Heparanase-neutralizing A54 monoclonal antibody

Abstract

The present invention relates to a heparanase-binding and heparanase-neutralizing monoclonal antibody (IgG-1 mAb A54), including its epitope (HBD-II) and mode of interaction with heparanase, to pharmaceutical compositions comprising same, and to its use for inhibiting or treating diseases or disorders associated with heparanase activity, including, but not limited to, cancer, inflammation, viral infection, diabetes, and related complications. The present invention further provides combination cancer treatments comprising a heparanase-neutralizing mAb and an additional anti-cancer treatment, such as chemotherapy or radiation.

Description

[Technical Field]

[0001] The present invention relates to heparanase-neutralizing monoclonal antibodies, pharmaceutical compositions containing the same, and their use for treating diseases or disorders related to heparanase activity in a subject. [Background technology]

[0002] Heparanase is an endo-β-D-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a limited number of sites. Heparanase activity correlates with the metastatic potential of tumor-derived cells, which is attributed to enhanced cell dissemination as a result of HS cleavage and remodeling of the extracellular matrix (ECM) and basement membrane beneath epithelial and endothelial cells. Heparanase expression is induced in all major types of human cancer, namely carcinomas, sarcomas, and hematological malignancies. Increased heparanase levels are most often associated with decreased patient survival after surgery, increased tumor metastasis, and elevated microvascular density. In addition, upregulation of heparanase was associated with larger tumor sizes. Similarly, heparanase overexpression was enhanced, while local delivery of anti-heparanase siRNA inhibited the proliferation of tumor xenografts. These results suggest that heparanase function is not limited to tumor metastasis but is involved in the progression of the primary lesion, thus definitively supporting the close involvement of heparanase in tumor progression and prompting the development of heparanase inhibitors as anticancer agents.

[0003] Heparanase has also been shown to promote cell infiltration associated with autoimmunity, inflammation (Lerner et al., J Clin Invest 2011;121(5):1709-21), and angiogenesis (Vlodavsky et al., Invasion & Metastasis 1992;12, 112-127). In addition, increased heparanase expression is associated with kidney (Levidiotis et al., Kidney Int. 60, 1287-1296, 2001; Abassi and Goligorsky MS. Adv Exp Med Biol. 2020;1221:685-702; van der Vlag and Buijsers. Adv Exp Med Biol. 2020;1221:647-667), liver (Xiao et al., Hepatology Res. 26, 192-198, 2003), and diabetic (Katz et al., Isr. Med. Assoc. 4, 996-1002, 2002; Gil et al., Diabetes 2012;61:208-16; Simeonovic et al., Adv Exp Med Biol. It has been observed in disorders of (2020;1221:607-630 and Ziolkowski et al., 2012;122:132-41). Heparanase is also involved in acute pancreatitis (Khamaysi et al., Adv Exp Med Biol. 2020;1221:703-719), cardiomyopathy (Shang et al., Adv Exp Med Biol. 2020;1221:721-745), amyloidosis (Li JP and Zhang X. Adv Exp Med Biol. 2020;1221:631-645), and viral infections (Agelidis A, Shukla D. Adv Exp Med Biol. 2020;1221:759-770).

[0004] The discovery that heparanase is involved in a wide variety of pathological processes has led to the development of therapeutic compounds that inhibit this enzyme, including PI-88, phosphomannopentaose sulfate (Chhabra and Ferro, Adv Exp Med Biol. 2020;1221:473-491), PG545 (Hammond and Dredge, Adv Exp Med Biol. 2020;1221:539-565), and roneparstat, a modified heparin that is 100% N-acetylated and 25% glycol-cleaved. Lonepalstat has little to no anticoagulant activity and exhibits a very reduced and undesirable release and activation of ECM-bound pro-angiogenic factor (i.e., bFGF) (Casu et al., Pathophysiol Haemost Thromb 2008;36(3-4):195-203; Naggi et al., J Biol Chem 2005;280(13):12103-13), and has been proven effective in several tumor model systems (Ritchie et al., 2011, ibid.; Yang et al., Blood 2007;110(6):2041-8; Noseda and Barbieri, Adv Exp Med Biol. 2020;1221:523-538), but may still exhibit properties unrelated to heparanase enzyme activity (Levidiotis et al., Nephrology (Carlton) 2005;10(2):167-73).

[0005] Three potential heparin-binding domains of heparanase were identified by the inventors and collaborators (Levy-Adam et al., Id J Biol Chem 2005;280(21):20457-66). The peptide corresponding to this sequence (called KKDC) physically interacts with heparin and HS with high affinity and inhibits heparanase enzyme activity, so Lys 158 -Asp 171The domain in question attracted particular attention. Furthermore, deletion constructs lacking this domain did not exhibit enzymatic activity, and a polyclonal antibody directed at this region (Ab#733) inhibited heparanase activity (Zetser et al., J Cell Sci 2004;117(11):2249-58).

[0006] Attempts to inhibit heparanase enzyme activity were initiated early in heparanase research, in parallel with the emerging clinical relevance of this activity. More recently, a variety of inhibitory molecules have been developed, including peptides, small molecules (Giannini et al., Adv Exp Med Biol. 2020;1221:567-603), modified non-anticoagulant species of heparin, and several other polyanionic molecules such as laminaran sulfate, suramin, PI-88, and PG545 (Dredge et al., Br J Cancer 2011, 635-42, Hammond and Dredge, Adv Exp Med Biol. 2020;1221:539-565). Similarly, anti-heparanase polyclonal antibodies have been developed, and it has been demonstrated that these antibodies neutralize heparanase enzyme activity and inhibit cell infiltration (He X et al., Cancer Res 2004;64(11):3928-33), proteinuria (Levidiotis V et al., Nephrology (Carlton) 2005;10(2):167-73), and neointima formation (Myler HA et al., J Biochem 2006;139:339-45). A neutralizing anti-heparanase monoclonal antibody has recently been reported (Weissmann et al., PNAS 113:704-709, 2016).

[0007] U.S. Patent No. 7,772,187 by some of the inventors of the present invention relates to the amino acid sequence derived from the N'-terminal region of the 50Kd subunit of heparanase, and in particular to the Lys of human heparanase. 158 -Asn 171 This patent relates to the sequence of [the sequence]. This 187 patent further discloses a polyclonal antibody against that sequence, as well as its composition and use as a heparanase inhibitor.

[0008] U.S. Patent No. 6,562,950 by the inventors and co - researchers of the present invention provides monoclonal antibodies induced by a heparanase protein or an immunogenic portion thereof that specifically inhibit heparanase activity. This '950 patent disclosed two monoclonal antibodies, HP - 130 and H - 239. Notably, HP - 239, which recognizes an internal epitope located at amino acids 130 - 230, did not cause inhibition of heparanase activity, while HP - 130, formed against the C - terminus of heparanase, almost completely inhibited its activity.

[0009] U.S. Patent No. 8,048,993 by the inventors and co - researchers of the present invention provides antibodies that specifically bind to an epitope of a heparanase protein on the condition that tyrosine at position 246 of the heparanase protein is substituted with phenylalanine.

[0010] International Publication No. 2017 / 064716A1 pamphlet describes a specific monoclonal antibody called 9E8 that binds to the Lys 158 -Asp 171 domain of heparanase, neutralizes heparanase enzyme activity, and attenuates tumor progression in lymphoma and myeloma (Weissmann et al., PNAS 113:704 - 709, 2016). IgG clones S9 - C1 and C6 - S4 - C3 are derived from IgM 9E8.

[0011] Therefore, the need for highly specific heparanase - neutralizing monoclonal antibodies that can be used for preventing and treating medical conditions related to heparanase activity is not satisfied.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Patent Document 2

Patent Document 3

[0013] [Non-Patent Document 1] Lerner et al., J Clin Invest 2011;121(5):1709-21 [Non-Patent Document 2] Vlodavsky et al., Invasion & Metastasis 1992;12, 112-127. [Non-Patent Document 3] Levidiotis et al., Kidney Int. 60, 1287-1296, 2001. [Non-Patent Document 4] Abassi and Goligorsky MS. Adv Exp Med Biol. 2020;1221:685-702 [Non-Patent Document 5] van der Vlag and Buijsers. Adv Exp Med Biol. 2020;1221:647-667 [Non-Patent Document 6] Xiao et al., Hepatology Res. 26, 192–198, 2003 [Non-Patent Document 7] Katz et al., Isr.Med.Assoc. 4, 996~1002, 2002 [Non-Patent Document 8] Gil et al., Diabetes 2012;61:208~16 [Non-Patent Document 9] Simeonovic et al. Adv Exp Med Biol. 2020;1221:607-630 [Non-Patent Document 10] Ziolkowski et al., 2012;122:132-41 [Non-Patent Document 11] Khamaysi et al. Adv Exp Med Biol. 2020;1221:703-719 [Non-Patent Document 12] Shang et al., Adv Exp Med Biol. 2020;1221:721-745 [Non-Patent Document 13] Li JP and Zhang X. Adv Exp Med Biol. 2020;1221:631-645 [Non-Patent Document 14] Agelidis A, Shukla D. Adv Exp Med Biol. 2020;1221:759-770 [Non-Patent Document 15] Chhabra and Ferro, Adv Exp Med Biol. 2020;1221:473-491 [Non-Patent Document 16] Hammond and Dredge, Adv Exp Med Biol. 2020;1221:539-565 [Non-Patent Document 17] Casu et al. Pathophysiol Haemost Thromb 2008;36(3-4):195-203 [Non-Patent Document 18] Naggi et al. J Biol Chem 2005;280(13):12103-13 [Non-Patent Document 19] Yang et al. Blood 2007;110(6):2041-8 [Non-Patent Document 20] Noseda and Barbieri, Adv Exp Med Biol. 2020;1221:523-538 [Non-Patent Document 21] Levidiotis et al., Nephrology (Carlton) 2005;10(2):167-73 [Non-Patent Document 22] Levy-Adam et al. Id J Biol Chem 2005;280(21):20457-66 [Non-Patent Document 23] Zetser et al. J Cell Sci 2004;117(11):2249-58 [Non-Patent Document 24] Giannini et al. Adv Exp Med Biol. 2020;1221:567-603 [Non-Patent Document 25] Dredge et al., Br J Cancer 2011, 635-42 [Non-Patent Document 26] He X et al., Cancer Res 2004;64(11):3928-33 [Non-Patent Document 27] Myler HA et al., J Biochem 2006;139:339-45 [Non-Patent Document 28] Weissmann et al., PNAS 113:704-709, 2016 [Overview of the project] [Means for solving the problem]

[0014] The present invention provides a heparanase-neutralizing IgG monoclonal antibody (mAb A54), a pharmaceutical composition containing the same, and its use for treating diseases or disorders related to heparanase activity, including but not limited to malignant proliferative disorders.

[0015] The mAb A54 of the present invention is useful for reducing and treating diseases and disorders associated with heparanase enzyme activity, either as a monotherapy or in combination with at least one further conventional therapy, including but not limited to chemotherapy or radiation therapy.

[0016] According to a first embodiment, the present invention provides a neutralizing monoclonal antibody (mAb), or an antibody fragment comprising at least an antigen-binding portion thereof. According to another embodiment, the mAb or the antibody fragment thereof has a heparanase neutralizing effect.

[0017] According to another embodiment, the antibody fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fd', Fv, dAb, isolated CDR region, single-chain antibody, diabody, and linear antibody.

[0018] In another aspect, the present invention provides an isolated polynucleotide sequence encoding an mAb or antibody fragment thereof of the present invention.

[0019] In another embodiment, a vector comprising a polynucleotide sequence encoding an mAb or antibody fragment thereof of the present invention is provided. In yet another embodiment, a host cell comprising the vector of the present invention is provided.

[0020] In another embodiment, a pharmaceutical composition is provided comprising an mAb or antibody fragment thereof of the present invention and a pharmaceutically acceptable carrier.

[0021] In another embodiment, a method is provided for treating a heparanase activity-related disease or disorder in a subject of interest, comprising the step of administering a therapeutically effective amount of the mAb or antibody fragment thereof of the present invention to the subject, thereby treating the heparanase-related disease or disorder in the subject.

[0022] In some embodiments, the method involves administering a pharmaceutical composition comprising the mAb or antibody fragment thereof of the present invention and a pharmaceutically acceptable carrier to a subject.

[0023] In some embodiments of the method of the present invention, the disease or disorder related to heparanase activity is selected from the group consisting of malignant proliferative disorders, inflammatory disorders, autoimmune disorders, viral infections, diabetes mellitus, and associated renal dysfunction.

