibodies, such useful fragments include, but are not limited to, the CDR sequences of one, two or three heavy or light chains, or all or a portion of an antibody chain including the variable or constant regions of a heavy or light chain.

[0109] As used herein, a "variant" of a polypeptide, e.g., antigen-binding fragment, protein, or antibody, is a polypeptide in which one or more amino acid residues have been inserted, deleted, added, and / or substituted relative to another polypeptide sequence, including fusion polypeptides. Protein variants also include those modified by enzymatic cleavage, phosphorylation, or other post-translational modifications, but which retain the biological activity of the antibodies disclosed herein, e.g., specific binding to CLL-1 and biological activity. A variant may be about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical to the sequence of an antibody or antigen-binding fragment thereof disclosed herein.

[0110] As used herein, the term "derivative" of a polypeptide refers to a polypeptide that has been chemically modified through conjugation with another chemical moiety, and is distinct from a mutant having an insertion, deletion, addition, or substitution.

[0111] As used herein, the term "recombinant" with respect to a polypeptide or polynucleotide refers to a form of a polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which is one that can be formed by combining polynucleotides or polypeptides that do not normally occur together.

[0112] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which can be aligned for purposes of comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, preferably less than 25% identity, with any one of the sequences of the present disclosure.

[0113] That a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a given percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that when the two sequences are compared and aligned, that percentage of bases (or amino acids) are identical.

[0114] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), virally derived RNA, or plasmid DNA (pDNA). A polynucleotide can contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0115] An "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention. Additional examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides that have been purified (partially or substantially) in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain the polynucleotide molecule, but where the polynucleotide molecule is present extrachromosomally or at a chromosomal location that differs from the natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the invention, as well as positive and negative stranded forms and double stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include synthetically produced molecules. Polynucleotides or nucleic acids may also be or include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators. The term "isolated" as used herein means that a nucleic acid or peptide is substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA technology, or is substantially free of chemical precursors or other chemicals if chemically synthesized. The term "isolated" is also used herein to indicate that a cell or polypeptide is isolated from other cellular proteins or tissues. An isolated polypeptide is meant to include both purified and recombinant polypeptides.

[0116] An "isolated polynucleotide (or nucleic acid) encoding [e.g., an antibody or bispecific antibody of the invention]" refers to one or more polynucleotide molecules encoding antibody heavy and light chains (or fragments thereof), including the polynucleotide molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present in one or more locations in a host cell.

[0117] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide with a series of defined nucleic acid elements that allow for transcription of a particular nucleic acid in a target cell. A recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plasmid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector specifically includes the nucleic acid sequence to be transcribed and a promoter. In some embodiments, the expression cassette includes a polynucleotide sequence encoding an antibody or bispecific antibody of the invention or a fragment thereof.

[0118] The term "vector" or "expression vector" refers to a DNA molecule used to introduce and express a particular gene operably linked to it in a cell. The term includes vectors as self-replicating nucleic acid structures as well as vectors introduced by incorporation into the genome of a host cell. The expression vector of the present invention comprises an expression cassette. The expression vector allows for the transcription of large amounts of stable mRNA. When the expression vector is present inside a cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding an antibody or bispecific antibody of the present invention or a fragment thereof.

[0119] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and its derived progeny, regardless of the number of transfers. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as selected or selected from the originally transformed cell are included herein. A host cell is any type of cell system that can be used to produce an antibody or bispecific antibody of the invention. Host cells include cultured cells, such as mammalian cultured cells, such as HEK cells, CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, as well as transgenic animals, transgenic plants, or cultured plants or cells contained within animal tissues. An "activating Fc receptor" is an Fc receptor that, upon binding to the Fc domain of an antibody, induces a signaling event that stimulates the receptor-bearing cell to perform an effector function. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0120] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism in which antibody-coated target cells are lysed by immune effector cells. The target cells are cells to which an antibody or derivative thereof containing an Fc region specifically binds, typically via a protein moiety at the N-terminus of the Fc region. As used herein, the term "ADCC reduction" is defined as a reduction in the number of target cells lysed in a given time at a given antibody concentration in the medium surrounding the target cells by the ADCC mechanism as defined above, or an increase in the antibody concentration in the medium surrounding the target cells required to effect lysis of a given number of target cells in a given time by the ADCC mechanism. The ADCC is reduced compared to ADCC mediated by the same antibody produced by the same type of host cell using the same standard production, purification, formulation and storage methods (well known to those skilled in the art), but not engineered. For example, ADCC mediated by an antibody containing an amino acid substitution in the Fc domain that reduces ADCC is reduced compared to ADCC mediated by the same antibody without such amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, PCT International Publication No. WO2006 / 082515 or PCT International Publication No. WO2012 / 130831).

[0121] An "effective amount" of an agent means the amount needed to effect a physiological change in a cell or tissue to which the agent is administered.

[0122] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, means an amount effective at administration for a period of time necessary to achieve a desired therapeutic or prophylactic result. For example, a therapeutically effective amount of an agent may, for example, eliminate, reduce, delay, minimize, or prevent the deleterious effects of a disease.

[0123] An "individual," "subject," or "patient" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, rats, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual, subject, or patient is a human.

[0124] The term "pharmaceutical composition" refers to a formulation in which the biological activity of the active ingredients contained therein is effective and which does not contain additional ingredients that are unacceptably toxic to a subject to which the composition is administered.

[0125] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0126] As used herein, "treatment" (and grammatical variations thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, which can be performed either prophylactically or during clinical pathology. Positive effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of a disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or alleviation of the disease state, and amelioration or improvement of prognosis. In some embodiments, the antibodies or bispecific antibodies of the invention are used to delay the onset of disease or slow the progression of a disease.

[0127] The term "package insert" refers to guidelines customarily included in commercial packages of therapeutic drugs, which contain information on the indications, usage, dosage, administration, concomitant therapy, restrictions and / or warnings pertaining to the use of that therapeutic drug.

[0128] <Anti-CLL-1 antibody>

[0129] The anti-CLL-1 antibody is a CLL-1 targeting moiety and can include an anti-CLL-1 antibody or an antigen-binding fragment thereof, which can exhibit potent binding and inhibitory activity against CLL-1 and can be useful in therapeutic and diagnostic applications.

[0130] In one embodiment, the anti-CLL-1 antibody or fragment thereof may have specificity for human CLL-1 protein.

[0131] In one embodiment, the anti-CLL-1 antibody or fragment thereof may comprise: (a) a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3; (b) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 5, and 6; (c) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, and 11; (d) a VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, and 15; (e) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, and 18; and (f) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, and 22.

[0132] The anti-CLL-1 CDR sequences contained in the heavy and light chain variable regions of an antibody or antigen-binding fragment according to one embodiment of the invention are shown in Table 2 below.

[0133] JPEG2024525916000003.jpg158122

[0134] In one embodiment, the CDRs of each light chain variable region and each heavy chain variable region disclosed in the table (Table 2) can be freely combined. In some embodiments, the antibody or fragment thereof can include no more than 1 substitution, no more than 2 substitutions, or no more than 3 substitutions.

[0135] In one embodiment, the anti-CLL-1 antibody or fragment thereof may comprise a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 and 24; or a peptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 and 24.

[0136] In one embodiment, the anti-CLL-1 antibody or fragment thereof may comprise a light chain constant region comprising the amino acid sequence consisting of SEQ ID NO:55.

[0137] In one embodiment, the anti-CLL-1 antibody or fragment thereof may comprise a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:23 and 24, and a light chain constant region comprising an amino acid sequence consisting of SEQ ID NO:55.

[0138] In one embodiment, the anti-CLL-1 antibody or fragment thereof can comprise a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 74; or a peptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 74.

[0139] In one embodiment, the anti-CLL-1 antibody or fragment thereof can comprise a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, and 75; or a peptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, and 75.

[0140] In one embodiment, the anti-CLL-1 antibody or fragment thereof can comprise a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 74; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, and 75.

[0141] In one embodiment, the anti-CLL-1 antibody or fragment thereof can comprise a heavy chain comprising an amino acid sequence consisting of SEQ ID NO:45; or a peptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence consisting of SEQ ID NO:45.

[0142] In one embodiment, the anti-CLL-1 antibody or fragment thereof may comprise a light chain comprising an amino acid sequence consisting of SEQ ID NO:46; or a peptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence consisting of SEQ ID NO:46.

[0143] In one embodiment, the anti-CLL-1 antibody or fragment thereof can comprise a heavy chain comprising the amino acid sequence consisting of SEQ ID NO:45; and a light chain comprising the amino acid sequence consisting of SEQ ID NO:46.

[0144] In one embodiment, the heavy chain constant region, the heavy and light chain variable regions, the heavy and light chains of the antibody or antigen-binding fragment can be illustrated in the table below (Table 3).

[0145] JPEG2024525916000004.jpg245162JPEG2024525916000005.jpg241162

[0146] In another embodiment, the heavy and light chain variable regions disclosed in the above table (Table 3) can be freely combined to produce various types of antibodies.

[0147] The heavy and light chain variable regions disclosed herein can be bound to a variety of targeting heavy and light chain constant regions to form intact antibody heavy and light chains, respectively, and the heavy and light chain sequences bound to such constant regions, respectively, can also be combined to form intact antibody structures.

[0148] Any variable region of the heavy and light chains of an antibody can be linked to at least a portion of a constant region. The constant region can be selected depending on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity are required. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not have cytotoxicity. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. For example, the heavy chain variable region can be bound to the constant region of IgG, such as IgG1, IgG2, IgG2a, IgG2b, IgG3, and IgG4, and the light chain variable region can be bound to a kappa or lambda constant region. In the case of the constant region, an appropriate one can be used as needed, for example, one derived from a human or mouse. In one embodiment, a human heavy chain constant region IgG1 is used. In another embodiment, a human lambda region can be used as the light chain constant region.

[0149] Any of the variable regions disclosed herein can be combined with a constant region to form heavy and light chain sequences. In one embodiment, a heavy chain variable region disclosed herein can be combined with a human IgG1 constant region to form a full-length heavy chain. In another embodiment, a light chain variable region disclosed herein can be combined with a human lambda constant region to form a full-length light chain. Light and heavy chains can be combined in a variety of combinations to form an intact antibody composed of two light chains and two heavy chains.

[0150] In another embodiment, the antibody can comprise or consist essentially of a combination of heavy and light chains, which are represented by the sequences SEQ ID NO:45 and SEQ ID NO:46.

[0151] However, these constant region sequences in combination with the variable regions disclosed herein are exemplary, and one of skill in the art will recognize that other constant regions may be used, including IgG1 heavy chain constant regions, IgG3 or IgG4 heavy chain constant regions, any kappa or lambda light chain constant regions, constant regions modified for stability, expression, manufacturability or other targeting properties, etc.

[0152] In some embodiments, the antigen-binding fragment of an anti-CLL-1 antibody may be any fragment containing the heavy chain CDRs and / or the light chain CDRs of the antibody, for example, but not limited to, it may be selected from the group consisting of Fab, Fab', F(ab')2, xFab, Fd (containing the heavy chain variable region and the CH1 domain), Fv (heavy chain variable region and / or light chain variable region), single chain Fv (scFv; containing or consisting essentially of the heavy chain variable region and the light chain variable region of any order, and a peptide linker between the heavy chain variable region and the light chain variable region), single chain antibody, disulfide-linked Fv (sdFv), scFab (single chain Fab), scFab-Fc (containing scFab and an Fc region), half IgG (containing one light chain and one heavy chain), and the like.

