Humanized monoclonal antibodies against cANGPTL4

Abstract

The present invention relates to humanized antibodies directed against the C-terminus of the angiopoietin-like 4 protein (cANGPTL4) and uses thereof, such as their use in the treatment of cancer and methods of cancer treatment.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to Singapore Patent Application No. 10202003838X, filed on April 27, 2020, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] This invention relates to humanized antibodies against the C-terminal portion (cANGPTL4) of angiopoietin-like 4 protein and their uses, such as their use in the treatment of cancer and cancer treatment methods. Background Technology

[0004] ANGPTL proteins belong to the angiogenesis regulatory secretory protein superfamily, which shares high similarity with members of the angiopoietin family. ANGPTL4 is a member of the angiopoietin-like protein family and functions through a Tie2-independent mechanism (Zhu, P., et al., Angiopoietin-like 4: a decade of research. Biosci Rep, 2012. 32(3): p. 211-9). The full-length ANGPTL4 protein undergoes proteolytic processing to generate N-terminal (nANGPTL4) and C-terminal (cANGPTL4) fragments in a tissue- and environment-dependent manner. Through this post-translational modification, ANGPTL4 has been shown to participate in energy balance, wound repair, tumorigenesis, angiogenesis, vascular permeability regulation, and redox regulation. cANGPTL4 mediates its function through interactions with specific matrix proteins, integrins, closure proteins, cadherins, and neuropilins (Sodhi, A., et al., Angiopoietin-like 4 binds neuropilins and cooperates with VEGF to induce diabetic macular edema. J Clin Invest, 2019. 129(11): p. 4593-4608; Huang, RL, et al., ANGPTL4 modulates vascular junction integrity by integrin signaling and disruption of intercellular VE-cadherin and claudin-5 clusters. Blood, 2011. 118(14): p. 3990-4002; Goh, YY, et al., Angiopoietin-like 4 interacts with integrins beta1 and beta5 to modulate keratinocyte migration. Am J Pathol, 2010.). 177(6): p. 2791-803; Goh,YY, et al., Angiopoietin-like 4 interacts with matrix proteins to modulatewound healing. J Biol Chem, 2010. 285(43): p. 32999-3009).mAbs that bind to cANGPTL4 and interfere with the interactions between cANGPTL4 and its various interacting partners can attenuate or eliminate the function of cANGPTL4 in various diseases.

[0005] A mouse antibody against cANGPTL4 has been disclosed in international patent publication WO2014 / 027959A1.

[0006] Recent interest and advances in antibody therapy have laid the foundation for engineering antibodies to treat and diagnose a variety of human conditions. Antibodies offer promising potential for improving disease, with fewer side effects than traditional chemotherapy or long-term medication, which is advantageous for recovery time.

[0007] Currently, four classes of monoclonal antibodies (mAbs) are available: mouse, chimeric, humanized, and human mAbs, each with its own advantages and disadvantages. Mouse mAbs are typically produced using traditional hybridoma technology, which involves injecting mice with a specific antigen, fusing isolated spleen cells with a mutated myeloma line, and then selecting hybridomas that secrete monoclonal antibodies into the culture supernatant. For chimeric mAbs, the same technique as for mouse monoclonal antibodies is used to produce hybridomas, but the genes encoding the variable regions of the mouse antibody heavy and light chains, as well as the genes encoding the constant regions of the human heavy and light chains, are inserted into a plasmid, which is then transfected into bacteria to subsequently produce chimeric antibodies as inclusion bodies.

[0008] However, mouse antibodies and chimeric antibodies have the drawbacks of human anti-mouse antibody (HAMA) reactions. In humans, mouse mAbs are often rejected by the host because the immune system perceives them as foreign. Therefore, long-term antibody therapy becomes ineffective due to the short half-life of mAbs caused by adverse immune responses and the side effects of immune complex formation. Even when human antibodies (such as chimeric mAbs) replace most of the non-antigen-binding portion, HAMA reactions still occur, typically causing similar, albeit less severe, immunogenic side effects compared to mouse mAbs.

[0009] To overcome the aforementioned problems, humanized antibodies have been developed that allow for minimization of the HAMA response through complementarity-determining region (CDR) transplantation. Humanized mAbs can be produced by transplanting six mouse CDRs along with human variable and constant region genes into an expression vector and expressing them in mammalian cells. This technology allows antibodies to possess properties nearly identical to human antibodies, which significantly reduces the immunogenicity of mouse mAbs and chimeric mAbs, thus extending the half-life of humanized mAbs for chronic therapy, which in turn allows for less frequent interventions.

[0010] The process involving CDR transplantation requires various design choices, such as determining the boundaries of the CDR, which human backbones to use, and which residues of the mouse mAb (if any) to replace in the human backbone region. This design has been found to be neither simple nor guaranteed to succeed, as the binding strength and specificity of the parental antibody and antibody stability must be preserved, while undesirable immunogenic properties in humans must be reduced or removed. Furthermore, the recombinant production of antibodies in a stable form is required.

[0011] Even with the necessary steps to convert mouse mAbs into humanized antibodies, warnings about humanized mAbs can still affect the efficacy of later human clinical studies and subsequent treatments, and may even lead to serious side effects. One issue may stem from insufficient transfer of the CDR loop, which could affect the retention of binding affinity compared to the original mAb construct, as the human backbone with variable and constant residues can influence loop orientation (George, AJT and CE Urch, Diagnostic and therapeutic antibodies. Methods in molecular medicine. 2000, Totowa, NJ: Humana Press. xiv, 477 p.), and may require further modifications and confirmation through in vivo assays to fully determine the specificity, affinity, and downstream immunogenic side effects (if any) of the humanized mAb.

[0012] These issues have hindered development, and therefore no humanized blocking monoclonal antibody against cANGPTL4 has been proposed to date. Summary of the Invention

[0013] The present invention addresses this need, and in a first aspect, provides a humanized antibody targeting the C-terminal region (cANGPTL4) of angiopoietin-like 4 protein, said humanized antibody comprising a heavy chain variable domain having a human backbone region and a light chain variable domain, said heavy chain variable domain comprising mouse V protein having the amino acid sequences shown in SEQ ID NO. 1-3. H CDR1, V H CDR2 and V H The CDR3 region, the light chain variable domain, contains mouse V amino acids having the amino acid sequences shown in SEQ ID NO. 4-6. L CDR1, V L CDR2 and V L CDR3 area, in which:

[0014] (1) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 7 (ANGH1) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 7 (ANGH1) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof;

[0015] (2) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 9 (ANGH2) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 9 (ANGH2) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof;

[0016] (3) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 11 (ANGH3) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 11 (ANGH2) or a variant thereof; the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof; or

[0017] (4) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 13 (ANGH4) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 13 (ANGH4) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof, wherein

[0018] The variants are unchanged for the CDR region defined in SEQ ID NO.1-6 and have at least 80% sequence identity with each reference sequence.

[0019] In various embodiments of the antibody of the present invention

[0020] (1) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 7 (ANGH1) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 7 (ANGH1) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof;

[0021] (2) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 9 (ANGH2) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 9 (ANGH2) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1) or 12 (ANGK3) or a variant thereof;

[0022] (3) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 11 (ANGH3) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 11 (ANGH3) or a variant thereof; the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 10 (ANGK2) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 10 (ANGK2) or 12 (ANGK3) or a variant thereof; or

[0023] (4) The heavy chain variable domain contains the amino acid sequence shown in SEQ ID NO: 13 (ANGH4) or a variant thereof or is composed of the amino acid sequence shown in SEQ ID NO: 13 (ANGH4) or a variant thereof, and the light chain variable domain contains the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof or is composed of the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof.

[0024] In various embodiments of the antibody of the present invention

[0025] (1) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof; or

[0026] (2) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 11 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 11 or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 12 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 12 or a variant thereof; or

[0027] (3) The heavy chain variable domain contains the amino acid sequence shown in SEQ ID NO: 13 or a variant thereof or is composed of the amino acid sequence shown in SEQ ID NO: 13 or a variant thereof, and the light chain variable domain contains the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof or is composed of the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof.

[0028] In one embodiment of the antibody of the present invention, the heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 11 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 11 or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 12 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 12 or a variant thereof.

[0029] In various embodiments, the variants of the light chain variable domain and the heavy chain variable domain are invariant to the CDR region as defined above, i.e., any variation occurs in the backbone region, and in various embodiments have at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with each reference sequence, i.e., any amino acid sequence shown in SEQ ID NO. 7-14.

[0030] Another aspect of the invention relates to a method for treating a proliferative disease or condition, preferably cancer, in a subject in need, said method comprising administering to said subject a therapeutically effective amount of an antibody as described herein. It also covers the use of the antibodies disclosed herein as medicines for treating or preventing cancer.

[0031] Another aspect of the invention relates to an in vitro method for reducing cell proliferation, the method comprising contacting proliferating cells with the antibody described herein.

[0032] Another aspect of the invention relates to nucleic acid molecules encoding heavy and / or light chains of antibodies as disclosed herein. It also covers recombinant host cells comprising the nucleic acids of the invention.

[0033] Another aspect of the invention relates to a composition, preferably a pharmaceutical composition, comprising an antibody as described herein and a pharmaceutically acceptable carrier and / or excipient. Attached Figure Description

[0034] In the following description, various embodiments of the present invention will be described with reference to the following drawings.

