Antibody-drug conjugates, including humanized antibodies targeting urokinase-type plasminogen activator receptor-associated protein (UPARAP)

Abstract

The present invention relates to humanized antibodies and molecular conjugates targeting urokinase-type plasminogen activator receptor-associated protein (uPARAP), in particular to antibody-drug conjugates (ADCs) comprising humanized antibodies directed against uPARAP and their use in delivering active agents to cells and tissues expressing uPARAP. The present invention further relates to the use of said ADCs in the treatment of diseases involving uPARAP-expressing cells, such as certain cancers.

Description

[Technical field]

[0001] The present invention relates to antibodies and molecular conjugates targeting the receptor uPARAP, in particular antibody-drug conjugates (ADCs) comprising humanized antibodies directed against uPARAP, and their use in delivering active agents to cells and tissues expressing uPARAP. The present invention further relates to the use of said ADCs in the treatment of diseases involving uPARAP-expressing cells, such as certain cancers. [Background technology]

[0002] Urokinase-type Plasminogen Activator Receptor Associated Protein (uPARAP), also known as CD280, Endo180, and mannose receptor type C2, is a member of the macrophage mannose receptor family of endocytic transmembrane glycoproteins. uPARAP is a membrane protein involved in matrix metabolism, particularly collagen uptake and intracellular degradation, during tissue remodeling. The uPARAP receptor consists of an N-terminal cysteine-rich domain (CysR), a fibronectin type II (FN-II) domain, and eight C-type lectin-like domains (CTLD 1-8).

[0003] The receptor uPARAP is upregulated in tumor cells of certain cancers, including sarcoma and late-stage glioblastoma. Furthermore, the receptor is most frequently upregulated in stromal cells surrounding solid tumors, and some literature suggests high expression of uPARAP in bone metastases from prostate cancer (Caley et al., 2012, J. Pathol 5:775-783). In healthy adult individuals, the receptor shows a restricted expression pattern (Melander et al., 2015, Int J Oncol 47:1177-1188).

[0004] Antibody-drug conjugates (ADCs) are a class of highly potent biopharmaceuticals designed as targeted therapies, especially for the treatment of cancer. ADCs are composite molecules composed of an antibody (whole mAb or antibody fragment) linked to an active agent, e.g., a biologically active drug or cytotoxic compound, via a stable chemical linker that may have a labile bond. By combining the unique targeting ability of antibodies with the cell-killing ability of cytotoxic agents, antibody-drug conjugates can sensitively distinguish between healthy and diseased tissues based on the expression of antibody antigens. This means that, in contrast to traditional chemotherapeutic agents, healthy cells with little or no antigen expression are not affected as severely, since antibody-drug conjugates actively target and attack cancer cells. Currently, more than 10 ADCs are approved on the market, and several ADCs are currently in clinical trials.

[0005] WO2010 / 111198 discloses a conjugate comprising an anti-uPARAP antibody, and suggests the use of such a conjugate in delivering a therapeutic agent to cells expressing uPARAP.

[0006] WO2017 / 133745 discloses ADCs directed against uPARAP.

[0007] Currently, treatments exist for most types of cancer. However, in many cases, they are inefficient or have adverse effects due to high doses of therapeutic drugs. Therefore, more efficient treatments with increased efficacy are needed. Summary of the Invention

[0008] Provided herein is a humanized form of the murine 9b7 antibody and its implementation in antibody drug conjugates (ADCs) targeting the uPARAP receptor. The murine 9b7 antibody was first described in WO2017 / 133745. The antibodies and ADCs described herein can specifically target cells and tissues expressing uPARAP and demonstrate enhanced potency compared to ADCs comprising the murine 9b7 antibody, and enhanced potency compared to other humanized forms of the murine 9b7 antibody.

[0009] In particular, the present disclosure provides an antibody that binds to uPARAP, a. an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:3, and / or b. The antibody as described above, comprising an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:6.

[0010] Further, the present disclosure provides a.An antibody as defined above, b. an active agent, and c. Optionally, an antibody drug conjugate (ADC) comprising a linker connecting a) to b).

[0011] Furthermore, the present disclosure relates to a method for treating a disease characterized by cells expressing uPARAP, said method comprising administering to a subject an antibody as defined above, an ADC as defined above, or a pharmaceutical composition comprising an antibody or ADC as defined above.

[0012] Further aspects of the present disclosure are polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO:2 and / or SEQ ID NO:5; isolated polynucleotides encoding the amino acid sequences defined herein; vectors comprising the polynucleotides defined above; and host cells comprising the polynucleotides defined above and / or the vectors defined above.

[0013] Yet another aspect of the present disclosure is a kit comprising an antibody as defined above, an ADC as defined above, or a pharmaceutical composition comprising an antibody or ADC as defined above, optionally further comprising a means for administering the antibody or antibody-drug conjugate to a subject and / or instructions for use. [Brief description of the drawings]

[0014] [Figure 1] In vitro cell viability assay of U937 cancer cell line exposed to MMAE-based ADCs containing either the LC0HC0 antibody (containing the variable domains of the original murine 9b7 antibody fused to a human IgG constant region) or the humanized LC4HC3 antibody, except in the case of the antibody, the two ADCs are identical and were generated by the same method. Cells were incubated for 96 hours and then analyzed by a colorimetric viability assay. The assay for the U937 cell line shows that the LC4HC3-based ADC has a significantly greater reduction in total cell viability compared to the LC0HC0 ADC. [Diagram 2] Internalization of humanized antibodies LC4HC3 and LC3HC3 in SAOS-2 osteosarcoma cells. A detailed protocol is shown in Example 2. The data show that LC4HC3 is not only internalized faster than LC3HC3, but also more extensively in SAOS-2 osteosarcoma cells. [Figure 3a] In vivo efficacy of vedotin-type ADCs based on LC4HC3 (LC4HC3-vc-MMAE). CB17 mice were seeded with U937 cells to induce tumor growth. Tumor size was closely monitored and treatment was initiated when tumors reached a size of approximately 80-150 mm3. However, for antibodies, the two ADCs were identical and produced by the same method. Each line represents the tumor size of mice treated with the reference ADC at a dose of 4 mg / kg twice daily for 7 days. The data show that ADCs based on the humanized 9b7 antibody LC4HC3 are superior antitumor agents compared to ADCs based on the different humanized 9b7 antibody LC3HC3. [Figure 3b]In vivo efficacy of vedotin-type ADCs based on LC3HC3 (LC3HC3-vc-MMAE). CB17 mice were seeded with U937 cells to induce tumor growth. Tumor size was closely monitored and treatment was initiated when tumors reached a size of approximately 80-150 mm3. However, for antibodies, the two ADCs were identical and produced by the same method. Each line represents the tumor size of mice treated with the reference ADC at a dose of 4 mg / kg twice daily for 7 days. The data show that ADCs based on the humanized 9b7 antibody LC4HC3 are superior antitumor agents compared to ADCs based on the different humanized 9b7 antibody LC3HC3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The antibodies of the present disclosure are internalized upon binding to the cell surface uPARAP receptor, thus enabling intracellular action of the active agent of the antibody drug conjugate.

[0016] Provided herein is a humanized version of the murine 9b7 antibody that binds to the uPARAP receptor.

[0017] Anti-uPARAP humanized antibody Methods for producing antibodies are well known in the art. For example, antibodies can be produced through any one of several methods utilizing inducing in vivo production of antibody molecules, screening immunoglobulin libraries, or producing monoclonal antibody molecules by cell lines in culture. These techniques include, but are not limited to, hybridoma technology, human B-cell hybridoma technology, and Epstein-Barr Virus (EBV)-hybridoma technology.

[0018] Humanized antibodies are generally preferred in pharmaceuticals intended for humans, and methods for humanizing antibodies are well known in the art. Although humanization techniques are known, it can be difficult to achieve a humanized antibody that retains the binding characteristics of the initial antibody, and even more difficult to achieve a humanized antibody with improved properties, such as improved ligand affinity and efficacy, compared to the initial antibody.

[0019] The inventors herein provide improved anti-uPARAP antibodies that are humanized versions of the 9b7 murine antibody and that exhibit improved ligand affinity and potency compared to the 9b7 murine antibody, as well as improved internalization and in vivo potency compared to other humanized versions of the 9b7 antibody.

[0020] The anti-uPARAP antibody of the present disclosure may be of any immunoglobulin type, including IgG, IgM, IgD, IgE, IgA and subclasses thereof. Subclasses of IgG are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3 and IgG4. In one embodiment, the antibody is an IgG monoclonal antibody. In one embodiment, the antibody is an IgG1κ.

[0021] The anti-uPARAP antibody of the present disclosure is a humanized 9b7 antibody that binds to the uPARAP receptor, and more specifically, the humanized 9b7 antibody disclosed herein binds at least to the fibronectin type II (FN-II) domain of the uPARAP receptor.

[0022] The humanized 9b7 antibody, also referred to herein as 980.2 LC4HC3, comprises a light chain variable region of amino acids comprising SEQ ID NO:3, which is the variable region of LC4, and a heavy chain variable region of amino acids comprising SEQ ID NO:6, which is the variable region of HC3.

[0023] The humanized 9b7 antibody, also referred to herein as 980.2 LC4HC3, can comprise a light chain of amino acids comprising or consisting of SEQ ID NO:1, which is LC4, and a heavy chain of amino acids comprising or consisting of SEQ ID NO:4, which is HC3.

[0024] In one embodiment of the disclosure, the anti-uPARAP antibody defined herein is a. an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:3, and / or b. comprises an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:6.

[0025] In one embodiment of the disclosure, an antibody that binds to uPARAP as defined herein is a. an immunoglobulin light chain (LC4) comprising the amino acid sequence of SEQ ID NO:1, and / or b. An immunoglobulin heavy chain (HC3) comprising the amino acid sequence of SEQ ID NO:4.

[0026] In one embodiment of the disclosure, an antibody that binds to uPARAP as defined herein is a. an immunoglobulin light chain (LC4) consisting of the amino acid sequence of SEQ ID NO:1; and b. Contains an immunoglobulin heavy chain (HC3) consisting of the amino acid sequence of SEQ ID NO:4.

[0027] Polypeptides, polynucleotides, vectors, and host cells One embodiment of the present disclosure is a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 and / or SEQ ID NO: 5. SEQ ID NO: 2 and SEQ ID NO: 5 correspond to SEQ ID NO: 1 and SEQ ID NO: 4, respectively, but additionally have an N-terminal signal peptide for expression.