[0024] In some embodiments of the method of the present invention, the disease or disorder related to heparanase activity is a malignant proliferative disorder such as cancer.

[0025] According to another embodiment, the proliferative disease is a solid malignant tumor, including but not limited to carcinomas (cytomas) and sarcomas. According to a particular embodiment, this solid malignant tumor is melanoma. According to a particular embodiment, the solid malignant tumor is selected from the group consisting of breast cancer, prostate cancer, skin cancer, colon cancer, lung cancer, pancreatic cancer, head and neck cancer, kidney cancer, ovarian cancer, cervical cancer, bone cancer, liver cancer, thyroid cancer, tongue cancer, and brain cancer.

[0026] According to a particular embodiment, the proliferative disease is a hematopoietic malignancy such as lymphoma, leukemia, and multiple myeloma. According to a particular embodiment, this hematopoietic malignancy is selected from the group consisting of acute myeloid leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphoblastic leukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma, Hodgkin lymphoma, and non-Hodgkin lymphoma.

[0027] In another embodiment, the method of the present invention reduces or inhibits tumor metastasis in the above-mentioned subject. In yet another embodiment, the method of the present invention inhibits tumor progression in the above-mentioned subject.

[0028] In some embodiments of the method of the present invention, the diseases or disorders associated with heparanase activity are inflammatory disorders, autoimmune disorders, viral infections, and renal disorders.

[0029] In certain embodiments of the method of the present invention, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a male. In some embodiments, the subject is a female. In some embodiments, the subject is an adult. In some embodiments, the subject is a child.

[0030] In another embodiment, a method is provided for treating a disease or disorder related to heparanase activity, comprising the step of administering an effective amount of the mAb or fragment thereof of the present invention to a subject in need thereof, in combination with at least one anticancer treatment, thereby treating the disease or disorder related to heparanase. In some embodiments, the anticancer treatment is selected from chemotherapy and radiotherapy.

[0031] In another embodiment, a method for neutralizing heparanase activity is provided, comprising the step of contacting cells with the mAb or antibody fragment thereof of the present invention.

[0032] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below.

[0033] In another embodiment, an antibody directed to a heparanase enzyme or an antigen-binding fragment, analog, or derivative thereof is provided, wherein the antibody, its antigen-binding fragment, analog, or derivative comprises at least one complementarity-determining region (CDR), the CDR comprising SEQ ID NOs: 1 to SEQ ID NOs: 6, or a combination thereof.

[0034] In another embodiment, an antibody or its antigen-binding fragment, analog, or derivative directed to the HBD-II region (heparin-binding domain 2) of a heparanase enzyme is provided, wherein the antibody, its antigen-binding fragment, analog, or derivative comprises at least one complementarity-determining region (CDR), the CDR comprising SEQ ID NOs: 1 to 6, or a combination thereof.

[0035] In some embodiments, the antibody or fragment is a mouse or human antibody or fragment, or a humanized antibody or fragment.

[0036] In another embodiment, a method is provided for suppressing, inhibiting, preventing or treating a disease or disorder related to heparanase activity in a subject of interest, comprising the step of administering to the subject a therapeutically effective amount of an antibody directed to the heparanase enzyme or to the HBD-II region (heparin-binding domain 2), or an antigen-binding fragment, analog, or derivative thereof, wherein the antibody, or its antigen-binding fragment, analog, or derivative thereof, comprises at least one complementarity-determining region (CDR), the CDR comprising SEQ ID NOs: 1 to SEQ ID NOs: 6, or a combination thereof.

[0037] In another embodiment, a method is provided for inhibiting or treating a disease or disorder related to heparanase activity in a subject of interest, comprising the step of administering to the subject a therapeutically effective amount of an antibody directed to the heparanase enzyme or an antigen-binding fragment, analog, or derivative thereof, wherein the antibody or antigen-binding fragment, analog, or derivative thereof comprises at least one complementarity-determining region (CDR), the CDR comprising SEQ ID NOs: 1 to SEQ ID NOs: 6, or a combination thereof.

[0038] In another embodiment, a method is provided for inhibiting or treating a disease or disorder related to heparanase activity in a subject of interest, comprising the step of administering to the subject a therapeutically effective amount of an antibody directed to the HBD-II region (heparin-binding domain 2) of the heparanase enzyme, or an antigen-binding fragment, analog, or derivative thereof, wherein the antibody, or its antigen-binding fragment, analog, or derivative thereof, comprises at least one complementarity-determining region (CDR), the CDR comprising SEQ ID NOs: 1 to SEQ ID NOs: 6, or a combination thereof. [Brief explanation of the drawing]

[0039] [Figure 1] Figure 1 is a line graph showing the activity assays of heparanase using various hybridomas. [Figure 2(1)] Figure 2A is a line graph showing the binding of mAb54 to immobilized latent heparanase at progressively increasing concentrations versus the binding of control mouse IgG. [Figure 2(2)] Figure 2B shows the SDS-PAGE of the A54 mAb heavy chain (HC) and light chain (LC), and Figure 2C shows the Western blot analysis of the A54 mAb heavy chain (HC) and light chain (LC). [Figure 3A] Figure 3A is a line graph showing the activity assay of purified recombinant active heparanase pre-incubated with 0.1 μg / ml purified mAb A54 versus purified recombinant active heparanase pre-incubated with control mouse IgG. [Figure 3B] Figure 3B is a line graph showing the activity assay of purified recombinant active heparanase pre-incubated with 1 μg / ml purified mAb A54 versus purified recombinant active heparanase pre-incubated with control mouse IgG. [Figure 4(1)] Figures 4A-4C illustrate the infiltration of U87 human glioma cells in the presence of mouse IgG or mAb A54. Figures 4A-4B show micrographs. [Figure 4(2)] Figures 4A–4C illustrate the infiltration of U87 human glioma cells in the presence of mouse IgG or mAb A54. Figure 4C is a bar graph showing the extent of cell infiltration through the reconstructed basement membrane. [Figure 5(1)] Figures 5A-5B show images of luciferase in vivo imaging (IVIS) of human myeloma tumors growing in NOS / SCID mice in the absence (PBS) and presence of mAb A54. [Figure 5(2)] Figure 5C is a bar graph showing the quantification of each luciferase signal. Figures 5D-5E are images showing luciferase in vivo imaging (IVIS) of human glioma tumors growing in NOS / SCID mice in the absence (PBS) and presence of mAb A54. [Figure 5(3)] Figure 5F is a bar graph showing the quantitative analysis of each luciferase signal. [Figure 6(1)] Figures 6A-6B show the effect of mAb A54 on attenuation of myeloma tumor growth in MPC-11 mice compared to tumor-bearing mice (controls) treated with PBS. [Figure 6(2)] Figure 6C shows the effect of mAb A54 on attenuation of myeloma tumor growth in MPC-11 mice compared to tumor-bearing mice (controls) treated with PBS. [Figure 7(1)] Figures 7A-7B show images of luciferase fluorescence in vivo imaging (IVIS) of mouse 4T1 breast cancer tumors growing in the mammary fat pad of Balb / c mice, in the absence (PBS) and in the presence of mAb A54. [Figure 7(2)] Figure 7C is a bar graph showing the quantitative analysis of each luciferase signal. [Figure 7(3)] Figure 7D is a bar graph showing the weight of 4T1 primary tumors excised from Balb / c mice treated with the vehicle (PBS) or mAb A54 as described in Figures 7A and 7B. Figures 7E and 7F are images showing luciferase fluorescence in vivo imaging (IVIS) of 4T1 metastatic lesions detected in Balb / c mice after removal of the primary tumor, treated with the vehicle (PBS) or mAb A54. [Figure 7(4)] Figure 7G is a bar graph showing the quantitative analysis of each luciferase signal. [Figure 8(1)] Figures 8A-8D show images of luciferase fluorescence in vivo imaging (IVIS) of human myeloma tumors growing in NOS / SCID mice in the absence (PBS) and presence (Figures 8A and 8B, respectively) of mAb A54, and in the presence of bortezomib and mAb A54 + bortezomib (Figures 8C and 8D, respectively). [Figure 8(2)] Figure 8E is a bar graph showing the quantitative analysis of each luciferase signal. [Figure 9(1)] Figures 9A and 9B are images showing luciferase fluorescence in vivo imaging (IVIS) of mouse mammary cancer tumors growing in the mammary fat pad of Balb / c mice in the absence (PBS) and presence of mAb A54. [Figure 9(2)]Figure 9C is a bar graph showing the quantification of each luciferase signal. Figure 9D is a bar graph showing the weight of tumors excised from Balb / c mice treated with the vehicle (PBS) or mAb A54 described in Figures 9A and 9B. [Figure 10] Figure 10 is a line graph showing the survival rates of C57BL / 6 mice treated with A54 mAb, Lonepalstat, or PBS. [Figure 11(1)] Figure 11A is a graph showing the size of tumors excised from C57BL / 6 mice treated with vehicle (PBS), mAb A54, gemcitabine, or a combination of mAb A54 and gemcitabine. [Figure 11(2)] Figure 11B is a graph showing the weight of tumors excised from C57BL / 6 mice treated with vehicle (PBS), mAb A54, gemcitabine, or a combination of mAb A54 and gemcitabine. Figure 11C shows a photograph of the tumors. [Figure 12(1)] Figure 12A is a schematic diagram of IgG digestion by papain and ficin protease. Figure 12B is an SDS-PAGE gel showing the cleavage and purification of A54 Fab. [Figure 12(2)] Figure 12C shows the size exclusion chromatography separation of the bound A54-HPSE complex (main peak) from the unbound Fab (upper right corner). [Figure 13] Figure 13 is a schematic diagram of the tertiary Fab structure of the A54 CDR loop. [Figure 14] Figures 14A and 14B are schematic diagrams showing the A54 bond interaction with HPSE(β / α)8-barrel from two different angles. Figure 14C is an electrostatic surface representation showing the strong contribution of static charge to the A54-HPSE bond. [Figure 15(1)] Figure 15A is a schematic diagram showing the interaction between the A54 H2 loop (green) and HPSE (blue). Figure 15B is a schematic diagram showing the interaction between the A54 H3 loop (green) and HPSE (blue). [Figure 15(2)]Figure 15C is a schematic diagram showing the interaction between the A54 L1 loop (red) and the HPSE (blue). Figure 15D is a schematic diagram showing the interaction between the A54 L3 loop (red) and the HPSE (blue). [Figure 15(3)] Figure 15E is a schematic diagram showing the interaction between the HPSE HBD-II residue (blue) and residues derived from A54 VH (green) and VL (red). Figure 15F is a schematic diagram showing the overlap between the dp4 tetrasaccharide (cyan) derived from PDB 5E9C and the A54-HPSE complex, illustrating how A54 sterically occludes the HPSE binding groove. [Modes for carrying out the invention]

[0040] The present invention relates to heparanase neutralizing monoclonal antibodies (mAbs), pharmaceutical compositions containing the same, and their use for treating diseases or disorders associated with heparanase activity, including but not limited to malignant diseases.

[0041] This invention is in part based on the development of an mAb that binds to heparanase protein (HPSE) (Figures 2A-2C) and neutralizes heparanase enzyme activity (Figures 1 and 3A-3B). As demonstrated herein, this mAb significantly inhibited cell invasion via Matrigel, a reconstituted basement membrane (Figures 4A-4C). Treatment with the mAb as a monotherapy resulted in smaller human myeloma (CAG) and glioma (U87) tumor xenografts proliferating in NOD / SCID mice (Figures 5A, 5B, 5D, and 5E) (Figures 5C and 5F), as revealed by the luciferase fluorescence in vivo imaging system (IVIS). Similarly, mAb A54 attenuated mouse myeloma (MPC-11) tumor growth in a syngeneic Balb / c mouse model (Figures 6A-6C). mAb A54 also inhibited spontaneous metastasis of 4T1 mouse mammary cancer in an orthotopic syngeneic Balb / c mouse model (Figures 7A-7D). Furthermore, the heparanase protein has multiple binding sites, including but not limited to HBD-I (heparin-binding domain 1) and HBD-II (heparin-binding domain 2). The majority of mAb A54 interactions are with HBD-II (detailed in Example 12). The mAb A54 CDR loop mediates almost all A54-HPSE binding interactions. A54 Fab binds to HPSE on the (β / α)8-barrel domain directly above HBD-II (Gln270-Lys280; Figures 14A and 14B). This interaction prevents HPSE from binding to its HS substrate by steric occlusion of the enzyme-binding groove. Protein surface charge shows a large electrostatic contribution to A54-HPSE interactions. HBD-II is substantially positively charged, while the bonding interface of A54 is negatively charged (Figure 14C).