[0035] Figure 1 The amino acid sequence alignment of the variable domain of the mouse monoclonal antibody mAb11F6C4 with the variable domain sequence of this invention is shown. Shaded amino acid regions (black letters) highlight the differences between the humanized and mouse-derived heavy and light chains. CDR regions are indicated by shaded white letters.

[0036] Figure 2 The ELISA assay results of different humanized monoclonal antibodies against human cANGPTL4 are shown. Mouse antibody mAb11F6C4 was used as a reference.

[0037] Figure 3 (A) ANGPTL4 secreted by MIO-M1 cells after hypoxia treatment (verified by Western blotting). The control medium was normoxic medium for MIO-M1 cells after 48 h. The treatment medium was hypoxic medium for MIO-M1 cells after 48 h. Measurements were taken at 6 h, 24 h, and 48 h; (B) Vascular leakage induced by hypoxia in MIO-M1 cells conditioned medium was inhibited after adding 10 ng of Antuzumab 1 to the conditioned medium from hypoxia-treated MIO-M1 cells.

[0038] Figure 4 The results of a simplified in vitro comparative immunogenicity assessment (IVCIA) of antibodies Antuzumab 1, 2, and 3 are shown.

[0039] Figure 5 The results of in vitro assays that can elicit antigen-specific effector T cell responses are shown, expressed as the percentage of total T cells induced by anti-VEGF, Antuzumab 1, 2 and 3.

[0040] Figure 6 Microscopic images of histological samples from an orthotopic bladder tumor xenograft model of high-risk NMIBC using human UMUC3 cells in an animal model (immunocompromised NSG mice) are shown at week 3 after intrabladder administration of Antuzumab1 (10 µg / mL) and without administration of Antuzumab1 (10 µg / mL). Detailed Implementation

[0041] The inventors previously demonstrated in international patent publication WO2014 / 027959A1 that a mouse monoclonal antibody targeting human cANGPTL4 (designated mAb11F6C4) showed reduced tumor vascular permeability and weakened tumor angiogenesis in mice, leading to reduced lung metastasis. Furthermore, treatment with the antibody (mAb11F6C4) significantly delayed melanoma growth in a mouse model, reproducing the RNAi effect. It was found that mAb11F6C4 targets an epitope located at the C-terminus (cANGPTL4) of human ANGPTL4 and does not affect the protein's mitochondrial activity or glucose regulation.

[0042] Based on these early results, the inventors developed humanized forms of the mouse antibodies by transferring the mouse CDR region into specific human backbone regions, and surprisingly found that certain CDR / backbone combinations, as claimed herein, were significantly more stable and effective than others. Overall, as stated above, the design of humanized antibodies is not merely conventional because it involves transferring the intact CDR region to a suitable backbone region location, maintaining the maximum binding strength and specificity of the parent antibody and its stability, while reducing undesirable immunogenic properties in humans.

[0043] Therefore, one aspect of the present invention relates to a humanized antibody targeting the C-terminal region (cANGPTL4) of angiopoietin-like 4 protein, said humanized antibody comprising a heavy chain variable domain having a human backbone region and a light chain variable domain, said heavy chain variable domain comprising mouse V protein having the amino acid sequences shown in SEQ ID NO. 1-3. H CDR1, V H CDR2 and V H The CDR3 region, the light chain variable domain, contains mouse V amino acids having the amino acid sequences shown in SEQ ID NO. 4-6. L CDR1, V L CDR2 and V L CDR3 area, in which:

[0044] (1) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 7 (ANGH1) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 7 (ANGH1) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof;

[0045] (2) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 9 (ANGH2) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 9 (ANGH2) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof;

[0046] (3) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 11 (ANGH3) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 11 (ANGH2) or a variant thereof; the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof; or

[0047] (4) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 13 (ANGH4) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 13 (ANGH4) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof, wherein

[0048] The variants are unchanged for the CDR region defined in SEQ ID NO.1-6 and have at least 80% sequence identity with each reference sequence.

[0049] The variable region of the heavy chain is also referred to as the VH domain in this paper, and the variable region of the light chain is referred to as the VL domain.

[0050] As used herein, the term "antibody" refers to a protein composed of one or more polypeptide chains substantially encoded by all or part of a known immunoglobulin gene. Known immunoglobulin genes in humans, for example, include kappa (κ), lambda (λ), and heavy chain loci, which together constitute a large number of variable region genes, and constant region genes mu (μ), delta (δ), gamma (γ), epsilon (ε), and alpha (α), which encode IgM, IgD, IgG (IgG1, IgG2, IgG3, and IgG4), IgE, and IgA (IgA1 and IgA2) isotypes, respectively. As used herein, the term "antibody" is intended to include both full-length antibodies and antibody fragments, and can refer to naturally occurring, engineered, or recombinant antibodies from any organism for experimental, therapeutic, or other purposes. However, antibody fragments or variants mentioned herein always include the heavy and light chain variable regions disclosed herein. Therefore, such fragments and variants include known scFv fragments or scFv antibodies.

[0051] The terms “antibody” and “immunoglobulin” are used interchangeably herein to refer to polypeptides encoded by immunoglobulin genes. As used herein, “IgG” refers to polypeptides belonging to a class of antibodies that are essentially encoded by the well-known immunoglobulin γ gene. In humans, these antibodies include IgG1, IgG2, IgG3, and IgG4. In mice, these antibodies include IgG1, IgG2a, IgG2b, and IgG3.

[0052] In various embodiments, the antibody of the present invention is an IgG antibody, such as an IgG1 antibody or an IgG1κ antibody. Such an antibody typically comprises two identical heavy chains and two identical light chains, both having an Ig domain as detailed below.

[0053] In this article, "immunoglobulin (Ig) domain" refers to a region of an immunoglobulin, which exists as a distinct structural entity as defined by those skilled in the art of protein structure. Ig domains typically exhibit a characteristic β-sandwich fold topology. Known Ig domains in IgG antibodies are VH, Cγ1, Cγ2, Cγ3, VL, and CL. As detailed above, VH and VL refer to the heavy chain variable region (VH) and light chain variable region (VL) and are defined herein by reference to their amino acid sequences. Cγ1, Cγ2, and Cγ3 refer to the Ig domains of the constant portion of the heavy chain, more commonly referred to as the CH1, CH2, and CH3 domains. The N-terminus of the CH1 or Cγ1 domain is linked to the C-terminus of the VH domain. CL relates to the constant portion of the light chain and is linked to the C-terminus of the VL domain. This linkage is typically via a peptide bond. Thus, the complete light chain contains both the VL and CL domains in the N-terminal to C-terminal direction. Therefore, a complete heavy chain contains VH, CH1, CH2, and CH3 domains in the direction from the N-terminus to the C-terminus. IgG antibodies contain two complete light chains and two complete heavy chains.

[0054] The variable region of an antibody contains the molecule's antigen-binding determinants, which determine the antibody's specificity for its target antigen. The variable region is so named because it is most distinct in sequence from other antibodies in its class. In the variable region, three loops are assembled in each variable (V) domain of the heavy and light chains to form an antigen-binding site. Each loop is called a complementarity-determining region (hereinafter referred to as "CDR"), where the amino acid sequence changes are most significant and are typically used to explain the antibody's binding specificity and affinity. There are a total of six CDRs, three for the heavy chain and three for the light chain, named V. H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2 and V LCDR3. The variable domain region outside the CDR is called the backbone region (FR). Although not as diverse as the CDR, sequence variability does exist in the FR region between different antibodies. Overall, this characteristic architecture of antibodies provides a stable scaffold (FR region) on which the immune system can explore a large number of antigen-binding diversity (CDR) to obtain specificity for a wide range of antigens. Many high-resolution structures are available for various variable region fragments from different organisms, some unbound and some complexed with antigens. Sequence and structural features of antibody variable regions are disclosed, for example, in Morea et al., 1997, Biophys Chem 68:9-16; Morea et al., 2000, Methods 20:267-279, and conserved features of antibodies are disclosed, for example, in Maynard et al., 2000, Annu Rev Biomed Eng 2:339-376.

[0055] As used herein, “variable region” refers to a region of an immunoglobulin that contains one or more Ig domains encoded by any VL (Vκ, Vλ) and / or VH genes that respectively constitute the κ, λ, and heavy chain immunoglobulin loci.

[0056] As defined in this article, the “constant region” of an antibody refers to the antibody region encoded by one of the constant region genes of light chain or heavy chain immunoglobulins.

[0057] As used in this article, a "constant light chain" or "light chain constant region" refers to a chain composed of κ (C κ ) or λ (C λ The antibody region encoded by the light chain. A constant light chain typically contains a single domain, which, as defined in this paper, refers to C... κ Or lambda C λ Positions 108-214 are numbered according to the EU index. Therefore, the IgG1κ antibody contains the κ light chain constant region.

[0058] As used herein, the “constant heavy chain” or “constant heavy chain region” refers to the region of an antibody encoded by the μ, δ, γ, α, or ε gene, to define the antibody isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For full-length IgG antibodies, the constant heavy chain, as defined herein, refers to the region from the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, thus encompassing positions 118–447, where the numbering is based on the EU index.