[0028] One embodiment of the disclosure is an isolated polynucleotide encoding any of the polypeptides disclosed herein, i.e., an isolated polynucleotide encoding the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5 and / or 6.

[0029] In one embodiment, the polynucleotide comprises SEQ ID NO:11 and / or SEQ ID NO:12, which encode SEQ ID NO:2 and SEQ ID NO:5, respectively.

[0030] In one embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:2, and optionally, the polypeptide further comprises the amino acid sequence of SEQ ID NO:5.

[0031] In one embodiment, the disclosure provides an isolated polynucleotide encoding the amino acid sequence of any one of SEQ ID NOs: 1, 2 or 3, optionally wherein the polynucleotide further encodes the amino acid sequence of any one of SEQ ID NOs: 4, 5 or 6.

[0032] In one embodiment, the disclosure provides an isolated polynucleotide comprising SEQ ID NO:11, optionally, said polynucleotide further comprising SEQ ID NO:12.

[0033] In one embodiment, the polypeptide is an isolated polypeptide.

[0034] One embodiment of the disclosure is a vector, e.g., an expression vector, comprising a polynucleotide defined herein.

[0035] In one embodiment of the present disclosure, the vector is a mammalian expression vector.

[0036] In one embodiment of the present disclosure, the vector is a plasmid vector, for example, a plasmid vector selected from the pD2610-v13(ATUM), pSV and pCMV series of plasmid vectors.

[0037] In one embodiment of the present disclosure, the vector is a viral vector, e.g., a viral vector selected from the group consisting of an adenoviral vector, a lentiviral vector, an adeno-associated viral vector, a herpes viral vector, a vaccinia viral vector, a pox viral vector, a baculoviral vector, and an oncolytic viral vector.

[0038] A further embodiment of the present disclosure is a host cell comprising a polynucleotide and / or a vector as defined herein.

[0039] In one embodiment of the present disclosure, the host cells comprising the polynucleotides and / or vectors described herein are selected from the group consisting of CHO (Chinese Hamster Ovary) cells, COS (derived from CV-1 (monkey) and carrying SV40 genetic material), HEK (human embryonic kidney) cells, and HeLa (Henrietta Lacks) cells.

[0040] In one embodiment, the host cell is CHO.

[0041] In one embodiment, the host cell is a recombinant host cell.

[0042] Antibody-drug conjugates (ADCs) containing anti-uPARAP humanized antibodies Our data surprisingly show that ADCs based on LC4HC3 (a humanized 9b7 antibody) result in a significant reduction in total cell viability compared to LC0HC0 based ADCs (having the variable domains of the 9b7 murine antibody fused to a human IgG constant region). LC4HC3 based ADCs also show improved internalization and in vivo efficacy compared to LC3HC3 based ADCs (another humanized 9b7 antibody).

[0043] A particularly preferred embodiment of the present disclosure comprises: a. an antibody as defined herein; b. an active agent, and c. Optionally, an antibody drug conjugate (ADC) comprising a linker linking a) to b).

[0044] In one embodiment of the disclosure, an antibody drug conjugate (ADC) as defined herein comprises: a. An antibody as defined herein, i) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 3, and / or ii) an antibody as defined herein, comprising an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:6; b. an active agent, and c. Optionally, including a linker connecting a) to b).

[0045] In one embodiment of the disclosure, an antibody drug conjugate (ADC) as defined herein comprises: a. An antibody as defined herein, i) an immunoglobulin light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 1, and / or ii) an antibody as defined herein above, comprising an immunoglobulin heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 4; b. an active agent, and c. Optionally, including a linker connecting a) to b).

[0046] In one embodiment of the disclosure, an antibody drug conjugate (ADC) as defined herein comprises: a. An antibody as defined herein, i) an immunoglobulin light chain consisting of the amino acid sequence of SEQ ID NO:1, and ii) an antibody as defined herein above, comprising an immunoglobulin heavy chain consisting of the amino acid sequence of SEQ ID NO: 4; b. an active agent, and c. Optionally, including a linker connecting a) to b).

[0047] Active Agent The ADC of the present disclosure comprises an active agent, such as a drug, that can be delivered intracellularly to the cell that expresses uPARAP.The active agent can be, for example, a therapeutic agent, a radioisotope or a detectable label.In a preferred embodiment, the active agent is a therapeutic agent.

[0048] In one embodiment, the active agent may be or may include a radioisotope. The radioisotope may function as a radiation emitter, either for the treatment of diseased tissue or for diagnostic purposes. In one embodiment, the radioisotope may be: 60 Co, 89 Sr, 90 Y, 99m Tc, 131 I, 137 Cs, 153 Sm or 223 Rd may consist of or include Rd. In one embodiment of the present disclosure, the radioisotope may be combined with a chelating agent such as DOTA or EDTA, or others known in the art.

[0049] In one embodiment, the active agent is a therapeutic agent, classes of therapeutic agents include DNA crosslinking agents, DNA alkylating agents, DNA strand breakers, anthracyclines, antimetabolites, anti-microtubule / mitotic inhibitors, histone deacetylase inhibitors, kinase inhibitors, metabolic inhibitors, peptide antibiotics, immune checkpoint inhibitors, platinum-based antineoplastic agents, topoisomerase inhibitors, DNA or RNA polymerase inhibitors, nucleotide-based drugs, and cytotoxic antibiotics.

[0050] In a preferred embodiment, the active agent is a cytotoxic drug that allows for efficient killing of cells expressing uPARAP.

[0051] In one embodiment, the active agent is a chemotherapeutic agent.

[0052] In one embodiment, the active agent is a DNA crosslinking agent, e.g., a DNA crosslinking agent selected from cisplatin or a derivative of cisplatin (e.g., carboplatin or oxaliplatin), mitomycin C (MMC), pyrrolobenzodiazepines, and dimeric pyrrolobenzodiazepine derivatives (e.g., SGD-1882), or a derivative of any of these.

[0053] In one embodiment of the present disclosure, the active agent is a DNA alkylating agent, e.g., a DNA alkylating agent selected from tris(2-chloroethyl)amine, pyridinobenzodiazepine or pyridinobenzodiazepine derivatives, indolinobenzodiazepine dimers, and nitrogen mustards such as duocarmycin SA, or a derivative of any of these.

[0054] In one embodiment, the active agent is a DNA strand cleaving agent, for example, a DNA strand cleaving agent selected from calicheamicin and hamirtron, or a derivative of any of these.

[0055] In one embodiment, the active agent is an anthracycline, such as an anthracycline selected from daunorubicin, doxorubicin, epirubicin, idarubicin, and PNU-159682, or a derivative of any of these.

[0056] In one embodiment, the active agent is an antimetabolite, e.g., an antimetabolite selected from an antifolate (e.g., methotrexate), a purine antimetabolite (e.g., 6-mercaptopurine or 6-thioguanine or fludarabine phosphate or pentostatin or cladribine), and a pyrimidine antimetabolite (e.g., 5-fluorouracil or 5-fluorodeoxyuridine or cytarabine or gemcitabine), or a derivative of any of these.

[0057] In one embodiment, the active agent is a mitotic inhibitor selected from the group consisting of, for example, a derivative of an auristatin or dolastatin (e.g., monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), etc.), a taxane (e.g., paclitaxel or docetaxel), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, or vinorelbine), a maytansinoid, colchicine, and podophyllotoxin, or a derivative of any of these.

[0058] In one embodiment, the active agent is monomethylauristatin E (MMAE) or a derivative thereof.

[0059] Due to its high toxicity, MMAE, which inhibits cell division by inhibiting the polymerization of tubulin, cannot be used as a single-agent chemotherapy drug. However, the combination of MMAE bound to an anti-CD30 monoclonal antibody (Brentuximab Vedotin, trade name Adcetris™) has been proven to be stable in extracellular fluids, cleavable by cathepsins, and safe for treatment.

[0060] In one embodiment, the active agent is a histone deacetylase inhibitor, such as a histone deacetylase inhibitor selected from trichostatin A, vorinostat, belinostat, panobinostat, gibinostat, resminostat, abexinostat, xinostat, rosilinostat, prasinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, entinostat, tacedinaline, 4SC202, mocetinostat, romidepsin, nicotinamide, sirtinol, cambinol, and EX-527, or any derivative thereof.

[0061] In one embodiment, the active agent is a kinase inhibitor, for example, a kinase inhibitor selected from genistein, lavendustin C, PP1-AG1872, PP2-AG1879, SU6656, CGP77675, PD166285, imatinib, erlotinib, gefitinib, lavendustin A, cetuximab, UCS15A, herbimycin A, and radicicol, or a derivative of any of these.

[0062] In one embodiment, the active agent is a metabolic inhibitor, such as a NAMPT inhibitor. Examples of NAMPT inhibitors include APO866, GMX-1777, GMX-1778, ATG-019, and OT-82, or any derivative thereof.

[0063] In one embodiment, the active agent is an immune checkpoint inhibitor, such as a PD-1 inhibitor or a PD-L1 inhibitor. Examples of PD-1 inhibitors include pembrolizumab, nivolumab, cemiplimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, AMP-224, and AMP-514. Examples of PD-L1 inhibitors include atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189, or any derivative thereof.

[0064] In one embodiment, the active agent is a platinum-based anti-cancer agent, such as a platinum-based anti-cancer agent selected from lipoplatin, cisplatin, carboplatin, oxaliplatin, nedaplatin, picoplatin, phenanthriplatin, satraplatin, and triplatin tetranitrate, or a derivative of any of these.

[0065] In one embodiment, the active agent is a topoisomerase inhibitor, e.g., a topoisomerase inhibitor selected from camptothecin or a derivative thereof, e.g., topotecan, belotecan, lurtotecan, irinotecan, SN-38, exatecan, and Dxd, or a derivative of any of these.

[0066] In one embodiment, the active agent is a DNA or RNA polymerase inhibitor, for example a polymerase inhibitor selected from amanitin or α-amanitin or a derivative thereof, actinomycin D, and aphidicolin, or a derivative of any of these.

[0067] In one embodiment, the active agent is a nucleotide-based agent, e.g., an RNA or DNA oligonucleotide, e.g., an siRNA or miRNA.

[0068] There may be one or more units of drug per antibody molecule. The ratio between the number of drug molecules per antibody is expressed as the drug-to-antibody ratio (DAR). In one embodiment, the DAR is between 1 and 10, such as between 2 and 8, for example between 2 and 6, such as 2 or 4.