[0042] mAb A54 exhibits high specificity, enabling targeting of heparanase enzyme activity only. Therefore, the antibody of the present invention is useful, either as a monotherapy or in combination with at least one further therapy, including but not limited to chemotherapy or radiation, for mitigating and treating diseases and disorders associated with heparanase activity, including but not limited to tumor progression, inflammation, type 1 diabetes, diabetic nephropathy, and viral infections.

[0043] According to another embodiment, an mAb or antibody fragment thereof is provided that contains at least one heavy chain CDR selected from the group consisting of a heavy chain CDR1 (CDR-H1) containing the sequence (GYTFTN) shown in SEQ ID NO: 1, a heavy chain CDR2 (CDR-H2) containing the sequence (YINPTTGYTEYNQKFKD) shown in SEQ ID NO: 2, and a heavy chain CDR3 (CDR-H3) containing the sequence (GGAGYDYDEDYAMDY) shown in SEQ ID NO: 3.

[0044] According to another embodiment, an mAb or antibody fragment thereof is provided, comprising a heavy chain CDR1 (CDR-H1) containing the sequence (GYTFTN) (short sequence) shown in SEQ ID NO: 1, a heavy chain CDR2 (CDR-H2) containing the sequence (YINPTTGYTEYNQKFKD) shown in SEQ ID NO: 2, and a heavy chain CDR3 (CDR-H3) containing the sequence (GGAGYDYDEDYAMDY) shown in SEQ ID NO: 3.

[0045] According to another embodiment, an mAb or antibody fragment thereof is provided that contains at least one light chain CDR selected from the group consisting of a light chain CDR1 (CDR-L1) containing the sequence shown in SEQ ID NO: 4 (RASESVEYFGTSYMN), a light chain CDR2 (CDR-L2) containing the sequence shown in SEQ ID NO: 5 (LASILES), and a light chain CDR3 (CDR-L3) containing the sequence shown in SEQ ID NO: 6 (QQSNEDPYT).

[0046] According to another embodiment, an mAb or antibody fragment thereof is provided, comprising a light chain CDR1 (CDR-L1) containing the sequence shown in SEQ ID NO: 4 (RASESVEYFGTSYMN), a light chain CDR2 (CDR-L2) containing the sequence shown in SEQ ID NO: 5 (LASILES), and a light chain CDR3 (CDR-L3) containing the sequence shown in SEQ ID NO: 6 (QQSNEDPYT).

[0047] According to another embodiment, an mAb or antibody fragment thereof is provided, comprising at least one heavy chain CDR selected from the group consisting of a heavy chain CDR1 (CDR-H1) containing the sequence (GYTFTN) shown in SEQ ID NO: 1, a heavy chain CDR2 (CDR-H2) containing the sequence (YINPTTGYTEYNQKFKD) shown in SEQ ID NO: 2, and a heavy chain CDR3 (CDR-H3) containing the sequence (GGAGYDYDEDYAMDY) shown in SEQ ID NO: 3, and at least one light chain CDR selected from the group consisting of a light chain CDR1 (CDR-L1) containing the sequence (RASESVEYFGTSYMN) shown in SEQ ID NO: 4, a light chain CDR2 (CDR-L2) containing the sequence (LASILES) shown in SEQ ID NO: 5, and a light chain CDR3 (CDR-L3) containing the sequence (QQSNEDPYT) shown in SEQ ID NO: 6.

[0048] According to another embodiment, an mAb or antibody fragment thereof is provided, comprising a heavy chain CDR1 (CDR-H1) containing the sequence (GYTFTN) shown in SEQ ID NO: 1, a heavy chain CDR2 (CDR-H2) containing the sequence (YINPTTGYTEYNQKFKD) shown in SEQ ID NO: 2, and a heavy chain CDR3 (CDR-H3) containing the sequence (GGAGYDYDEDYAMDY) shown in SEQ ID NO: 3, and a light chain CDR1 (CDR-L1) containing the sequence (RASESVEYFGTSYMN) shown in SEQ ID NO: 4, a light chain CDR2 (CDR-L2) containing the sequence (LASILES) shown in SEQ ID NO: 5, and a light chain CDR3 (CDR-L3) containing the sequence (QQSNEDPYT) shown in SEQ ID NO: 6.

[0049] According to another embodiment, the heavy chain CDR1 (CDR-H1) containing the short sequence (GYTFTN) shown in SEQ ID NO: 1 may be replaced by the heavy chain CDR1 (CDR-H1) containing the long sequence (GYTFTNYWMH) shown in SEQ ID NO: 17.

[0050] According to several embodiments, the present invention provides an mAb or antibody fragment thereof comprising a heavy chain variable domain sequence having the amino acid sequence shown in SEQ ID NO: QVQLQQSGAELAKPGASVRMSCKASGYTFTNYWMHWVKQRPGQGLEWIGYINPTTGYTEYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARGGAGYDYDEDYAMDYWGQGTSVTVSS, or an analog or derivative thereof having at least 70% sequence identity with this heavy chain sequence. In some embodiments, the analog or derivative has at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 7.

[0051] According to another embodiment, the present invention provides an mAb or antibody fragment thereof comprising a light chain variable domain sequence having the amino acid sequence shown in SEQ ID NO: DIVLTQSPASLAVSLGQRATISCRASESVEYFGTSYMNWYQQKPGQPPKLLIYLASILESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK, or an analog or derivative thereof having at least 70% sequence identity with this light chain sequence. In some embodiments, the analog or derivative has at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 8.

[0052] According to a specific embodiment, the antibody or fragment thereof includes a heavy chain variable domain having the sequence shown in SEQ ID NO: 7 and a light chain variable domain having the sequence shown in SEQ ID NO: 8.

[0053] Analogues and derivatives of the monoclonal antibody or its fragment having at least 70% sequence identity with the antigen-binding portion of the reference sequence are also within the scope of the present invention. According to some embodiments, analogues and derivatives of the monoclonal antibody or its fragment having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the antigen-binding portion of the reference sequence are provided.

[0054] The term "having at least X percent identity" refers to the percentage of amino acid residues that are identical in two sequences being compared when the sequences are optimally aligned. Therefore, 70% amino acid sequence identity means that 70% of the amino acids in two or more optimally aligned polypeptide sequences are identical.

[0055] The monoclonal antibodies according to the present invention may contain constant regions derived from any mammalian species, including but not limited to mouse, rat, and human. The monoclonal antibodies according to the present invention include chimeric antibodies, humanized antibodies, fully human antibodies, heterologous antibodies, and antibody fragments that include at least an antigen-binding portion of an antibody.

[0056] The present invention encompasses monoclonal antibodies isolated from hybridoma cells or other biological systems, as well as monoclonal antibodies produced by recombination or synthesis. These hybridomas may be prepared by any method known in the art (e.g., Kohler, G. and Milstein, C., Nature, 256:495-497 (1975)). The supernatant of the hybridoma cell line is typically screened for antibody-binding activity by any method known in the art, such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). The supernatant may also be screened for the production of mAbs that inhibit heparanase enzyme activity.

[0057] DNA sequences encoding any of the amino acid sequences of the heavy or light chains of the above mAb are also included within the scope of the present invention. As will be undoubtedly clear to those skilled in the art, due to the degeneracy of the genetic code, multiple nucleic acid sequences may encode the mAb of the present invention other than those shown in SEQ ID NO: 9 or SEQ ID NO: 10. The present invention also provides expression vectors such as plasmids having the above DNA sequences, and host cells containing one or more of these expression vectors.

[0058] An antibody or immunoglobulin comprises two heavy chains and two light chains linked to each other by disulfide bonds, and each light chain is linked to its respective heavy chain by disulfide bonds in a "Y" - shaped configuration. Protein digestion of an antibody yields Fv (variable fragment) and Fc (crystalline fragment) domains. The antigen - binding domain, Fab, contains regions where the polypeptide sequence varies. The term F(ab’)2 represents two Fab’ arms linked by disulfide bonds. The central axis of an antibody is called the Fc fragment. Each heavy chain has a variable domain (V H ) at one end, followed by several constant domains (C H ). Each light chain has a variable domain (V L ) at one end and a constant domain (C L ) at the other end. The light - chain variable domain is aligned with the variable domain of the heavy chain, and the light - chain constant domain is aligned with the first constant domain (CH1) of the heavy chain. Each pair of variable domains of the light and heavy chains forms an antigen - binding site. The domains on the light and heavy chains have the same general structure, and each domain contains four framework regions, the sequences of which are relatively conserved and are linked by three hyper (high - frequency) variable domains known as complementarity - determining regions (CDR1 - 3). These domains contribute to the specificity and affinity of the antigen - binding site. The isotype of the heavy chain (γ, α, δ, ε or μ) determines the class of the immunoglobulin (IgG, IgA, IgD, IgE or IgM, respectively). The light chain is either one of two isotypes (kappa, κ or lambda, λ) found in all classes of antibodies.

[0059] The term "antibody" is used in its broadest sense and includes monoclonal antibodies (including full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity.

[0060] The antibody according to the present invention is a molecule containing at least an antigen-binding portion of the antibody. In specific embodiments, the antibody(s) according to the present invention are monoclonal antibodies (mAbs) and their proteolytic fragments such as Fab or F(ab')2 fragments. Furthermore, chimeric antibodies, human antibodies and humanized antibodies, recombinant and engineered antibodies, and their fragments are included within the scope of the present invention. Moreover, chimeric antibodies can be produced by inserting DNA encoding the variable region of an antibody into DNA encoding another antibody. Single-chain antibodies are also included within the scope of the present invention.

[0061] An "antibody fragment" contains a portion of an intact antibody and generally includes the antigen-binding site of an intact antibody, thus retaining its ability to bind to an antigen. Examples of antibody fragments included in this definition include: (i) Fab fragments having a VL domain, CL domain, VH domain, and CH1 domain; (ii) Fab' fragments having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) Fd fragments having a VH domain and a CH1 domain; (iv) Fd' fragments having a VH domain and a CH1 domain, and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) Fv fragments having a single arm of the antibody with a VL domain and a VH domain; (vi) dAb fragments consisting of a VH domain (Ward et al., Nature 1989, 341, 544-546); (vii) isolated CDR region; (viii) F(ab')2 fragments, which are bivalent fragments containing two Fab' fragments linked by disulfide crosslinks at the hinge region; and (ix) single-chain antibody molecules (e.g., single-chain Fv; scFv) (Bird et al., Science (1988, 242, 423-426; Huston et al., PNAS (USA) 1988, 85, 5879-5883), (x) a "diabody (bispecific antibody)" having two antigen-binding sites including a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., European Patent Application Publication No. 404,097; International Publication No. 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448); (xi) a "linear antibody" containing a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with a complementary light chain polypeptide (Zapata et al., Protein Examples include "Eng., 1995, 8, 1057-1062; and U.S. Patent No. 5,641,870."

[0062] Single-chain antibodies possess antigen-binding ability and have amino acid sequences homologous or similar to the variable regions of immunoglobulin light and heavy chains, i.e., linked V H -V L Alternatively, it can be a single-chain complex polypeptide containing a single-chain Fv (scFv).

[0063] As used herein, “neutralizing antibody” refers to a molecule having an antigen-binding site to a specific ligand target (e.g., heparanase or HBD-II) that can reduce or inhibit (block) the activity or signaling mediated by the target, as determined by an in vivo or in vitro assay in accordance with this specification.

[0064] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations, which may be present in small amounts. Monoclonal antibodies are highly specific and directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. The modifier “monoclonal” should not be construed as requiring the production of the antibody by any particular method. mAbs may be obtained by several methods known to those skilled in the art. For example, monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described by Kohler et al., Nature 1975, 256, 495, or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). The "monoclonal antibody" may be isolated from a phage antibody library using techniques such as those described by Clackson et al., Nature 1991, 352, 624-628 or Marks et al., J.Mol.Biol., 1991, 222:581-597.

[0065] The mAbs of the present invention may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and any subclass thereof. Hybridomas producing mAbs may be cultured in vitro or in vivo. High-titer mAbs can be obtained by in vivo production, in which cells from individual hybridomas are intraperitoneally injected into pristine-primed Balb / c mice to generate ascites containing high concentrations of the desired mAb. Isotype IgM or IgG mAbs may be purified from such ascites or culture supernatant using column chromatography methods well known to those skilled in the art.

[0066] The monoclonal antibodies in the present invention specifically include “chimeric” antibodies and fragments of such antibodies, insofar as the fragments of such antibodies exhibit the desired biological activity, wherein in chimeric antibodies, a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass (U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). In addition, complementarity-determining region (CDR) transplantation may be performed to modify specific properties of the antibody molecule, including affinity or specificity. Non-limiting examples of CDR transplantation are disclosed in U.S. Patent No. 5,225,539.