[0153] The present invention can include one or more amino acid sequences that have substantial sequence identity to one or more of the amino acid sequences disclosed herein, where substantial identity means that the effects of the disclosure are maintained even in the presence of sequence variations.

[0154] In some embodiments, the anti-CLL-1 antibody or antigen-binding fragment thereof can be a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0155] In one embodiment, the anti-CLL-1 antibody or antigen-binding fragment thereof can be fused to a polypeptide(s) to form a fusion protein. The polypeptide(s) can be an antibody or an antigen. In one embodiment, the fusion protein can be in the form of a multispecific antibody (e.g., a bispecific antibody).

[0156] In one embodiment, the present disclosure provides a fusion protein comprising (a) one or more single domain antibodies or antigen-binding fragments thereof described herein (e.g., one or more CDRs described herein), and (b) one or more additional polypeptides. For example, the fusion protein can comprise one or more single domain antibodies or antigen-binding fragments thereof described herein and a constant region or Fc region described herein. In one embodiment, one or more single domain antibodies or antigen-binding fragments thereof described herein (e.g., one or more CDRs described herein) can be non-covalently or covalently conjugated, e.g., fused, to an antibody or antigen.

[0157] <Anti-CD3 antibody>

[0158] The anti-CD3 antibody is a CD3 targeting moiety and can include an anti-CD3 antibody or an antigen-binding fragment thereof, which can exhibit potent binding and inhibitory activity against CD3 and can be useful in therapeutic and diagnostic applications.

[0159] In one embodiment, the anti-CD3 antibody or fragment thereof may have specificity for the human CD3 protein, preferably the human CD3E polypeptide.

[0160] In one embodiment, the anti-CD3 antibody or fragment thereof may comprise (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 47; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 48; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 50; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 51; and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0161] In one embodiment, the CDRs of each of the light chain variable regions and the CDRs of each of the heavy chain variable regions disclosed above can be freely combined.

[0162] In some embodiments, the antibody or fragment thereof can include no more than 1 substitution, no more than 2 substitutions, or no more than 3 substitutions.

[0163] In one embodiment, the anti-CD3 antibody or fragment thereof can comprise a heavy chain comprising an amino acid sequence consisting of SEQ ID NO:53 or a peptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence consisting of SEQ ID NO:53, and a light chain comprising an amino acid sequence consisting of SEQ ID NO:54 or a peptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence consisting of SEQ ID NO:54.

[0164] In another embodiment, the heavy and light chain variable regions disclosed above can be freely combined to produce antibodies of various types.

[0165] The heavy and light chain variable regions disclosed herein can be bound to a variety of targeting heavy and light chain constant regions to form intact antibody heavy and light chains, respectively, and the heavy and light chain sequences bound to such constant regions can also be combined to form intact antibody structures.

[0166] Any variable region of the heavy and light chains of an antibody can be linked to at least a portion of a constant region. The constant region can be selected depending on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity are required. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not have cytotoxicity. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. For example, the heavy chain variable region can be bound to the constant region of IgG, for example, IgG1, IgG2, IgG2a, IgG2b, IgG3, and IgG4, and the light chain variable region can be bound to a kappa or lambda constant region. In the case of the constant region, an appropriate one can be used as needed, for example, one derived from a human or mouse. In one embodiment, a human heavy chain constant region IgG1 is used. In another embodiment, a human lambda region can be used as the light chain constant region.

[0167] Any of the variable regions disclosed herein can be combined with a constant region to form heavy and light chain sequences. In one embodiment, a heavy chain variable region disclosed herein can be combined with a human IgG1 constant region to form a full-length heavy chain. In another embodiment, a light chain variable region disclosed herein can be combined with a human lambda constant region to form a full-length light chain. Light and heavy chains can be combined in a variety of combinations to form an intact antibody composed of two light chains and two heavy chains.

[0168] In another embodiment, the antibody can comprise or consist essentially of a combination of heavy and light chains, which are represented by the sequences SEQ ID NO:53 and SEQ ID NO:54.

[0169] However, these constant region sequences in combination with the variable regions disclosed herein are exemplary, and one of skill in the art will recognize that other constant regions can be used, including IgG1 heavy chain constant regions, IgG3 or IgG4 heavy chain constant regions, any kappa or lambda light chain constant regions, constant regions modified for stability, expression, manufacturability or other targeting properties, etc.

[0170] In some embodiments, the antigen-binding fragment of an anti-CD3 antibody may be any fragment containing the heavy and / or light chain CDRs of the antibody, for example, but not limited to, it may be selected from the group consisting of Fab, Fab', F(ab')2, xFab, Fd (containing the heavy chain variable region and the CH1 domain), Fv (heavy chain variable region and / or light chain variable region), single chain Fv (scFv; containing or consisting essentially of the heavy chain variable region and the light chain variable region of any order, and a peptide linker between the heavy chain variable region and the light chain variable region), single chain antibody, disulfide-linked Fv (sdFv), scFab (single chain Fab), scFab-Fc (containing scFab and an Fc region), half IgG (containing one light chain and one heavy chain), and the like.

[0171] The present invention includes one or more amino acid sequences that have substantial sequence identity to one or more of the amino acid sequences disclosed herein, meaning that the effects of the disclosure are maintained even in the presence of sequence variations.

[0172] In one embodiment, the present disclosure provides a fusion protein comprising (i) one or more single domain antibodies or antigen-binding fragments thereof described herein (e.g., one or more CDRs described herein), and (b) one or more additional polypeptides. For example, the fusion protein can comprise one or more single domain antibodies or antigen-binding fragments thereof described herein and a constant region or Fc region described herein. In one embodiment, one or more single domain antibodies or antigen-binding fragments thereof described herein (e.g., one or more CDRs described herein) can be non-covalently or covalently joined, e.g., fused, to an antibody or antigen.

[0173] <Anti-CLL-1 / anti-CD3 bispecific antibody>

[0174] The anti-CLL-1 antibody / anti-CD3 bispecific antibody may comprise an anti-CLL-1 antibody, or an antigen-binding fragment thereof, and an anti-CD3 antibody, or an antigen-binding fragment thereof. The anti-CLL-1 antibody / anti-CD3 bispecific antibody may be useful for therapeutic and diagnostic applications.

[0175] In one embodiment, the bispecific antibody comprises a CLL-1 targeting moiety and a CD3 targeting moiety.

[0176] In one embodiment, the anti-CLL-1 antibody, or antigen-binding fragment thereof, and the anti-CD3 antibody, or antigen-binding fragment thereof, can be fused to each other directly or via a peptide linker.

[0177] In one embodiment, the anti-CLL-1 antibody, or antigen-binding fragment thereof, and the anti-CD3 antibody, or antigen-binding fragment thereof, can each independently be a Fab molecule.

[0178] In one embodiment, the Fab molecule can be a human Fab molecule or a chimeric Fab-like domain comprising a TCR constant region. In one embodiment, the Fab molecule can be chimeric or humanized.

[0179] In one embodiment, (a) an anti-CLL-1 antibody or antigen-binding fragment thereof can be fused to the N-terminus of the Fab heavy chain of an anti-CD3 antibody or antigen-binding fragment thereof at the C-terminus of the Fab heavy chain of the anti-CLL-1 antibody or antigen-binding fragment thereof, or (b) an anti-CD3 antibody or antigen-binding fragment thereof can be fused to the N-terminus of the Fab heavy chain of an anti-CLL-1 antibody or antigen-binding fragment thereof at the C-terminus of the Fab heavy chain of the anti-CD3 antibody or antigen-binding fragment thereof.

[0180] In one embodiment, the anti-CLL-1 / anti-CD3 bispecific antibody can comprise an Fc domain comprising a first subunit and a second subunit.

[0181] In one embodiment, the Fc domain can be a human Fc domain with or without additional mutations, including, but not limited to, the N297A mutation, which has no ADCC activity, or the knob-into-hole (KIH) mutation, which promotes correct antibody pairing.

[0182] In one embodiment, the anti-CLL-1 antibody or antigen-binding fragment thereof and the anti-CD3 antibody or antigen-binding fragment thereof can each be a Fab molecule, and (a) the anti-CD3 antibody or antigen-binding fragment thereof can be fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain of the anti-CD3 antibody or antigen-binding fragment thereof, and (b) the anti-CLL-1 antibody or antigen-binding fragment thereof can be fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain of the anti-CLL-1 antibody or antigen-binding fragment thereof.

[0183] In one embodiment, the anti-CLL-1 antibody or antigen-binding fragment thereof may be a first anti-CLL-1 antibody or antigen-binding fragment thereof and may further comprise a second anti-CLL-1 antibody or antigen-binding fragment thereof.

[0184] In one embodiment, the first anti-CLL-1 antibody or antigen-binding fragment thereof, the anti-CD3 antibody or antigen-binding fragment thereof, and, if present, the second anti-CLL-1 antibody or antigen-binding fragment thereof, can each be a Fab molecule; (a) the anti-CD3 antibody or antigen-binding fragment thereof can be fused at the C-terminus of the Fab heavy chain of the anti-CD3 antibody or antigen-binding fragment thereof to the N-terminus of the Fab heavy chain of the first anti-CLL-1 antibody or antigen-binding fragment thereof, and the first anti-CLL-1 antibody or antigen-binding fragment thereof can be fused at the C-terminus of the Fab heavy chain of the first anti-CLL-1 antibody or antigen-binding fragment thereof to the N-terminus of the first subunit of the Fc domain. or (b) a first anti-CLL-1 antibody, or antigen-binding fragment thereof, can be fused at the C-terminus of the Fab heavy chain of the first anti-CLL-1 antibody, or antigen-binding fragment thereof, to the N-terminus of the Fab heavy chain of the anti-CD3 antibody, or antigen-binding fragment thereof, and the anti-CD3 antibody, or antigen-binding fragment thereof, can be fused at the C-terminus of the Fab heavy chain of the anti-CD3 antibody, or antigen-binding fragment thereof, to the N-terminus of the first subunit of the Fc domain, and a second anti-CLL-1 antibody, or antigen-binding fragment thereof, if present, can be fused at the C-terminus of the Fab heavy chain of the second anti-CLL-1 antibody, or antigen-binding fragment thereof, to the N-terminus of the second subunit of the Fc domain.

[0185] In one embodiment, the first anti-CLL-1 antibody or antigen-binding fragment thereof, the anti-CD3 antibody or antigen-binding fragment thereof and the second anti-CLL-1 antibody or antigen-binding fragment thereof are each a Fab molecule, the first anti-CLL-1 antibody or antigen-binding fragment thereof is fused at the C-terminus of the Fab heavy chain of the first anti-CLL-1 antibody or antigen-binding fragment thereof to the N-terminus of the Fab heavy chain of the anti-CD3 antibody or antigen-binding fragment thereof, the anti-CD3 antibody or antigen-binding fragment thereof is fused at the C-terminus of the Fab heavy chain of the anti-CD3 antibody or antigen-binding fragment thereof to the N-terminus of the first subunit of the Fc domain, and the second anti-CLL-1 antibody or antigen-binding fragment thereof, if present, is fused at the C-terminus of the Fab heavy chain of the second anti-CLL-1 antibody or antigen-binding fragment thereof to the N-terminus of the second subunit of the Fc domain. With the shielded CD3 binding site in such a structure, the bispecific antibody is able to strongly induce T cell activation and cytokine expression of IFN-γ and IL-2, but weakly induce CRS-associated cytokines such as TNF-α and IL-6, thus demonstrating less off-tumor toxicity.