[0069] Linker A stable bond between the antibody and the active agent is a key aspect of ADC technology. The linker may be based on chemical motifs, including, for example, disulfides, hydrazones or peptides (cleavable), or thioethers (non-cleavable), to control the distribution and delivery of cytotoxic drugs to target cells. Both cleavable and non-cleavable types of linkers have been proven safe in preclinical and clinical trials. For example, brentuximab vedotin contains an enzyme-sensitive cleavable linker that delivers the synthetic anti-cancer drug monomethyl auristatin E (MMAE), a potent and highly toxic anti-microtubule agent, to cells.

[0070] Another approved ADC, trastuzumab emtansine, is a combination of the microtubule inhibitor mertansine (DM-1), a derivative of maytansine, and the antibody trastuzumab (Herceptin™, Genentech / Roche) linked by a stable non-cleavable linker.

[0071] The type of cleavable or non-cleavable linker provides certain properties to the delivered drug. For example, cleavable linkers can be cleaved, for example, by enzymes in target cells, resulting in efficient intracellular release of active agents, for example, cytotoxic drugs. In contrast, ADCs that contain non-cleavable linkers have no mechanism for drug release and must rely on mechanisms such as the degradation of targeting antibodies for drug release. Furthermore, as those skilled in the art will appreciate, the linker composition can affect important factors such as the solubility and pharmacokinetic properties of the ADC as a whole.

[0072] For both types of linkers, drug release is important for cellular effects: drugs that can diffuse freely across the cell membrane can escape from the target cell and attack neighboring cells, such as cancer cells, in the vicinity of the uPARAP-expressing target cell, in a process called "bystander killing."

[0073] In a preferred embodiment of the present disclosure, the uPARAP-targeting ADCs disclosed herein comprise a linker that attaches the antibody to the active agent.

[0074] In one embodiment of the present disclosure, the linker may be cleavable or non-cleavable.

[0075] Cleavable groups include disulfide bonds, amide bonds, substituted amide bonds in the form of peptide bonds, thioamide bonds, ester bonds, thioester bonds, vicinal diol bonds, or hemiacetals. These or other cleavable bonds can include enzymatically cleavable bonds such as peptide bonds (cleaved by peptidases), phosphate bonds (cleaved by phosphatases), nucleic acid bonds (cleaved by endonucleases), and sugar bonds (cleaved by glycosidases).

[0076] In a further embodiment of the present disclosure, the linker is a cleavable linker that allows for intracellular release of the active agent inside the target cell.

[0077] In further embodiments, the linker is a peptide linker. The selection of the peptide sequence is important for the success of the conjugate. In some embodiments, the linker is stable to serum proteases, but is cleaved by lysosomal enzymes in the target cell.

[0078] In further embodiments, the linker is an enzyme-cleavable peptide-containing linker, such as a cathepsin-cleavable peptide-containing linker. Cathepsin is one of several types of cathepsins, which are a group of lysosomal proteases.

[0079] In further embodiments of the present disclosure, the linker comprises or consists of a dipeptide, such as valine-citrulline (VC) or valine-alanine (VA).

[0080] In one embodiment, the linker comprises or consists of a dipeptide, such as valine-citrulline (VC) or valine-alanine (VA), which may be further linked to other structural elements via an amide bond. The valine-citrulline based linker, in which the citrulline carboxyl functional group is modified to a substituted amide, can be cleaved by lysosomal cathepsins, whereas the valine-alanine based linker, in which the alanine carboxyl functional group is modified to a substituted amide, can be cleaved by other lysosomal proteases, including other cathepsins.

[0081] In further embodiments of the disclosure, the antibody drug conjugate defined herein further comprises a spacer, e.g., a spacer comprising p-aminobenzoic acid (PAB), p-aminobenzylcarbamate (PABC), p-aminobenzoyloxycarbonyl, or polyethylene glycol (PEG).

[0082] In one embodiment of the disclosure, an antibody-drug conjugate as defined herein comprises p-aminobenzylcarbamate (PABC).

[0083] In further embodiments of the disclosure, the antibody drug conjugates defined herein further comprise a linking group, such as maleimide and caproic acid (MC), N-hydroxysuccinimide, reactive linking groups directed to modified or unmodified protein-bound carbohydrates, peptide sequences required for enzymatic reactions, azides or alkynes, or derived therefrom by reaction with an antibody, or chemically or enzymatically generated derivatives thereof, or alternatively comprise a linking group consisting of these.

[0084] In one embodiment of the disclosure, the ADC of the disclosure further comprises a linking entity. The linking entity may, for example, link an antibody to a cleavable linker, the linking entity being the reaction product of an antibody amino acid side chain in the linker precursor with a reactive linking group. In one embodiment, the reactive linking group comprises or consists of maleimide and caproic acid (MC), the maleimide preferably reacting with cysteine ​​thiol during coupling. In other embodiments, the linking group comprises or consists of N-hydroxysuccinimide, reactive linking groups directed to modified or unmodified protein-bound carbohydrates, peptide sequences required for enzymatic reactions, azides or alkynes, or derived therefrom by reaction with an antibody, or chemically or enzymatically generated derivatives thereof.

[0085] In one embodiment of the disclosure, an ADC comprises an antibody targeting uPARAP, as defined herein, and the linker-drug conjugate vedotin. Vedotin is a linker-drug conjugate that includes the cytotoxic drug MMAE, a spacer (p-aminobenzoic acid), a cathepsin-cleavable linker (valine-citrulline dipeptide), and a linking group consisting of caproic acid and maleimide. Vedotin is MC-VC-PAB-MMAE.

[0086] In one embodiment, an ADC of the disclosure that targets uPARAP comprises an antibody as defined herein and a linker-spacer-toxin unit that is VC-PAB-MMAF.

[0087] In one embodiment, an ADC of the disclosure that targets uPARAP comprises an antibody as defined herein and a linker-spacer-toxin unit that is VC-PABC-MMAF.

[0088] In one embodiment, an ADC of the disclosure that targets uPARAP comprises: a. An antibody as defined herein, i) an immunoglobulin light chain consisting of the amino acid sequence of SEQ ID NO:1, and ii) an antibody as defined herein above, comprising an immunoglobulin heavy chain consisting of the amino acid sequence of SEQ ID NO: 4; b. VC linker, c.MC binding group, d. PAB or PABC spacer, and e. Comprising or consisting of MMAE as the active agent.

[0089] In one embodiment, an ADC of the disclosure that targets uPARAP comprises: a. An antibody as defined herein, i) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 3, and / or ii) an antibody as defined herein, comprising an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:6; b. VC linker, c.MC binding group, d. PAB or PABC spacer, and e. Comprising or consisting of MMAE as the active agent.

[0090] therapeutic use The ADCs directed against uPARAP described herein are useful for the delivery of active agents, e.g., therapeutic or cytotoxic drugs, to cells that express uPARAP and similar proteins, and are therefore useful for the treatment of a variety of diseases and disorders characterized by either the expression or overexpression of said proteins.

[0091] Thus, one embodiment of the present disclosure is an antibody or antibody-drug conjugate as defined herein for use as a medicament.

[0092] One embodiment of the disclosure is a pharmaceutical composition comprising an effective amount of an antibody or antibody-drug conjugate as defined herein, and a pharma- ceutically acceptable buffer, diluent, carrier, adjuvant or excipient.

[0093] As used herein, "therapeutically effective amount" or "effective amount" or "therapeutically effective" refers to an amount that produces a therapeutic effect for a given condition and administration regimen. It is a predetermined amount of active material calculated to produce a desired therapeutic effect in association with necessary additives and diluents, i.e., carriers or administration vehicles. Moreover, it is intended to mean an amount sufficient to reduce, and most preferably prevent, a clinically significant deficiency in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to improve a clinically significant condition in the host. As will be appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. An appropriate dosage may include a predetermined amount of active composition calculated to produce a desired therapeutic effect in association with necessary diluents.

[0094] The ADCs of the disclosure can be formulated into any type of pharmaceutical composition known in the art suitable for their administration.

[0095] Pharmaceutical compositions can be prepared by methods known in the art that are sufficiently storage stable and suitable for administration to humans and / or animals, for example, pharmaceutical compositions can be lyophilized, for example, by freeze drying, spray drying, spray chilling, or by using particle formation from supercritical particle formation.

[0096] "Pharmaceutically acceptable" means a non-toxic material that does not reduce the efficacy of the ADC. Such pharma- ceutically acceptable buffers, carriers, or excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), the disclosures of which are incorporated herein by reference).

[0097] The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture for the purpose of stabilizing the pH. Pharmaceutically acceptable buffers are well known in the art.

[0098] The term "diluent" is intended to mean an aqueous or non-aqueous solution intended for diluting a drug in a pharmaceutical formulation.

[0099] The term "adjuvant" is intended to mean any compound added to the formulation to enhance the biological effect of the agent of the present invention.Adjuvants can be one or more of zinc, copper or silver salts with different anions, such as, but not limited to, fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetate with different acyl composition.Adjuvants can also be cationic polymers, such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly(vinylimidazole), and cationic polypeptides, such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

[0100] The excipient may be one or more of carbohydrates, polymers, lipids, and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrin, which are added to the composition, for example, to facilitate lyophilization. Examples of polymers are starch, cellulose ether, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, alginates, carrageenans, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonates, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate with different hydrolysis degrees, and polyvinylpyrrolidone, all with different molecular weights, which are added to the composition, for example, to adjust viscosity, bioadhesion, or to protect lipids from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di- and triglycerides, ceramides, sphingolipids and glycolipids of all different acyl chain lengths and degrees of saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those of the polymers. Examples of minerals are talc, magnesium oxide, zinc oxide, and titanium oxide, which are added to the composition to obtain benefits such as reduced liquid accumulation or favorable pigment properties.

[0101] Another embodiment of the present disclosure is a method of treating a disease in a subject characterized by cells expressing uPARAP, said method comprising administering to the subject an antibody or antibody-drug conjugate defined herein.

[0102] The expression and role of uPARAP in cancer has been investigated by several research groups, see the review by Melander et al. (Melander et al., 2015, Int J Oncol 47:1177-1188) and the paper by Engelholm et al. (Engelholm et al., 2016, J. Pathol. 238, 120-133).

[0103] In one embodiment of the disclosure, the method is a method defined herein, wherein the disease characterized by cells expressing uPARAP is selected from cancer, a bone degradative disease, e.g., osteoporosis, fibrosis, and a macrophage-associated disease or disorder, e.g., atherosclerosis, arthritis, or chronic inflammation.

[0104] In one embodiment of the disclosure, the method is a method defined herein, wherein the arthritis is selected from osteoarthritis, inflammatory arthritis, rheumatoid arthritis, psoriatic arthritis, lupus, Lyme disease-induced arthritis, e.g., Lyme arthritis, gout or pseudogout, and ankylosing spondylitis.

