Megalin-targeting drug conjugates for treatment of cancers

Abstract

The present invention relates to LRP2 / megalin-targeting conjugates comprising a megalin-targeting moiety and a conjugated, preferably an antibody-drug conjugate (ADC) for use in the treatment and / or alleviation of cancers expressing LRP2 / megalin, preferably a pleural mesothelial cancer in a subject.

Description

[0001] MEGALIN-TARGETING DRUG CONJUGATES FOR TREATMENT OF CANCERS

[0002] Technical field of the invention

[0003] The present invention relates to megalin-targeting drug conjugates for treatment of cancers, in particular mesothelial cancers. In particular, the present invention relates to megalin targeting antibody-drug conjugates for use in the treatment of mesothelial cancers.

[0004] Background of the invention

[0005] Mesothelial cancer is a highly aggressive cancer with poor prognosis (5-year survival rate of 10%). Current treatment methods include surgery and chemotherapeutic regimens, but the efficacy is unsatisfactory and involves unfavorable side effects.

[0006] Low density lipoprotein receptor-related protein 2 (LR.P2), also known as megalin, and gp330, is a large endocytic membrane receptor involved in ligand uptake in absorptive epithelial cells. In healthy adults, megalin displays a highly restricted expression pattern, with kidney proximal tubule epithelial cells being the major site of expression.

[0007] Andersen et al. (Melanoma tumors frequently acquire LRP2 / megalin expression, which modulates melanoma cell proliferation and survival rates. Pigment Cell Melanoma Res. 2015 May;28(3):267-80) discloses that megalin, is frequently expressed in malignant melanoma samples.

[0008] Rasmussen et al. (Epigenetic silencing of LRP2 is associated with dedifferentiation and poor survival in multiple solid tumor types. Cancers 2023, 15, 1830) discloses (based on RNA data) that low LRP2 / megalin expression is associated with tumor cell dedifferentiation and poor outcome in clear cell renal cell carcinoma, papillary renal cell carcinoma, mesothelioma, papillary thyroid carcinoma, and invasive breast carcinoma. Rasmussen et al. speculates that LRP2 / megalin targeted drug delivery might be effective in some cancer types. Wang et al. (Design and in vivo characterization of kidney-targeting multimodal micelles for renal drug delivery. Nano Research 2018, 11(10): 5584-5595) discloses design and in vivo characterization of kidney-targeting multimodal micelles for renal drug delivery. Wang et al. is silent in respect of mesothelioma.

[0009] Ordikhani et al. (Selective trafficking of light chain-conjugated nanoparticles to the kidney and renal cell carcinoma. Nano Today. 2020 December; 35) discloses synthesized LC-NPs, nanocarriers with the capability to target selectively both the PTECs of the kidney and RCC through the binding of light chains to megalin. Ordikhani et al. is silent in respect of mesothelioma.

[0010] WO 2005 / 070965 A2 discloses pharmaceutical compositions containing antagonists to LRP4, LRP8 or megalin (LRP2) for treatment of diseases. WO 2005 / 070965 A2 is completely devoid of any data supporting the stated effects.

[0011] Hence, an improved method to treat mesothelioma would be advantageous, and in particular a more efficient and / or reliable method to treat mesothelioma would be advantageous.

[0012] Summary of the invention

[0013] The present invention relates to the realization that megalin protein is present in the cell membrane of a subset of cancers, in particular pleural mesothelial cancer cells. It has further been discovered that antibodies targeting megalin can be endocytosed on such megalin-presenting cancer cells and thereby be used for cancer cell specific drug delivery.

[0014] In addition, it has been identified that megalin is primarily localized to the apical / luminal membrane of tumor cells facing the pleural space, the antibodydrug conjugates according to the invention can be administered directly to the pleural cavity, thereby avoiding side effects of e.g. systemic drug delivery.

[0015] Thus, an object of the present invention relates to an improved method to treat or alleviate pleural mesothelial cancer. As outlined above, Rasmussen et al. speculates that LRP2 / megalin targeted drug delivery might be effective in some cancer types. However, the skilled person reviewing Rasmussen et al. will realize the following:

[0016] Data relating to mesotheliom is based on RNA sampling from a heterogeneous population of cells. Thus, Rasmussen does not provide information on protein levels or location of any such protein in the sample containing, among other cells, the cancer cells. Also, it is unclear from which cells the RNA is derived since the tissue sample analyzed contains heterogenous subset of cells including normal cells.