[0186] In one embodiment, the first anti-CLL-1 antibody, or antigen-binding fragment thereof, and the second anti-CLL-1 antibody, or antigen-binding fragment thereof, may be identical to one another.

[0187] In one embodiment, the bispecific antibody is capable of simultaneously binding to the activating T cell antigens CLL-1 and CD3. In one embodiment, the bispecific antibody is capable of crosslinking the T cell and the target cell by simultaneously binding to CLL-1 and CD3. In one embodiment, such simultaneous binding results in lysis of the target cell, particularly a CLL-1 expressing tumor cell. In one embodiment, such simultaneous binding results in activation of the T cell. In another embodiment, such simultaneous binding results in a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, the cellular response being selected from the group of proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers.

[0188] In one embodiment, the bispecific antibody is capable of redirecting the cytotoxic activity of a T cell to a target cell. In certain embodiments, the redirection is independent of MHC-mediated peptide antigen presentation by the target cell and / or the specificity of the T cell. Specifically, the T cell according to any embodiment of the invention is a cytotoxic T cell. In some embodiments, the T cell is a CD4 + or CD8 + T cells, specifically CD8+ T cells.

[0189] In one embodiment, a bispecific antibody according to an embodiment is capable of inducing cytokine expression, granzyme B and / or perforin in the presence of U937 and HL-60 cell lines.

[0190] <Conjugate>

[0191] The invention provides immunoconjugates in which the anti-CLL-1 antibodies or anti-CLL-1 / anti-CD3 bispecific antibodies described herein are conjugated (chemically linked) to one or more therapeutic agents, such as a cytotoxic agent, chemotherapeutic agent, drug, growth inhibitory agent, toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof) or a radioisotope.

[0192] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), in which an antibody is conjugated to one or more of the therapeutic agents described above. The antibody is typically linked to one or more therapeutic agents using a linker. An overview of ADC technology, including examples of therapeutic agents and drugs and linkers, is provided in Pharmacol Review 68:3-19 (2016).

[0193] In another embodiment, the immunoconjugate is selected from the group consisting of diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria The toxins may include the antibodies described herein conjugated to enzymatically active toxins or fragments thereof, including, but not limited to, C. officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecenes.

[0194] In another embodiment, the immunoconjugate can include a radioconjugate formed by conjugating an antibody described herein to a radioactive atom. A variety of radioisotopes are available for the production of radioconjugates. For example, At 211 , I 131 , I 125 , Y 90 , Re 186 , Re188 , Sm 153 , Bi 212 , P 32 , Pb 212and radioisotopes of Lu. When radial conjugates are used for detection, they can include radioactive nuclear agents for scintigraphic studies, such as tc99m or I123, and spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0195] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexanecarboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate hydrochloride), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug inside the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); US Pat. No. 5,208,020) can be used.

[0196] Immunoconjugates or ADCs of the present application may be, but are not limited to, conjugates prepared using crosslinker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which is commercially available (e.g., Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0197] Also conjugable cytotoxic agents include, for example, pyrrolobenzodiazepines (PBDs), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), camptothecin, doxorubicin, cisplatin, verapamil, fluorouracil, oxaliplatin, daunorubicin, irinotecan, topotecan, paclitaxel, carboplatin, gemcitabine, methotrexate, docetaxel, acivicin, aclarubicin, acodazole, acronisine, adzelesin, alanosine, aldesleukin, allopurinol sodium, altretamine, aminoglutethimide, amonafide, ampligen, amsacrine, androgen, anguidine, aphidicolin glycinate, asarly, asparaginase, 5-azacytidine, azathioprine, BCG (Bacillus Calmette-Guerin, Baker's Antifol, β-2-deoxythioguanosine, Bisantrene hydrochloride, Bleomycin sulfate, Busulfan, Buthionine sulfoximine, BWA773U82, BW502U83 / HCl, BW7U85 mesylate, Caracemide, Carbetimer, Carboplatin, Carmustine, Chlorambucil, Chloroquinoxaline sulfonamide sulfonamide), chlorozotocin, chromomycin A3, cisplatin, cladribine, corticosteroids, Corynebacterium parvum, CPT-11, crisnatol, cyclocytidine, cyclophosphamide, cytarabine, sitembena, davismaleate, dacarbazine, dactinomycin, daunorubicin hydrochloride, deazauridine, dexrazoxane, dianhydrogalactitol, diaziquone, dibromodulcitol, didemnin B, diethyldithiocarbamic acid, diglycoaldehyde, dihydro-5-azacytidine, echinomycin, edatrexate, edelfosine, eflornithine, Elliott's solutionsolution), elsamitrucin, esorubicin, estramustine phosphate, estrogen, etanidazole, etiophos, etoposide, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavone acetic acid, floxuridine, fludarabine phosphate, 5-fluorouracil, Fluosol TM, flutamide, gallium nitrate, gemcitabine, goserelin acetate, hepsulfame, hexamethylene bisacetamide, homoharringtonine, hydrazine sulfate, 4-hydroxyandrostenedione, hydroxyurea, idarubicin hydrochloride, ifosfamide, interferon alpha, interferon beta, interferon gamma, interleukin-1alpha and interleukin-1beta, interleukin-3, interleukin-4, interleukin-6,4-Ipomeanol, iproplatin, isotretinoin, leucovorin calcium, leuprolide acetate, levamisole, liposomal daunorubicin, liposomal daunorubicin, lomustine, lonidamine, maytansine, mechlorethamine hydrochloride, melphalan, menogaril, melbarone, 6-mercaptopurine, Bacillus Calmette-Guerin calmette-guerin), methotrexate, N-methylformamide, mifepristone, mitoguazone, mitomycin C, mitotane, mitoxantrone hydrochloride, monocyte-macrophage colony-stimulating factor, nabilone, nafoxidine, neocarzinostatin, octreotide acetate, ormaplatin, oxaliplatin, paclitaxel, N-(phosphonoacetyl)-L-aspartic acid (PALA), pentostatin, piperazinedione, pipobroman, pirarubicin, piritrexim, piroxantrone hydrochloride, PIXY-321, plicamycin, porfimer sodium, prednimustine, procarbazine, progestin, pirazofurin, lazo xanthane, sargramostim, semustine, spirogermanium, spiromustine, streptonigrin, streptozocin, sulofenur, suramin sodium, tamoxifen, taxotere, tegafur, teniposide, terephthalamidine, teroxylon, thioguanine, thiotepa, thymidine injection, tiazofurin, topotecan, toremifene, tretinoin, trifluoperazine hydrochloride, trifluridine, trimetrexate, tumor necrosis factor (TNF), uracil mustard, vinblastine sulfate, vincristine sulfate, vindesine, vinorelbine, vinzolidine, Yoshi 864, zorubicin, pharma- ceutical acceptable salts thereof, and mixtures thereof.

[0198] <Glycosylation mutants>

[0199] In certain embodiments, the antibodies provided herein can be altered to increase or decrease the extent to which the antibody is glycosylated. Adding or deleting glycosylation sites to an antibody can be accomplished simply by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0200] If the antibody comprises an Fc region, the oligosaccharide attached thereto can be altered. Natural antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide, generally attached by an N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide can comprise a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention can be made to form antibody variants with certain improved properties.

[0201] In one embodiment, antibody variants are provided that have nonfucosylated oligosaccharides, i.e., oligosaccharide structures lacking fucose attached (directly or indirectly) to the Fc region. Such nonfucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are N-linked oligosaccharides lacking the fucose residue attached to the first GlcNAc, particularly in the stem of the biantennary oligosaccharide structure. In one embodiment, antibody variants are provided that have an increased proportion of nonfucosylated oligosaccharides in the Fc region compared to the native or parent antibody. For example, the proportion of nonfucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). The proportion of nonfucosylated oligosaccharides is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g., complexes, hybrids and their mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO2006 / 082515. Asn297 refers to the asparagine residue located at about position 297 (EU numbering of Fc region residues) in the Fc region, however, Asn297 can also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, depending on minor sequence changes in the antibody. Such antibodies with an increased proportion of nonfucosylated oligosaccharides in the Fc region can have improved FcγRIIIa receptor binding and / or improved effector function, particularly ADCC function. See, for example, US2003 / 0157108; US2004 / 0093621.

[0202] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US2003 / 0157108; and WO2004 / 056312, especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107), or cells with reduced or eliminated activity of GDP-fucose synthesis or transporter proteins (see, e.g., US2004259150, US2005031613, US2004132140, US2004110282).

[0203] In a further embodiment, antibody variants are provided that have bisected oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechno Biotechn Bioeng 117, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO99 / 54342; WO2004 / 065540, WO2003 / 011878.

[0204] Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants can have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087; WO1998 / 58964; and WO1999 / 22764.

[0205] <Fcドメイン>

[0206] In certain embodiments, an antibody or bispecific antibody of the invention may comprise an Fc domain consisting of a first subunit and a second subunit, it being understood that the features of the Fc domain described herein in relation to an antibody or bispecific antibody may equally be applied to the Fc domain comprised in the antibody of the invention.

[0207] The Fc domain of an antibody or bispecific antibody may be composed of a pair of polypeptide chains comprising the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, with each subunit comprising the CH2 and CH3 IgG heavy chain constant domains. The two subunits of an Fc domain can stably bind to each other. In one embodiment, an antibody or bispecific antibody of the invention comprises no more than one Fc domain.

[0208] In one embodiment, the Fc domain of the antibody or bispecific antibody may be an IgG Fc domain. In a further particular embodiment, the Fc domain may be a human Fc domain.

[0209] <Fc domain modifications that promote heterodimerization>

[0210] The antibody or bispecific antibody according to the invention can contain various antigen-binding moieties, which can be fused to one or the other of the two subunits of the Fc domain, so that the two subunits of the Fc domain are typically contained in two non-identical polypeptide chains. Recombinant co-expression of such polypeptides and subsequent dimerization leads to many possible combinations of the two polypeptides. To improve the yield and purity of the antibody or bispecific antibody during recombinant production, it is advantageous to introduce modifications to the Fc domain of the antibody or bispecific antibody that facilitate the binding of the polypeptide of interest.

[0211] Thus, in a particular embodiment, the Fc domain of an antibody or bispecific antibody according to the invention may comprise a modification that facilitates binding of the first and second subunits of the Fc domain. The most extensive protein-protein interaction site between the two subunits of a human IgG Fc domain is present in the CH3 domain of the Fc domain. Thus, in one embodiment, said modification may be present in the CH3 domain of the Fc domain.