[0067] Chimeric antibodies are molecules in which different parts originate from different animal species, such as those possessing a variable region derived from a mouse mAb and a constant region from a human immunoglobulin. Antibodies that substantially possess variable region framework residues derived from a human antibody (called an acceptor antibody) and substantially possess a complementarity-determining region derived from a mouse antibody (called a donor antibody) are also called humanized antibodies. Chimeric antibodies are primarily used to reduce immunogenicity in application and increase yield in production. For example, human / mouse chimeric mAbs are used when mouse mAbs have a higher yield from hybridomas but are more immunogenic in humans. Chimeric antibodies and methods for producing them are publicly known in the art (for example, International Publication No. 86 / 01533, International Publication No. 97 / 02671, International Publication No. 90 / 07861, International Publication No. 92 / 22653, and U.S. Patent Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101, and 5,225,539).

[0068] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capability. In some examples, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, in which case all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are from the human immunoglobulin sequence. Humanized antibodies may optionally include at least a portion of the constant region (Fc) of an immunoglobulin, typically that of human immunoglobulin. For further details, see Jones et al., Nature 1986, 321, 522-525; Riechmann et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2, 593-596.

[0069] A “human antibody” is one that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or is produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art. In one embodiment, a human antibody is selected from a phage library expressing human antibodies (Vaughan et al., Nature Biotechnology 1996 14, 309-314; Sheets et al., PNAS (USA), 1998, 95, 6157-6162; Hoogenboom and Winter, J.Mol. Biol., 1991, 227, 381; Marks et al., J.Mol. Biol., 1991, 222, 581). Human antibodies can also be produced by introducing the human immunoglobulin locus into a transgenic animal, such as a mouse in which the endogenous immunoglobulin gene is partially or completely inactivated. Upon challenge, the production of human antibodies is observed, which closely resemble those found in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).Alternatively, human antibodies may be prepared by immortalizing human B lymphocytes (such B lymphocytes may be recovered from an individual or immunized in vitro) that produce antibodies directed toward a target antigen. See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991); and U.S. Patent No. 5,750,373.

[0070] The term "single-chain variable (region) fragment (scFv)" refers to a fusion of the variable regions of the heavy and light chains of an immunoglobulin, linked together with a short (usually serine, glycine) linker. Single-chain antibodies possess antigen-binding ability and are linked to the variable regions of the immunoglobulin light and heavy chains (scFv). H -V L Alternatively, it may be a single-chain complex polypeptide containing an amino acid sequence homologous or similar to that of a single-chain Fv(scFv). H and V L Both may copy a natural monoclonal antibody sequence, or one or both chains may contain a CDR-FR construct of the type described in U.S. Patent No. 5,091,513. The entire contents of U.S. Patent No. 5,091,513 are incorporated herein by reference. Separate polypeptides similar to the variable regions of the light and heavy chains are held together by a polypeptide linker. Such a method for producing single-chain antibodies is particularly V H Chain and V L If the DNA encoding the polypeptide structure of the chain is known, it may be achieved, for example, by following the methods described in U.S. Patent No. 4,946,778, U.S. Patent No. 5,091,513 and U.S. Patent No. 5,096,815, the entire contents of each of these patent documents are incorporated herein by reference.

[0071] As used herein, “molecule having an antigen-binding moiety of an antibody” refers to any isotype and includes, but is not limited to, intact immunoglobulin molecules produced by any animal cell line or microorganism, as well as its antigen-binding reactive fraction, its heavy and / or light chain variable portions, and Fab mini-antibodies (the entire contents of which are incorporated herein by reference in International Publication No. 93 / 15210, U.S. Patent Application No. 08 / 256,790, International Publication No. 96 / 13583, and U.S. Patent Application No. 08 / 256,790). The invention is intended to include dimeric bispecific miniantibodies (see International Publication No. 08 / 817,788, International Publication No. 96 / 37621, and U.S. Patent Application No. 08 / 999,554), as well as chimeric antibodies or single-chain antibodies incorporating such reactive fractions, and any other type of molecule or cell into which such antibody-reactive fractions are physically inserted, such as chimeric T cell receptors or T cells having such receptors, or molecules developed to deliver the therapeutic portion by a portion of a molecule containing such reactive fractions. Such molecules may be provided by any known technique, including but not limited to enzymatic cleavage, peptide synthesis, or recombination techniques.

[0072] For the preparation of monoclonal antibodies, any technique known in the art that provides antibodies produced by serial cell line culture can be used. Examples include Kohler, G. and Milstein, C., Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); "Monoclonal antibodies and cancer therapy," and the technique described by Cole et al. in Alan R. Liss, Inc. (1985), pp. 77-96.

[0073] In addition to conventional methods for producing antibodies in vivo, antibodies can be generated in vitro using phage display technology. Such recombinant antibody generation is much faster than conventional antibody production, and such recombinant antibodies can be generated against a vast number of antigens. Furthermore, when using conventional methods, many antigens are revealed to be non-immunogenic or highly toxic and therefore cannot be used for antibody generation in animals. Moreover, affinity maturation (i.e., increase in affinity and specificity) of recombinant antibodies is very simple and relatively rapid. Finally, it is possible to generate a large number of different antibodies against a particular antigen in a single selection procedure. To produce recombinant monoclonal antibodies, a large pool of antibodies with different antigen recognition sites can be created using various methods, all based on display libraries. Such libraries can be prepared in several ways. A synthetic repertoire can be generated by cloning synthetic CDR3 regions in a pool of heavy chain germline genes, thus generating a large antibody repertoire from which recombinant antibody fragments with various specificities can be selected. A human lymphocyte pool can be used as a starting material for constructing antibody libraries. It is possible to construct a naive repertoire of human IgM antibodies and thus create a human library with high diversity. This method has been successfully and widely used to select a large number of antibodies against different antigens. Protocols for constructing bacteriophage libraries and selecting recombinant antibodies are provided in the well-known reference *Current Protocols in Immunology*, Colligan et al. (eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1.

[0074] Non-human antibodies may be humanized by any method known in the art. In one method, a non-human complementarity-determining region (CDR) is inserted into a human antibody or consensus antibody framework sequence. Further modifications can then be introduced into the antibody framework to modulate affinity or immunogenicity.

[0075] Composition, dosage, and dosage For use in the method of the present invention, the monoclonal antibody may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers, stabilizers, or excipients (vehicles) to form a pharmaceutical composition known in the art, particularly with respect to protein activators. The carrier is "acceptable" in the sense that it is compatible with the other raw materials (components) of the composition and is not harmful to the recipient. Suitable carriers typically include physiological saline or ethanol, polyols, such as glycerol or propylene glycol.

[0076] The antibody may be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups), which are formed using inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, and maleic acid. Salts formed with free carboxyl groups may be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine.

[0077] The composition may be appropriately formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal administration, and conveniently, preferably, comprises a sterile aqueous solution of an antibody that isotonic with the recipient's blood. Such formulations are typically prepared by dissolving a solid active ingredient in water containing a physiologically compatible substance, such as sodium chloride or glycine, and having a buffered pH compatible with physiological conditions, to produce an aqueous solution, and then sterilizing this solution. These may be prepared in unit dose or multi-dose containers, such as sealed ampoules or vials.

[0078] The composition may incorporate stabilizers, such as polyethylene glycol, proteins, sugars (e.g., trehalose), amino acids, inorganic acids, and mixtures thereof. The stabilizers are used in aqueous solutions at appropriate concentrations and pH. The pH of the aqueous solution is adjusted to be within the range of 5.0 to 9.0, preferably 6 to 8. Antiadsorbents may be used in antibody formulation. Other suitable excipients typically include antioxidants such as ascorbic acid.

[0079] The composition may be formulated as a controlled-release preparation, which may be achieved by using a polymer to complex with or absorb the protein. Suitable polymers for controlled-release formulations include, for example, polyester, polyamino acids, polyvinylpyrrolidone, ethylene vinyl acetate, and methylcellulose. Another possible method for controlled release is to incorporate the antibody into particles of polymer material such as polyester, polyamino acids, hydrogel, poly(lactic acid), or ethylene vinyl acetate copolymer. Alternatively, instead of incorporating these drugs into polymer particles, these materials can be encapsulated in microcapsules prepared, for example, by coacervation technology or interfacial polymerization, such as hydroxymethylcellulose microcapsules, gelatin microcapsules, and poly(methyl methacrylate) microcapsules, or in colloidal drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, or in macroemulsions.

[0080] If an oral preparation is preferred, the composition may be combined with a carrier such as lactose, sucrose, starch, talc, magnesium stearate, crystalline cellulose, methylcellulose, carboxymethylcellulose, glycerin, sodium alginate, or gum arabic.

[0081] The mAbs of the present invention may be administered parenterally, generally by intravenous infusion. Administration may be via intraperitoneal, oral, subcutaneous, or intramuscular routes. Antibodies are generally administered in the range of about 0.1 to about 20 mg / kg patient body weight, usually about 0.5 to about 10 mg / kg, and often about 1 to about 5 mg / kg. In this regard, it is preferable to use antibodies with a circulating half-life of at least 12 hours, preferably at least 4 days, and more preferably up to 21 days. Chimeric antibodies and humanized antibodies are expected to have circulating half-lives of up to 4 days and 14 to 21 days, respectively. In some cases, it may be advantageous to administer a large loading dose followed by a regular (e.g., weekly) maintenance dose over the treatment period. Antibodies can also be delivered by sustained-release delivery systems, pumps, and other known delivery systems for continuous infusion. Dosage regimens may be modified to provide a desired circulating level of a particular antibody based on the pharmacokinetics of that particular antibody. Thus, doses are calculated to maintain a desired circulating level of the therapeutic agent.

[0082] Typically, the effective dose is determined by the patient's condition and the patient's body weight or surface area. The dose size and administration regimen are also determined by the presence, nature, and severity of any adverse side effects associated with the administration of the mAb or combination of additional therapeutic agents in the particular patient. When determining the effective amount of therapeutic composition to be administered, the physician should, among other things, assess circulating plasma levels, toxicity, and disease progression.

[0083] chemotherapy In yet another embodiment, a combination cancer therapy is provided comprising the administration of the mAb of the present invention and at least one chemotherapeutic agent.

[0084] Chemotherapy drugs are divided into several groups based on their effect on cancer cells, the cellular activity or process they interfere with, or the specific stage of the cell cycle they affect. Therefore, chemotherapeutic drugs fall into one of the following categories: alkylating agents, nitrosoureas, antimetabolites, anthracyclines, topoisomerase I and II inhibitors, mitotic inhibitors, platinum-based drugs in particular, steroids, and anti-angiogenic agents.

[0085] Antimetabolites, also known as "nucleoside analogs," replace natural substances as building blocks (basic units) in DNA molecules, thereby altering the function of enzymes necessary for cellular metabolism and protein synthesis. When antimetabolites mimic nutrients necessary for cell proliferation, cells ultimately undergo lysis. When nucleosides are replaced with non-functional nucleoside analogs, these analogs are incorporated into DNA and RNA, ultimately inducing cell cycle arrest and apoptosis by inhibiting the cell's ability to synthesize DNA. Because antimetabolites primarily act on cells undergoing the synthesis of new DNA for the formation of new cells, they are cell cycle specific and most effective during the S phase of cell division. The toxicity associated with these drugs is observed in rapidly growing and dividing cells. Examples of antimetabolites include purine antagonists, pyrimidine antagonists, and folate antagonists. These drugs damage cells during the S phase and are commonly used to treat leukemia, tumors of the breast, ovaries, and gastrointestinal tract, as well as other cancers. Specific examples of antimetabolites include 5-fluorouracil (also known as 5FU), capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, and pemetrexed.

[0086] Platinum-based chemotherapeutic agents cross-link DNA in several different ways, interfering with mitotic cell division. Damaged DNA triggers DNA repair mechanisms, which then activate apoptosis when repair proves impossible. The most prominent of these DNA modifications are 1,2-intrachain cross-links with purine bases. These include 1,2-intrachain d(GpG) adducts, which form nearly 90% of adducts, and the less common 1,2-intrachain d(ApG) adducts. 1,3-intrachain d(GpXpG) adducts also occur, but are readily excised by nucleotide excision repair (NER). Other adducts include interchain cross-links and non-functional adducts, which are hypothesized to contribute to the activity of platinum-based drugs. Interactions with cellular proteins, particularly HMG domain proteins, are also evolving as a mechanism for interfering with mitosis, although this is probably not their primary mode of action. Platinum-based chemotherapy drugs include cisplatin (also known as cisplatinum or cis-diammine dichloride platinum II (CDDP)), carboplatin, and oxaliplatin. Although cisplatin is often specified as an alkylating agent, it does not have an alkyl group and cannot perform alkylation reactions. More accurately, cisplatin is classified as an alkylating-like agent. Platinum-based chemotherapy drugs are used to treat various types of cancer, including sarcomas, some carcinomas (cell tumors) (e.g., small cell lung cancer and ovarian cancer), lymphomas, and germ cell tumors.