[0017] Rasmussen states that low LRP2 / megalin expression is associated with tumor cell dedifferentiation and poor outcome in clear cell renal cell carcinoma, papillary renal cell carcinoma, mesothelioma, papillary thyroid carcinoma, and invasive breast carcinoma. Since low LRP2 / megalin expression is associated with poor outcome, LRP2 may not be a preferred target.

[0018] Rasmussen shows no data in relation to antibody-drug conjugates, thus no data substantiates that drugs can indeed be internalized via LRP2 targeting or for that matter kill cancer cells. Thus, statements in that direction are purely speculative.

[0019] Rasmussen is silent in respect of direct delivery of drugs to the pleural cavity thereby targeting LRP2 / megalin presenting mesothelioma cancer cells, which the data in here shows is a surprisingly feasible route considering that LRP2 / megalin is localized to the apical membrane of tumor cells facing the pleural space and thus directly reachable via the pleural cavity. Importantly, for this group of patients, access to their pleural space has very often already been established, since these patients often need to have intermittent drainage of excess fluid from the pleural space to reduce pressure on lungs and to facilitate breathing.

[0020] Rasmussen et al. is incorporated in here in its entirety.

[0021] In the example section presented below, the following data is presented supporting the invention.

[0022] Example 1 shows generation of megalin-targeting antibodies. Example 2 shows specificity of the megalin-targeting antibodies.

[0023] Example 3 shows generation of megalin-targeting antibody-drug conjugates (ADCs).

[0024] Example 4 shows that the ADC's bind megalin both before and after conjugation.

[0025] Example 5 demonstrates expression of megalin protein in mesothelioma tumors. Further, polarization of megalin to the apical membrane of mesothelioma cells facing the pleural space is shown, suggesting that efficient megalin targeting can be achieved with administration to the pleural space.

[0026] Example 6 shows specific binding and uptake of anti-megalin antibodies by megalin-expressing cancer cell lines.

[0027] Example 7 shows specific killing of megalin-expressing cancer cells by antibody drug-conjugates targeting megalin.

[0028] In particular, it is an object of the present invention to provide an effective treatment protocol for pleural mesothelioma that solves the above-mentioned problems of the prior art with low efficacy, delivery and unfavorable side effects.

[0029] Thus, one aspect of the invention relates to a megalin-targeting conjugate comprising a megalin-targeting moiety and a conjugated drug, preferably being an antibody-drug conjugate (ADC), for use in the treatment and / or alleviation of mesothelial cancer and / or breast cancer in a subject.

[0030] In a preferred embodiment, the megalin-targeting conjugate being an antibodydrug conjugate (ADC) for use in the treatment and / or alleviation of pleural mesothelial cancer in a subject.

[0031] Another aspect of the present invention relates to a megalin-targeting antibodydrug conjugate (ADC), for use in the treatment and / or alleviation of mesothelial cancer and / or breast cancer in a subject.

[0032] Yet another aspect of the present invention relates to a method for determining if a mesothelial cancer or a breast cancer from a subject is susceptible to treatment with a megalin-targeting drug, such as an ADC, the method comprising a) determining in a mesothelial cancer sample or breast cancer sample from the subject the level of megalin protein, preferably membrane bound megalin; b) comparing said determined level of megalin protein to a reference level; and c)

[0033] - determining that said cancer from a subject is susceptible to treatment with a megalin targeting drug; when said determined level is equal to or above said reference level, or

[0034] - determining that said cancer from a subject is not susceptible to treatment with a megalin targeting drug; when said determined level is below said reference level.

[0035] In a further aspect, the invention relates to a method for determining if a mesothelial cancer from a subject is susceptible to treatment with a megalin- targeting antibody-drug conjugate (ADC), via administration to the pleural cavity, the method comprising a) determining in a mesothelial cancer sample from the subject the level of megalin protein, preferably membrane bound megalin; b) comparing said determined level of megalin protein to a reference level; and c)

[0036] - determining that said cancer from a subject is susceptible to treatment with a megalin targeting antibody-drug conjugate (ADC),; when said determined level is equal to or above said reference level, or

[0037] - determining that said cancer from a subject is not susceptible to treatment with a megalin targeting antibody-drug conjugate (ADC),; when said determined level is below said reference level; wherein the reference level is based on megalin levels from one or more corresponding samples from healthy subjects, or from neighboring corresponding healthy samples from the same subject; wherein the reference level is determined using immunohistochemical staining; wherein the level of megalin protein determined in step a) is the level in the apical / luminal membrane facing the pleural cavity.