[0212] There are many approaches for modification of the CH3 domain of an Fc domain to effect heterodimerization, well described, for example, in WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012058768, WO2013157954, WO2013096291. Typically, in all such approaches, the CH3 domain of the first Fc domain subunit and the CH3 domain of the second Fc domain subunit are both engineered in a complementary manner such that each CH3 domain (or the heavy chain containing it) can no longer homodimerize with itself, but heterodimerize with the other CH3 domain that has been engineered in a complementary manner (thus, the first and second CH3 domains are heterodimerized and no homodimers are formed between the two first or second CH3 domains, respectively). These various approaches to improve heavy chain heterodimerization are considered as various alternatives in combination with heavy-light chain modifications (e.g., VH and VL exchanges / substitutions in one binding arm and introduction of oppositely charged amino acid substitutions at the CH1 / CL interface) that reduce heavy / light chain mispairing and Bence Jones by-products in antibodies or bispecific antibodies.

[0213] In one particular embodiment, the modification that promotes binding of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which comprises a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain.

[0214] The knob-into-hole technology is described, for example, in U.S.Pat.Nos.5,731,168;7,695,936;Ridgway et al.,Prot Eng9,617-621(1996) and Carter, J Immunol Meth 248,7-15(2001). Generally, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, positioning the protrusion in the cavity to promote the formation of heterodimers and prevent the formation of homodimers. The protrusion is created by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A compensatory cavity having the same or similar size as the protrusion is formed by replacing large amino acid side chains at the interface of the second polypeptide with smaller side chains (e.g., alanine or threonine).

[0215] <Modification of the Fc domain that reduces Fc receptor binding and / or effector function>

[0216] The Fc domain confers suitable pharmacokinetic properties, including a long serum half-life that contributes to a favorable tissue-blood distribution ratio and excellent accumulation in the target tissue, to an antibody or bispecific antibody. However, at the same time, it can lead to the undesirable result that the antibody or bispecific antibody targets cells that express Fc receptors rather than the desired antigen-bearing cells. Furthermore, co-activation of the Fc receptor signaling pathway can induce cytokine release, which, in combination with the long half-life of the antibody or bispecific antibody (e.g., in embodiments of bispecific antibodies where the second antigen-binding moiety binds to an activated T cell antigen), can lead to excessive activation of cytokine receptors and serious side effects upon systemic administration. Activation of (Fc receptor-bearing) immune cells other than T cells can reduce the efficacy of bispecific antibodies (specifically, bispecific antibodies where the second antigen-binding moiety binds to an activated T cell antigen) by potential destruction by T cells, such as NK cells.

[0217] Thus, in a particular embodiment, the Fc domain of an antibody or bispecific antibody according to the invention may exhibit reduced binding affinity to an Fc receptor and / or reduced effector function compared to a native IgG1 Fc domain. In one such embodiment, the Fc domain (or an antibody or bispecific antibody comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the binding affinity to an Fc receptor compared to a native IgG1 Fc domain (or an antibody or bispecific antibody comprising a native IgG1 Fc domain), and / or less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the effector function compared to a native IgG1 Fc domain (or an antibody or bispecific antibody comprising a native IgG1 Fc domain). In one embodiment, the Fc domain (or an antibody or bispecific antibody comprising said Fc domain) does not substantially bind to an Fc receptor and / or does not induce effector function. In one particular embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor may be a human Fc receptor. In one embodiment, the Fc receptor may be an activating Fc receptor. In one particular embodiment, the Fc receptor may be an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI or FcγRIIa, most particularly human FcγRIIIa. In one embodiment, the effector function may be one or more selected from the group of CDC, ADCC, ADCP and cytokine secretion. In one particular embodiment, the effector function may be ADCC. In one embodiment, the Fc domain may exhibit a substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to a native IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or an antibody or bispecific antibody comprising said Fc domain) exhibits more than about 70%, particularly more than about 80%, more particularly more than about 90% of the binding affinity of the native IgG1 Fc domain (or an antibody or bispecific antibody comprising the native IgG1 Fc domain) to FcRn.

[0218] In certain embodiments, the Fc domain can be engineered to have reduced binding affinity to the Fc receptor and / or reduced effector function compared to a non-engineered Fc domain. In certain embodiments, the Fc domain of an antibody or bispecific antibody can include one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to the Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutations can reduce the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination can reduce the binding affinity of the Fc domain to the Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, an antibody or bispecific antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor compared to an antibody or bispecific antibody comprising a non-engineered Fc domain. In a particular embodiment, the Fc receptor can be an Fcγ receptor. In some embodiments, the Fc receptor can be a human Fc receptor.

[0219] In some embodiments, the Fc receptor may be an activating Fc receptor. In one particular embodiment, the Fc receptor may be an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI or FcγRIIa, most particularly human FcγRIIIa. Desirably, binding to each of these receptors is reduced. In some embodiments, the binding affinity to complement components is also reduced, in particular the binding affinity to C1q. In one embodiment, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e., preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or an antibody or bispecific antibody comprising said Fc domain) exhibits more than about 70% of the binding affinity of a non-engineered form of the Fc domain (or an antibody or bispecific antibody comprising a non-engineered form of the Fc domain) to FcRn. The Fc domain, or a bispecific antibody of the invention comprising said Fc domain, may exhibit more than about 80%, even more than about 90%, of such affinity. In certain embodiments, the Fc domain of the bispecific antibody can be engineered to have reduced effector function compared to a non-engineered Fc domain. Reduced effector function can include, but is not limited to, any one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immunoconjugate-mediated antigen uptake by antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling to induce apoptosis, reduced cross-linking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, the reduced effector function can be any one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment, the reduced effector function can be reduced ADCC.In one embodiment, the ADCC reduction may be less than 20% of the ADCC induced by the non-engineered Fc domain (or a bispecific antibody comprising a non-engineered Fc domain).

[0220] In one embodiment, the amino acid mutation that reduces the binding affinity and / or effector function of the Fc domain to an Fc receptor can be an amino acid substitution. In one embodiment, the Fc domain can comprise an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numbering according to the Kabat EU index). In a more specific embodiment, the additional amino acid substitution can be E233P, L234A, L235A, L235E, N297A, N297D or P331S, preferably L234A or / and L235A.

[0221] <Compositions, formulations and routes of administration>

[0222] In an additional aspect, the invention provides a pharmaceutical composition comprising any of the antibodies or bispecific antibodies, e.g., for use in any one of the therapeutic methods described below. In one embodiment, the pharmaceutical composition comprises any of the antibodies or bispecific antibodies provided herein and a pharma- ceutical acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the antibodies or bispecific antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

[0223] Also provided is a method of producing an antibody or bispecific antibody of the invention in a form suitable for in vivo administration, said method comprising the steps of: (a) obtaining an antibody or bispecific antibody according to the invention; and (b) formulating the antibody or bispecific antibody with at least one pharma- ceutically acceptable carrier, thereby formulating the antibody or bispecific antibody formulation for in vivo administration.

[0224] The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of the antibody or bispecific antibody dissolved or dispersed in a pharma- ceutically acceptable carrier. The term "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that are normally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce any harmful, allergic or other adverse reactions when administered to animals, if, for example, preferably humans. The preparation of pharmaceutical compositions containing an antibody or bispecific antibody and, optionally, additional active ingredients, will be known to those of skill in the art in light of this disclosure, as exemplified in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Furthermore, it will be understood that if administered to animals (e.g., humans), the formulation must meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards or the appropriate government of other countries. A preferred composition is a lyophilized formulation or an aqueous solution. As used herein, "a pharma- ceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonicity agents, absorption retardants, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and the like, and combinations thereof known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except in the event that any existing carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0225] The antibodies or bispecific antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and, for localized treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., injections, e.g., intravenous or subcutaneous injections, depending in part on whether administration is brief or chronic.

[0226] Parenteral compositions include those designed for administration by injection, e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection. For injection, the antibody or bispecific antibody of the invention can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, e.g., Hanks' solution, Ringer's solution, or physiological saline buffer. The solution can contain formulatory agents, e.g., suspending, stabilizing and / or dispersing agents. Alternatively, the antibody or bispecific antibody can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the antibody or bispecific antibody of the invention in the required amount in an appropriate solvent with various other ingredients as enumerated below, as required. Sterilization can be readily accomplished, for example, by filtration through sterile filtration membranes. In general, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and / or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred preparation method is vacuum drying or freeze-drying technology to obtain a powder of the active ingredient and any additional desired ingredient from a liquid medium that has already been sterile filtered. The liquid medium should be appropriately buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose before injection. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination must be kept to a minimum, such as at a safe level, e.g., less than 0.5 ng / mg protein.Suitable pharma- ceutically acceptable carriers include buffers, such as phosphate, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, and the like. Injectable agents include, but are not limited to, glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG). Aqueous injection suspensions can contain compounds which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran, and the like. Optionally, the suspension can also contain suitable stabilizers or agents which increase the solubility of the active compounds so that highly concentrated solutions can be prepared. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes. Pharmaceutical compositions containing the antibody or bispecific antibody of the invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliary agents that facilitate processing of the protein into a pharma-ceutically usable preparation. Appropriate formulations will vary depending on the chosen route of administration.

[0227] Treatment Methods and Compositions

[0228] Any of the antibodies or bispecific antibodies provided herein can be used in therapeutic methods. The antibodies or bispecific antibodies of the invention can be used as immunotherapeutic agents, for example, in the treatment of cancer.

[0229] When used in a method of treatment, the antibodies or bispecific antibodies of the invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to the physician.

[0230] In one aspect, an antibody or bispecific antibody of the invention is provided for use as a medicament. In an additional aspect, an antibody or bispecific antibody of the invention is provided for use in the treatment of a disease. In certain embodiments, an antibody or bispecific antibody of the invention is provided for use in a method of treatment.

[0231] In one embodiment, the invention provides an antibody or bispecific antibody described herein for use in treating a disease in an individual in need thereof. In certain embodiments, the invention provides an antibody or bispecific antibody for use in a method of treating an individual having a disease, comprising administering to the individual a therapeutically effective amount of the antibody or bispecific antibody. In one embodiment, the disease may be cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, for example an anti-cancer agent where the disease being treated is cancer. In additional embodiments, the invention provides an antibody or bispecific antibody described herein for use in inducing lysis of target cells, particularly tumor cells. In certain embodiments, the invention provides an antibody or bispecific antibody for use in a method of inducing lysis of target cells, particularly tumor cells, in an individual, comprising administering to the individual an effective amount of the antibody or bispecific antibody to induce lysis of the target cells. In any of the above embodiments, the "individual" is a mammal, preferably a human.

[0232] In one embodiment, the disease to be treated can be cancer.

[0233] In one embodiment, the cancer can be a solid cancer or a hematological cancer.

[0234] In one embodiment, the cancer is leukemia, rectal cancer, endometrial cancer, nephroblastoma, basal cell carcinoma, nasopharyngeal carcinoma, bone tumor, esophageal cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular thyroid carcinoma, hepatocellular carcinoma, oral cancer, renal cell carcinoma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, mesenchymal tumor, soft tissue sarcoma, liposarcoma, gastrointestinal stromal tumor, malignant peripheral nerve sheath tumor (MPNST), Ewing's sarcoma, leiomyosarcoma, mesenchymal chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, teratoma, neuroblastoma, medulloblastoma, glioma, benign skin tumor, Burkitt's lymphoma, mantle cell lymphoma, diffuse large intestine, pulmonary arterial tumor, pulmonary arterial tumor, pulmonary sarcoma ... The cancer may be selected from the group consisting of: DLBCL, follicular lymphoma, marginal zone lymphoma, neuroectodermal tumors, epithelial tumors, cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), pancreatic cancer, hematopoietic malignancies, renal cancer, tumor vasculature, breast cancer, renal cancer, ovarian cancer, epithelial ovarian cancer, gastric cancer, liver cancer, lung cancer, colon cancer, pancreatic cancer, skin cancer, bladder cancer, testicular tumor, uterine cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, adrenal cancer.