[0087] Mitotic inhibitors interfere with cell division. The most well-known chemotherapeutic agent in this category is paclitaxel (Taxol®, also known as "plant alkaloid," "taxane," and "anti-microtubule agent"). Along with docetaxel, paclitaxel forms the taxane drug category. However, other mitotic inhibitors are known, including but not limited to etoposide, vinblastine, and vincristine. Paclitaxel works by interfering with normal microtubule growth during cell division, thereby halting their function. Paclitaxel over-stables their structure. This destroys the cell's ability to flexibly use its cytoskeleton. Specifically, paclitaxel binds to the β-subunit of tubulin, the "building block" of microtubules, and the binding of paclitaxel fixes these building blocks in place. The resulting microtubule / paclitaxel complex has no ability to be degraded. This has adverse effects on cellular function. This is because the shortening and lengthening of microtubules (known as dynamic instability) is necessary for their function as a mechanism for transporting other cellular components. For example, during mitosis, microtubules position all chromosomes through their replication and subsequent separation into two daughter cell nuclei. Furthermore, paclitaxel induces programmed cell death (apoptosis) in cancer cells by binding to the apoptosis arrest protein Bcl-2 (B-cell leukemia 2) and thus halting its function.

[0088] Another group of DNA-interacting drugs widely used in anticancer chemotherapy is the anthracycline antibiotic group, including, among others, daunorubicin, doxorubicin (also known as Adriamycin® and doxorubicin hydrochloride), respinomycin D, and idarubicin. These drugs interact with DNA by inhibiting intercalation and macromolecular biosynthesis, thereby inhibiting the progression of topoisomerase II, the enzyme that unwinds DNA for transcription. They stabilize the topoisomerase II complex after it has broken down the DNA strand for replication, preventing the DNA double helix from being re-encapsulated, thereby halting the replication process. These are commonly used in the treatment of a wide range of cancers.

[0089] Alkylating antitumor agents directly attack DNA. They attach alkyl groups to DNA, crosslinking the guanine nucleic acid bases in the DNA double helix. This prevents the chain from unraveling and separating. Since this is necessary for DNA replication, the cell can no longer divide. These drugs act nonspecifically. Cyclophosphamide is an alkylating agent, but it is also a very potent immunosuppressant.

[0090] Topoisomerase I and II inhibitors interfere with the enzymatic activity of topoisomerase I and topoisomerase II, respectively, ultimately leading to the inhibition of both DNA replication and transcription. Examples of topoisomerase I inhibitors include topotecan and irinotecan. Irinotecan is a prodrug converted by carboxylesterase-converting enzyme to the biologically active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38). SN-38 is 1000 times more potent than its parent compound, irinotecan, and inhibits topoisomerase I activity by stabilizing the cleavable complex between topoisomerase I and DNA, resulting in DNA cleavage that inhibits DNA replication and induces apoptotic cell death. Because irinotecan requires ongoing DNA synthesis to exert its cytotoxic effects, it is also classified as an S-phase specific drug. Examples of topoisomerase II inhibitors include etoposide and teniposide.

[0091] Anti-angiogenic agents inhibit the formation of new blood vessels, ultimately leading to tumor "starvation." Non-exclusive examples of anti-angiogenic agents include the monoclonal antibody bevacizumab, dopamine, and tetrathiomolybdate (salt).

[0092] Vascular endothelial growth factor (VEGF) is a 32-42 kDa dimeric glycoprotein that mediates vasodilation, increased vascular permeability, and mitotic induction in endothelial cells. Differential exon splicing of the VEGF gene results in three major mRNA species encoding three secretory isoforms (subscripts indicate amino acid numbers): VEGF189, VEGF165, and VEGF121. Numerous minor splice variants have also been described (VEGF206, VEGF183, VEGF145, and VEGF148). Variants of VEGF polypeptides and their use in cancer therapy are disclosed, for example, in International Publication No. 2003 / 012105.

[0093] According to various embodiments, the above-mentioned at least one chemotherapeutic agent is selected from the group consisting of antimetabolites, platinum-based drugs, mitotic inhibitors, anthracycline antibiotics, topoisomerase inhibitors, anti-angiogenic agents, and combinations thereof.

[0094] According to some embodiments, the at least one chemotherapeutic agent is an antimetabolite comprising a purine antagonist, a pyrimidine antagonist, and a folic acid antagonist. According to some embodiments, the antimetabolite is a pyrimidine antagonist. According to some embodiments, the antimetabolite is selected from the group consisting of methotrexate, pemetrexed, cladribine, clofarabine, fludarabine, 6-mercaptopurine, nerarabine, pentostatin, capecitabine, cytarabine, 5-fluorouracil, uracil mustard, uracil, gemcitabine, hydroxyurea, and fludarabine.

[0095] According to some embodiments, the at least one chemotherapeutic agent is a platinum-based drug, including but not limited to cisplatin, carboplatin, and oxaliplatin.

[0096] In further embodiments, the at least one chemotherapeutic agent is a mitotic inhibitor, including but not limited to paclitaxel, docetaxel, etoposide, vinblastine, vincristine, and vinorelbine.

[0097] In further embodiments, the at least one chemotherapeutic agent is an anthracycline antibiotic, including but not limited to daunorubicin, respinomycin D, and idarubicin.

[0098] According to some embodiments, the at least one chemotherapeutic agent is an anti-angiogenic agent that includes, but is not limited to, bevacizumab, dopamine, tetrathiomolybdate (salt), and anti-angiogenic variants of VEGF.

[0099] According to some embodiments, the at least one chemotherapeutic agent is a topoisomerase inhibitor, including but not limited to daunorubicin, doxorubicin, epirubicin, irinotecan, topotecan, etoposide, and mitoxantrone.

[0100] According to some embodiments, the above-mentioned at least one chemotherapeutic agent is an alkylating agent that includes, but is not limited to, carmustine, lomustine, bendamustine, dacarbazine, and procarbazine.

[0101] Brachytherapy In yet another embodiment, a combination cancer therapy comprising the mAb and radiotherapy of the present invention is provided. The radiotherapy is administered according to well-known standard techniques using standard devices manufactured for this purpose, such as the AECL Theratron and Varian Clinac.

[0102] The distance between the external radiation source and the point of entry into the patient may be any distance that represents an acceptable balance between target cell death and minimizing side effects. Typically, the external radiation source is located 70–100 cm from the point of entry into the patient.

[0103] The radiation source, which may be used in combination with the mAb and chemotherapeutic agent of the present invention, can be located either outside or inside the patient being treated. When the radiation source is outside the patient, this therapy is known as external beam radiation therapy (EBRT). When the radiation source is inside the patient, this therapy is called brachytherapy (BT).

[0104] Brachytherapy is generally performed by placing a radioactive source within the patient. Typically, the radioactive source is placed approximately 0–3 cm from the tissue being treated. Known techniques include intratissue brachytherapy, intracavitary brachytherapy, and surface brachytherapy. Radioactive seeds can be implanted permanently or temporarily. Some typical radioactive atoms used in permanent implants include iodine-125 and radon. Some typical radioactive atoms used in temporary implants include radium, cesium-137, and iridium-192. Some additional radioactive atoms used in brachytherapy include americium-241 and gold-198.

[0105] Radiation doses depend on numerous factors, as is well known in the art. These factors include the organ being treated, healthy organs in the path of radiation that could be inadvertently adversely affected, the patient's tolerance to radiation therapy, and the area of ​​the body requiring treatment. Dose ranges typically from 1 to 100 Gy, more specifically from 2 to 80 Gy. Some reported doses include 35 Gy to the spinal cord, 15 Gy to the kidneys, 20 Gy to the liver, and 65 to 80 Gy to the prostate. However, it should be emphasized that the present invention is not limited to any specific dose. The dose will be determined by the treating physician according to the specific factors in a given situation, including the factors mentioned above.

[0106] The radiation dose for brachytherapy can be the same as the dose for external beam radiation therapy described above. In addition to the factors described above for determining the dose for external beam radiation therapy, the properties of the radioactive atoms used are also taken into consideration when determining the dose for brachytherapy.

[0107] In another embodiment, the anticancer treatment is a heparanase inhibitor, which includes, but is not limited to, a glycol-cleaved heparin compound (e.g., lonepalstat).

[0108] In various embodiments of the combination method of the present invention, the mAb and at least one drug or treatment (e.g., chemotherapy, radiotherapy) may be administered according to any of several treatment schedules, also called “dosage schedules” and “administration regimens,” which refer to the frequency and order of administration of each active agent. For example, the mAb and at least one chemotherapy agent may be administered substantially simultaneously, i.e., at the same time, for example, using a combined dosage form or separate dosage forms. This mode of administration may also be called “concomitant” administration. Concurrent administration refers to the administration of active agents within the same overall period, for example, on the same day but not necessarily at the same time. For example, one active agent may require administration with food, and the other may require administration in a semi-fasted state. Alternate administration includes the administration of one agent over a specific period, for example, over a course of several days or a week, followed by the administration of the other agent over the same period thereafter, and then repetition of this pattern over one or more cycles. Sequential or continuous administration includes the administration of one drug during a first period using one or more doses, followed by the administration of other drugs during a second period using one or more doses. Overlapping schedules, including administration of active drugs on different days throughout the treatment period, may also be employed, not necessarily in a regular order. Variations of these general guidelines may be adopted depending on the drugs used and the patient's condition.

[0109] Proteasome inhibitors In yet another embodiment, a combination cancer therapy comprising the mAb and proteasome inhibitor of the present invention is provided. A proteasome inhibitor is a drug that blocks proteasomes. Proteasomes are enzymes that break down unwanted or damaged proteins.

[0110] In some embodiments, the proteasome inhibitor includes bortezomib, carfilzomib, ixazomib, or a combination thereof. In some embodiments, the proteasome inhibitor is bortezomib.

[0111] Method of the present invention The mAbs of the present invention are useful for treating diseases or disorders related to heparanase activity in the target population. In some embodiments, the use of the mAbs of the present invention for the preparation of pharmaceuticals for treating diseases or disorders related to heparanase activity is provided.

[0112] As used herein, the terms “heparanase activity,” “heparanase enzyme activity,” or “heparanase catalytic activity” refer to animal endoglycosidase hydrolytic activity specific to heparin or heparan sulfate substrates, in contrast to the activity of bacterial enzymes (heparanases I, II, and III) that degrade heparin or heparan sulfate by β-elimination.

[0113] The heparanase activity inhibited or neutralized according to the present invention may be either recombinant heparanase or native heparanase activity. Such activities are disclosed, for example, in U.S. Patent No. 6,177,545 and U.S. Patent No. 6,190,875, which are incorporated by reference as if they were fully described herein. Methods for determining heparanase activity and antibody neutralizing effect are known in the art. In some embodiments, the heparanase neutralizing effect may be measured by activity assays described herein (e.g., Examples 1 and 3). In another embodiment, human heparanase is used as a neutralizing agent. 158 -Asp 171 The affinity of an antibody to heparan sulfate (HS) binding domains such as domains may be measured. In a further embodiment, the cellular uptake of heparanase may be measured.

[0114] As used herein, the term “related to heparanase catalytic activity” refers to a condition that is at least partially dependent on the catalytic activity of heparanase. While the catalytic activity of heparanase can be normal under many such conditions, it is understood that its inhibition under such conditions leads to improvement in affected individuals.

[0115] The pharmaceutical composition according to the present invention may be administered as a standalone treatment or in addition to treatment with any other therapeutic agent. In specific embodiments, the antibody according to the present invention is administered to a subject requiring the antibody as part of a treatment regimen in combination with at least one anticancer agent. The pharmaceutical composition according to the present invention may be administered together with other agents or separately.

[0116] As used herein to describe the present invention, “malignant proliferative disorder,” “cancer,” “tumor,” and “malignant tumor” are all equivalently related to the hyperplasia of tissue or organ. All types of tumors may be treated by the methods of the present invention. Tumors may be solid or non-solid.

[0117] According to some embodiments, the mAb or compositions comprising the present invention can be used to treat or inhibit non-solid tumors, such as hematopoietic malignancies, such as all types of leukemia, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), mast cell leukemia, hairy cell leukemia, Hodgkin's disease, non-Hodgkin lymphoma, Burkitt lymphoma, and multiple myeloma.