[0105] In one embodiment of the disclosure, the method is as defined herein and the disease is cancer.

[0106] Examples of cancers characterized by overexpression of uPARAP include sarcomas, including osteosarcoma (Engelholm et al., 2016, J Pathol 238(1):120-33) and other sarcomas, glioblastoma (Huijbers et al., 2010, PLoS One 5(3):e9808), prostate cancer and bone metastases from prostate cancer (Kogianni et al., 2009, Eur J Cancer 45(4):685-93), breast cancer, particularly "basal-like" breast cancer (Wienke et al., 2007, Cancer Res 1;67(21):10230-40), head and neck cancer (Sulek et al., 2007, J Histochem Cytochem 55(4):347-53), and mesothelioma (Cakilkaya et al., 2021, Int J Mol Sci 22(21):11452), but are not limited to these.

[0107] In one embodiment of the disclosure, the method is a method defined herein, wherein the cancer is selected from sarcoma, glioblastoma, mesothelioma, colon cancer, prostate cancer, bone metastasis from prostate cancer, breast cancer, head and neck cancer, and leukemia.

[0108] In one embodiment of the disclosure, the method is a method defined herein, and the cancer is leukemia, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), or subtypes thereof.

[0109] In one embodiment of the disclosure, the method is a method defined herein, wherein the cancer is a sarcoma, such as osteosarcoma or soft tissue sarcoma (STS), or a subtype thereof.

[0110] In one embodiment of the disclosure, the method is a method defined herein, wherein the soft tissue sarcoma (STS) is selected from epithelioid sarcoma, clear cell sarcoma, alveolar soft part sarcoma, extraskeletal myxoid chondrosarcoma, epithelioid hemangioendothelioma, inflammatory myofibroblastic tumor, undifferentiated embryonal sarcoma, alveolar soft part sarcoma (ASPS), angiosarcoma, chondrosarcoma, dermatofibrosarcoma protuberans (DFSP), desmoid sarcoma, Ewing's sarcoma, fibrosarcoma, myxofibrosarcoma, gastrointestinal stromal tumor (GIST), non-uterine leiomyosarcoma, uterine leiomyosarcoma, liposarcoma, malignant fibrous histiocytoma (MFH), malignant peripheral nerve sheath tumor (MPNST), rhabdomyosarcoma, synovial sarcoma, and / or leiomyosarcoma (LMS).

[0111] In one embodiment of the disclosure, the method is as defined herein and the cancer is metastatic cancer.

[0112] In one embodiment of the disclosure, the method is as defined herein, and the cancer is a solid tumor.

[0113] In one embodiment of the disclosure, the method is as defined herein, wherein the cancer is glioblastoma.

[0114] In one embodiment of the present disclosure, the cancer is not a solid tumor. For example, the ADCs of the present disclosure may be used to treat uPARAP-expressing leukemias, e.g., derived from macrophage-monocyte cell lines.

[0115] In other embodiments of the present disclosure, the disease or disorder characterized by cells that express uPARAP is not cancer.

[0116] uPARAP is involved in bone growth and homeostasis (Madsen et al., 2013, PLoS One 5;8(8):e71261). Thus, in one embodiment, the ADCs of the disclosure can be used to treat diseases characterized by bone degradation, where the bone degradation is mediated by non-malignant cells, such as osteoporosis.

[0117] Due to its role in collagen accumulation, a role for uPARAP has also been demonstrated in fibrosis (Madsen et al., 2012, J Pathol 227(1):94-105). Thus, in one embodiment, the ADCs of the present disclosure may be used to treat fibrosis, e.g., in the kidney, lung, and liver.

[0118] In one embodiment of the disclosure, the ADCs of the disclosure may be used to treat macrophage-associated diseases and disorders, including atherosclerosis, arthritis, and chronic inflammation.

[0119] The ADCs or pharmaceutical compositions comprising the ADCs of the present disclosure can be administered by any suitable route known to those of skill in the art. Possible routes of administration thus include parenteral (intravenous, subcutaneous, and intramuscular), topical, ocular, nasal, pulmonary, buccal, buccal, vaginal, and rectal. Administration from an implant is also possible.

[0120] In a preferred embodiment, pharmaceutical compositions can be administered parenterally, for example, intravenously, intraventricularly, intraarticularly, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly, or subcutaneously, or by infusion techniques.They are conveniently used in the form of a sterile aqueous solution, which may contain other substances, for example, sufficient salts or glucose to make the solution isotonic with blood.The aqueous solution should be suitably buffered, if necessary.

[0121] In one embodiment of the disclosure, the method is a method defined herein, wherein the antibody drug conjugate is administered parenterally, e.g., intravenously, intracerebroventricularly, intraarticularly, intraarterially, intraperitoneally, intrathecally, intraventricularly, intrasternal, intracranially, intramuscularly or subcutaneously, or by infusion techniques.

[0122] Preparations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the preparation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents.The preparations may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid carrier, such as water, for injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0123] In one embodiment of the disclosure, the method is a method defined herein, wherein the antibody drug conjugate or antibody is administered intravenously.

[0124] In one embodiment of the disclosure, the method is a method defined herein, wherein the antibody drug conjugate or antibody is administered subcutaneously.

[0125] In one embodiment of the disclosure, the method is a method defined herein, wherein the antibody drug conjugate or antibody is administered in combination with one or more further agents, such as one or more further therapeutic agents.

[0126] In one embodiment of the disclosure, the ADC or an antibody of the disclosure is administered in combination with additional reagents and / or therapeutic agents that may increase the functional efficiency of the ADC, for example, an established or novel agent that increases lysosomal membrane permeability, thereby facilitating entry of molecules from inside the lysosome into the cytoplasm, or an agent that increases the permeability of the blood-brain barrier.

[0127] In one embodiment of the disclosure, the ADCs or antibodies described herein may be administered in combination with a variety of anti-cancer agents, such as antimetabolites, alkylating agents, anthracyclines and other cytotoxic antibiotics, vinca alkaloids, anti-microtubule / anti-mitotic agents, histone deacetylase inhibitors, kinase inhibitors, peptide antibiotics, immune checkpoint inhibitors, platinum-based antineoplastic agents, etoposide, taxanes, topoisomerase inhibitors, antiproliferative immunosuppressants, corticosteroids, sex hormones and hormone antagonists, cytotoxic antibiotics, and other therapeutic agents.

[0128] Thus, in one embodiment of the disclosure, the method is a method defined herein, wherein the cells expressing uPARAP exhibit uPARAP overexpression.

[0129] In one embodiment of the disclosure, the method is a method defined herein, wherein the cell expressing uPARAP is a tumor cell.

[0130] In one embodiment of the disclosure, the method is a method defined herein, wherein the cells expressing uPARAP are tumor-associated cells.

[0131] Tumor-associated cells include, but are not limited to, activated fibroblasts, myofibroblasts, angiogenic and infiltrating cells of the macrophage-monocyte lineage or other leukocytic cell types, and cells of the stromal tissue surrounding the tumor.

[0132] In one embodiment of the disclosure, the method is a method defined herein, wherein the antibody or antibody drug conjugate induces cell death and / or inhibits the growth and / or proliferation of uPARAP-expressing cells.

[0133] In one embodiment of the disclosure, the method is a method defined herein, wherein the antibody or antibody-drug conjugate induces the release of a cytotoxin released from uPARAP-expressing cells, resulting in cell death and / or inhibiting the growth and / or proliferation of adjacent cancer cells.

[0134] In one embodiment of the disclosure, the method is a method defined herein and the treatment is remission or cure.

[0135] A further embodiment of the present disclosure is a method for inhibiting tumor progression in a subject, comprising administering to the subject an antibody or antibody-drug conjugate, or pharmaceutical composition defined herein.

[0136] A further embodiment of the present disclosure is a method for inhibiting, reducing or eliminating the metastatic potential of a tumor in a subject, comprising administering to said subject an antibody or antibody-drug conjugate, or pharmaceutical composition defined herein.

[0137] Yet another embodiment of the present disclosure is a kit comprising an antibody or antibody-drug conjugate, or pharmaceutical composition as defined herein, and optionally further comprising a means for administering said antibody-drug conjugate or pharmaceutical composition to a subject and / or instructions for use.

[0138] In one embodiment, the disclosure relates to an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, for use in the manufacture of a medicament for the treatment of a disease characterized by cells expressing uPARAP, e.g., cancer.

[0139] In one embodiment, the disclosure relates to an antibody or antibody-drug conjugate, or a pharmaceutical composition comprising said antibody or antibody-drug conjugate, for use in the manufacture of a medicament for the treatment of a disease characterized by cells expressing uPARAP, e.g., cancer, wherein said antibody or antibody-drug conjugate is a. an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:3, and b. An antibody comprising, or including, an immunoglobulin heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO:6.

[0140] In one embodiment, the disclosure relates to an antibody or antibody-drug conjugate, or a pharmaceutical composition comprising said antibody or antibody-drug conjugate, for use in the manufacture of a medicament for the treatment of a disease characterized by cells expressing uPARAP, e.g., cancer, wherein said antibody or antibody-drug conjugate is a. an immunoglobulin light chain comprising or consisting of the amino acid sequence of SEQ ID NO:1; and b. is or comprises an antibody comprising an immunoglobulin heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:4.

[0141] In one embodiment, the disclosure relates to an antibody or antibody-drug conjugate, or a pharmaceutical composition comprising said antibody or antibody-drug conjugate, for use in the manufacture of a medicament for the treatment of a disease characterized by cells expressing uPARAP, e.g., cancer, wherein said antibody or antibody-drug conjugate is a. an immunoglobulin light chain consisting of the amino acid sequence of SEQ ID NO:1, and b. Is or comprises an antibody comprising an immunoglobulin heavy chain consisting of the amino acid sequence of SEQ ID NO:4. EXAMPLES

[0142] Example 1: Humanization of the murine 9b7 antibody and efficacy of ADCs based thereon Materials and Methods Humanization of the murine antibody 9b7 directed against uPARAP The amino acid sequence of murine antibody 9b7 and data regarding its CDR regions are available in published patent application WO2017 / 133745.

[0143] Humanized variants of the 9b7 antibody were constructed by a third party (Fusion Antibodies, Belfast, UK). Briefly, the mouse parent antibody (clone 9b7) was sequenced and the consensus CDR sequences were grafted in silico onto the human donor sequences.