[0059] As used herein, the antibody-related term "humanization" generally refers to an antibody containing a human backbone region (FR) and one or more complementarity-determining regions (CDRs) derived from a non-human (typically mouse or rat) antibody. The non-human antibody providing the CDR is called the "donor," and the human immunoglobulin providing the backbone is called the "recipient." Humanization primarily relies on transplanting the donor CDR onto the recipient (human) VL and VH backbones (Winter US 5,225,539). This strategy is called "CDR transplantation." Typically, selected recipient backbone residues are "reverted" to the corresponding donor residues to regain the affinity lost in the initial transplanted construct (US 5,693,762). Ideally, the humanized antibody will also contain at least a portion of the immunoglobulin constant region, typically at least a portion of the constant region of a human immunoglobulin, and therefore will usually contain the human Fc region. Various techniques and methods for humanizing and reshaping nonhuman antibodies are well known in the art (see Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of BCells, 533-545, Elsevier Science (USA), and the references cited therein). Other methods for humanizing or reducing the immunogenicity of variable regions of nonhuman antibodies may include resurfacing methods, such as those described in Roguska et al. (1994, Proc Natl Acad Sci USA 91 969-973). In one embodiment, a selection-based approach may be used to humanize and / or mature the antibody variable region for affinity, i.e., to increase the affinity of the variable region for its target antigen. Other humanization methods may involve transplanting only portions of the CDRs, including but not limited to those described in Tan et al., 2002, J Immunol 169 1119-1125, and De Pascalis et al., 2002, J Immunol 169 3076-3084. Structure-based methods can be employed for humanization and affinity maturation, such as those described in U.S. Patent 7,117,096 and related applications. The antibodies of this invention are humanized because they comprise human FRs and all six CDRs from the mouse antibody mAb11F6C4 as described in International Patent Publication WO2014 / 027959A1.

[0060] In various embodiments, the antibodies of the present invention comprise a heavy chain and a light chain. In specific embodiments, they comprise a human IgG1 constant region within the immunoglobulin heavy chain and a human constant region within the immunoglobulin light chain. The immunoglobulin is contained within the variable domain of the heavy chain as defined by the amino acid sequence described herein and within the human backbone region of the variable domain of the light chain. The remaining portions of the antibody, namely the constant regions of the heavy and light chains, may be selected by those skilled in the art based on their common knowledge.

[0061] The antibodies of this invention target the C-terminal region (cANGPTL4) of angiopoietin-like 4 protein. In various embodiments, this means that they specifically recognize and bind to cANGPTL4. As used herein, "specifically binding" and "specific binding" refer to the antibody binding to its target, namely human cANGPTL4, based on the recognition of an epitope on the target molecule. The antibody recognizes and binds to the target molecule human cANGPTL4 with a binding affinity higher than that of other compounds that may be present. In various embodiments of this invention, "specific binding" may mean that the affinity of the antibody for binding to the target molecule cANGPTL4 is at least about 10 times that of the affinity of the antibody for binding to molecules unrelated to the target molecule (e.g., albumin). 6 Multiples, preferably at least about 10 7 Times, more preferably at least 10 8 The optimal ratio is at least approximately 10 times. 9 10 times. Typically, specific binding refers to non-specific binding of approximately 10 times. 6 times to about 10 9 Affinity in the range of 10 times. In some embodiments, the characteristic of specific binding can be that the affinity is 10 times that of nonspecific binding. 9 The binding affinity can be determined by any suitable method. Such methods are known in the art and include, but are not limited to, surface plasmon resonance and isothermal titration calorimetry. In specific embodiments, the antibody uniquely recognizes and binds to the target analyte. According to a preferred embodiment of the invention, the binding affinity of the humanized antibody disclosed herein is at least equal to the binding affinity of the mouse antibody mAb11F6C4 described in WO2014 / 027959A1, preferably tested according to the scheme described in the Examples section of this application.

[0062] The antibody of this invention comprises a heavy chain and a light chain variable region, wherein the heavy chain variable region comprises V H CDR1, V H CDR2 and V H The CDR3 region, the light chain variable region contains V L CDR1, V L CDR2 and V L CDR3, wherein the V HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, and is substantially composed of or consisting of the amino acid sequence shown in SEQ ID NO:1; the V H CDR2 contains the amino acid sequence shown in SEQ ID NO:2, and is substantially composed of or consisting of the amino acid sequence shown in SEQ ID NO:2; and the V H CDR3 comprises the amino acid sequence shown in SEQ ID NO:3, is substantially composed of or consists of the amino acid sequence shown in SEQ ID NO:3; and the V L CDR1 comprises the amino acid sequence shown in SEQ ID NO:4, and is substantially composed of or consisting of the amino acid sequence shown in SEQ ID NO:4; the V L CDR2 contains the amino acid sequence shown in SEQ ID NO:5, and is substantially composed of or consisting of the amino acid sequence shown in SEQ ID NO:5; the V L CDR3 comprises the amino acid sequence shown in SEQ ID NO:6, and is substantially composed of or consisting of the amino acid sequence shown in SEQ ID NO:6. In various embodiments, V having the amino acid sequence shown in SEQ ID NO:6 L CDR3, at the C-terminus of the sequence segment, i.e. after the W residue, also contains a T residue.

[0063] As defined herein, the antibodies of the present invention may also comprise variants of the amino acid sequences of the heavy and light chain variable domains as defined by the amino acid sequences shown in SEQ ID NO. 7-14. These variants are invariant to the CDR region as defined above; that is, any changes occur in the backbone region. In various embodiments, the variants have at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO. 7-12. Variations, typically amino acid substitutions or deletions, occur in the backbone region rather than in the CDR. Possible variations include, for example, short truncations at the C-terminus or N-terminus of the variable domain, typically 1, 2, 3, or 4 amino acids in length and / or single amino acid substitutions. In various implementations, in variants of the heavy chain variable structural domain, the positions corresponding to positions 2, 10, 11, 12, 15, 16, 19, 23, 24, 43, 46, 68, 72, 77, 78, 83, 84, 86, 87, 89, 90, 99, 114, and 115 in SEQ ID NO: 7 (i.e., using the position numbers of SEQ ID NO: 7) remain unchanged, except for the CDR region. In several embodiments, in variants of the light chain variable structural domain, the positions corresponding to positions 1, 3, 4, 9, 10, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 40, 46, 47, 49, 62, 64, 78, 80, 81, 82, 83, 84, 87, 88, 89, 104, 107, and 108 in SEQ ID NO:8 (i.e., position numbers using SEQ ID NO:8) remain unchanged, except for the CDR region.

[0064] As used herein, "amino acid" and "amino acid identity" refer to one of 20 naturally occurring amino acids or any non-natural analogues that may be present at a specific, defined position. Therefore, "amino acid" as used herein refers to both naturally occurring and synthetic amino acids. For example, for the purposes of this invention, homophenylalanine, citrulline, and leucine are considered amino acids. "Amino acid" also includes imino acid residues, such as proline and hydroxyproline. Side chains may be (R) or (S) configurations. In a preferred embodiment, the amino acid is in (S) or L configuration. If non-naturally occurring side chains are used, such non-amino acid substituents may be used, for example, to prevent or delay in vivo degradation. Amino acid identity can be readily determined using well-established comparisons and methods in the art. For example, the determination of sequence identity between nucleic acid or amino acid sequences can be accomplished through sequence alignment based on the well-established and commonly used BLAST algorithm (see, for example, Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment searchtool." J. Mol. Biol. 215:403-410 and Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J. Lipman (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs"; Nucleic Acids Res., 25, pp.3389-3402). This alignment is based on comparing similar nucleotide or amino acid sequence segments to each other. Another algorithm known in the art for this purpose is the FASTA algorithm. Alignment, especially multiple sequence comparison, is typically performed using computer programs.Commonly used algorithms include the Clustal series (see, for example, Chenna et al. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31, 3497-3500), T-Coffee (see, for example, Notredame et al. (2000): T-Coffee: A novel method for multiple sequence alignments. J. Mol. Biol. 302, 205-217), or programs based on these known programs or algorithms. Sequence alignment can also be performed using the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA, USA), which has set standard parameters, with the AlignX module for sequence comparison based on ClustalW. Unless otherwise stated, the BLAST algorithm is used to determine sequence identity. This comparison allows for the determination of the similarity between the sequences being compared. The similarity is typically expressed as a percentage of identity, which is the portion of the same nucleotide / amino acid at the same or corresponding (in alignment) sequence position compared to the total number of aligned nucleotides / amino acids. For example, if, in an alignment, 90 amino acids of a 100-amino acid-long query sequence are identical to the corresponding amino acids in the template sequence, then the sequence identity is 90%. Unless otherwise stated, “sequence identity” refers to the full length of the aligned sequence.

[0065] In various embodiments of the antibody of the present invention

[0066] (1) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 7 (ANGH1) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 7 (ANGH1) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof;

[0067] (2) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 9 (ANGH2) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 9 (ANGH2) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1) or 12 (ANGK3) or a variant thereof;

[0068] (3) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 11 (ANGH3) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 11 (ANGH3) or a variant thereof; the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 10 (ANGK2) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 10 (ANGK2) or 12 (ANGK3) or a variant thereof; or

[0069] (4) The heavy chain variable domain contains the amino acid sequence shown in SEQ ID NO: 13 (ANGH4) or a variant thereof or is composed of the amino acid sequence shown in SEQ ID NO: 13 (ANGH4) or a variant thereof, and the light chain variable domain contains the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof or is composed of the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof.

[0070] In various embodiments of the antibody of the present invention, the heavy chain variable domain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable domain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 8.

[0071] In various other embodiments, the heavy chain variable domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 11, and the light chain variable domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 12.