[0118] According to another embodiment, proliferative disorders include, but are not limited to, solid malignant tumors such as carcinomas, sarcomas, gliomas, and melanomas. According to a further embodiment, the mAb of the present invention or compositions comprising the same are tumors of the lips and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectal colon, anal canal, liver, gallbladder, extrahepatic bile duct, ampulla of Vater, exocrine part of the pancreas, lung tumors, pleural mesothelioma, bone tumors, soft tissue sarcomas, skin, breast, vulva, vagina, cervix, uterine body, ovaries, fallopian tubes, and Fallopian tubes. It can be used to treat or inhibit solid tumors such as carcinomas and malignant melanomas of the fallopian tubes, gestational trophoblastic tumors, carcinomas and malignant melanomas of the penis, prostate, testes, kidneys, renal pelvis, ureters, bladder, and urethra, carcinomas of the eyelids, carcinomas of the conjunctiva, malignant melanoma of the conjunctiva, malignant melanoma of the uvea, retinoblastoma, carcinomas of the lacrimal gland, orbital sarcomas, sarcomas of the brain, spinal cord, and vascular system, angiosarcoma, and Kaposi's sarcoma.

[0119] Therefore, it should be understood that the compositions of the present invention are useful for treating or inhibiting tumors at all stages, namely tumor formation, primary tumors, tumor progression, or tumor metastasis.

[0120] In another embodiment, the mAb of the present invention can be used to inhibit angiogenesis and is therefore useful in the treatment of diseases and disorders associated with angiogenesis or vascularization, such as, but not limited to, tumor angiogenesis, ophthalmic disorders such as diabetic retinopathy and macular degeneration, particularly age-related macular degeneration, and reperfusion of gastric ulcers.

[0121] The mAb or any composition thereof of the present invention is useful for inhibiting or treating other proliferative diseases or disorders such as psoriasis, hypertrophic scarring, acne and sclerosis / scleroderma, and for inhibiting or treating other diseases or disorders such as polyps, multiple exostosis, hereditary exostosis, post-lens fibroproliferation, hemangioma, and arteriovenous malformations.

[0122] Heparanase catalytic activity correlates with the ability of activated immune system cells to detach from the circulatory system and induce both inflammatory and autoimmune responses. Interactions between platelets, granulocytes, T lymphocytes and B lymphocytes, macrophages, and mast cells with the subendothelial extracellular matrix (ECM) are associated with the degradation of heparan sulfate (HS) by heparanase catalytic activity (Vlodavsky, I. et al., Invasion & Metastasis 12, 112-127 (1992)). This enzyme is released from intracellular compartments (e.g., lysosomes, specific granules) in response to various activation signals (e.g., thrombin, calcium ionophores, immune complexes, antigens, mitogens), suggesting its regulated involvement and presence in inflammatory sites and autoimmune lesions. Heparanase released by platelets and macrophages is likely to be present in atherosclerotic lesions (Campbell, KH et al., Exp. Cell Res. 200, 156-167 (1992)). Therefore, the mAb or any composition thereof of the present invention is also useful for inhibiting or treating autoimmune diseases and inflammatory diseases.

[0123] Therefore, in another embodiment, the compositions of the present invention may be useful in treating or improving inflammatory symptoms in any disease, condition or disorder in which immunosuppression and / or suppression of inflammation is beneficial, for example, but not limited to, inflammatory symptoms in the joints (Li et al., Arthritis Rheum 2008, 58:1590-600), musculoskeletal disorders and connective tissue disorders, or inflammatory symptoms associated with hypersensitivity (Edovitsky et al., Blood 2005, 3609-16), allergic reactions, asthma, atherosclerosis (Planer et al., Plos ONE 2011;6(4):e18370), otitis and other otolaryngological diseases, dermatitis and other skin diseases, posterior uveitis and anterior uveitis, conjunctivitis, optic neuritis, scleritis, and other immune and / or inflammatory eye diseases.

[0124] In another embodiment, the compositions of the present invention are used to treat autoimmune diseases, such as, but are not limited to, Eaton-Lambert syndrome, Goodpasture syndrome, Graves' disease, Guillain-Barr syndrome, autoimmune hemolytic anemia (AIHA), hepatitis, insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus (SLE), multiple sclerosis (MS), myasthenia gravis, nerve plexus disorders, such as acute brachial neuritis, polyglandular dysfunction syndrome, primary biliary cirrhosis, and rheumatoid arthritis (L). It is useful in the treatment or improvement of scleroderma, thrombocytopenia, thyroiditis, for example, Hashimoto's disease, Sjögren's syndrome, allergic purpura, psoriasis, mixed connective tissue disease, polymyositis, dermatomyositis, vasculitis, polyarteritis nodosa, polymyalgia rheumatica, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, pemphigus, bullous pemphigoid, herpetic dermatitis, insulin-dependent diabetes mellitus, inflammatory bowel disease, ulcerative colitis, and Crohn's disease (Lerner et al., J Clin Invest 2011, 121:1709-21).

[0125] Furthermore, it has been proposed that heparanase is involved in the pathogenesis of proteinuria by selectively degrading the negatively charged side chains of heparan sulfate proteoglycans within the glomerular basement membrane. The loss of negatively charged heparan sulfate proteoglycans may lead to changes in the permeability selectivity of the glomerular basement membrane, loss of glomerular epithelial and endothelial cell anchoring points, and release of growth factors, potentially resulting in different renal disorders such as passive Heymann nephritis (PHN) and puromycin aminonucleoside nephrotic syndrome (PAN). As described by Levidiotis, V. et al. (Levidiotis, V. et al., J. Am. Soc. Nephrol. 15, 68-78 (2004)), polyclonal antibodies against heparanase significantly reduce proteinuria without affecting the histological appearance of the glomeruli and the immune mechanisms that produce PHN; therefore, inhibition of heparanase may be used to reduce proteinuria. In particular, the heparanase inhibitor lonepalstat reduced proteinuria associated with type 1 and type 2 diabetes (Gil et al., Diabetes 2012;61:208-16). Therefore, in another embodiment, the compositions and mAbs described herein are useful for the treatment or improvement of any renal impairment and organ fibrosis (Abassi and Goligorsky MS. Adv Exp Med Biol. 2020;1221:685-702; van der Vlag and Buijsers. Adv Exp Med Biol. 2020;1221:647-667; Masola et al., Adv Exp Med Biol. 2020;1221:669-684).

[0126] In another embodiment, the compositions and mAbs described herein are useful for the treatment or improvement of diabetic nephropathy. In another embodiment, the compositions and mAbs described herein are useful for the treatment or improvement of type 1 diabetes (Ziolkowski et al., 2012;122:132-41; Simeonovic et al., Adv Exp Med Biol. 2020;1221:607-630).

[0127] In another embodiment, the compositions and mAbs described herein are useful for the treatment or improvement of amyloidosis (Li JP and Zhang X. Adv Exp Med Biol. 2020;1221:631-645) and viral infections (Agelidis A, Shukla D. Adv Exp Med Biol. 2020;1221:759-770). In another embodiment, the compositions and mAbs of the present invention are useful for the treatment or improvement of sepsis.

[0128] The term "mammal" means any mammal, including pet animals such as dogs and cats, livestock such as pigs, cows, sheep and goats, laboratory animals such as mice and rats, primates such as monkeys, apes and chimpanzees, and preferably humans.

[0129] The present invention has been outlined above, but it will be more readily understood by referring to the following examples, which are provided as illustrations and are not intended to limit the present invention. [Examples]

[0130] Materials and methods Generation of mouse monoclonal antibodies (mAbs). BALB / c mice were immunized with 50 μg of recombinant 65 kDa latent pro-heparanase conjugated to keyhole limpet hemocyanin (KLH) in complete Freund's adjuvant (CFA; Sigma), followed by five injections of KLH-heparanase in incomplete Freund's adjuvant (IFA) every two weeks (50 μg). After tail vein injection, splenocytes were isolated and fused with NSO myeloma cells, and the hybridomas were screened for their ability to bind to proheparanase by ELISA as essentially described (Gingis-Velitski et al., Faceb J 2007; Levy-Adam et al., J Biol Chem 2010;285(36):28010-9; Shafat et al., Biochem Biophys Res Commun 2006;341(4):958-63). Positive hybridomas were selected, grown, and cloned. Hybridoma subclasses were determined by isotyping kits according to the manufacturer's instructions (Serotec, Oxford, UK). mAb A54 was characterized as IgG1-κ and purified by affinity chromatography with Protein G Sepharose 4 according to the manufacturer's instructions (Pierce Biotechnology, Rockford, Illinois).

[0131] Purified mAb A54 antibody was isolated by SDS-PAGE, and the heavy and light chain protein bands were extracted and sequenced. The corresponding nucleotide sequences were then determined, the genes were amplified by RT-PCR, and cloned. Briefly, total RNA was isolated from the above hybridoma cells using TRIzol® reagent. This total RNA was then reverse transcribed into cDNA using isotype-specific antisense primers. VH and VL antibody fragments were amplified according to standard rapid amplification of cDNA ends (RACE). The amplified antibody fragments were separately cloned into standard cloning vectors. Colony PCR was performed to screen for clones with correctly sized insertion fragments. Five or more colonies with correctly sized insertion fragments were sequenced for each fragment. The sequences of different clones were aligned. The consensus nucleotide and amino acid sequences of these clones are provided in Table 1. Mouse IgG (Sigma) was used as a control.

[0132] [Table 1(1)] [Table 1(2)]

[0133] Cells and cell cultures. Human HEK293, U87-MG glioma, and CAG myeloma cells, as well as mouse 4T1 breast cancer and MPC-11 myeloma cells, were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia). CAG myeloma (Ramani et al., Matrix Biol. 2016;55:22-34), U87 glioma (Barash et al., Int J Cancer, 2019;145(6):1596-1608), and 4T1 breast cancer (Hammond et al., PLoS One 2012;7(12):e52175) cells have been previously described. Cells were grown in Dulbecco's modified Eagle medium (Biological Industries, Beit Haemek, Israel) supplemented with 10% fetal bovine serum and antibiotics.

[0134] Cell lysates, heparanase activity, and protein blotting. Preparation of cell lysates, protein blotting, and measurement of heparanase enzyme activity were performed as described (Arvatz et al., Faceb J 2011;24(12):4969-76). For inhibition studies, highly purified recombinant heparanase (200 ng) was pre-incubated with the indicated antibody (2 μg) on ​​ice for 30 minutes under neutral pH conditions (pH 7.2) and then used as a naturally occurring substrate for heparanase production. 35 It was added to S-labeled ECM (Gingis-Velitski et al., 2007, previously cited).

[0135] ECM degradation assay. The extracellular matrix (ECM) substrate is deposited by cultured endothelial cells and thus closely resembles the subendothelial basement membrane in its composition, biological function, and barrier properties. Years of experience indicate that compounds that effectively inhibit the above enzymes in this assay are also effective in preclinical animal models. Further details regarding the preparation of this substrate and its use for the heparanase assay can be found below: Current Protocols in Cell Biology (Vlodavsky 2001; pages 10.4.1 - 10.4.14). Briefly, the surface of a 35 mm tissue culture dish is coated with sulfate 35 S]-labeled ECM and incubated with recombinant human heparanase (200 ng / ml) in the absence and presence of A54 mAb (4 hours, 37 °C, pH 6.0, final volume 1 ml). The reaction mixture contains: 50 mM NaCl, 1 mM DTT, 1 mM CaCl2, and 10 mM phosphate - citrate buffer, pH 6.0. To assess the occurrence of proteoglycan degradation, the incubation medium is recovered and applied (loaded) onto a Sepharose 6B column (0.9 × 30 cm) for gel filtration. Fractions (0.2 ml) are eluted with PBS and counted for radioactivity. The excluded volume (Vo) is marked with blue dextran, and the total inclusion volume (Vt) is marked with phenol red. Degradation fragments of HS side chains are eluted from Sepharose 6B at 0.5 < Kav < 0.8 (peak II). The results are best represented by the actual gel filtration pattern (Vlodavsky et al., Nature Med. 5:793 - 802, 1999).