[0144] For this purpose, a number of human framework sequences (see search procedure below) were used as acceptor frameworks for the CDR sequences. All of these acceptor sequences were derived from mature human IgG of human origin and not from phage display or other techniques. The humanized variants generated from the antibody 9b7 sequence are light and heavy chain combinations designated Ab980.2 LCXHCX (light chain X, heavy chain X), where LC0HC0 refers to a chimeric antibody in which the variable regions of the original mouse antibody are fused to the same human IgG constant region used in the humanized antibody. The mature humanized antibodies are complete IgG molecules of the IgG1κ type.

[0145] For the heavy chain, an online database of human IgG sequences was searched for comparison with the mouse VH domain using the BLAST search algorithm, and candidate human variable regions selected from the top 200 BLAST results were retrieved. These were reduced to four candidates based on a combination of framework homology, maintaining key framework residues and canonical loop structures.

[0146] For the light chain, an online database of human IgK sequences was searched for comparison with the mouse VL domain using the BLAST search algorithm, and candidate human variable regions selected from the top 200 BLAST results were retrieved. These were reduced to four candidates based on a combination of framework homology, maintaining key framework residues and canonical loop structures.

[0147] Thus, in total, DNA sequences encoding four humanized light chains and four humanized heavy chains were selected. All 16 resulting light and heavy chain combinations were used for protein expression in CHO cells. To enable protein expression, each of the variable light chain domains was placed in frame with a human IgK isotype constant domain sequence, while each of the variable heavy chain domains was placed in frame with a human IgG1 isotype constant domain sequence. A chimeric antibody LC0HC0, in which the variable domains of the mouse protein were fused to the same human IgG constant region, was expressed for comparison.

[0148] For protein expression (performed by a third party (Fusion Antibodies, Belfast, UK)), mammalian expression vectors encoding each variant were transfected into CHO cells and batch cultures of each variant were grown for up to 7 days. Expressed antibodies were then purified from cell culture supernatants by affinity chromatography. Concentration and purity were determined for the purified antibody products.

[0149] The resulting sequence was cloned into the mammalian transient expression plasmid pD2610-v13(ATUM). The humanized antibody variants were expressed using a CHO-based transient expression system and the resulting antibody-containing cell culture supernatant was clarified by centrifugation and filtration. The humanized variants were then purified from the cell culture supernatant by affinity chromatography (using a prior art AKTA chromatography device). The purified antibody was dialyzed / buffer exchanged into phosphate buffered saline. The purity of the antibody was assessed by sodium dodecyl sulfate polyacrylamide gel and determined to be greater than 95%.

[0150] Among the 16 resulting humanized antibodies, a combination named LC4HC3 was selected for further experiments based on favorable protein expression yields and antigen-binding properties. Another humanized antibody named LC3HC3 was selected for comparison with LC4HC3 on important parameters such as manufacturability, internalization, and in vivo efficacy.

[0151] SPR analysis for the determination of antibody-ligand affinity Once suitable antibodies are obtained, they can be tested for antigen specificity, for example, by surface plasmon resonance (SPR) or ELISA. When a soluble recombinant protein consisting of the three N-terminal domains of uPARAP (CysR, FN-II and CTLD-1) is immobilized in a BIAcore setup, mAb 9b7 binds to this construct.

[0152] SPR analyses were performed to determine the affinity of the resulting antibodies to uPARAP. These analyses were performed using a Biacore 2000 instrument (Biaffin GmbH, Kassel, Germany) using a CM5 sensor chip with an anti-human Fc capture surface for antibody binding. The analysis temperature was set at 25°C. After the antibodies were bound to the surface, soluble full-length uPARAP was passed over the chip and the association and dissociation rates were derived from the resulting binding curves. For kinetic interaction analyses, a flow rate of 30 μL / min was used and the analysis buffer consisted of 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20.

[0153] Preparation and Evaluation of Antibody Drug Conjugates (ADC) The ADCs used in these experiments were generated using well-established conjugation methods. Briefly, the target antibody was subjected to conjugation to a "vedotin" type payload (MC-VC-PABC-MMAE) by mild reduction of the interchain disulfides, followed by conjugation to excess payload via the maleimide group, resulting in a moderate average drug-to-antibody ratio (DAR) of about 4. The ADCs were then purified using a PD-10 desalting column (GE Healthcare).

[0154] cell line The U937 cell line was obtained from ATCC and maintained in RPMI (10% fetal bovine serum, 1% penicillin / streptomycin) in a 37° C. incubator with a 5% CO 2 atmosphere.

[0155] In vitro cytotoxicity of ADCs – cell viability assay U937 cells were seeded at low density (20% confluence, 2x103 cells per well) in 90μL of medium in flat-bottom 96-well plates and incubated overnight. The next day, MMAE-based ADCs of LC4HC3 and LC0HC0 antibodies, synthesized comparably using the method described above, were prepared as serial dilutions (1:4) in PBS and added to each well in a volume of 10μL with a final maximum ADC concentration (mAb component) of 0.1μg / mL ADC. Cells were incubated for 96 hours before adding 12μL of CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS, Promega) and incubating for a time appropriate for color formation (approximately 60 minutes). Plates were then read at 490nm with background subtraction at 630nm using a plate reader to obtain viability estimates. Cells treated with PBS alone were used as untreated controls to which the ADC-treated wells were normalized for viability.

[0156] result Manufacturability and expression of LC4HC3 and LC3HC3: The humanized antibodies designated LC4HC3 and LC3HC3 were expressed in CHO cells and purified as described above. The same procedure was carried out for both antibodies. The results are summarized in Table 1 below and clearly show that LC4HC3 can be produced in significantly larger quantities with sufficient purity. [Table 1]

[0157] SPR analysis: LC4HC3 and LC0HC0 were analyzed by SPR as described in Materials and Methods above. In particular, the binding kinetics of LC4HC3 were compared to that of LC0HC0 (Table 2). These analyses reveal a low K D exhibits approximately 1.7-fold higher ligand affinity than the parent antibody LC0HC0. [Table 2]

[0158] In vitro potency analysis of ADC: MMAE-containing ADCs containing either the antibody LC4HC3 or the antibody LC0HC0 were prepared as described above. The in vitro cytotoxicity of these ADCs was tested against uPARAP-positive U937 cells using a concentration series of the ADCs (Figure 1). The viability curves obtained from treatment with LC4HC3-vc-MMAE are shifted several-fold to lower concentrations compared to those obtained from treatment with LC0HC0-vc-MMAE, demonstrating that less ADC was required for cell eradication with the LC4HC3-based than with the LC0HC0-based ADCs.

[0159] conclusion The humanized antibody 980.2 LC4HC3 has been developed from the mouse monoclonal antibody mAb 9b7. The properties of this novel antibody can be directly compared to those obtained from the parent variable sequences by comparing with the chimeric antibody 980.2 LC0HC0, in which the entire mouse variable sequences are otherwise kept in the setting of human IgG. This comparison reveals that 1) humanized 980.2 LC4HC3 has a higher ligand affinity than 980.2 LC0HC0, and 2) ADCs based on 980.2 LC4HC3 are more efficient in terms of cytotoxicity than other comparable ADCs based on 980.2 LC0HC0.

[0160] Example 2: Internalization of humanized variants of murine 9b7 antibody, LC3HC3 and LC4HC3 Materials and Methods antibody labeling Iodogen (Thermo Fischer) was dissolved in chloroform at 120 μg / ml and used to coat the bottom of glass tubes by evaporation. In the coated tubes, 200 μg / ml of humanized antibody (either LC3HC3 or LC4HC3) was reacted with 588 ng / ml of I-125 (Perkin Elmer) in 0.1 M Tris buffer at pH 7.6 for 10 min. The reaction was stopped by adding a 9-fold excess of 0.1 M Tris pH 8.1 buffer containing 0.01% Tween-80. Unbound iodine was separated from the collagen on a PD-10 column and the labeled antibody was eluted in 0.1 M Tris / HCl buffer at pH 8.1 containing 0.01% Tween-80. Assuming that all the antibody is eluted in this buffer, this would result in a concentration of 8 μg / ml. The integrity and radioactivity of the labeled collagen was routinely confirmed by SDS-PAGE followed by Coomassie staining and phosphorimaging.

[0161] Cell culture and antibody internalization procedures SAOS-2 osteosarcoma cells (Finsenlab; 98.7% viability, density 1.07x10^6 / ml) were diluted to 1x10^5 / ml and 1 ml was seeded per 24 wells for experiments. Cells were allowed to adhere overnight. At least 30 min before the addition of radiolabeled antibodies, this medium was replaced with internalization medium consisting of DMEM / F12 with 1.5% FBS and 20 mM HEPES. Internalization medium without cells was seeded in separate wells as controls. Radioactivity from these samples is considered to represent the amount of radiolabeled protein that adhered to the plastic and was recovered upon trypsinization. These measurements can be considered as "baseline levels" and can be subtracted from measurements of samples containing cells. 5 μl of LC4HC3 or LC3HC3, estimated to be just under 40 ng based on the above assumptions, was added to each well. After 1 or 4 hours, the medium was removed by aspiration and the cells were washed three times with 500 μl ice-cold PBS. 500 μl of trypsin-EDTA containing 50 μg / ml proteinase K was added to each well for 2 min. Cells were harvested, transferred to Eppendorf tubes and spun at 1000 g for 1 min at 4° C. Supernatants (containing cell-bound antibodies) and pellets were collected separately and analyzed in a gamma counter. 2 μl of labeled antibody stock was analyzed simultaneously to assess labeling efficiency.

[0162] result The results shown in FIG. 2 clearly demonstrate that humanized antibody LC4HC3 is not only internalized significantly faster but also to a greater extent than humanized antibody LC3HC3.

[0163] conclusion The humanized antibody LC4HC3 was internalized to the greatest extent by SAOS-2 osteosarcoma cells in a time-dependent manner. LC3HC3 was also internalized, but to a much lesser extent and not as rapidly as LC4HC3. The two reference antibodies contain the same heavy chain, and the difference in internalization can only be attributed to differences in the amino acid sequences of the light chains.

[0164] Example 3: In vivo efficacy of ADCs based on humanized murine 9b7 antibodies LC3HC3 and LC4HC3 Materials and Methods Cell culture and preparation U937 cells (above) were passaged according to standard procedures until sufficient cells were obtained for this experiment. Cells were spun down at 150g for 5 min and washed three times in cold PBS (Gibco). Cell concentration was adjusted to 3.6 x 106 cells / ml. This translates to approximately 3 million viable cells per 100 μl of intended inoculation volume.