[0072] In various embodiments, the heavy chain variable domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 14.

[0073] In the above embodiments, "comprising" means that the variable domains of the heavy chain and / or light chain may contain additional amino acids at their C or N ends. However, typically, these extensions are only 1-10 amino acids long, for example, 1, 2, 3, 4, 5, 6, 7, or 8 amino acids.

[0074] In various embodiments, the aforementioned heavy chain variable domain consists of a specified amino acid sequence, i.e., without any C or N-terminal extensions. This similarly or alternatively applies to the light chain variable domain in various embodiments.

[0075] The humanized antibody of the present invention is derived from a monoclonal antibody, more specifically, the mouse monoclonal antibody mAb11F6C4 as described in WO2014 / 027959A1.

[0076] The described techniques for generating single-chain antibodies (US Patent No. 4,946,778; Bird (1988), Science 242: 423-26; Huston, et al. (1988), Proc. Natl. Acad. Sci. USA, 85: 5879-83; and Ward, et al. (1989), Nature, 334: 544-46) are applicable to generating gene-single-chain antibodies comprising the heavy chain variable domain and light chain variable domain of the present invention. Single-chain antibodies are typically formed by linking heavy chain and light chain fragments of the Fv region via amino acid bridges, thereby generating single-chain polypeptides.

[0077] Antibody fragments that recognize specific epitopes can be generated using known techniques. For example, such fragments include, but are not limited to, F(ab')2 fragments generated by pepsin digestion of antibody molecules and Fab fragments generated by reducing the disulfide bonds of F(ab')2 fragments. The antibody can be a monovalent antibody. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves the recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is typically truncated at any point in the Fc region to prevent heavy chain cross-linking. Alternatively, the associated cysteine ​​residue may be substituted with or deleted by another amino acid residue to prevent cross-linking.

[0078] In vitro methods are also suitable for preparing monovalent antibodies. Antibody digestion to produce its fragments, particularly Fab fragments, can be achieved using conventional techniques known in the art.

[0079] Alternatively, antibodies can be of various structures, including but not limited to antibody fragments. Antibody fragments include, but are not limited to, bispecific antibodies, microantibodies, domain antibodies, synthetic antibodies, antibody mimics, chimeric antibodies, antibody fusions (sometimes referred to as “antibody conjugates”), and their respective fragments. Specific antibody fragments include, but are not limited to: (i) Fab fragments consisting of VL, VH, CL, and CH1 domains; (ii) Fv fragments consisting of the VL and VH domains of a single antibody; (iii) F(ab')2 fragments, which are bivalent fragments containing two linked Fab fragments; (iv) single-chain Fv molecules (scFv) in which the VH and VL domains are linked by a peptide linker that allows the two domains to associate to form an antigen-binding site; (v) bispecific single-chain Fv dimers; and (vi) “dimeric antibodies” or “trimeric antibodies”, multivalent or multispecific fragments constructed by gene fusion. In all these antibody variants, the CDR region as defined herein is invariant and includes at least one heavy chain variable region and at least one light chain variable region as defined herein.

[0080] Antibody fragments can be modified. For example, the molecule can be stabilized by incorporating disulfide bonds that link the VH and VL domains. Examples of antibody forms and architectures are described in Holliger & Hudson, 2006, Nature Biotechnology 23(9):1126-1136, and Carter 2006, Nature Reviews Immunology 6:343-357.

[0081] The antibodies of this invention may include multispecific antibodies, particularly bispecific antibodies, sometimes also referred to as “bispecific antibodies.” These are antibodies that bind two (or more) different antigens. Bispecific antibodies can be manufactured in a variety of ways known in the art, such as chemical preparation or preparation from hybridomas. In one embodiment, the antibody is a microantibody. A microantibody is a minimized antibody-like protein containing an scFv linked to a CH3 domain. In some cases, the scFv may be linked to an Fc region and may include some or all of the hinge region. For a description of multispecific antibodies, see Holliger & Hudson, 2006, Nature Biotechnology 23(9): 1126-1136.

[0082] In various embodiments, the antibodies of the present invention are antibody fragments. Particular interest is found in antibodies comprising an Fc region, an Fc fusion complex, and a heavy chain constant region (CH1-hinge-CH2-CH3). The antibodies of the present invention may comprise an Fc fragment. The Fc fragments of the present invention may comprise 1%-90% of the Fc region, for example, 10%-90%, 30%-90%, etc. Thus, for example, the Fc fragments of the present invention may comprise an IgG1 Cγ2 domain, an IgG1 Cγ2 domain and a hinge region, an IgG1 Cγ3 domain, etc. In one embodiment, the Fc fragment of the present invention further comprises a fusion partner, thereby effectively making it an Fc fragment fusion complex. The Fc fragment may or may not contain additional polypeptide sequences.

[0083] In one embodiment, the antibody of the present invention is an antibody "fusion protein," sometimes referred to herein as an "antibody conjugate." The fusion conjugate or conjugate conjugate can be protein- or non-protein-based; the latter is typically generated using functional groups on the antibody and the conjugate conjugate. Conjugates and fusion conjugates can be any molecule, including small molecule compounds and peptides. For example, a variety of antibody conjugates and methods are described in Trail et al., 1999, Curr. Opin. Immunol. 11:584-588. Possible conjugate conjugates include, but are not limited to, cytokines, cytotoxic agents, toxins, radioisotopes, chemotherapeutic agents, anti-angiogenic agents, tyrosine kinase inhibitors, and other therapeutically active agents.

[0084] In various embodiments, a conjugate conjugate can be considered a payload, meaning the conjugate aims to target and deliver the conjugate conjugate to target cells, such as cancer cells or immune cells, via an antibody. Thus, for example, the conjugation of a toxin to an antibody aims to deliver the toxin to cells expressing a target antigen. As those skilled in the art will understand, the concepts and definitions of fusion and conjugation actually overlap. Referring to an antibody as a fusion or conjugate does not imply limitation to any particular embodiment of the invention. Rather, these terms are used loosely to convey the broad concept that any antibody of the present invention can be genetically, chemically, or otherwise linked to one or more polypeptides or molecules to provide some desired properties.

[0085] Suitable conjugates include, but are not limited to: markers as described below; pharmaceuticals; and cytotoxic agents, including but not limited to cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, absinin A chain, lacrimal, crotonin, phenolmycin, enoxacin, etc. Cytotoxic agents also include radiochemical substances prepared by conjugating a radioisotope to an antibody or by binding a radionuclide to a chelating agent covalently linked to an antibody. Other embodiments use calicheamicin, aunstatins, geldanamycin, maytansine, and duocarmycins and analogues; for the latter, see U.S. Patent Application 2003 / 0050331.

[0086] In various embodiments, the antibodies of the present invention are fused or conjugated with cytokines. As used herein, "cytokine" is a general term for proteins released by a population of cells that act as intercellular mediators to another cell. For example, as described in Penichet et al., 2001, J Immunol Methods 248-91-101, cytokines can be fused with antibodies to provide a range of desired properties. Examples of such cytokines are lymphokines, monokines, and conventional peptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; pro-relaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental prolactin; tumor necrosis factor-α and tumor necrosis factor-β; Müllerian-inhibiting substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-derived growth factor; and transforming growth factor. Factors (TGFs), such as TGF-α and TGF-β; insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); bone-inducing factor; interferons, such as interferon-α, interferon-β, and interferon-γ; colony-stimulating factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, and IL-15; tumor necrosis factors, such as TNF-α or TNF-β; C5a; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes biologically active equivalents of proteins and natural sequence cytokines derived from natural sources or from recombinant cell cultures.

[0087] In various other embodiments, the antibodies of the present invention are fused, conjugated, or operatively linked to toxins, including but not limited to small molecule toxins and enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof. For example, various immunotoxins and immunotoxin methods are described in Thrush et al., 1996, Ann. Rev. Immunol. 14:49-71. Small molecule toxins include, but are not limited to, calichiomycin, maytansine (US5,208,020), monosporine, and CC1065. Analogs of auristatin 10, such as auristatin E (AE) and monomethylauristatin E (MMAE), have been found to be usable as conjugates of the antibodies of the present invention. Useful enzymatically active toxins include, but are not limited to, diphtheria A chain, non-bound active fragments of diphtheria toxin, exotoxin A chain (from *Pseudomonas aeruginosa*), ricin A chain, abrin A chain, modizol A chain, α-salicylicin, *Aleurites fordii* protein, caryophyllin protein, *Phytolacca americana* protein (PAPI, PAPII, and PAP-S), *Momordica charantia* inhibitor, lactudin, crotonin, saponin (*Sapaonaria officinalis*) inhibitor, white tree toxin, mitogellin, restriction bacteria, phenolmycin, enomycin, and trichosporins. This invention further encompasses conjugates between the antibodies of this invention and compounds with nucleolytic activity (e.g., ribonucleases or DNA endonucleases such as deoxyribonuclease (DNase)).

[0088] In further embodiments, the antibodies of the present invention can be fused, conjugated, or operatively linked to radioisotopes to form radioconjugates. A variety of radioisotopes can be used to produce radioconjugate antibodies. Examples include, but are not limited to, radioisotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and Lu.