[0136] Matrigel invasion assay. An invasion assay was performed using a modified Boyden chamber with a polycarbonate Nucleopore membrane, essentially as described (Arvatz et al., 2011, supra). Briefly, a filter (diameter 6.5 mm, pore size 8 μm) was coated with Matrigel (30 μl). Cells (2 × 10 5The cells were seeded in triplicate at the top of each chamber in the presence of the indicated antibody, and the lower compartment was filled with 600 μl of medium supplemented with 10% FCS. After incubation at 37°C for 5 hours in a 5% CO2 incubator, non-invasive cells were wiped from the top surface of the filter with a cotton swab, migratory cells were fixed from the bottom surface of the filter, stained with 0.5% crystal violet (Sigma), and counted by examining at least seven microscopic fields (Barash et al., J Natl Cancer Inst. 110:1102-1114, 2018).

[0137] tumorigenicity U87 glioma. Cells from exponential cultures of luciferase-labeled U87 glioma cells were detached with trypsin / EDTA, washed with PBS, and 5 × 10⁻⁶ cells were removed. 7 The concentration was adjusted to cells / ml. Cell suspension (5 × 10 6 Luciferin (0.1 ml) was subcutaneously inoculated into the right flank of 5-week-old female SCID / Beige mice (n=7). Three days after cell inoculation, the mice were randomly assigned to two cohorts (5-10 mice each) receiving (a) vehicle (PBS) and (b) A54 mAb (500 μg / mouse, 3 times / week). Tumor formation was examined by IVIS imaging after luciferin administration (once a week) as described (Barash et al., FASEB J 2010;24:1239-48, Barash et al., J Natl Cancer Inst. 110:1102-1114, 2018) (see below). At the end of the experiment, the mice were euthanized, and xenografts were excised, weighed, and fixed in formalin for pathological examination.

[0138] 4T1 mouse breast cancer (experimental metastasis). Luciferase-labeled 4T1 breast cancer cells (1 × 10⁻¹⁰ 5Intravenous injection of / Balb / c mice (n=10 mice / group; 2 groups: untreated control, A54 mAb). Inject antibody 20 minutes before cell inoculation (intraperitoneal, 500 μg / mouse, 3 times / week). IVIS bioluminescence imaging is performed 6, 10, and 14 days after cell inoculation. At the end of the study, mice are euthanized and their lungs are subjected to pathological examination and counting of cell colonies per 5 microscopic fields. Cells metastasize mainly to the lungs, and the luminescence signal is reproducible, quantitative, and reliable.

[0139] 4T1 mouse breast cancer (spontaneous metastasis). Luciferase-labeled 4T1 breast cancer cells (1 × 10⁻¹⁰ 5 / Balb / c mice) were injected directly into the third mammary fat body, and treatment of the mice (PBS control vs. mAb A54 500 μg / mouse, 3 times / week) was started 3 days after 4T1 cell inoculation. Four days after inoculation (day 0), the mice were randomized into two groups of 6 mice each. On day 15 of the study, the mammary fat body containing the primary tumor was excised from all mice under 2% isoflurane anesthesia. The mice were treated with mAb A54 as described above, and IVIS bioluminescence imaging was performed 12 and 18 days after mastectomy. At the end of the study, the mice were euthanized, and the number of overt macrometastases on the surface of the lungs was manually counted (Hammond et al., PLoS One 2012;7(12):e52175).

[0140] CAG myeloma. Luciferase-labeled CAG human myeloma cells (5 × 10) 6 The drug is injected into the tail vein of NOD / SCID mice. Three days after cell inoculation, the mice are randomly assigned to two cohorts (5-10 mice each) receiving (a) vehicle (PBS) and (b) A54 mAb (500 μg / mouse, 3 times / week). Tumor formation is examined by IVIS imaging after luciferin administration (once a week) (see below). At the end of the study, the mice are euthanized, their skeletons are excised, fixed in formalin, decalcified with 10% EDTA solution, embedded in paraffin, and subjected to histological and immunohistochemical analysis.

[0141] MPC-11 myeloma. Mouse MPC-11 myeloma cells were detached with trypsin / EDTA, washed with PBS, and 5 × 10⁶ cells were collected. 5 The solution is administered subcutaneously to the right flank of 6-8 week old Balb / c mice at a concentration of cells / 0.2 ml. Three days after cell inoculation, the mice are randomly assigned to two cohorts (6 mice each) receiving (a) vehicle (PBS) and (b) A54 mAb (500 μg / mouse, 3 times / week). Xenograft size is determined on days 7, 10, and 14 by externally measuring the tumor in two dimensions using calipers. At the end of the experiment, the mice are euthanized, and the tumor xenografts are removed and weighed.

[0142] B16 melanoma. B16-BL6 mouse melanoma cells (2 × 10⁻¹⁶) 5 The compound was injected into the tail vein of C57 / BL mice along with the indicated compound, and lung metastases were measured on day 18. All animal studies were approved by the Animal Care Committee of Technion, Haifa, Israel.

[0143] IVIS imaging. Bioluminescence imaging of luciferase-expressing tumors is performed using a highly sensitive cooled charge-coupled device (CCD) camera mounted in a light-shielding specimen box (IVIS; Xenogen Corp., Waltham, Massachusetts). Imaging is performed in real time, is non-invasive, and provides quantitative data. Briefly, mice are anesthetized by intraperitoneal injection of 150 mg / kg of D-luciferin substrate and placed on a heated stage in a light-shielding camera box while being continuously exposed to isoflurane (EZAnesthesia, Palmer, Pennsylvania). Light emitted from bioluminescent cells is detected by the IVIS camera system, and images are produced at 5 × 10⁻¹⁶ pixels. 4 ~1 × 10 7The tumor volume is quantified using a logarithmic color range, and the total photon counts per second (PPS) are measured using Living Image software (Xenogen).

[0144] Statistics. Data are presented as mean ± SE. Statistical significance was analyzed using a two-tailed Student's t-test. A value of P < 0.05 is considered statistically significant.

[0145] Example 1. Identification and selection of the most active heparanase-neutralizing monoclonal antibody. To select the hybridoma clone that best inhibits heparanase enzyme activity, supernatants from various hybridomas produced against 65kDa latent heparanase protein were collected, pre-incubated with recombinant heparanase, and examined for their ability to inhibit the release of heparan sulfate (HS) degradation fragments from sulfate-labeled ECM. For this purpose, purified recombinant active heparanase (200 ng) was pre-incubated with the supernatants of various hybridomas in serum-free RPMI medium on ice for 2 hours. This mixture was then prepared. 35 In addition to the S-labeled ECM-coated dish, heparanase activity was determined as described in "Materials and Methods" above. As shown in Figure 1, pre-incubation with hybridoma #A54 yielded the best heparanase inhibitory activity (a nearly 80% reduction in the amount of sulfate-labeled HS degradation products released) compared to all other hybridoma supernatants.

[0146] Example 2. mAb A54 preferentially recognizes heparanase. ELISA was used to characterize the preferential recognition of heparanase by mAb A54 versus control mouse IgG (Figure 2A). For this purpose, a 96-well microtiter plate was coated with latent 65 kDa heparanase (Hpa65). Purified (Protein G Sepharose) mAb A54 or control mouse IgG was then added at the indicated concentrations, and the degree of binding to immobilized heparanase was determined by ELISA. Although there was no interaction with non-immunized mouse IgG, the A54 antibody showed high affinity binding to this enzyme (IC50 = 0.45 nM). Next, the purified A54 mAb was subjected to SDS-PAGE (Figure 2B) and Western blotting (Figure 2C, secondary antibody: goat anti-mouse κ-HRP) analysis to further characterize the purity, molecular weight, and isotype of the antibody light and heavy chains. Lanes M1 and M2: protein markers; Lane 1: reducing conditions; Lane 2: non-reducing conditions; Lane P: mouse IgG1, κ as positive control. (Ab: Antibody: Goat anti-mouse IgG-HRP). Briefly, mAb A54 was expressed in 293F cells using a pcDNA3.4 expression vector and serum-free (Expi293F) medium. The antibody isotype was characterized as IgG1-κ.

[0147] Example 3. mAb A54 inhibits heparanase enzyme activity. Next, we investigated the ability of purified mAb A54 to inhibit heparanase enzyme activity. For this purpose, recombinant active heparanase (200 ng) was pre-incubated for 1 hour in serum-free RPMI medium on ice with control mouse IgG or purified mAb A54 (0.1 and 1 μg / ml). Then, this mixture was analyzed. 35 In addition to dishes coated with S-labeled ECM, heparanase enzyme activity was determined as described in "Methods". As shown in Figure 3A, pre-incubation with 0.1 μg / ml of the above antibody resulted in nearly 75% inhibition of this enzyme, and complete inhibition (i.e., release of HS degradation fragments eluted in fractions 15-30 when subjected to gel filtration with Sepharose 6B) was obtained in the presence of 1 μg / ml of the above antibody, as shown in Figure 3B. There was no inhibitory effect on non-immunized mouse IgG.

[0148] Example 4. mAb A54 attenuates cell invasion. Subsequently, the effect of the above antibodies on cell infiltration was investigated. In short, U87 glioma cells (1 × 10⁶) 5 The cells were seeded onto an 8 μm Transwell filter coated with Matrigel in the presence of control mouse IgG or mAb A54 (2 μg). Infiltrating cells adhering to the underside of the membrane were visualized after 6 hours (Figure 4A-B) and quantified as described in "Methods" (Figure 4C; number of infiltrating cells per high-magnification field of view). * (p=0.001). As shown in Figures 4A-C, the infiltration of U87 glioma cells through Matrigel (reconstituted basement membrane) was reduced by almost 90% in the presence of mAb A54.

[0149] Example 5. The anti-heparanase mAb A54 attenuates the growth of human myeloma and human glioma tumors. The ability of mAb A54 to attenuate myeloma tumor formation over time was investigated. In short, NOD / SCID mice (n=5) were inoculated with Luc-CAG human myeloma cells (5×10⁶). 6 The drug was inoculated intravenously (iv), and mice were treated with A54 mAb (500 μg / mouse, 3 times / week) or PBS as a control, starting 3 days after cell inoculation. Tumor growth was evaluated by IVIS imaging after 3 weeks (Figures 5A and 5B). Quantification of luciferase signaling is also shown in graph (Figure 5C). As shown in Figure 5B, a significant decrease in the myeloma growth rate was observed (P=0.02).

[0150] Similarly, Luc-U87 human glioma cells (5 × 10 6 0.1 ml of mAb A54 was subcutaneously (sc) inoculated into the right flank of 5-week-old female NOD / SCID mice (n=5). Mice were treated with mAb A54 (500 μg / mouse, 3 times / week) or PBS as a control. Tumor growth was evaluated by IVIS imaging (Figures 5D and 5E), and the quantification of luciferase signaling is shown graphically (Figure 5F). As shown in Figure 5E, a significant inhibition of glioma tumor growth rate was observed in antibody-treated mice (P=0.04).

[0151] Example 6. The anti-heparanase mAb A54 attenuates mouse myeloma tumor growth. Having demonstrated the inhibitory effect of mAb A54 in an immunodeficient mouse model, we then investigated the effect of the above antibody in a syngeneic (immunely qualified) mouse model. In short, Balb / c mice were inoculated with MPC-11 mouse myeloma cells (0.5 × 10⁶). 6 Cells were inoculated with mAb A54 (sc). Treatment with mAb A54 (250 or 500 μg / mouse every other day) was started two days after cell inoculation. Control cells were administered PBS. Xenograft size was determined twice a week by measuring the tumor externally in two dimensions using calipers, and tumor volume was calculated (Figure 6A). At the end of the experiment (day 14), the xenografts were removed, weighed (Figure 6B), fixed in formalin, and photographed (Figure 6C). The results show that treatment with mAb A54 significantly (4-fold) attenuated myeloma tumor growth, and that higher doses of antibody were more effective. These results suggest that the anti-heparanase mAb A54 can suppress the formation of primary tumors.

[0152] Example 7. mAb A54 attenuates spontaneous metastasis of mouse mammary cancer. In subsequent studies, the effect of mAb A54 on cancer metastasis, a characteristic of heparanase activity, was investigated. A syngeneic breast cancer model similar to the human scenario was applied. Briefly, luciferase-labeled 4T1 breast cancer cells (0.5 × 10⁶) 5mAb A54 was orthotopically injected into the third mammary fat body of Balb / c mice. Mouse treatment (PBS control vs. mAb A54; 500 μg / mouse, 3 times / week) was initiated 3 days after 4T1 cell inoculation. IVIS imaging performed on day 12 showed no difference in luciferase signaling produced by the primary tumor (Figures 7A and 7B). On day 15 of the study, the mammary fat bodies containing this primary tumor were excised and weighed from all mice (under 2% isoflurane anesthesia). There was no weight difference between tumors from A54-treated mice and those from untreated mice (Figure 7C). Mice were further treated with mAb A54 as described above, and IVIS bioluminescence imaging was performed on day 35 (e.g., 23 days after mastectomy). As shown in Figures 7E and 7F, only 2 out of 5 untreated mice developed lung metastases, compared to the almost undetectable signaling observed in A54-treated mice (one mouse died on day 15 of the experiment). Repeated experiments yielded similar results, further indicating almost complete inhibition of lung colony formation in this model system.