[0165] Xenograft tumor inoculation Recipient CB17 mice were anesthetized with Zoletil (AEM), administered Viscotears eye drops, and ear-tagged. The right flank was shaved and disinfected with 70% ethanol. Using a 25G needle, 100 μl of resuspended U937 cells were injected into the subcutaneous space (no incision or suture required). Mice were allowed to recover from anesthesia in their cages. Recovery was monitored until the mice were mobile. Mice were monitored again the next day and tumor size was closely monitored until treatment initiation.

[0166] Treatment was initiated as soon as tumors reached an appropriate size (approximately 80-150 mm3).

[0167] ADC Treatment and Monitoring Vedotin-type (MMAE) ADCs containing LC3HC3 or LC4HC3 humanized antibodies were prepared as described above. Mice were divided into groups with 3-5 animals (N=3-5) per group, and groups differed by the ADC or dose of said ADC used. One cohort of mice was tested with LC3HC3 ADCs receiving concentrations of 2 mg / kg, 4 mg / kg, or 6 mg / kg, and another cohort was tested with LC4HC3 ADCs receiving the same concentration range.

[0168] For each group, mice were administered a controlled amount of ADC intravenously (tail vein) once a week for a total of two injections (qd7x2), and tumor growth was closely monitored after treatment. Illustratively, Figures 3a and 3b show tumor growth in groups treated with LC4HC3 and LC3HC3 ADC at a dose of 4 mg / kg, respectively.

[0169] Monitoring consisted of inspecting the overall health condition as well as measuring the width and length dimensions of the tumors using digital calipers. All observations and measurements were recorded manually and transferred to a digital data sheet after examination. Animals were euthanized if tumor size exceeded 12 mm in one dimension, if tumor volume (calculated as (length × width2) / 2) exceeded 1000 mm3, or if severe effects on general health were observed. Animals were euthanized by cervical dislocation.

[0170] result U937 tumor volumes following treatment with ADCs based on LC4HC3 and LC3HC3 are shown in Figures 3a and 3b, respectively: Figure 3a shows the tumor volumes for a group of four mice (N=4), each administered LC4HC3 ADC at 4 mg / kg once per week for a total of two injections (qd7x2), and Figure 3b shows the tumor volumes for a different group of four mice (N=4), each administered LC3HC3 ADC at 4 mg / kg once per week for a total of two injections (qd7x2).

[0171] conclusion As can be seen by comparing Figures 3a and 3b, LC4HC3 ADC-based treatment completely cured all mice without tumor regrowth during the post-treatment period, particularly at all doses tested. In contrast, the same LC3HC3-based treatment failed to kill all tumor cells, e.g., aggressive tumor growth was observed during the post-treatment monitoring period.

[0172] The data show that the humanized antibody LC4HC3 and ADCs containing said humanized antibody are potent antitumor agents with improved in vivo efficacy compared to another humanized antibody, LC3HC3. The two reference antibodies contain the same heavy chain, and the difference in efficacy can only be attributed to differences in the amino acid sequence of the light chain.

[0173] [Table 3] TIFF2024528496000004.tif230162TIFF2024528496000005.tif231162TIFF2024528496000006.tif229162TIFF2024528496000007.tif221162

[0174] Clause 1 Item 1. An antibody that binds to uPARAP, a. an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:3, and / or b. The antibody comprises an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:6.

[0175] Item 2. The antibody, a. an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO:1, and / or b. The antibody of item 1, comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO:4.

[0176] Item 3. The antibody, a. an immunoglobulin light chain consisting of the amino acid sequence of SEQ ID NO:1, and b. The antibody of any one of the preceding items, comprising an immunoglobulin heavy chain consisting of the amino acid sequence of SEQ ID NO:4.

[0177] Item 4. An antibody-drug conjugate (ADC), a. The antibody of any one of the preceding items, b. an active agent, and c. The antibody-drug conjugate optionally comprising a linker connecting a) to b).

[0178] Item 5. The antibody-drug conjugate of item 4, wherein the active agent is selected from a therapeutic agent, a radioisotope, and a detectable label.

[0179] Item 6. The antibody-drug conjugate according to any one of Items 4 to 5, wherein the active agent is a cytotoxic drug.

[0180] Item 7. The antibody-drug conjugate according to any one of Items 4 to 6, wherein the active agent is a therapeutic agent, for example, a therapeutic agent selected from the group consisting of microtubule inhibitors / mitosis inhibitors, DNA crosslinking agents, DNA alkylating agents, DNA strand breakers, anthracyclines, metabolic antagonists, histone deacetylase inhibitors, kinase inhibitors, metabolic inhibitors, peptide antibiotics, immune checkpoint inhibitors, platinum-based antineoplastic agents, topoisomerase inhibitors, DNA or RNA polymerase inhibitors, nucleotide-based agents, and cytotoxic antibiotics.

[0181] Item 8. The antibody-drug conjugate according to any one of items 4 to 7, wherein the active agent is a mitotic inhibitor selected from the group consisting of a derivative of an auristatin or dolastatin (e.g., monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), etc.), a taxane (e.g., paclitaxel or docetaxel), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine, etc.), a maytansinoid, colchicine, and podophyllotoxin, or a derivative of any of these.

[0182] Item 9. The antibody-drug conjugate of any one of items 4 to 8, wherein the active agent is monomethylauristatin E (MMAE).

[0183] Item 10. The antibody-drug conjugate according to any one of items 4 to 7, wherein the active agent is a DNA crosslinking agent, for example, a DNA crosslinking agent selected from cisplatin or a derivative of cisplatin (e.g., carboplatin or oxaliplatin), mitomycin C (MMC), pyrrolobenzodiazepines, and dimeric pyrrolobenzodiazepine derivatives (e.g., SGD-1882).

[0184] Item 11. The antibody-drug conjugate according to any one of items 4 to 7, wherein the active agent is a DNA alkylating agent, for example, a DNA alkylating agent selected from tris(2-chloroethyl)amine, pyridinobenzodiazepine or pyridinobenzodiazepine derivatives, indolinobenzodiazepine dimers, and nitrogen mustards such as duocarmycin SA.

[0185] Item 12. The antibody-drug conjugate according to any one of Items 4 to 7, wherein the active agent is a DNA strand cleaving agent, for example, a DNA strand cleaving agent selected from calicheamicin and hamirtron.

[0186] Item 13. The antibody-drug conjugate according to any one of items 4 to 7, wherein the active agent is an anthracycline, e.g., an anthracycline selected from daunorubicin, doxorubicin, epirubicin, idarubicin, and PNU-159682.

[0187] Item 14. The antibody-drug conjugate according to any one of items 4 to 7, wherein the active agent is an antimetabolite selected from an antimetabolite, for example, an antifolate (e.g., methotrexate), a purine antimetabolite (e.g., 6-mercaptopurine or 6-thioguanine or fludarabine phosphate or pentostatin or cladribine), and a pyrimidine antimetabolite (e.g., 5-fluorouracil or 5-fluorodeoxyuridine or cytarabine or gemcitabine).

[0188] Item 15. The antibody-drug conjugate according to any one of Items 4 to 7, wherein the active agent is a histone deacetylase inhibitor selected from the group consisting of trichostatin A, vorinostat, belinostat, panobinostat, gibinostat, resminostat, abexinostat, xinostat, rosilinostat, pracinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, entinostat, tacedinaline, 4SC202, mocetinostat, romidepsin, nicotinamide, sirtinol, cambinol, and EX-527.

[0189] Item 16. The antibody-drug conjugate according to any one of Items 4 to 7, wherein the active agent is a kinase inhibitor, for example, a kinase inhibitor selected from genistein, lavendustin C, PP1-AG1872, PP2-AG1879, SU6656, CGP77675, PD166285, imatinib, erlotinib, gefitinib, lavendustin A, cetuximab, UCS15A, herbimycin A, and radicicol.

[0190] Item 17. The antibody-drug conjugate according to any one of items 4 to 7, wherein the active agent is a metabolic inhibitor such as a NAMPT inhibitor selected from APO866, GMX-1777, GMX-1778, ATG-019 and OT-82.

[0191] Item 18. The antibody-drug conjugate according to any one of items 4 to 7, wherein the active agent is an immune checkpoint inhibitor, for example a PD-1 inhibitor selected from pembrolizumab, nivolumab, cemiplimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, AMP-224 and AMP-514; or a PD-L1 inhibitor selected from atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170 and BMS-986189.

[0192] Item 19. The antibody-drug conjugate according to any one of Items 4 to 7, wherein the active agent is a platinum-based anticancer agent, for example, a platinum-based anticancer agent selected from lipoplatin, cisplatin, carboplatin, oxaliplatin, nedaplatin, picoplatin, phenanthriplatin, satraplatin, and triplatin tetranitrate.

[0193] Item 20. The antibody-drug conjugate according to any one of items 4 to 7, wherein the active agent is a topoisomerase inhibitor, e.g., a camptothecin or a derivative thereof, e.g., a topoisomerase inhibitor selected from topotecan, belotecan, lurtotecan, irinotecan, SN-38, exatecan, and Dxd.

[0194] Item 21. The antibody-drug conjugate according to any one of Items 4 to 7, wherein the active agent is a DNA polymerase or RNA polymerase inhibitor, for example a polymerase inhibitor selected from amanitin or α-amanitin or a derivative thereof, actinomycin D, and aphidicolin.

[0195] Item 22. The antibody-drug conjugate according to any one of Items 4 to 21, wherein the active agent comprises a radioisotope selected from 60Co, 89Sr, 90Y, 99mTc, 131I, 137Cs, 153Sm, and 223Rd.

[0196] Item 23. The antibody-drug conjugate according to any one of items 4 to 22, wherein the drug-antibody ratio (DAR) is between 1 and 10, such as between 2 and 8, for example between 2 and 6, such as 2 or 4.

[0197] Item 24. The antibody-drug conjugate according to any one of Items 4 to 23, wherein the antibody-drug conjugate comprises a linker selected from a cleavable linker and a non-cleavable linker.

[0198] Item 25. The antibody-drug conjugate according to any one of Items 4 to 24, wherein the linker is a peptide linker.

[0199] Item 26. The antibody-drug conjugate according to any one of items 4 to 25, wherein the linker comprises or consists of a dipeptide, such as valine-citrulline (VC) or valine-alanine (VA).

[0200] Item 27. The antibody-drug conjugate according to any one of items 4 to 26, further comprising a spacer, for example a spacer comprising p-aminobenzoic acid (PAB), p-aminobenzylcarbamate (PABC), p-aminobenzoyloxycarbonyl, or polyethylene glycol (PEG).