[0089] In various other embodiments, the antibodies of the present invention can be conjugated to a "receptor" (e.g., streptavidin) for tumor pre-targeting, wherein the antibody-receptor conjugate is administered to a patient, unbound conjugates are then removed from circulation using a scavenger, and a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radioactive nucleotide) is then administered. In alternative embodiments, the antibody is conjugated to or operably linked to an enzyme to use antibody-dependent enzyme-mediated prodrug therapy (ADEPT). ADEPT can be used by conjugating or operably linking the antibody to a prodrug-activating enzyme that converts a prodrug (e.g., a peptide-based chemotherapeutic agent, see PCT application WO81 / 01145, the contents of which are incorporated herein by reference in their entirety) into an active anticancer drug. See, for example, PCT application WO88 / 07378 or U.S. Patent 4,975,278, each of which is incorporated herein by reference in its entirety. The enzyme component of the immunoconjugates that can be used for ADEPT includes any enzyme that can act on the prodrug in a way that converts the prodrug into a more active cytotoxic form. Enzymes that can be used in the methods of the present invention include, but are not limited to: alkaline phosphatase, which can be used to convert phosphate-containing prodrugs into free drugs; arylsulfatase, which can be used to convert sulfate-containing prodrugs into free drugs; cytosine deaminase, which can be used to convert non-toxic 5-fluorocytosine into the anticancer drug 5-fluorouracil; proteases, such as Serratia protease, thermophilic protease, subtilis protease, carboxypeptidase, and cathepsins (e.g., cathepsins B and L), which can be used to convert peptide-containing prodrugs into free drugs; D-alanyl carboxypeptidase, which can be used to convert prodrugs containing D-amino acid substituents; carbohydrate lyases, such as β-galactosidase and neuramimidase, which can be used to convert glycosylated prodrugs into free drugs, and β-lactamase, which can be used to convert drugs derived from α-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, which can be used to convert drugs derived from drugs having phenoxyacetyl or phenylacetyl groups on their amine nitrogen into free drugs. Alternatively, antibodies with enzymatic activity, also referred to in the art as "abzymes," can be used to convert prodrugs into free active drugs (see, for example, Massey, 1987, Nature 328. 457-458, the contents of which are incorporated herein by reference in their entirety). Antibody-abzyme conjugates can be prepared to deliver the abzyme to tumor cell populations. A variety of other conjugates are contemplated for the antibodies of the present invention. Various chemotherapeutic agents, antiangiogenic agents, tyrosine kinase inhibitors, and other therapeutic agents are described below as potential antibody conjugates.

[0090] Preferably, the antibodies described herein are suitable for treating proliferative diseases, particularly tumors. A tumor, as defined herein, is a cancer or growth that may include melanoma, prostate cancer, colon cancer, liver cancer, bladder cancer, breast cancer, or lung cancer. The term tumor or cancer includes any cancer cell line in which elevated Angptl4 expression is found. Any cancer derived from these cancer cell lines is also included in the term tumor or cancer. Metastatic cancer is included in the term tumor or cancer.

[0091] Therefore, if one aspect of the invention relates to a method for treating a patient suffering from a proliferative disease or condition, preferably cancer, the method may include administering a therapeutically effective amount of an antibody as described herein to the patient. It also covers the antibodies of the invention used as medicines, for example, in methods for treating proliferative diseases or conditions such as cancer or tumors.

[0092] "Treatment" and "performing treatment," and their synonyms, refer to therapeutic treatment aimed at stopping or reducing cell proliferation in (cancerous) cells. Preferably, stopping or reducing cell proliferation will thereby stop or halt tumor growth, reduce tumor size, or stop or slow metastasis. Those who require such treatment include individuals or patients who have been diagnosed with cancer, tumors, or metastatic cancer. Patients or individuals refer to animals, such as mammals, preferably humans. However, in various embodiments, the cells are in vitro and the method is an in vitro method. In various other embodiments, the cells are in vivo and antibodies are applied to the subject, such as patients or individuals requiring treatment.

[0093] Therefore, one aspect of the invention also relates to an in vitro method for reducing cell proliferation, the method comprising the step of contacting proliferating cells with an antibody described herein. In various embodiments, the in vitro cells are cell lines. In various other cases, the antibody may be applied to cells to reduce cell proliferation in cancer cells.

[0094] Preferably, the antibody administered to the subject is a therapeutically effective amount. A therapeutically effective amount will be able to block the angiopoietin-like 4 (ANGPTL4) polypeptide in proliferating cells in the culture medium or at the tumor site.

[0095] As used herein, a “therapeutic effective amount” of an antibody will be an amount capable of stopping or preventing cell proliferation, which may result in stopping or preventing tumor growth or reducing (decreasing) tumor size. It may also refer to prevention. The dosage and administration of the antibodies of the present invention in pharmaceutical compositions can be determined by those skilled in the art of clinical pharmacology or pharmacokinetics. The effective amount of an antibody used therapeutically (e.g., the antibody described herein) will depend on, for example, the therapeutic target, the route of administration, and the condition of the mammal. Therefore, it is necessary for therapists to adjust the dosage and modify the route of administration as needed to obtain the best therapeutic effect. Typical daily dosages range from about 10 ng to up to 100 mg per day, preferably from about 1 μg to 10 mg per day. Dosages may include any amount of antibody in the range of 10 μg to 100 μg per day, or more preferably 25 μg, 50 μg, or 75 μg.

[0096] Another aspect of the present invention relates to nucleic acid molecules encoding heavy or light chain variable domains of the antibodies described herein.

[0097] The terms “polynucleotide” and “nucleic acid (molecule)” are used interchangeably herein to refer to polymeric forms of nucleotides of any length, including naturally occurring and non-naturally occurring nucleic acids. Polynucleotides may comprise deoxyribonucleotides, ribonucleotides, and / or their analogues. Methods for selecting and preparing nucleic acids are diverse and are described in detail in standard biomolecular protocols. Typical methods include preparative PCR and chromatographic purification or stepwise synthesis of artificial nucleic acids starting from existing template DNA. Generally, the nucleic acid molecule referred to herein is a DNA molecule.

[0098] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a variable domain of the light chain shown in SEQ ID NO:16, 18, 20 or 22.

[0099] Similarly, in various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a variable domain of the heavy chain shown in SEQ ID NO:15, 17, 19 or 21.

[0100] The present invention may also include an expression system comprising:

[0101] (i) The first gene encoding the variable region of the light chain or the complete light chain; and

[0102] (ii) A second gene encoding the variable region of the heavy chain or the complete heavy chain;

[0103] The expression system may optionally be inducible to express antibodies as described herein.

[0104] Preferably, the expression system is a cell-based expression system in prokaryotic or eukaryotic cells as described above.

[0105] Another aspect of the invention relates to a composition for treating cancer, the composition comprising an antibody and a carrier and / or excipient as described herein.

[0106] The antibody formulations of the present invention can be prepared for storage by mixing antibodies of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington Pharmaceutical Sciences (16th edition, Osol, A. editor, 1980)), for example, in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are pharmaceutically acceptable at the amounts and concentrations used, i.e., non-toxic to receptors, and include: buffers, such as phosphates, citrates, acetates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives, such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzyl ammonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or other immunoglobulins. Proteins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as 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; sweeteners and other flavorings; fillers, such as microcrystalline cellulose, lactose, corn, and other starches; binders; additives; colorants; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0107] In one embodiment, the pharmaceutical composition comprising the antibody of the present invention may be in a water-soluble form, for example, in the form of certain components comprising pharmaceutically acceptable salts (e.g., acid and / or base addition salts). Formulations for in vivo administration are typically sterile. This is readily achieved by known methods.

[0108] The antibodies disclosed herein can also be formulated into immunoliposomes or microcapsules. Remington Pharmaceutical Sciences (16th edition, Osol, A. editor, 1980) discloses techniques for preparing such formulations.

[0109] The pharmaceutical composition comprising the antibody of the present invention can be administered in a variety of ways (e.g., in the form of a sterile aqueous solution), including but not limited to oral, subcutaneous, intravenous, intranasal, intraocular, transdermal, topical (e.g., gel, ointment, lotion, cream, etc.), intraperitoneal, intramuscular, intrapulmonary, vaginal, parenteral, rectal, or intraocular administration. As is known in the art, the pharmaceutical composition can be formulated accordingly based on the method of administration.

[0110] Protein therapeutic agents are typically delivered via intravenous infusion or bolus. In various embodiments, the antibodies of the present invention can also be delivered using such methods. For example, they can be administered via intravenous infusion of 0.9% sodium chloride as an infusion solvent or carrier.

[0111] In various implementations, the dosage and frequency of administration are selected to be effective in treatment or prevention. As is known in the art, adjustments may need to be made based on protein degradation, systemic versus local delivery, as well as age, weight, general health condition, sex, diet, timing of administration, drug interactions, and severity of the condition, and such adjustments will be determined by those skilled in the art through routine experiments.

[0112] The concentration of the therapeutically active antibody in the formulation can vary from about 0.1% by weight to 100% by weight. In one embodiment, the antibody concentration is in the range of 0.003 μM to 1.0 M.

[0113] For the purpose of treating a patient, a therapeutically effective dose of the antibody of the present invention may be administered. "Therapeutically effective dose" as used herein refers to the dose that produces an effect. The exact dose will depend on the purpose of treatment and can be determined by those skilled in the art using known techniques. Dosage ranges may be from 0.0001 to 100 mg / kg body weight or greater, for example 0.1, 1, 10, or 50 mg / kg body weight, for example 1 to 10 mg / kg body weight.