[0235] In one embodiment, the leukemia may be selected from the group consisting of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML) and acute myeloid leukemia (AML), preferably AML.

[0236] In one embodiment, the cancer can be a CLL-1 expressing cancer.

[0237] Those skilled in the art will readily recognize that in many cases an antibody or bispecific antibody will not provide a cure, but may only provide a partial benefit. In some embodiments, a physiological change that has some benefit is also considered to be therapeutically beneficial. Thus, in some embodiments, the amount of antibody or bispecific antibody that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount." The subject, patient, or individual in need of treatment is typically a mammal, more particularly a human.

[0238] In some embodiments, an effective amount of an antibody or bispecific antibody of the invention is administered to a cell, hi other embodiments, a therapeutically effective amount of an antibody or bispecific antibody of the invention is administered to an individual to treat a disease.

[0239] The appropriate dosage of the antibody or bispecific antibody of the invention for the prevention or treatment of a disease (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease to be treated, the route of administration, the weight of the patient, the type of antibody or bispecific antibody, the severity and course of the disease, whether the antibody or bispecific antibody is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, the clinical history of the patient, and response to the antibody or bispecific antibody, and the discretion of the attending physician. The physician responsible for administration will in any event determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for the individual subject. A variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time points, bolus administration, and pulse infusion.

[0240] The antibody or bispecific antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 ng / kg to 100 mg / kg of the antibody or bispecific antibody can be administered to the patient.

[0241] The present disclosure will now be described in more detail with reference to examples.

[0242] The following examples are merely illustrative of the present disclosure and are not to be construed as limiting the present disclosure.

[0243] Example 1: Production of anti-CLL-1 antibody

[0244] <1.1. Monoclonal antibody screening by mouse immunization>

[0245] To produce anti-CLL-1 antibodies, mice were immunized to screen for monoclonal antibodies.

[0246] Briefly, two groups of mice (SJL and Balb / c mice, Charles River Laboratories, Hollister, CA) were immunized with a mixture of human and cynomolgus CLL1 and N-terminal human IgG1Fc fusion proteins (produced by the mice themselves, and the amino acid sequences of the antigens are shown in Table 4). Through ELISA (enzyme-linked immunosorbent assay) screening and FACS (fluorescence-activated cell sorting) analysis, three hybridoma clones were screened for human and cynomolgus CLL1 cross-reactive clones. The three clones were 16C6, 33C2 and 84A2 from immunized Balb / c mice. All clones also positively bound to CLL1-expressing cell lines (U937, HL60) and CLL-1-overexpressing cell lines (cynomolgus CLL1 and human CLL1 overexpressed in HEK293E cells).

[0247] JPEG2024525916000006.jpg128154

[0248] <1.2. Sequence analysis of monoclonal antibodies from hybridoma cells and chimerization of antibodies>

[0249] Monoclonal antibodies from hybridoma cells were sequenced to sequence 16C6, 33C2 and 84A2 from the group of Balb / c mice immunized in Example 1.1.

[0250] Briefly, murine 16C6, 33C2 and 84A2 total RNA was isolated from hybridoma cells according to the technical manual of Trizol Reagent (Ambion, Cat. No.: 15596-026). Then, the total RNA was reverse transcribed into cDNA through RT-PCR using isotype-specific antisense primers or universal primers. VH (variable heavy chain) and VL (variable light chain) antibody fragments were amplified using rapid amplification of cDNA ends (RACE) method. The amplified antibody fragments were cloned into cloning vectors and then sequenced. The variable regions and CDRs of the three CLL1 positive clones are shown in Tables 5, 6 and 7. In the tables, HCDR is the CDR of the heavy chain and LCDR is the CDR of the light chain.

[0251] JPEG2024525916000007.jpg91152

[0252] JPEG2024525916000008.jpg91152

[0253] JPEG2024525916000009.jpg90152

[0254] To generate chimeric antibodies, the VH and VL of the murine antibodies were combined with the human IgG1 heavy chain constant region and the human kappa light chain constant region, respectively, and cloned into an expression vector. The constant regions of the chimeric antibodies were modified by introducing one or more mutations or modifications to human IgG1, such as N297A (also referred to as "NA"), which has no ADCC activity. The three modified chimeric antibodies of the three clones were then further modified to remove N-glycosylation residues or posttranslational modifications (PTMs) using site-directed mutagenesis. The modified sequences of 16C6, 33C2, and 84A2 described above are shown in Tables 8, 9, and 10. Hereinafter, ch16C6 refers to a chimeric antibody of 16C6 or 16C6(NA) comprising a heavy chain having an N297A mutation, ch33C2 refers to a chimeric antibody of 33C2 or 33C2(NA) comprising a heavy chain having an N297A mutation, and ch84C2 refers to a chimeric antibody of 84C2 or ch84A2(NA / N12S) comprising a heavy chain having an N297A mutation and a light chain having an N12S mutation.

[0255] JPEG2024525916000010.jpg200152

[0256] JPEG2024525916000011.jpg195152

[0257] JPEG2024525916000012.jpg200152

[0258] <1.3. Antibody humanization>

[0259] To humanize the chimeric antibody of Example 1.2, homology modeling was performed.

[0260] Briefly, through homology modeling, modeled structures of murine antibodies were obtained and the residues required for back mutation were analyzed. Human acceptor frameworks for VH and VL with maximum sequence identity with mouse counterparts were selected. Then, the complementarity determining regions (CDRs) of murine antibodies were grafted onto the human acceptor framework. Rational reverse mutation design of the grafted antibodies was then performed. The designed humanized antibodies were produced and their binding properties were evaluated. Among the murine and chimeric clones, 16C6 and 33C2 were selected as lead candidate antibodies. In particular, the humanized antibodies with CDRs of the chimeric antibodies (i.e., further modifications to remove PTM or N-glycosylation residues) are named 16C6(M14) and 33C2(M12). The sequences of the humanized heavy and light chains of 16C6, 16C6(M14), 33C2 and 33C2(M12) are shown in Tables 11-14. Hereinafter, hu16C6 and hu16C6(M14) refer to the humanized antibodies 16C6 and 16C6(M14), respectively, and hu33C2 and 33C2(M12) refer to the humanized antibodies 33C2 and 33C2(M12), respectively.

[0261] JPEG2024525916000013.jpg129152

[0262] JPEG2024525916000014.jpg91152

[0263] JPEG2024525916000015.jpg149149

[0264] JPEG2024525916000016.jpg91149

[0265] Example 2. Evaluation of activity of anti-CLL-1 monospecific antibodies

[0266] <2.1. Ligand binding activity test (ELISA)>

[0267] The antibody candidates of Example 1.2 were analyzed for comparative ligand binding activity by ELISA using huCLL1-His and hFc-huCLL1 as ligands.

[0268] Briefly, antigen (ligand) was coated overnight at 4°C. The antigen-coated plate was blocked with 1% BSA dissolved in PBS for 2 hours at 4°C and incubated with antibody for 2 hours at 4°C. To determine the EC50 (nM) of each antibody, antibody solutions were prepared at 50nM to 0.2pM concentrations in 4-fold serial dilutions, and the diluted antibodies were treated in each well, and the plate was washed with 1XPBST. Then, a chlorine anti-human IgG F(ab')2 cross-adsorbed secondary antibody conjugated with HRP (horseradish peroxidase) (Thermo, 31414) was added to each well and incubated for 1 hour at 4°C. After washing, TMB (3,3',5,5'-tetramethylbenzidine) substrate (Sigma, T0440) was added to each well, and the OD at 450 nm was measured with an ELISA plate reader. The results are shown in Figures 1 and 2.

[0269] FIG. 1 is a graph showing the ligand binding activity of three chimeric antibodies according to one embodiment using huCLL1-His as the ligand.

[0270] FIG. 2 is a graph showing the ligand binding activity of three chimeric antibodies according to one embodiment using hFc-huCLL1 as the ligand.

[0271] As shown in Figures 1 and 2, it was confirmed that all three chimeric antibodies according to one embodiment strongly bound to both human CLL1-His and hFc-human CLL1 antigens.

[0272] The antibody candidates of Example 1.2 were then analyzed to compare their ligand binding activity by ELISA using hFc-cynomolgus CLL at 150 ng / well and 100 ng / well as the ligand in the same manner as described above. The anti-CLL-1 antibody 6E7 from Genentech was used as a reference. The results are shown in Figures 3 and 4.

[0273] FIG. 3 is a graph showing the ligand binding activity of three chimeric antibodies according to one embodiment using 150 ng / well of hFc-cynomolgus CLL as the ligand.

[0274] FIG. 4 is a graph showing the ligand binding activity of three chimeric antibodies according to one embodiment using 100 ng / well of hFc-cynomolgus CLL1 as the ligand.

[0275] As shown in Figures 3 and 4, all three chimeric antibodies according to one embodiment were found to strongly bind to hFc-cynomolgus CLL1 regardless of antigen concentration. In addition, all three chimeric antibodies according to one embodiment were found to have stronger ligand binding activity than antibody 6E7 manufactured by Genentech.

[0276] The ligand binding activities of the chimeric antibody of Example 1.2. and the humanized antibody of Example 1.3. were compared in a manner similar to that described above, and the results are shown in Figures 5, 6 and 7.

[0277] FIG. 5 is a graph showing the ligand binding activity of chimeric and humanized antibodies hu16C6 according to one embodiment.

[0278] FIG. 6 is a graph showing the ligand binding activity of a chimeric antibody and humanized antibody hu33C2 according to one embodiment.

[0279] FIG. 7 is a graph showing the ligand binding activity of various humanized antibody hu33C2, according to one embodiment.

[0280] As shown in Figures 5 to 7, the humanized antibody according to one embodiment exhibits binding affinity equivalent to that of the chimeric antibody according to one embodiment. These results demonstrate the advantage of the humanized antibody according to one embodiment, since humanization of murine or chimeric antibodies usually reduces or even loses binding affinity.

[0281] <2.2. Cell binding activity test (FACS)>

[0282] To assess cell binding properties, the chimeric antibody candidates from Example 1.2. were analyzed on CLL1-negative cells (HEK293E and Jurkat) and various CLL1-expressing cancer cells using FACS.

[0283] Briefly, cells were incubated with antibodies for 1 hour at 4°C. First, cells were washed with assay buffer (1% BSA in PBS). Then, anti-human IgGFc conjugated with FITC (Fluorescein isothiocyanate) (Sigma, F9512) was added to each well and incubated for 1 hour at 4°C. After washing, the MFI (Median Fluorescence Intensity) of FITC was measured with a BD FACS Calibur (BD Biosciences). The results are shown in Table 15 and Figure 8.

[0284] JPEG2024525916000017.jpg85163

[0285] FIG. 8 is a graph showing the cell binding activity of a chimeric antibody according to one embodiment in CLL1-negative cells and various CLL1-expressing cancer cells. As shown in Table 15 and FIG. 8, all of the chimeric antibodies according to one embodiment were confirmed to have binding potency in various CLL1-expressing cell lines.