[0153] Example 8. The combination of A54 mAb and bortezomib attenuates myeloma tumor growth. In subsequent studies, the effect of mAb A54 in combination with bortezomib on myeloma tumor growth was investigated. NOD / SCID mice (n=5) were used to study CAG luciferase cells (5×10 6 Mice were inoculated with (iv) A54 mAb (360 μg / mouse, twice / week), or bortezomib (Brot; 0.5 mg / kg twice weekly), or A54 mAb + bortezomib (as described), or PBS as a control. Tumor growth was evaluated by IVIS imaging (Figures 8A-D). Quantification of luciferase signaling is shown graphically (Figure 8E). These results indicate that treatment of myeloma with a combination of A54 mAb and bortezomib attenuates tumor growth.

[0154] Example 9. A54 mAb attenuates breast cancer tumor growth. In subsequent studies, the effect of mAb A54 on breast cancer tumor growth was investigated. Balb / c mice (n=3) were subjected to EMT-6 luciferase cells (0.5 × 10⁶). 6 Mice were inoculated with A54 mAb (360 μg / mouse, twice / week) or treated with PBS as a control after inoculation of the mammary fat pad. Tumor growth was evaluated by IVIS imaging (Figures 9A and 9B). Quantification of luciferase signaling is shown graphically (Figure 9C). At the end of the experiment, the tumors were excised and weighed (Figure 9D). These results indicate that A54 mAb treatment attenuates the growth of mammary cancer.

[0155] Example 10. A54 mAb effectively protects mice from LPS-induced sepsis. In a subsequent study, the effect of mAb A54 on LPS-induced sepsis was investigated. C57BL / 6 mice (n=8) were treated with A54 mAb (500 μg / mouse), lonepalstat (SST) (1.2 mg / mouse), or PBS as a control, followed by LPS injection (15 mg / kg, ip) 30 minutes later. SST was administered again 16 hours after LPS administration (1.2 mg / mouse). Mouse survival was monitored every 12 hours for 8 days (Figure 10). The results showed that A54 mAb effectively protected mice from LPS-induced sepsis.

[0156] Example 11. The combination of A54 mAb and gemcitabine attenuates pancreatic tumor growth. The effect of mAb A54 on pancreatic tumor growth was investigated. Panc02 cells (1 × 10⁶) were introduced into C57BL / 6 mice (n=7). 6 Mice were inoculated with (sc) and treated with A54 mAb (360 μg / mouse, twice / week), gemcitabine (Gem; 30 mg / kg twice / week), A54 mAb + gemcitabine (as described), or vehicle alone as a control (PBS). Tumor formation was calculated from external caliper tumor measurements (Figure 11A). At the end of the experiment on day 32, tumors were excised, photographed (Figure 11C), and weighed (Figure 11B). The results indicate that treatment of pancreatic cancer with a combination of A54 mAb and gemcitabine attenuates tumor growth.

[0157] Example 12. Interaction between A54 mAb and heparanase. The interaction between A54 mAb and heparanase was investigated by X-ray crystallography. Fab fragments were prepared from intact A54 antibody using Thermo Scientific mouse IgG1 Fab and F(ab')2 preparation kits and standard manufacturer protocols. Isolated Fab fragments were further purified by size exclusion chromatography to remove impurities, and the buffer was replaced with 20 mM HEPES pH 7.4, 200 mM NaCl, and 1 mM DTT (Figures 12A-12B). Purified A54 was mixed with purified heparanase (=HPSE) in a Fab:HPSE ratio of approximately 2:1. This mixture was incubated at room temperature for 2 hours and then purified again by size exclusion chromatography to remove unbound Fab fragments (Figure 12C).

[0158] The purified A54+Fab complex was concentrated to 5.4 mg / mL and tested for crystallization using a commercially available screen. Crystals were found on the PACT Premier crystallization screen (Molecular Dimensions) under condition E5 (0.2 M sodium nitrate, 20% polyethylene glycol 3350). The crystals were transferred to a cryoprotection solution (0.2 M sodium nitrate, 20% polyethylene glycol 3350, 25% ethylene glycol), then collected for X-ray data acquisition and flash-cooled in liquid nitrogen.

[0159] X-ray diffraction data were collected at the Diamond Light Source, beamline I04-1 in the UK, and processed to 3.54 Å using the XDS2 and STARANISO3 pipelines. Structures were phase-determined by molecular substitution using the structures of unliganded HPSE (PDB accession code 5E8M) and an unrelated mouse IgG Fab fragment (PDB accession code 1AE6). Structures were further refined by iterative rounds of manual model construction and maximum-likelihood refinement using COOT and REFMAC5, respectively. Refinement was performed using TLS constraints, jelly body constraints, and Prosmart constraints, with 5E8M and 1AE6 as reference models. The final models were checked using the wwPDB validation server.

[0160] Crystallographic data show that A54 Fab binds to HPSE on the (β / α)8-barrel domain directly above HBD-II (Gln270-Lys280; Figures 14A and 14B). This interaction prevents HPSE from binding to its HS substrate by steric occlusion of the enzyme binding groove (illustrated in Figure 15F). Examination of protein surface charges reveals a large electrostatic contribution to the A54-HPSE interaction. HBD-II is substantially positively charged, while the binding site of A54 is negatively charged (Figure 14C).

[0161] This result indicates that the single most important region of HPSE at the A54-HPSE junction (interface) is HBD-II. Several negatively charged A54 amino acids (Asp123VH, Asp125VH, Glu78VH, Asp20VL, Glu116VL, Asp117VL) cluster at the A54-HPSE junction and form a network of salt bridges that strongly stabilize the binding interaction. Tyr124VH and Tyr55VL also have strong charge components, and the cation-π interactions involving Tyr124VH and Tyr55VL are also dominant. As expected, the CDR loops mediate almost all of the A54-HPSE binding interactions. Figure 13 is a schematic diagram of the tertiary Fab structure of the A54 CDR loops. Each CDR loop interaction is described in more detail below.

[0162] H1 interaction The A54 H1 loop has no interaction with HPSE.

[0163] H2 interaction (Figure 15A) H2 (Tyr68-Asp85) interacts with the HPSE region around Tyr298-Asp309 and several nearby residues (Tyr348, Lys231). A54 Lys93 does not strictly belong to the H2 loop but appears to be spatially close to the H2 interaction. Particularly important interactions in this region include Glu78 A54 and Lys231 HPSE and between Lys293 A54 and Asp309 HPSE and include salt bridges between them.

[0164] H3 interaction (Figure 15B) H3 (Gly118-Tyr132) interacts with the HPSE α-helix around Asn312-Lys325 and several residues (Pro271-Ala276) of HBD-II. Asp123 A54 and Lys325 HPSE and between Asp125 A54 and Arg273 HPSE and there are obvious electrostatic interactions between them.

[0165] L1 interaction (Figure 15C) The L1 (Arg43-Asn57) interaction is primarily with the HBD-II region (Arg272-Ser281) of HPSE and several nearby residues. Asp20-Ile21 of A54 is also included because they are close to the L1 loop. There are no obvious salt bridges or H bonds involved in the L1 interaction, but Tyr55 is well-positioned to form a cation-π interaction with Arg273.

[0166] L2 interaction The A54 L2 loop has no interaction with HPSE.

[0167] L3 interaction (Figure 15D) The L3 (Gln111-Thr120) interaction is primarily an interaction with the HBD-II region (Arg272-Ser281) of the HPSE. The important salt bridge is Glu116. A54 and Lys277 HPSE , and Asp117 A54 and Arg273 HPSE It is formed between.

[0168] Figure 15E is a schematic diagram showing the interaction between the HPSE HBD-II residue (blue) and the residues derived from A54 VH (green) and VL (red).

[0169] Figure 15F is a schematic diagram showing the overlap between the dp4 tetrasaccharide (cyanide) from PDB 5E9C and the A54-HPSE complex, illustrating how A54 sterically occludes the HPSE binding groove. A54 binds to HPSE very close to the dp4 site. Native HS substrates extending beyond the dp4 site would be sterically blocked from accessing the HPSE active site groove (red arrow).

[0170] Overview of the interaction Tables 2 and 3 summarize the A54 and HPSE residues involved in the binding site, as determined by PISA5 analysis. The analysis is categorized by antibody chain. Table 2 presents heavy chain (VH) interactions, and Table 3 presents light chain (VL) interactions. Some HPSE residues interact with both VH and VL and therefore appear in both tables. This HPSE model includes two chains corresponding to the 50kDa (A) and 8kDa (B) chains of this protein. The HPSE residues involved in binding originate from chain A. The A54 residues are grouped according to the CDR loop of the residues. Some A54 interacting residues do not belong to the CDR loop.

[0171] The report also shows whether the residue is involved in H-binding or salt bridge interactions (H / S), the estimated accessible surface area / buried surface area of ​​the interaction (ASA / BSA), and the estimated contribution to the binding energy (ΔG). 1. ASA: Accessible Surface Area - The area of ​​monomer units, residues, or atoms that can access the solvent. ASA is measured in square angstroms. 2. BSA: Buried Surface Area - The surface area that becomes inaccessible to the solvent, for example, during protein folding or mating site formation. Measured in square angstroms. 3. ΔG: Increase in solvation energy during humming site formation, kcal / mol. Solvation energy (SE) is the energy difference between the bonded and unbonded states of a monomer unit, residue, or atom due to the solvation effect. In the bonded (humming) state, a portion of the structure's surface becomes inaccessible to the solvent. If the bonded surface has a positive solvation effect, the total energy decreases upon bonding; this is known as hydrophobic interaction.

[0172] [Table 2(1)] [Table 2(2)]

[0173] [Table 3]

[0174] The above-described descriptions of specific embodiments fully illustrate the general nature of the invention; therefore, those skilled in the art can readily modify and / or adapt such specific embodiments to various applications without excessive experimentation and without departing from the overarching concept, by applying their current knowledge. Such adaptations and modifications should therefore be, and are intended to be, included within the meaning and scope of the equivalents of the disclosed embodiments. It should be understood that any expressions or terms used herein are for illustrative purposes only and not for limitation. Means, materials, and processes for performing various disclosed functions may take various alternative forms, without departing from the invention.

Claims

1. An antibody or antigen-binding fragment directed to a heparanase enzyme, wherein the antibody or antigen-binding fragment comprises a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 1, an HCDR2 containing the amino acid sequence of SEQ ID NO: 2, an HCDR3 containing the amino acid sequence of SEQ ID NO: 3, and a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 4, an LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and an LCDR3 containing the amino acid sequence of SEQ ID NO:

6.

2. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or fragment is a mouse antibody or a fragment thereof, or a humanized antibody or a fragment thereof.

3. The antibody or antigen-binding fragment according to claim 2, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, and the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fd', Fv, single-chain antibody, diabody, and linear antibody.

4. An isolated polynucleotide encoding an antibody according to any one of claims 1 to 3.

5. An isolated polynucleotide according to claim 4, comprising any nucleotide sequence from SEQ ID NO: 9 to SEQ ID NO:

16.

6. A vector comprising the polynucleotide described in claim 4.

7. A cell comprising an antibody or antigen-binding fragment according to any one of claims 1 to 3, an isolated polynucleotide according to claim 4, or a vector according to claim 6.

8. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 3, an isolated polynucleotide according to claim 4, a vector according to claim 6, or a cell according to claim 7, and a pharmaceutically acceptable carrier.

9. An antibody or antigen-binding fragment according to any one of claims 1 to 3 for use as a pharmaceutical.

10. An antibody or antigen-binding fragment according to any one of claims 1 to 3, for use in inhibiting or treating a disease or disorder related to heparanase activity in a subject.

11. The antibody or antigen-binding fragment for use according to claim 10, wherein the disease or disorder is a malignant proliferative disorder such as carcinoma, sarcoma, melanoma, or hematological malignancy, or the disease or disorder is type 1 diabetes, inflammatory disorder, kidney disease, or a combination thereof.

12. The antibody or antigen-binding fragment for use according to claim 10, wherein the antibody or antigen-binding fragment inhibits tumor progression, inhibits tumor metastasis, or a combination thereof.

13. The antibody or antigen-binding fragment for use according to claim 10, wherein the subject has a malignant proliferative disease and further anti-cancer treatment such as chemotherapy or radiotherapy is administered to the subject.

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