[0114] In some embodiments, only a single dose of antibody is used. In other embodiments, multiple doses of antibody are administered. The elapsed time between administrations can be less than 1 hour, about 1 hour, about 1-2 hours, about 2-3 hours, about 3-4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 2-4 days, about 4-6 days, about 1 week, about 2 weeks, or more than 2 weeks.

[0115] In other embodiments, the antibodies of the present invention are administered using a metronome dosing regimen, by continuous infusion or frequent administration without extended rest periods. Such metronome dosing may involve administration at constant intervals without rest periods. Typically, such regimens cover long-term low-dose or long-term continuous infusions, such as 1-2 days, 1-2 weeks, 1-2 months, or up to 6 months or longer. Using lower doses can minimize side effects and the required rest periods.

[0116] In some embodiments, the antibody of the present invention and one or more other preventive or therapeutic agents are periodically administered to a patient. Periodic therapy includes administering a first agent once, a second agent a second time, and optionally, another agent at a separate time or during a rest period, and then repeating this sequence of administration one or more times. The number of cycles is typically 2 to 10. Periodic therapy can reduce the development of resistance to one or more drugs, minimize side effects, or improve therapeutic efficacy.

[0117] The antibodies of the present invention can be administered concurrently with one or more other treatment regimens or agents. Additional treatment regimens or agents can be used to improve the efficacy or safety of the antibodies. Furthermore, additional treatment regimens or agents can be used to treat the same disease or concomitant diseases, rather than altering the function of the antibody. For example, the antibodies of the present invention can be administered to a patient in conjunction with chemotherapy, radiotherapy, or both chemotherapy and radiotherapy. The antibodies of the present invention can be administered in combination with one or more other preventative or therapeutic agents, including but not limited to cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitors, anti-hormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotective agents, immunostimulants, immunosuppressants, agents that promote blood cell proliferation, angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors, other antibodies, FcγRIIb or other Fc receptor inhibitors, or other therapeutic agents.

[0118] The terms "in combination with" and "to be administered together" are not limited to administering the prophylactic or therapeutic agent at exactly the same time. Rather, it means that the antibodies of the present invention and one or more other agents are administered sequentially and at time intervals, so that they can act together to provide increased benefit compared to treatment using only the antibodies or one or more other agents of the present invention. In one embodiment, the antibodies and one or more other agents act in combination; in another embodiment, they act synergistically. Such molecules are appropriately present in combination in amounts effective for the intended purpose. A skilled medical practitioner can determine, by experience or by considering the pharmacokinetics and mode of action of the agents, one or more appropriate doses of each therapeutic agent, as well as appropriate timing and method of administration.

[0119] In various implementations, this co-administration also requires that the active agents be co-formulated in a pharmaceutical composition, as defined herein.

[0120] In various embodiments, the antibodies of the present invention are administered in combination with one or more additional molecules, such as other antibodies or Fc molecules. The antibodies of the present invention may be co-administered with one or more other antibodies effective in treating the same disease or other concomitant diseases; for example, two antibodies recognizing two antigens overexpressed in a given type of cancer may be administered.

[0121] In various embodiments, the antibodies of the present invention are administered in combination with chemotherapeutic agents. As used herein, "chemotherapeutic agent" refers to a compound that can be used to treat cancer. Examples of chemotherapy agents include, but are not limited to: alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; androgens, such as capprotestone, drotalonone propionate, cyclothionol, methanone, and testosterone; antiadreners, such as aminoglutethimide, mitotane, and trilostane; antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprorelin, and goserelin; and antibiotics, such as aclacinomysins, actinomycin, autramycin, diazoserine, bleomycin, cactinomycin, carlicycin, carabicin, carminomycin, carzinophilm, and chromomycin. s), daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogamycin, oligomycin, perpriomycin, potfiromycin, puromycin, quelamycin n), rodorubicin, streptometin, etreptozocin, tuberculin, ubenimex, zinostatin, zorubicin; anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitor of 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, Onapristone and Fareston; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as folate, methotrexate, pteroxate, and trimetrexate; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa.Ethyleneimides and methylmelamines, including hexamethylmelamine, triethylamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide, and trimethylol melamine; folic acid supplements, such as frolinic acid; nitrogen mustards, such as chlorambucil, naphthiamethoxam, chophosphatamide, estramustine, ifosfamide, nitrogen mustard, methoxynitric acid, melphalan, novobichin, benzyl mustard cholesterol, prednimustine, trazophosphatamide, and uracil mustard; nitroureas, such as carmustine, chloramphenicol, fotemustine, lomustine, nimustine, and ranimnustin. e); platinum analogs, such as cisplatin and carboplatin; vincristine; platinum; proteins, such as arginine deiminase and asparaginase; purine analogs, such as fludarabine, 6-mercaptopurine, thioimidazoline, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, fluorouridine, and 5-FU; taxanes, such as paclitaxel (TAXOL®). Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); topoisomerase inhibitor RFS 2000; thymidine synthase inhibitors (such as Tomudex); other chemotherapy drugs, including: acetylglucuronide; aldehyde phosphoramidide; aminolevulinic acid; acridine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; difluoromethylornithine (DMFO); elfornithine; elliptinium acetate); etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; mitoxantrone; mopidanmol; nitracrine; pentostatin; phenamet; pirarubicin; podophylloic acid; 2-acetylhydrazine;Procarbazine; PSK®; Razoxane; Sizofiran; Germanium spiroamine; Tenuazonic acid; Triaminequinone; 2,2',2"-Trichlorotriethylamine; Uranium; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactalol; Pipobroman; Gacytosine; Arabinoside (“Ara-C”); Cyclophosphamide; Thiotepa; Chlorobenzide; Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Etoposide (e Toposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; retinoic acid; esperamicin; capecitabine. Pharmaceutically acceptable salts, acids, or derivatives of any of the above substances may also be used.

[0122] As described above, such chemotherapeutic agents or other cytotoxic agents can be administered as prodrugs. As used herein, a "prodrug" refers to a precursor or derivative form of a drug that is less cytotoxic to tumor cells than the parent drug and can be enzymatically activated or converted into a more active parent form. See, for example, Wilman, 1986, Biochemical Society Transactions, 615th Meeting Belfast, 14:375-382; Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," DirectedDrug Delivery; and Borchardt et al., (ed.): 247-267, Humana Press, 1985. Prodrugs discovered that can be used in this invention include, but are not limited to: phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, prodrugs containing optionally substituted phenoxyacetamides or optionally substituted phenylacetamides, 5-fluorocytosine and other 5-fluorouridine prodrugs, which can be converted into more active, non-cytotoxic drugs. Examples of cytotoxic drugs that can be derived into prodrug forms for use with the antibodies of this invention include, but are not limited to, any of the above-described chemotherapeutic agents.

[0123] In various other embodiments, the antibody is administered in conjunction with one or more immunomodulatory agents. Such agents can increase or decrease the production of one or more cytokines, upregulate or downregulate self-antigen presentation, mask MHC antigens, or promote the proliferation, differentiation, migration, or activation of one or more types of immune cells.Immunomodulators include, but are not limited to: nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, celecoxib, diclofenac, etodoxacin, fenprofen, indomethacin, ketolaccoside, oxamylene, nabumentone, sulindac, tolmentin, rofecoxib, naproxen, ketoprofen, and nabumetone; steroids (such as glucocorticoids, dexamethasone, cortisone, hydroxycortisone, methylprednisolone, prednisone, prednisolone, triamcinolone); azulfidineicosanoids, such as prostaglandins, thromboxanes, and leukotrienes; and topical steroids, such as anthraquinone, calcipotriol, clobetasol, and tazarotene); and cytokines, such as TGFβ, IFNα, etc. IFNβ, IFNγ, IL-2, IL-4, IL-10, cytokines, chemokines, or receptor antagonists, including those targeting BAFF, B7, CCR2, CCR5, CD2, CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28, CD40, CD40L, CD44, CD45, CD402, CD64, CD80, CD86, CD147, CD152, complement factor (C5, D) CTLA4, eosinophil activation chemokine, Fas, ICAM, ICOS, IFNα, IFNβ, IFNγ, IFNAR, IgE, IL-1, IL-2, IL-2R, IL-4, IL-5R, IL-6, IL-8, and IL-9. IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, integrin, LFA-1, LFA-3, MHC, selectins, TGFβ, TNFα, TNFβ, TNF-R1, antibodies against T-cell receptors, soluble receptors and receptor-Fc fusions, including Enbrel® (etanercept), Humira® (adalimumab), and Remicade® (infliximab), heterologous anti-lymphocyte globulins; other immunomodulatory molecules, Examples include anti-idiotypic antibodies against 2-amino-6-aryl-5-substituted pyrimidines, MHC-binding peptides and MHC fragments, azathioprine, brequinar, bromocryptine, cyclophosphamide, cyclosporine A, D-penicillamine, deoxyguanidine, FK506, glutaraldehyde, gold, hydroxychloroquine, leflunomide, malononitriloamides (such as leflunomide), methotrexate, minocycline, imidazoribine, rapamycin, and sulfasalazine.