[0286] The cell binding properties of the three chimeric antibodies of Example 1.2 were then evaluated in the CLL1-expressing tumor cell lines HL60 and U937 using FACS in a manner similar to that described above, and the results are shown in Figures 9 and 10.

[0287] FIG. 9 is a graph showing the cell binding activity of a chimeric antibody according to one embodiment in HL60.

[0288] FIG. 10 is a graph showing the cell binding activity of a chimeric antibody according to one embodiment in U937.

[0289] As shown in Figures 9 and 10, it was confirmed that all the chimeric antibodies according to one embodiment have binding ability to the leukemia cell lines, and in particular, the ch16C6 and ch33C2 clones have excellent binding ability.

[0290] The cell binding properties of the three chimeric antibodies of Example 1.2. were then evaluated in CynoCLL1_HEK293E using FACS in a similar manner as described above. The anti-CLL-1 antibody 6E7 from Genentech was used as a reference. The results are shown in Figure 11.

[0291] FIG. 11 is a graph showing the cell binding activity of a chimeric antibody according to one embodiment in HEK293E overexpressing cynomolgus CLL-1.

[0292] As shown in Figure 11, all the chimeric antibodies according to one embodiment were confirmed to have higher binding ability than 6E7 to HEK293E cells overexpressing cynomolgus monkey CLL-1. In addition, all three chimeric antibodies according to one embodiment were confirmed to have higher cell binding activity than 6E7, an antibody manufactured by Genentech.

[0293] To compare the antigen-binding properties of the chimeric antibody of Example 1.2. and the humanized candidate substance of Example 1.3., the antibodies were analyzed using the PL21 cell line in a similar manner as described above, and the results are shown in Figures 12 and 13.

[0294] FIG. 12 is a graph showing the cell binding activity of a chimeric antibody according to one embodiment and various humanized antibodies hu33C2.

[0295] FIG. 13 is a graph showing the cell binding activity of a chimeric antibody according to one embodiment and various humanized antibodies hu16C6.

[0296] As shown in Figures 12 and 13, the humanized antibody according to one embodiment exhibited cell binding activity equivalent to that of the chimeric antibody according to one embodiment.

[0297] 2.3. Evaluation of antibody-dependent cellular cytotoxicity (ADCC)

[0298] To assess the in vitro activity of an anti-CLL-1 antibody according to one embodiment in tumor cell killing by effector-mediated immunity, an ADCC reporter bioassay kit (Promega, G7102) was used.

[0299] Briefly, target cells (HEK293-huCLL-1, HEK293, which is a cell line expressing exogenous human CLL-1) were plated in 96-well flat white bottom plates in assay buffer (RPMI1640 with 0.5% low IgGFBS) on the day of the experiment. Then, serial dilutions of antibodies were added to the plates. ADCC effector cells were added to each well, and the plates were incubated at 37°C in a CO2 incubator for 6 hours. After incubation, the plates were left to stand at room temperature for about 10 minutes, and then Bio-Glo luciferase assay reagent (Promega, G7940) was added to each well. The luminescence level was measured with a PHERAster FS BMG LABTECH to analyze the degree of ADCC induction. The dose-response curve was fitted with a 4-parameter model using GraphPad Prism 8. The results are shown in Figure 14.

[0300] FIG. 14 is a graph showing ADCC of ch84A2, hu16C6 and hu33C2 according to one embodiment.

[0301] As shown in Figure 14, it was confirmed that antibodies hu16C6 (VH1 / VL1 (M14)), hu33C2 (VH3 / VL6 (M12)), and ch84A2 (N12S) according to one embodiment induce ADCC by effector cells. In addition, the EC50 of hu16C6 (VH1 / VL1 (M14)), hu33C2 (VH3 / VL6 (M12)), and ch84A2 (N12S) were 130.1 pM, 140.5 pM, and 416.5 pM, respectively.

[0302] <2.4. Evaluation of cytotoxic activity of ADC (Antibody Drug Conjugation)>

[0303] To evaluate the cytotoxicity of ADCs containing the chimeric antibody of Example 1.2, various ADCs were produced using the chimeric antibody and their cytostatic activity was analyzed.

[0304] Briefly, the valine (V) at position 205 of the existing antibody light chain (according to Kabat numbering, which also applies below) was mutated to cysteine ​​(C), and reacted with a reducing agent, such as dithiothreitol (DTT), to generate a thiol group on the antibody light chain V205C (V205CT), and the antibody was conjugated to the drug through a thioether bond generated between the thiol group and the drug. Hereinafter, the three ADSs prepared for each chimeric antibody are referred to as "ch16C6(V205C)-T-AB009", "ch33C2(V205C)-T-AB009", and "ch84A2(V205C)-T-AB009". In addition, the anti-CLL-1 antibody 6E7 manufactured by Genentech was used as a reference, and an ADC of 6E7 was prepared in a similar manner (hereinafter referred to as "6E7(N54A / V205C)-T-AB009").

[0305] The cancer cell proliferation inhibitory activity of the prepared ADCs was then measured using commercially available cancer cell lines (EOL-1, THP-1 cell lines (ATCC)). 5,000 cells of each cancer cell line were sorted into each well of a 96-well plate. After culturing for 24 hours, they were treated with ADCs at 5 to 100,000 pM concentrations (3-fold serial dilution) as shown in Table 16. After 6 days, the number of live cells was measured using WST-8 (Dojindo Molecular Technology Inc.) dye. The results are shown in Table 17, Figure 15, and Figure 16.

[0306] JPEG2024525916000018.jpg87156

[0307] JPEG2024525916000019.jpg33157

[0308] FIG. 15 is a graph showing the cytostatic activity of an ADC according to an embodiment at EOL-1.

[0309] FIG. 16 is a graph showing the cytostatic activity of an ADC according to an embodiment in THP-1.

[0310] As shown in Table 17, Figures 15 and 16, anti-CLL-1 monoclonal antibodies 16C6, 33C2 and 84A2 were confirmed to have improved ability to kill cancer cells compared to 6E7 in EOL-1 and THP-1 cell lines.

[0311] Example 3. Preparation of anti-CLL-1 / anti-CD3 bispecific antibody

[0312] The anti-CLL-1 clone hu33C2 (VH3 / VL6(M12)) prepared in Example 1.3 and the CD3 clone disclosed in PCT / CN2018 / 106618 were selected to prepare an anti-CLL-1 / anti-CD3 bispecific antibody. The bispecific antibody was produced by the WuXiBody generation method disclosed in PCT / CN2018 / 106766.

[0313] Briefly, the DNA fragment of VLA-CL was inserted into a linearized vector containing the CMV promoter and human light chain signal peptide. The DNA fragment of VHB-CH1(33C2)-(G4S)3-VHA-CH1(CD3) or VHB-CH1(33C2)-(G4S)2-VHA-CH1(CD3) was inserted into a linearized vector containing human IgG1 constant region CH2-CH3 with knob mutations. The DNA fragment of VHB-CH1 was inserted into a linearized vector containing human IgG1 constant region CH2-CH3 with a cavity and N297A mutation. All vectors contain the CMV promoter and human antibody heavy chain signal peptide. The DNA fragment of VHB-CL was inserted into a linearized vector containing the CMV promoter and human light chain signal peptide. Here, VLA-CL is the light chain variable domain (VLA) of an anti-CD3 antibody-light chain constant domain (CL) of an anti-CD3 antibody, VHB-CH1 is the heavy chain variable domain (VHB) of an anti-CLL-1 antibody-first constant domain (CH1) of the heavy chain of an anti-CLL-1 antibody, G4S is a peptide linker composed of the amino acid sequence GGGGS, VHA-CH1 is the heavy chain variable domain (VHB) of an anti-CD3 antibody-first constant domain (CH1) of the heavy chain of an anti-CD3 antibody, and VHB-CL is the heavy chain variable domain (VHB) of an anti-CLL-1 antibody-light chain constant domain (CL) of an anti-CLL-1 antibody.

[0314] The DNA ratio for expression of anti-CD3 partial light chain: anti-CD3 partial heavy chain: anti-CLL1 partial heavy chain: anti-CLL1 partial light chain was 4:1:1:2. Expi293F cells with viability >95% 2.94 x 10 6 / mL was prepared in 300 mL of cell culture medium. Plasmid DNA and ExpiFectamine TMThe 293 reagent was mixed and then added to the cell culture medium. The cell cultures were cultured on a platform shaker at a rotation speed of 150 rpm. The temperature was maintained at 37°C and the CO2 level was maintained at 8%. After 6 days of culture, the cells were pelleted by centrifugation at 4000 rpm at 25°C for 10 minutes. The supernatant was collected for purification and gel electrophoresis. The supernatant was analyzed by NuPAGE. TM Following the instructions for 4%-12% Bis-Tris Protein Gels (ThermoFisher), the gels were loaded onto SDS-PAGE gels and analyzed using the PageRuler. TM The molecular weight of the antibody was determined using Unstained Protein Ladder (ThermoFisher) with the antibody samples. The remaining supernatant of each mutant was used for subsequent purification. A Protein A column was pre-packed with 1 mL of MabSelect Sure resin. The column was equilibrated with 0.1 M Tris, pH 7.0 before loading with cell culture medium. Subsequently, after loading, the column was washed with 0.1 M Tris, pH 7.0, and eluted with 0.1 M citric acid, pH 3.5. Then, 0.1 M Tris, pH 9.0 was added to neutralize the eluted solution. The sample was then dialyzed against PBS buffer (Sangon Biotech, B548117-0500). Finally, the antibody was filtered through a 0.2 μm filter and sterilely divided into 1.5 mL tubes in aliquots of 0.2 mL or 0.5 mL. Antibodies were frozen, stored at -80°C, shipped by ABL, and analyzed in vitro at WuXi Biologics.

[0315] Consequently, two bispecific antibodies were prepared, differing only in the type of linker ((G4S)2 or (G4S)3). The bispecific antibody with the (G4S)2 linker is designated R2, and the bispecific antibody with the (G4S)3 linker is designated R3. The amino acid sequences of the bispecific antibodies are shown in Table 18. Hereinafter, the bispecific antibody with the R2 linker is also referred to as 33C2 / CD3-R2, and the bispecific antibody with the R3 linker is also referred to as 33C2 / CD3-R3.

[0316] JPEG2024525916000020.jpg251158JPEG2024525916000021.jpg59158

[0317] Example 4. Evaluation of activity of anti-CLL1 / anti-CD3 bispecific antibodies

[0318] <4.1. Evaluation of binding activity in CLL-1 expressing cells (FACS)>

[0319] To evaluate the tumor antigen binding properties of the bispecific antibodies of Example 3, the binding ability of the CLL1 targeting moieties of 33C2 / CD3-R2 and 33C2 / CD3-R3 to CLL1 expressed in mammalian cells was analyzed using FACS. Briefly, CLL1 positive cells (U937 and HL60) were cultured with 33C2 / CD3-R2 and 33C2 / CD3-R3 antibodies. After washing with FACS buffer (1% BSA in PBS), PE-anti-human IgG antibody was added to each well and incubated at 4°C for 60 min. The MFI (Median Fluorescence Intensity) of PE (phycoerythrin) was evaluated by FACS Calibur. BsAbmock / CD3, which has anti-CD3 antigen-binding fragment in one arm and no antigen-binding fragment in the other arm, was used as a control group. Merus' anti-CLL-1 / anti-CD3 bispecific antibody MCLA-117 was used as a reference. The results are shown in Figure 17 and Table 19.