[0201] Item 28. The antibody-drug conjugate according to any one of items 4 to 27, further comprising a linking group, such as maleimide and caproic acid (MC), N-hydroxysuccinimide, reactive linking groups directed to modified or unmodified protein-bound carbohydrates, peptide sequences required for enzymatic reactions, azides or alkynes, or those derived therefrom by reaction with an antibody, or chemically or enzymatically generated derivatives thereof, or a linking group consisting of these.

[0202] Item 29. The antibody-drug conjugate a.An antibody as defined in item 3; b. VC linker, c.MC binding group, d. PAB or PABC spacer, and e. The antibody-drug conjugate according to any one of items 4 to 28, comprising or consisting of MMAE as an active agent.

[0203] Item 30. The antibody-drug conjugate according to any one of items 4 to 9 and 23 to 29, wherein the antibody-drug conjugate consists of the antibody defined in item 3 and MC-VC-PAB-MMAE.

[0204] Item 31. The antibody-drug conjugate according to any one of items 4 to 9 and 23 to 29, wherein the antibody-drug conjugate consists of the antibody defined in item 3 and MC-VC-PABC-MMAE.

[0205] Item 32. A polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 and / or SEQ ID NO: 5.

[0206] Item 33. An isolated polynucleotide encoding any one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5 and / or 6.

[0207] Item 34. The isolated polynucleotide according to Item 33, wherein the polynucleotide comprises SEQ ID NO:11 and / or SEQ ID NO:12.

[0208] Item 35. A vector comprising a polynucleotide defined in any one of items 33 to 34.

[0209] Item 36. The vector according to Item 35, wherein the vector is a mammalian expression vector.

[0210] Item 37. The vector according to any one of Items 35 to 36, wherein the vector is a plasmid vector, for example, a plasmid vector selected from pD2610-v13(ATUM), pSV and pCMV series plasmid vectors.

[0211] Item 38. The vector according to any one of Items 35 to 37, wherein the vector is a viral vector selected from the group consisting of an adenovirus vector, a lentivirus vector, an adeno-associated virus vector, a herpes virus vector, a vaccinia virus vector, a poxvirus vector, a baculovirus vector, and an oncolytic virus vector.

[0212] Item 39. A host cell comprising the polynucleotide according to any one of Items 32 to 33 and / or the vector according to any one of Items 35 to 38.

[0213] Item 40. The host cell according to item 39, wherein the host cell is selected from the group consisting of CHO (Chinese Hamster Ovary) cells, COS (derived from CV-1 (monkey) and carrying SV40 genetic material), HEK (human embryonic kidney) cells, and HeLa (Henrietta Lacks) cells.

[0214] Item 41. The antibody according to any one of items 1 to 3, or the antibody-drug conjugate according to any one of items 4 to 31, for use as a drug.

[0215] Item 42. A pharmaceutical composition comprising the antibody according to any one of items 1 to 3, or the antibody-drug conjugate according to any one of items 4 to 31, and a pharma- ceutically acceptable buffer, diluent, carrier, adjuvant or excipient.

[0216] Item 43. A method for treating a disease characterized by cells expressing uPARAP, the method comprising administering to a subject the antibody described in any one of items 1 to 3, the antibody-drug conjugate described in any one of items 4 to 31, or the pharmaceutical composition described in item 42.

[0217] Item 44. The method of item 43, wherein the disease characterized by cells expressing uPARAP is selected from cancer, bone degradative diseases such as osteoporosis, fibrosis, and macrophage-related diseases or disorders such as atherosclerosis, arthritis, or chronic inflammation.

[0218] Item 45. The method of item 44, wherein the arthritis is selected from osteoarthritis, inflammatory arthritis, rheumatoid arthritis, psoriatic arthritis, lupus, Lyme disease-induced arthritis, e.g., Lyme arthritis, gout or pseudogout, and ankylosing spondylitis.

[0219] Item 46. The method according to any one of Items 43 to 44, wherein the disease is cancer.

[0220] Item 47. The method of item 46, wherein the cancer is selected from sarcoma, glioblastoma, mesothelioma, colon cancer, prostate cancer, bone metastasis from prostate cancer, breast cancer, head and neck cancer, and leukemia.

[0221] Item 48. The method according to any one of Items 46 to 47, wherein the cancer is a solid tumor.

[0222] Item 49. The method of any one of items 46 to 47, wherein the cancer is leukemia, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML).

[0223] Item 50. The method according to any one of items 46 to 48, wherein the cancer is glioblastoma.

[0224] Item 51. The method according to any one of items 46 to 48, wherein the cancer is a sarcoma, such as osteosarcoma or soft tissue sarcoma (STS).

[0225] Item 52. The method of item 51, wherein the soft tissue sarcoma (STS) is selected from epithelioid sarcoma, clear cell sarcoma, alveolar soft part sarcoma, extraskeletal myxoid chondrosarcoma, epithelioid hemangioendothelioma, inflammatory myofibroblastic tumor, undifferentiated embryonal sarcoma, alveolar soft part sarcoma (ASPS), angiosarcoma, chondrosarcoma, dermatofibrosarcoma protuberans (DFSP), desmoid sarcoma, Ewing's sarcoma, fibrosarcoma, myxofibrosarcoma, gastrointestinal stromal tumor (GIST), non-uterine leiomyosarcoma, uterine leiomyosarcoma, liposarcoma, malignant fibrous histiocytoma (MFH), malignant peripheral nerve sheath tumor (MPNST), rhabdomyosarcoma, synovial sarcoma, and / or leiomyosarcoma (LMS).

[0226] Item 53. The method according to any one of items 46 to 52, wherein the cancer is a metastatic cancer.

[0227] Item 54. The method according to any one of items 43 to 53, wherein the antibody according to any one of items 1 to 3, the antibody-drug conjugate according to any one of items 4 to 31, or the pharmaceutical composition according to item 42 is administered parenterally, for example intravenously, intracerebroventricularly, intraarticularly, intraarterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, or by injection techniques.

[0228] Item 55. The method according to any one of items 43 to 54, wherein the antibody according to any one of items 1 to 3, the antibody-drug conjugate according to any one of items 4 to 31, or the pharmaceutical composition according to item 42 is administered intravenously.

[0229] Item 56. The method according to any one of items 43 to 55, wherein the antibody according to any one of items 1 to 3, the antibody-drug conjugate according to any one of items 4 to 31, or the pharmaceutical composition according to item 42 is administered in combination with one or more further agents, e.g., one or more further therapeutic agents.

[0230] Item 57. The method according to any one of Items 43 to 56, wherein the cells expressing uPARAP exhibit uPARAP overexpression.

[0231] Item 58. The method according to any one of Items 43 to 57, wherein the cells expressing uPARAP are tumor cells and / or tumor-associated cells.

[0232] Item 59. The method according to any one of items 43 to 58, wherein the antibody according to any one of items 1 to 3, the antibody-drug conjugate according to any one of items 4 to 31, or the pharmaceutical composition according to item 42 induces cell death and / or inhibits the growth and / or proliferation of the uPARAP-expressing cells.

[0233] Item 60. The method according to any one of items 43 to 59, wherein the antibody according to any one of items 1 to 3, the antibody-drug conjugate according to any one of items 4 to 31, or the pharmaceutical composition according to item 42 induces release of a cytotoxin released from the uPARAP-expressing cells, resulting in cell death and / or inhibiting the growth and / or proliferation of adjacent cancer cells.

[0234] Item 61. The method according to any one of items 43 to 60, wherein the treatment is remission or cure.

[0235] Item 62. A method for suppressing tumor progression in a subject, comprising administering to the subject the antibody according to any one of items 1 to 3, the antibody-drug conjugate according to any one of items 4 to 31, or the pharmaceutical composition according to item 42.

[0236] Item 63. A method for inhibiting, reducing or eliminating the metastatic potential of a tumor expressing uPARAP in a subject, the method comprising administering to the subject the antibody according to any one of items 1 to 3, the antibody-drug conjugate according to any one of items 4 to 31, or the pharmaceutical composition according to item 42.

[0237] Item 64. A kit comprising the antibody according to any one of items 1 to 3, the antibody-drug conjugate according to any one of items 4 to 31, or the pharmaceutical composition according to item 42, optionally further comprising a means for administering the antibody or the antibody-drug conjugate to a subject and / or instructions for use.

[0238] Item 65. The antibody according to any one of items 1 to 3, the antibody-drug conjugate according to any one of items 4 to 31, or the pharmaceutical composition according to item 42, for use in the manufacture of a drug for the treatment of a disease characterized by cells expressing uPARAP, such as cancer.

[0239] Clause 2 Item 1. An antibody that binds to uPARAP, a. an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:3, and / or b. The antibody comprises an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:6.

[0240] Item 2. An antibody-drug conjugate (ADC), a. The antibody according to item 1, b. an active agent, and c. An antibody-drug conjugate, optionally comprising a linker connecting a) to b).

[0241] Item 3. The antibody-drug conjugate of item 2, wherein the active agent is a therapeutic agent, e.g., a therapeutic agent selected from the group consisting of microtubule-anti / mitosis inhibitors, DNA crosslinking agents, DNA alkylating agents, DNA strand breakers, anthracyclines, antimetabolites, histone deacetylase inhibitors, kinase inhibitors, metabolic inhibitors, peptide antibiotics, immune checkpoint inhibitors, platinum-based antineoplastic agents, topoisomerase inhibitors, DNA or RNA polymerase inhibitors, nucleotide-based agents, and cytotoxic antibiotics.

[0242] Item 4. The antibody-drug conjugate according to any one of Items 2 to 3, wherein the antibody-drug conjugate comprises a linker selected from a cleavable linker and a non-cleavable linker.

[0243] Item 5. The antibody-drug conjugate according to any one of items 2 to 4, further comprising a spacer, for example, a spacer comprising p-aminobenzoic acid (PAB), p-aminobenzylcarbamate (PABC), p-aminobenzoyloxycarbonyl, or polyethylene glycol (PEG).

[0244] Item 6. The antibody-drug conjugate, a.An antibody as defined in item 1; b. VC linker, c.MC binding group, d. PAB or PABC spacer, and e. The antibody-drug conjugate according to any one of items 2 to 5, comprising or consisting of MMAE as an active agent.

[0245] Item 7. A polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:2 and / or SEQ ID NO:5.

[0246] Item 8. An isolated polynucleotide encoding any one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5 and / or 6.

[0247] Item 9. The antibody according to item 1, or the antibody-drug conjugate according to any one of items 2 to 6, for use as a drug.