[0124] In various embodiments, the antibodies of the present invention are administered in conjunction with cytokines. As used herein, "cytokine" is a general term for proteins released by a population of cells that act as intercellular mediators to another cell. Examples of such cytokines are lymphokines, monokines, and conventional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; pro-relaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); epidermal growth factor; liver growth factor; fibroblast growth factor; prolactin; placental prolactin; tumor necrosis factor-α and tumor necrosis factor-β; Müllerian inhibitory substances; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors, such as NGF-α; platelet-derived growth factor; and transforming growth factor. Growth factors (TGFs), such as TGF-α and TGF-β; insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); bone-inducing factor; interferons, such as interferon-α, interferon-β, and interferon-γ; colony-stimulating factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, and IL-15; tumor necrosis factors, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes biologically active equivalents of proteins and natural sequence cytokines derived from natural sources or from recombinant cell cultures.

[0125] In various embodiments, co-administered cytokines or other agents are used to stimulate cells of the immune system. This modality of treatment can enhance desired effector functions. For example, agents that stimulate NK cells, including but not limited to IL-2, can be co-administered. In another embodiment, agents that stimulate macrophages, including but not limited to C5a and formyl peptides such as N-formyl-methionyl-leucyl-phenylalanine (Beigier-Bompadre et al. (2003) Scand J. Immunol. 57. 221-8), can be co-administered. Furthermore, agents that stimulate neutrophils, including but not limited to G-CSF, GM-CSF, etc., can be administered. Additionally, agents that promote the migration of such immunostimulatory cytokines can be used. Furthermore, other agents, including but not limited to interferon-γ, IL-3, and IL-7, can promote one or more effector functions.

[0126] Alternatively, cytokines or other agents that inhibit effector cell function can be co-administered with the antibodies of the present invention. This modality of treatment can limit unwanted effector function.

[0127] The antibodies of the present invention can be combined with other treatment regimens. For example, in one embodiment, a patient treated with the antibodies of the present invention may also receive radiotherapy. Radiotherapy can be administered according to regimens commonly used in the art and known to those skilled in the art. Such therapy includes, but is not limited to, cesium, iridium, iodine, or cobalt radiation. Radiotherapy can be whole-body irradiation or can be locally directed to a specific site or tissue within or on the body, such as the lungs, bladder, or prostate. Typically, radiotherapy is administered in pulses over a period of about 1 to 2 weeks. However, radiotherapy can be administered over a longer period. For example, radiotherapy can be administered to a patient with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple consecutive doses. A skilled medical practitioner can empirically determine one or more appropriate doses of radiotherapy used herein. According to another embodiment of the invention, the antibodies of the present invention and one or more other anticancer therapies are used to treat cancer cells ex vivo. Such ex vivo treatment is intended for use in bone marrow transplantation, particularly autologous bone marrow transplantation. For example, antibodies and one or more other anticancer therapies (such as those described above) can be used to treat cells or tissues containing cancer cells to deplete or substantially deplete the cancer cells before transplantation to a recipient patient.

[0128] Of course, the antibodies of the present invention can be used in combination with other treatment techniques such as surgery or phototherapy.

[0129] The present invention is further illustrated by the following embodiments. However, it should be understood that the present invention is not limited to the exemplary embodiments.

[0130] "Including" means including, but not limited to, anything that follows the word "including". Therefore, the use of the term "including" indicates that the listed elements are necessary or mandatory, but other elements are optional and may or may not be present.

[0131] "Composed of" means including but not limited to anything that follows the phrase "composed of". Therefore, the phrase "composed of" indicates that the listed elements are necessary or mandatory, and no other elements may exist.

[0132] The invention described illustratively in this application can be suitably implemented by removing any or more elements or limitations not specifically disclosed herein. Therefore, terms such as "comprising," "including," and "containing" should be interpreted broadly and without limitation. Furthermore, the terms and expressions used herein are intended to describe rather than limit, and their use is not intended to exclude any equivalent forms or portions thereof of the shown and described features, but rather to understand that various modifications may be made within the scope of the invention claimed. Therefore, it should be understood that although the invention has been specifically disclosed through preferred embodiments and optional features, those skilled in the art can still adopt optional features, modifications, and variations of the specific embodiments of the invention disclosed herein, and such modifications and variations are considered to be within the scope of the invention.

[0133] "Approximately" relative to a given value refers to a value within a range of 10% of the specified value.

[0134] This document provides a broad and general description of the invention. Each narrower group of species and subgenus falling within the scope of this general disclosure also constitutes part of the invention. This includes general descriptions of the invention with incidental conditions or negative limitations that remove any subject matter from that genus, regardless of whether the excluded material is specifically enumerated herein.

[0135] Other embodiments are described within the scope of the following claims and non-limiting examples. Furthermore, since features or aspects of the invention are described according to the Markush group, those skilled in the art will recognize that the invention is therefore also described according to any individual member or subgroup of the Markush group.

[0136] Example

[0137] Materials & Methods

[0138] Indirect ELISA: Purified cANGPTL4 was immobilized on carboxylated polystyrene microplates (Biomat) at 37°C for 2 hours using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)-mediated amination. Purified BSA was used as a control. The microplates were washed three times with 1xPBST (PBS containing 0.1% Tween 20) at 300 µL / well. The wells were blocked with 2% BSA in PBS solution (300 µL / well) at 37°C for 1 hour. The microplates were washed three times with 1xPBST at 300 µL / well. Serially diluted Antuzumab (starting from 10 µg / mL) was added and incubated at 37°C for 1 hour. The microplates were washed four times with 1xPBST at 300 µL / well. Next, add 100 µL of anti-human Fc IgG-HRP in PBS solution (1:2K dilution) and incubate for 1 hour to detect the bound humanized antibody. Wash the plate five times with 1xPBST at 300 µL / well. Finally, add one-step TMB (3,3',5,5'-tetramethylbenzidine) and incubate at room temperature for 30 minutes. Stop the reaction by adding 100 µL of 2M H2SO4. Measure the absorbance at 450 nm.

[0139] Surface plasmon resonance (SPR): The CM5 sensor chip and amine coupling kit were from GE Healthcare. Purified cANGPTL4 was immobilized on the CM5-carboxydextran sensor chip via amine coupling using the surface preparation module of the BIACORE 3000 recommended by the manufacturer (BIAcore). Antiuzumab (0.16, 0.32, 0.63, 1.25, 2.50, and 5.0 µm) diluted and buffered with 50 mM Tris (pH 8.0) and running buffer (50 mM Tris, pH 8.0, 100 mM NaCl) was introduced into the cANGPTL4-conjugated CM5 chip at a flow rate of 5 µl / min for 10 min. After incubation for 45 seconds, the chamber was washed with the same buffer, followed by elution of bound molecules with 10 mM glycine (pH 6.0). The CM5 chip was reused after washing with running buffer at 20 µl / min for 10 min. Each sensor map was corrected by subtracting the sensor map obtained from a reference flow cell without protein fixation. Global fitting of the SPR data was used to determine association with the Scrubber 2 software (K-axis). ass ) and dissociation (K diss Values ​​are given as the mean ± SD of the three independent formulations of Antuzumab.

[0140] 3D Culture of Tumor-like Spheroids with Micropatterned Agarose Hydrogel: Micromolds were cast using MicroTissues® 3DPetri Dish® micromolds with 1% agarose (w / v in PBS). Each micromold had 256 wells, producing 256 spheroids. The micromolds were transferred to 12-well plates and irradiated with UV for 45 minutes before use. 120 µL of 1.5 x 10⁻⁶ microspheres were then cultured. 6 A suspension of cancer cells dispersed in DMEM was seeded onto each micromold. After settling for 5 minutes, 1.5 mL of culture medium was added to each well, and the cells were cultured for 20 hours prior to treatment. For treatment, 512 spheres (from 2 micromolds) were flushed from the wells using culture medium and centrifuged at 200 g for 4 minutes, then resuspended in 4.8 mL of DMEM. 400 µL of the suspended spheres were pipetted into agarose-coated 12-well plates for monoclonal antibody treatment.

[0141] Antuzumab treatment under normoxic and hypoxic conditions: Tumor spheroids were treated with different concentrations of 1–8 µg / mL of Antuzumab for 16 hours. For the negative control, 6 µg / mL of IgG was used. Hypoxia was induced by treating the spheroids in a hypoxic chamber with a 1% O2 concentration (Stem Cell Technology, USA).

[0142] Cell viability assay & dose-response curve establishment: Cancerous tumor cells were collected from wells and trypsinized in 300 µL of 0.05% trypsin for 4 min. The dispersed cells were resuspended in 50 µL of culture medium. Cell viability was determined using the ADAM-MC automated cell counter (NanoEnTek, Singapore). Total cells and non-viable cells were measured using ADAM-MC software, and cell viability was tabulated. Half-maximal inhibitory concentration (IC50) was also determined. 50 IC50 is defined as the drug concentration administered to a 3D in vitro cell model that induces 50% total cell death. 50 The results were calculated using the built-in nonlinear regression model and sigmoid dose-response (variable slope) analysis in Graphpad PRISM 7.

[0143] Microscopy and immunofluorescence for live / dead cell imaging: 3D cell cultures were monitored and confirmed using the JuLI Stage: Real-Time Cell History Recorder (NanoEnTek, Singapore). Immunofluorescence imaging was performed in parallel using Hoechst 33342 fluorescent dye and 1 µg / mL PI (Thermo Fisher Scientific, USA) to stain live and dead cells separately.

[0144] Orthotopic xenograft for bladder tumor. This protocol is as described by Huebner et al. (An orthotopic xenograft model for high-risk non-muscle invasive bladder cancer in mice: influence of mouse strain, tumor cell count, dwell time and bladder pretreatment. BMCCancer 17, 790 (2017)).