[0320] JPEG2024525916000022.jpg39140

[0321] FIG. 17 is a graph showing the cell binding activity of a bispecific antibody according to one embodiment in CLL1-expressing cancer cells.

[0322] As shown in FIG. 17 and Table 19, the binding activity of the bispecific antibody according to one embodiment in CLL1-positive cancer cell lines was confirmed to be significantly higher than the binding activity of the MCLA-117 antibody.

[0323] <4.2. Evaluation of binding activity in CD3-expressing cells (FACS)>

[0324] To assess the CD3 binding properties of the bispecific antibodies of Example 3, the binding activity of the CD3 targeting moieties of 33C2 / CD3-R2 and 33C2 / CD3-R3 to CD3-expressing mammalian cells was analyzed using FACS.

[0325] Briefly, CD3-expressing Jurkat cell lines were incubated with 33C2 / CD3-R2 and 33C2 / CD3-R3 antibodies. After washing with FACS buffer (1% BSA in PBS), PE-anti-human IgG antibodies were added to each well and incubated at 4°C for 60 min. The MFI (Median Fluorescence Intensity) of PE was evaluated by FACS Calibur. The results are shown in Figure 18.

[0326] FIG. 18 is a graph showing the cell binding activity of a bispecific antibody according to one embodiment.

[0327] As shown in FIG. 18, it was confirmed that a bispecific antibody according to one embodiment strongly bound to a CD3-expressing Jurkat cell line in a dose-dependent manner.

[0328] <4.3. In vitro T cell activation of bispecific antibodies under various CLL1 expression conditions>

[0329] To examine the CLL1-specific T cell activating activity of 33C2 / CD3-R2 and 33C2 / CD3-R3 bispecific antibodies (BsAbs), a Promega kit was used with AML cell lines (U937 and HL-60).

[0330] Briefly, 2 × 10 cells of U937 or HL-60 cancer cells were cultured in a 30-well plate. 4 Individual cells were sorted into 96-well plates in Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% FBS. BsAbs and 1 × 10 5TCR / CD3 effector cells (Nuclear factor of activated T-cells, NFAT) were added. T-cell activation was assessed after 6 hours of incubation at 37°C in 5% CO2 atmosphere. Bio-Glo reagent (Promega) was then added and luminescence was measured with a luminescence plate reader. BsAbs U1R2, a bispecific antibody (unrelated antibody) with anti-Claudin18.2, and an anti-CD3 antigen-binding fragment were used as negative controls. The results are shown in Figure 19 and Table 20. JPEG2024525916000023.jpg33161

[0331] FIG. 19 is a graph showing T cell activation of a bispecific antibody according to one embodiment.

[0332] As shown in FIG. 19 and Table 20, a bispecific antibody according to one embodiment significantly induced CLL1-specific T-cell activation in a dose-dependent manner, whereas the control BsAb had no effect.

[0333] 4.4. T cell activation and cytolytic activity of bispecific antibodies in various CLL1-expressing AML cell lines

[0334] To examine the T cell activation and cytolytic activity of the bispecific antibodies of Example 3, FACS was performed on AML cell lines (U937 or HL60).

[0335] Briefly, U937 or HL60 suspension target cells (2 × 10 4 Cells) were sorted into 96-well U-bottom plates. Various concentrations of BsAbs or control molecules (U1R2) and human purified T cells (purified from PBMCs) were added to the plates at an effector:target ratio of 5:1. After 48 hours, T cell activation and the number of remaining CD33 positive target cells were quantified by flow cytometry. The percentage of specific cytolysis was calculated as follows:

[0336] 100 - [100 x (number of target cells treated with BsAbs) / (number of target cells treated with PBS)].

[0337] T cells were analyzed by flow cytometry using cell surface levels of CD25 and CD69 as T cell activation markers. BsAbsmock / CD3 was used as a control and Merus anti-CLL-1 / anti-CD3 bispecific antibody MCLA-117 was used as a reference. EC50 results for HL60 and U937 are shown in Figures 20 and 21, respectively, and Tables 21 and 22, respectively.

[0338] JPEG2024525916000024.jpg52162

[0339] JPEG2024525916000025.jpg52162

[0340] FIG. 20 is a graph showing T cell activation and cytolytic activity of a bispecific antibody according to one embodiment in HL-60.

[0341] FIG. 21 is a graph showing T cell activation and cytolytic activity of a bispecific antibody according to one embodiment in U937.

[0342] As shown in Figures 20 and 21 and Tables 21 and 22, it was confirmed that a bispecific antibody according to one embodiment significantly induced CLL1-specific T cell activation (as measured by CD25 and CD69 upregulation) and cancer cell lysis in a dose-dependent manner compared to MCLA-117, while the control BsAb had no effect.

[0343] <4.5. In vitro cytotoxicity in various CLL1-expressing cell lines>

[0344] To determine the ability of bispecific antibodies according to one embodiment to promote antigen-dependent lysis, the in vitro cytotoxicity of BsAbs was analyzed with FarRed-labeled U937 or HL60.

[0345] Briefly, FarRed-labeled U937 or HL60 suspension target cells (1.5 × 10 4 Cells) were sorted into 96-well U-bottom plates. Various concentrations of BsAbs or control molecule (U1R2) and human PBMC effector cells obtained from various donors were added to the plates at effector:target ratios of 10:1 or 5:1. All experiments were performed in triplicate. Target cell killing was assessed after 48 hours by flow cytometry using the Zombie Violet™ Fixable Viability kit (BioLegend, Cat# 423114). The percentage of specific cytolysis was calculated as follows:

[0346] 100×[FarRed pos dead cells / (FarRed pos live cells + FarRed pos dead cells)]

[0347] The results are shown in Table 23 and FIG.

[0348] JPEG2024525916000026.jpg124149

[0349] FIG. 22 is a graph showing antigen-dependent cytolytic activity of a bispecific antibody according to one embodiment.

[0350] As shown in FIG. 22 and Table 23, it was found that a bispecific antibody according to one embodiment significantly induced T cell-mediated lysis of CLL1-positive U937 and HL60 cells in a concentration-dependent manner, whereas the control BsAb (U1R2) had no effect.

[0351] <4.6. In vivo effects in xenograft model (U937 model)>

[0352] The effect of the bispecific antibody of Example 3 was evaluated in a subcutaneous U937 xenograft model.

[0353] Specifically, the in vivo antitumor activity was evaluated in NOG mice bearing U937 subcutaneous xenografts. The brief experimental design is as follows: Tumor cell line: U937 Mice: NOG group = 9 Tumor cell sc injection: 5 × 10 6 cells / animal Day 5 expanded T cell iP injection: 1 x 10 7 Cells / animal, donor (ABL02T) (E:T=2:1) hIgG1(Fc block) ip injection on day 6 On the 7th day, ip injection of drug (BsAbs) was started (1mpk, 2QW (2 times / week), total 6 times) Grouping: Day 7; Group 1 (control group): PBS (vehicle); Group 2: 33C2 / CD3-R2; and Group 3: 33C2 / CD3-R3

[0354] Tumor size, median tumor growth inhibition (% TGI), and body weight were measured on days 5, 7, 10, 13, 17, 20, and 24.

[0355] Because the CD3 arm of the bispecific antibody does not cross-react with mouse CD3, human T cells were injected intraperitoneally into the mice on day 5, after which 33C2 / CD3-R2 (group 2) and 33C2 / CD3-R3 (group 3), or PBS (vehicle) (group 1) were administered at a dose of 1 mg / kg twice a week for 3 weeks. 3 ) (=(W)×(L)×(H)×0.5), median tumor growth inhibition (% TGI), and mouse body weight were measured and the results are shown in FIG. 23.

[0356] FIG. 23 is a graph showing the in vivo efficacy of a bispecific antibody according to an embodiment in a U937 xenograft model.

[0357] As shown in Figure 23, there was no significant change in mouse body weight in either the experimental groups (Groups 2 and 3) or the control group (Group 1). In addition, the control group had significantly larger tumor volumes than the experimental groups, 33C2 / CD3-R2 and 33C2 / CD3-R3BsAbs-treated mice. In particular, a bispecific antibody according to an embodiment induced complete tumor regression in a U937 xenograft model.

[0358] <4.7. In vivo efficacy in xenograft model (HL60-Lu orthotopic AML model)>

[0359] Efficacy evaluation of the bispecific antibody of Example 3 was carried out in the IV HL60-Lu orthotopic AML model.

[0360] Specifically, in vivo antitumor activity was evaluated in NOG mice bearing HL60-LuIV (intravenous) xenografts. The brief experimental design is as follows: Tumor cell line: HL 60luc Mice: NOG group = 9 Tumor cell IV injection: 1 × 10 7 cells / animal Day 5 expanded T cell iP injection: 1.5 x 10 7 Cells / animal, donor (ABL14) (E:T=1.5:1) hIgG1 (Fc block) ip injection on day 6, 30mpk On the 7th day, ip injection of drug (BsAbs) was started (2QW (2 times / week), total 7 times) Grouping: Day 7; Group 1 (control group): PBS (vehicle); Group 2: 33C2 / CD3-R2; and Group 3: 33C2 / CD3-R3

[0361] Bioluminescence index (BLI) was performed on days 7, 14, 21, and 28.

[0362] Efficacy evaluation of bispecific antibodies according to one embodiment was performed in the HL60-Lu orthotopic AML model. Specifically, in an established disseminated HL60-luc model, 33C2 / CD3-R2 or 33C2 / CD3-R3 treatment was initiated after AML cell homing to the bone marrow was confirmed following IV injection.

[0363] Briefly, to generate a whole-body luciferase-labeled HL60 orthotopic model, 1 x 10 7 HL60-luc cells were injected intravenously into the tail vein on day 0. Animals were randomly assigned to seven groups on day 6 according to bioluminescence intensity.

[0364] Starting on day 7, mice were administered 0.5 mg / kg of 33C2 / CD3-R2, 33C2 / CD3-R3 or vehicle by intraperitoneal injection twice weekly for a total of seven doses. Bioluminescence index (BLI) analysis was performed on days 7, 14, 21, and 28, the results of which are shown in Figure 24, and the quantitative analysis is shown in Figure 25. Median TGI was also measured on days 14, 21, and 28, the results of which are shown in Table 24.

[0365] FIG. 24 is an image showing bioluminescence index (BLI) upon administration of a bispecific antibody according to one embodiment in an HL60-Lu orthotopic AML model.

[0366] FIG. 25 is a graph showing quantitative analysis of BLI upon administration of a bispecific antibody according to one embodiment in an HL60-Lu orthotopic AML model (statistical analysis: Two-way ANOVA (Bonferroni's multiple comparisons test), *p<0.05, **p<0.01, ***p<0.001).