[0248] Item 10. A pharmaceutical composition comprising the antibody according to Item 1, or the antibody-drug conjugate according to any one of Items 2 to 6, and a pharma- ceutically acceptable buffer, diluent, carrier, adjuvant or excipient.

[0249] Item 11. The antibody according to item 1, the antibody-drug conjugate according to any one of items 2 to 6, or the pharmaceutical composition according to item 10, for use in a method for treating a disease characterized by cells expressing uPARAP.

[0250] Item 12. The antibody according to item 1, the antibody-drug conjugate according to any one of items 2 to 6, or the pharmaceutical composition according to item 10 for use according to item 11, wherein the disease characterized by cells expressing uPARAP is selected from cancer, bone degradative diseases such as osteoporosis, fibrosis, and macrophage-related diseases or disorders such as atherosclerosis, arthritis, or chronic inflammation.

[0251] Item 13. The antibody according to item 1, the antibody-drug conjugate according to any one of items 2 to 6, or the pharmaceutical composition according to item 10, for use in a method for inhibiting tumor growth in a subject.

[0252] Item 14. The antibody according to item 1, the antibody-drug conjugate according to any one of items 2 to 6, or the pharmaceutical composition according to item 10, for use in a method for inhibiting, reducing or eliminating the metastatic ability of a tumor expressing uPARAP.

[0253] Item 15. A kit comprising the antibody according to Item 1, the antibody-drug conjugate according to any one of Items 2 to 6, or the pharmaceutical composition according to Item 10, optionally further comprising a means for administering the antibody or the antibody-drug conjugate to a subject and / or instructions for use.

Claims

1. An antibody that binds to uPARAP, a. an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 3, and b. an antibody comprising an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:

6.

2. The antibody is a. an immunoglobulin light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 1, and b. the antibody according to claim 1, comprising an immunoglobulin heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:

4.

3. An antibody-drug conjugate (ADC), a. the antibody defined in claim 1, b. an active agent, and c. optionally, an antibody-drug conjugate comprising a linker that binds a) to b).

4. The active agent is selected from therapeutic agents, radioisotopes and detectable labels, the antibody-drug conjugate according to claim 3.

5. The active agent is a therapeutic agent selected from the group consisting of microtubule inhibitors / mitotic inhibitors, DNA cross-linking agents, DNA alkylating agents, DNA strand breakers, anthracyclines, antimetabolites, histone deacetylase inhibitors, kinase inhibitors, metabolic inhibitors, peptide antibiotics, immune checkpoint inhibitors, platinum-based antineoplastic agents, topoisomerase inhibitors, DNA or RNA polymerase inhibitors, nucleotide-based agents, and cytotoxic antibiotics, the antibody-drug conjugate according to claim 3.

6. The active agent is - a mitotic inhibitor, such as a derivative of auristatin or dolastatin (e.g., monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF)), taxane (e.g., paclitaxel or docetaxel), vinca alkaloid (e.g., vinblastine, vincristine, vindesine or vinorelbine), maytansinoid, colchicine or podophyllotoxin, - a DNA cross-linking agent, such as cisplatin or a derivative of cisplatin (e.g., carboplatin or oxaliplatin), mitomycin C (MMC), pyrrolobenzodiazepine, and a dimeric pyrrolobenzodiazepine derivative (e.g., SGD-1882), a DNA cross-linking agent selected from the group consisting of - DNA alkylating agents, such as DNA alkylating agents selected from nitrogen mustard (e.g., tris(2-chloroethyl)amine), pyridobenzo-diazepine or pyridobenzo-diazepine derivatives, indolinobenzo-diazepine dimers, and duocarmycin SA, - DNA strand breakers, such as DNA strand breakers selected from calicheamicin and hamiltomycin, - Anthracyclines, such as anthracyclines selected from daunorubicin, doxorubicin, epirubicin, idarubicin, and PNU-159682, - Antimetabolites, such as antimetabolites selected from folic acid antagonists (e.g., methotrexate), purine antimetabolites (e.g., 6-mercaptopurine or 6-thioguanine or fludarabine phosphate or pentostatin or cladribine), and pyrimidine antimetabolites (e.g., 5-fluorouracil or 5-fluorodeoxyuridine or cytarabine or gemcitabine), - Histone deacetylase inhibitors, such as histone deacetylase inhibitors selected from trichostatin A, vorinostat, belinostat, panobinostat, givinostat, resminostat, abexinostat, xeno stat, roslinostat, plasinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, entinostat, tacedinaline, 4SC202, mocetinostat, romidepsin, nicotinamide, sirtinol, cambinol, and EX-527, - Kinase inhibitors, such as kinase inhibitors selected from genistein, lavendustin C, PP1-AG1872, PP2-AG1879, SU6656, CGP77675, PD166285, imatinib, erlotinib, gefitinib, lavendustin A, cetuximab, UCS15A, herbimycin A, and radicicol, - Metabolic inhibitors, such as Nampt inhibitors selected from APO866, GMX-1777, GMX-1778, ATG-019, and OT-82, - An immune checkpoint inhibitor, for example, a PD-1 inhibitor selected from pembrolizumab, nivolumab, semipramab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, AMP-224, and AMP-514; or a PD-L1 inhibitor selected from atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189, - A platinum-based anti-cancer agent, for example, a platinum-based anti-cancer agent selected from lipoplatin, cisplatin, carboplatin, oxaliplatin, nedaplatin, picoplatin, phenanthriplatin, satraplatin, and triplatin tetranitrate, - A topoisomerase inhibitor, for example, a topoisomerase inhibitor selected from camptothecin or its derivatives, for example, topotecan, belotecan, lurtecan, irinotecan, SN-38, exatecan, and Dxd, or - A DNA polymerase or RNA polymerase inhibitor, for example, a polymerase inhibitor selected from amanitin or α-amanitin or its derivatives, actinomycin D, and aphidicolin The antibody-drug conjugate according to claim 3, which is as described above.

7. The antibody-drug conjugate according to claim 3, wherein the active agent contains a radioisotope selected from 60Co, 89Sr, 90Y, 99mTc, 131I, 137Cs, 153Sm, and 223Rd.

8. The antibody-drug conjugate according to claim 3, wherein the drug-antibody ratio (DAR) is between 1 and 10, such as between 2 and 8, for example, between 2 and 6, such as 2 or 4.

9. The antibody-drug conjugate according to claim 3, which contains a linker selected from a cleavable linker and a non-cleavable linker, and optionally, the linker is a peptide linker. For example, the linker contains or consists of a dipeptide, for example, valine-citrulline (VC) or valine-alanine (VA).

10. The antibody-drug conjugate according to claim 3, which further contains a spacer, for example, a spacer containing p-aminobenzoic acid (PAB), p-aminobenzyl carbamate (PABC), p-aminobenzoyl oxycarbonyl, or polyethylene glycol (PEG).

11. The antibody-drug conjugate of claim 3 further comprises a linking group, such as maleimide and caproic acid (MC), N-hydroxysuccinimide, a reactive linking group directed to a modified or unmodified protein-binding carbohydrate, a peptide sequence required for an enzymatic reaction, an azide or alkyne, or a derivative derived therefrom by reaction with an antibody, or a chemically or enzymatically produced derivative thereof, or a linking group comprising or consisting of these.

12. A polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO:

5.

13. An isolated polynucleotide encoding any one of the amino acid sequences of SEQ ID NO: 1, 2, or 3, wherein the polynucleotide further encodes any one of the amino acid sequences of SEQ ID NO: 4, 5, or 6, for example, the polynucleotide comprises SEQ ID NO: 11 and SEQ ID NO: 12, an isolated polynucleotide.

14. A vector comprising the polynucleotide defined in claim 13.

15. A host cell comprising the polynucleotide defined in claim 13 and / or the vector defined in claim 14.

16. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 2, or the antibody-drug conjugate according to any one of claims 3 to 11, and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient.

17. A pharmaceutical composition for use in the treatment of a disease characterized by cells expressing uPARAP, comprising the antibody according to any one of claims 1 to 2, or the antibody-drug conjugate according to any one of claims 3 to 11, for example, the disease characterized by cells expressing uPARAP is selected from cancer, bone resorption diseases, such as osteoporosis, fibrosis, and macrophage-related diseases or disorders, such as atherosclerosis, arthritis or chronic inflammation.

18. The disease is cancer, for example, the cancer is selected from sarcoma, glioblastoma, mesothelioma, colon cancer, prostate cancer, bone metastasis from prostate cancer, breast cancer, head and neck cancer, and leukemia, a pharmaceutical composition for use according to claim 17.

19. The cancer is a solid tumor, a pharmaceutical composition for use according to claim 18.

20. The pharmaceutical composition for use according to claim 18, wherein the cancer is leukemia, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML).

21. The pharmaceutical composition for use according to claim 18, wherein the cancer is glioblastoma.

22. The pharmaceutical composition for use according to claim 18, wherein the cancer is a sarcoma, such as osteosarcoma or soft tissue sarcoma (STS).

23. The pharmaceutical composition for use according to claim 22, wherein the soft tissue sarcoma (STS) is selected from epitheloid sarcoma, clear cell sarcoma, alveolar soft part sarcoma, extraskeletal myxoid chondrosarcoma, epitheloid hemangioendothelioma, inflammatory myofibroblastic tumor, embryonal undifferentiated sarcoma, alveolar soft part sarcoma (ASPS), angiosarcoma, chondrosarcoma, dermatofibrosarcoma protuberans (DFSP), desmoid sarcoma, Ewing sarcoma, fibrosarcoma, myxofibrosarcoma, gastrointestinal stromal tumor (GIST), non-uterine leiomyosarcoma, uterine leiomyosarcoma, liposarcoma, malignant fibrous histiocytoma (MFH), malignant peripheral nerve sheath tumor (MPNST), rhabdomyosarcoma, synovial sarcoma, and / or leiomyosarcoma (LMS).

24. The pharmaceutical composition for use according to claim 18, wherein the cancer is metastatic cancer.

25. The pharmaceutical composition for use according to claim 17, wherein the administration is parenteral, such as intravenous, intraventricular, intra-articular, intra-arterial, intraperitoneal, intrathecal, intraventricular, intrasternal, intracranial, intramuscular or subcutaneous, or by infusion techniques.

26. The pharmaceutical composition for use according to claim 17, wherein the antibody or the antibody-drug conjugate is administered in combination with one or more additional agents, such as one or more additional therapeutic agents.

27. A kit comprising the antibody according to any one of claims 1 to 2, or the antibody-drug conjugate according to any one of claims 3 to 11, optionally further comprising means for administering the antibody or the antibody-drug conjugate to a subject and / or instructions for use.

Images