[0145] Example 1

[0146] Starting with the known mouse monoclonal antibody mAb11F6C4, four humanized heavy chain variable regions (ANGH1-4) and light chain variable regions (ANGK1-4) were synthesized. Their DNA sequences and amino acid sequences are shown in [the diagram]. Figure 1 It is also listed in SEQ ID NO.7-14. Figure 1 The amino acid sequences of AGH1-4 and ANGK1-4 are shown compared with the heavy and light chain amino acid sequences of mAb11F6C4 from mice. Shaded amino acid regions (black letters) highlight the differences between the humanized and mouse-derived heavy and light chains. CDR regions are indicated by shaded boxes with white letters.

[0147] Sixteen pairs of ANGH+ANGK combinations were transfected into CHO cells, of which six pairs did not produce stable humanized monoclonal antibody cell lines. The table is shown below.

[0148] Table 1: Humanized monoclonal antibodies against human cANGPTL4

[0149]

[0150] X indicates an unstable monoclonal antibody cell line.

[0151] Example 2

[0152] Indirect ELISA and surface plasmon resonance (SPR) assays of the stable antibodies in Example 1 identified three humanized mAbs that showed an affinity for the human cANGPTL4 antigen that was greater than that of the original mouse-derived mAb (11F6C4; V). H ::SEQ IDNO: 23;V L (SEQ ID NO:24) 7, 5, and 2 times. The results of the ELISA assay are shown in... Figure 2The results of the SPR assay are shown in Table 2 below. These three humanized mAbs with higher affinity (named Antuzumab 1, 2 and 3) were selected for stable cell line production.

[0153] Table 2

[0154]

[0155] Example 3

[0156] Antitumor assays were performed using in vitro 3D tumor models of bladder cancer (T24, UMUC3), gastric cancer (MKN74), and glioblastoma (U87) to evaluate the antitumor activity of these three higher-affinity humanized antibodies and a mouse antibody as a reference. IC50 values ​​(µg / mL) are shown in Table 3 below.

[0157] Table 3:

[0158]

[0159] Example 4

[0160] The growth and maintenance of hybridomas of Antuzumab 1, 2, and 3 were determined in two batches of production. The results are shown in Table 4.

[0161] Table 4

[0162]

[0163] Example 5

[0164] ANGPTL4 erodes cell-cell integrity, leading to retinal vascular leakage, an early sign of diabetic retinopathy and age-related macular degeneration. Antuzumab 1 can block the action of ANGPTL4 and prevent retinal vascular leakage.

[0165] Following hypoxia treatment, MIO-M1 cells secreted ANGPTL4 (verified by Western blotting). The control medium was normoxic medium for MIO-M1 cells after 48 hours. The treatment medium was hypoxic medium for MIO-M1 cells after 48 hours. Measurements were taken at 6, 24, and 48 hours.

[0166] MIO-M1 (Muller cells) secrete high levels of ANGPTL4 under hypoxic conditions, resulting in decreased transendothelial impedance at 6 hours (the first measurement time point). Transendothelial impedance reaches saturation between 24 and 48 hours. Figure 3A). Next, 10 ng Antuzumab 1 was added to the conditioned medium of hypoxic-treated MIO-M1 cells. Antuzumab 1 was able to inhibit vascular leakage induced by hypoxia in the conditioned medium of MIO-M1 cells. Figure 3 B).

[0167] Example 6

[0168] Compared with the original mAb 11F6C4, Antuzumab 1, 2, and 3 showed reduced immunogenicity in both early and late responses, as determined by a simplified in vitro comparative immunogenicity assessment (IVCIA). Results are as follows... Figure 4 As shown.

[0169] Example 7

[0170] When therapeutic agents are processed by antigen-presenting cells and presented to T helper cells, an immune response to the biotherapy can be induced. The total percentage of T cells induced by anti-VEGF, Antuzumab 1, 2, and 3 was assessed using in vitro assays that elicit antigen-specific effector T cell responses. Results are as follows: Figure 5 As shown.

[0171] Antuzumab 1 and 2 showed immunogenicity comparable to humanized anti-VEGF (bevacizumab), as indicated by the percentage of T cell populations from five human PBMCs. Antuzumab 3 had a higher percentage of T cells in patients 2 and 4 compared to bevacizumab. However, Antuzumab 3 was comparable to published data for adalimumab, infliximab (anti-TNFα), and rituximab (anti-CD20).

[0172] Example 8

[0173] A high-risk NMIBC orthotopic bladder tumor xenograft model was developed in an animal model (immune-impaired NSG mice) using human UMUC3 cells. Intravesical administration of Antuzumab 1 (10 µg / mL) at week 3 following cancer cell infusion attenuated bladder tumor growth. Results are as follows... Figure 6 As shown in the figure. This experiment demonstrated the effectiveness of Antuzumab 1 in the treatment of bladder cancer by inhibiting cANGPTL4.

Claims

1. A humanized antibody targeting the C-terminal region (cANGPTL4) of angiopoietin-like 4 protein, said humanized antibody comprising a heavy chain variable domain and a light chain variable domain having a human backbone region, said heavy chain variable domain comprising mouse V protein having the amino acid sequence shown in SEQ ID NO. 1-3. H CDR1, V H CDR2 and V H The CDR3 region, the light chain variable domain, contains mouse V amino acids having the amino acid sequences shown in SEQ ID NO. 4-6. L CDR1, V L CDR2 and V L CDR3 area, in which: (1) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 11 (ANGH3) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 11 (ANGH3) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 12 (ANGK3) or a variant thereof; or (2) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 7 (ANGH1) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 7 (ANGH1) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof; (3) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 9 (ANGH2) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 9 (ANGH2) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof; (4) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 11 (ANGH3) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 11 (ANGH3) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in either SEQ ID NO: 8 (ANGK1) or 10 (ANGK2) or a variant thereof, or is composed of the amino acid sequence shown in either SEQ ID NO: 8 (ANGK1) or 10 (ANGK2) or a variant thereof; or (5) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 13 (ANGH4) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 13 (ANGH4) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof; wherein The variants are unchanged for the CDR region defined in SEQ ID NO.1-6 and have at least 80% sequence identity with each reference sequence.

2. The antibody according to claim 1, wherein... (1) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 7 (ANGH1) or a variant thereof or is composed of the amino acid sequence shown in SEQ ID NO: 7 (ANGH1) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof or is composed of the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1), 10 (ANGK2) or 12 (ANGK3) or a variant thereof; (2) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 9 (ANGH2) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 9 (ANGH2) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 8 (ANGK1) or 12 (ANGK3) or a variant thereof; (3) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 11 (ANGH3) or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 11 (ANGH3) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 10 (ANGK2) or 12 (ANGK3) or a variant thereof, or is composed of the amino acid sequence shown in any one of SEQ ID NO: 10 (ANGK2) or 12 (ANGK3) or a variant thereof; or (4) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 13 (ANGH4) or a variant thereof or is composed of the amino acid sequence shown in SEQ ID NO: 13 (ANGH4) or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof or is composed of the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof.

3. The antibody according to claim 1 or 2, wherein (1) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof; or (2) The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 11 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 11 or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 12 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 12 or a variant thereof; or (3) The heavy chain variable domain contains the amino acid sequence shown in SEQ ID NO: 13 or a variant thereof or is composed of the amino acid sequence shown in SEQ ID NO: 13 or a variant thereof, and the light chain variable domain contains the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof or is composed of the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof.

4. The antibody according to any one of claims 1 to 3, wherein, The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 11 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 11 or a variant thereof, and the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 12 or a variant thereof, or is composed of the amino acid sequence shown in SEQ ID NO: 12 or a variant thereof.

5. The antibody according to any one of claims 1 to 4, wherein, The antibody is IgG immunoglobulin, preferably IgG1 immunoglobulin, and more preferably IgG1κ immunoglobulin.

6. The antibody according to any one of claims 1 to 5, wherein, The antibody comprises a human IgG1 constant region within the heavy chain of the immunoglobulin and a human constant region within the light chain of the immunoglobulin.

7. The antibody according to any one of claims 1 to 6, wherein the antibody is used to treat proliferative diseases or conditions, preferably cancer.

8. A method for treating a patient suffering from a proliferative disease or condition, preferably cancer, the method comprising administering to the patient a therapeutically effective amount of an antibody according to any one of claims 1 to 6.

9. An in vitro method for reducing cell proliferation, comprising contacting proliferating cells with an antibody according to any one of claims 1 to 6.

10. A nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain and / or light chain variable domain of an antibody according to any one of claims 1 to 6.

11. The nucleic acid molecule according to claim 10, wherein, The nucleic acid molecule comprises a nucleotide sequence encoding a variable domain of a heavy chain as shown in any one of SEQ ID NO: 15, 17, 19 and 21.

12. The nucleic acid molecule according to claim 10 or 11, wherein, The nucleic acid molecule comprises a nucleotide sequence encoding a variable domain of a light chain as shown in any one of SEQ ID NO:16, 18, 20 and 22.

13. The nucleic acid molecule according to any one of claims 10 to 12, wherein, The nucleic acid molecule is a carrier, preferably a plasmid.

14. A host cell comprising the nucleic acid molecule of any one of claims 10 to 13.

15. A composition for treating cancer, comprising the antibody of any one of claims 1 to 6 and a pharmaceutically acceptable carrier and / or excipient.

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