[0367] JPEG2024525916000027.jpg32137

[0368] 24 and 25, and Table 24, administration of 0.5 mg / kg of a bispecific antibody according to one embodiment was confirmed to significantly inhibit tumor growth (90% and 80%, respectively) compared to the vehicle-treated group as assessed by bioluminescence on day 28. Additionally, administration of a bispecific antibody according to one embodiment was confirmed to significantly reduce tumor burden in the bone marrow, spine, and hind limbs as observed by bioluminescence.

[0369] <4.8. Evaluation of effective dose in xenograft model (HL60-Lu orthotopic AML model)>

[0370] The effective dose of the 33C2 / CD3-R3 bispecific antibody was evaluated in the HL60-Lu orthotopic AML model in a similar manner to Example 4.7., except that the dose of the bispecific antibody was changed to 0.5 mg / kg, 0.05 mg / kg, and 0.005 mg / kg.

[0371] Bioluminescence index (BLI) analysis was performed on days 7, 14, 21, and 28, the results of which are shown in Figure 26 and the quantitative analysis is shown in Figure 27, and median TGI was measured on days 14, 21, and 28, the results of which are shown in Table 25. Intramedullary tumor cells were also measured using flow cytometry and IHC staining, the results of which are shown in Figures 28 and 29, respectively.

[0372] FIG. 26 is an image showing bioluminescence index (BLI) upon administration of a bispecific antibody according to one embodiment in an HL60-Lu orthotopic AML model.

[0373] FIG. 27 is a graph showing quantitative analysis of BLI upon administration of a bispecific antibody according to an embodiment in an HL60-Lu orthotopic AML model (***=P<0.005).

[0374] FIG. 28 is a graph showing the results of FACS measurement of intramedullary tumor cells after administration of a bispecific antibody according to one embodiment.

[0375] FIG. 29 is an image showing the results of IHC staining of intramedullary tumor cells after administration of a bispecific antibody according to one embodiment.

[0376] JPEG2024525916000028.jpg32153

[0377] 26 and 27, and Table 25, administration of a bispecific antibody according to one embodiment at 0.5 mg / kg, 0.05 mg / kg, and 0.005 mg / kg significantly inhibited tumor growth (91%, 78%, and 32%, respectively) compared to vehicle-treated mice, as assessed by bioluminescence on day 28. Additionally, administration of a bispecific antibody according to one embodiment significantly reduced tumor burden in the bone marrow, spine, and hind limbs in a dose-dependent manner, as observed by bioluminescence.

[0378] 28 and 29, it was confirmed that intramedullary tumor cells were significantly reduced by administration of the bispecific antibody according to one embodiment. Furthermore, such results indicate that the antibody according to one embodiment can inhibit cancer metastasis.

[0379] 4.9. T cell activation and cytotoxicity in primary patient-derived AML blasts

[0380] AML blasts derived from PBMCs obtained from primary patients were used to test the activity of the bispecific antibody of Example 3. AML blasts represent a more similar condition to the clinical environment than animal models or cell lines.

[0381] First, PBMCs were isolated by density gradient separation using Ficoll (GE Healthcare), stained with antibodies, and analyzed immediately after isolation using a FACSLSRFortessa (BD Biosciences) to examine the expression of CLL1 and CD33 in AML blasts from four primary patients. Data were analyzed using FlowJo (BD Biosciences) and GraphPad Prism ver.9 (GraphPad Software, Inc.) software. The percentage of positive cells and MFI of each IgG negative control group staining in the AML blast population were determined. The results of CLL1 and CD3 expression in whole blood cells and AML blasts are shown in Table 26.

[0382] JPEG2024525916000029.jpg52163

[0383] In four AML patient samples, the median CLL1 expression in AML blasts was similar to the median CD33 expression in terms of both percentage of positive cells and mean fluorescence intensity (MFI) profiles.

[0384] The ability of bispecific antibodies according to one embodiment to induce cytotoxicity and T cell activation was assessed using PBMC from 11 AML patients in an ex vivo cytotoxicity assay.

[0385] Briefly, AMLPBMCs (2 × 10 ) were isolated from fresh blood from AML patients as previously described. 5 ) was aliquoted in triplicate into 96-well U-bottom plates. BsAbs were added to the plates in 10-fold serial dilutions starting from 50 nM. U1R2 was used as a control. After 72 hours, CLL1-positive AML blasts were analyzed for cytolysis (%) and T cell activation (%) by flow cytometry, and the results are shown in Figures 30 and 31, respectively.

[0386] FIG. 30 is a graph showing the cytolytic activity of a bispecific antibody according to one embodiment in AML blasts.

[0387] FIG. 31 is a graph showing the T cell activation activity of a bispecific antibody according to one embodiment in AML blasts.

[0388] As shown in Figure 30, it was confirmed that the bispecific antibody according to one embodiment significantly induced cytotoxicity in AML blasts in a concentration-dependent manner (EC50: 0.07-0.2 nM). It was also confirmed that the above cytotoxicity results correlated with increased T cell activation in four patient samples (EC50: 0.0003-0.04 nM) as shown in Figure 31. These data indicate that the bispecific antibody according to one embodiment was effective in killing CLL1 positive AML cells in an ex vivo environment more similar to in vivo conditions.

Claims

**Claim 1** An isolated anti-CLL-1 antibody or an antigen-binding fragment thereof, comprising: (a) VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3; (b) VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 5, and 6; (c) VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, and 11; (d) VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, and 15; (e) VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, and 18; and (f) VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, and 22. **Claim 2** The anti-CLL-1 antibody or fragment thereof according to claim 1, wherein the anti-CLL-1 antibody or fragment thereof comprises a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 and 24. **Claim 3** The anti-CLL-1 antibody or fragment thereof according to claim 1, wherein the anti-CLL-1 antibody or fragment thereof comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO:

55. **Claim 4** The anti-CLL-1 antibody or fragment thereof according to claim 1, wherein the anti-CLL-1 antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 74. **Claim 5** The anti-CLL-1 antibody or fragment thereof according to claim 1, wherein the anti-CLL-1 antibody or fragment thereof comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, and 75. **Claim 6** The anti-CLL-1 antibody or fragment thereof according to claim 1, wherein the anti-CLL-1 antibody or fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:

45. **Claim 7** The anti-CLL-1 antibody or fragment thereof according to claim 1, wherein the anti-CLL-1 antibody or fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO:

46. **Claim 8** The anti-CLL-1 antibody or fragment thereof comprises the sequences of CDRH1, CDRH2, and CDRH3 of the heavy chain variable region, and CDRL1, CDRL2, and CDRL3 of the light chain variable region. (a) CDRH1, CDRH2, and CDRH3 are SEQ ID NO: 1, 4, and 7 respectively, and CDRL1, CDRL2, and CDRL3 are SEQ ID NO: 12, 16, and 19 respectively; or (b) CDRH1, CDRH2, and CDRH3 are SEQ ID NO: 2, 5, and 8 respectively, and CDRL1, CDRL2, and CDRL3 are SEQ ID NO: 13, 17, and 20 respectively; or (c) CDRH1, CDRH2, and CDRH3 are SEQ ID NO: 3, 6, and 9 respectively, and CDRL1, CDRL2, and CDRL3 are SEQ ID NO: 14, 18, and 21 respectively; or (d) CDRH1, CDRH2, and CDRH3 are SEQ ID NO: 1, 4, and 10 respectively, and CDRL1, CDRL2, and CDRL3 are SEQ ID NO: 15, 16, and 22 respectively; or (e) CDRH1, CDRH2, and CDRH3 are SEQ ID NO: 2, 5, and 11 respectively, and CDRL1, CDRL2, and CDRL3 are SEQ ID NO: 13, 17, and 20 respectively. The anti-CLL-1 antibody according to claim 1.

9. The anti-CLL-1 antibody according to claim 1, wherein the anti-CLL-1 antibody or its antigen-binding fragment is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

10. The anti-CLL-1 antibody or its antigen-binding fragment is whole IgG, Fab, Fab', F(ab') ２ , xFab, scFab, dsFv, Fv, scFv, scFv-Fc, scFab-Fc, diabody, minibody, scAb, dAb, semi-IgG, and combinations thereof, and the anti-CLL-1 antibody according to claim 1 selected from the group consisting of.

11. The anti-CLL-1 antibody according to claim 1, which is in the form of IgG1.

12. The anti-CLL-1 antibody according to claim 1, wherein the anti-CLL-1 antibody or its antigen-binding fragment is a Fab molecule.

13. The anti-CLL-1 antibody according to claim 1, wherein the anti-CLL-1 antibody or antigen-binding fragment has one or more of the following characteristics: (a) Binding to human CLL-1; (b) Binding to cynomolgus monkey CLL-1; (c) Binding to CLL-1 on the surface of human peripheral blood mononuclear cells (PBMC); (d) Binding to CLL-1 on the surface of cynomolgus monkey PBMC; and (e) Binding to CLL-1 on the surface of cancer cells.

14. The anti-CLL-1 antibody according to claim 1, wherein a cytotoxic agent is conjugated to at least a part of the anti-CLL-1 antibody or antigen-binding fragment.

15. The anti-CLL-1 antibody according to claim 14, wherein the cytotoxin is attached to the anti-CLL-1 antibody or antigen-binding fragment via a linker.

16. The anti-CLL-1 antibody according to claim 15, wherein the linker is cleavable by a protease.

17. The anti-CLL-1 antibody according to any one of claims 1 to 16, which is for use as a medicament.

18. The anti-CLL-1 antibody according to any one of claims 1 to 16, which is for use in treating or preventing cancer.

19. An immunoconjugate comprising the formula Ab-(L-D)p, wherein (a) Ab is the anti-CLL-1 antibody according to any one of claims 1 to 16, (b) L is a linker, (c) D is a cytotoxin, and (d) p ranges from 1 to 8.

20. An isolated nucleic acid encoding the anti-CLL-1 antibody according to any one of claims 1 to 16.

21. A vector comprising the isolated nucleic acid according to claim 20.

22. A host cell comprising the vector according to claim 21.

23. A pharmaceutical composition comprising the anti-CLL-1 antibody according to any one of claims 1 to 16.

24. The pharmaceutical composition according to claim 23, for treating or preventing cancer.

25. The pharmaceutical composition according to claim 24, wherein the cancer is a cancer that expresses CLL-1.

26. The cancer is selected from the group consisting of leukemia, rectal cancer, endometrial cancer, nephroblastoma, basal cell carcinoma, nasopharyngeal carcinoma, bone tumor, esophageal cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular thyroid cancer, hepatocellular carcinoma, oral cancer, renal cell carcinoma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, mesenchymal tumor, soft tissue sarcoma, liposarcoma, gastrointestinal stromal tumor, malignant peripheral nerve sheath tumor (MPNST), Ewing sarcoma, leiomyosarcoma, mesenchymal chondrosarcoma, lymphoma, fibrosarcoma, rhabdomyosarcoma, teratoma, neuroblastoma, medulloblastoma, glioma, benign skin tumor, Burkitt's lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma, neuroectodermal tumor, epithelial tumor, cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), peripheral T-cell lymphoma, pancreatic cancer, hematological malignancies, renal cancer, tumor vasculature, breast cancer, renal cancer, ovarian cancer, epithelial ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular tumor, uterine cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colorectal cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, adrenal cancer, and the pharmaceutical composition according to claim 24 selected therefrom.

27. The leukemia according to claim 26 is selected from the group consisting of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy-cell leukemia, myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML), and acute myeloid leukemia (AML).

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