[0038] In a preferred embodiment, the cancer is a mesothelial cancer.

[0039] Brief description of the figures

[0040] Figure 1

[0041] Figure 1 shows antibody binding to human and murine megalin. A) Binding to human megalin of megalin antibody either free (solid line) or conjugated with duocarmycin (dotted line). B) Binding to murine megalin of megalin antibody either free (solid line) or conjugated with duocarmycin (dotted line).

[0042] Figure 2

[0043] Figure 2 shows immunohistochemical detection of megalin protein expression in different cancers. (A and E) Representative image of bulk mesothelioma tumor region from two different epithelioid mesothelioma tumors. Megalin is observed in mesothelioma cells (single arrowhead) but not in adjacent normal cells (double arrowhead). Magnification 5x. Scale bar = 250 jim. (B-D) High magnification images of mesothelioma tumor cells from panel A. Magnification 80x. Scale bar = 25 |im. (F-H) High magnification images of mesothelioma tumor cells from panel E. Mesothelioma cells appear polarized with megalin localized to the apical membrane of tumor cells facing the pleural space (single arrowhead).

[0044] Magnification 80x. Scale bar = 25 jim. (I) Image of a luminal breast invasive carcinoma with megalin observed in breast cancer cells (single arrowhead). Magnification 20x. Scale bar = 60 jim. (J) Image of a primary cutaneous melanoma with megalin observed in melanoma cells (single arrowhead).

[0045] Magnification 40x. Scale bar = 50 jim. (K) Image of a melanoma brain metastasis with megalin observed in melanoma cells (single arrowhead). Magnification 40x. Scale bar = 50 jim.

[0046] Figure 3

[0047] Immunohistochemical analysis of megalin protein expression in mesothelioma pleural effusions. (A-D) Representative high magnification images of cells in pleural effusion sample 1 (A-B) and sample 2 (C-D). Megalin is observed at the plasma membrane of mesothelioma cells (single arrowhead) but not in normal cells (double arrowhead). Magnification 80x. Scale bar = 25 jim.

[0048] Figure 4

[0049] Figure 4 shows specific binding of anti-megalin antibodies to cancer cells measured by flow cytometry. (A) Flow cytometry analysis of AF647-labeled anti- megalin antibody binding / uptake to FM3 sgCtrl (left panel) and FM3 sgLRP2 #3 (middle panel) and FM92 (right panel) cells. (B) Flow cytometry analysis of AF647-labeled anti-megalin antibody binding / uptake to MDAMB-134VI (left panel) or T47D (right panel) cells.

[0050] Figure 5

[0051] Figure 5 shows specific binding and internalization of anti-megalin antibodies to cancer cells measured by imaging flow cytometry. Brightfield images (left panel), cell surface HLA-A / B / C-FITC staining (second panel from left), anti-megalin-AF647 staining (second panel from right) and merged images (FITC and AF647) are shown for 5 representative FM3 cells in the experiment. HLA-A / B / C staining is observed at the cell surface. Megalin staining is observed in intracellular compartments.

[0052] Figure 6

[0053] Figure 6 shows cell-killing effect of anti-megalin ADC on human melanoma cell lines. (A-C) Dose-response curves of IgGl-duocarmycin (IgGl-duo) control or anti-megalin-duocarmycin (aMegalin-duo) in FM3 (A), FM3 sgLRP2 #3 (B) and FM92 (C) cells. Relative cell numbers are calculated compared to untreated control cells. Means (colored squares) are shown (n = 3 technical replicates). (D) Comparison of cell-killing effect of 500 pg / ml anti-megalin ADC in FM3, FM3 sgLRP2 #3 and FM92 melanoma cells. Means (bars) + / - standard error of mean (error bars) and individual values (colored squares) are shown (n = 3 technical replicates, unpaired t test). ****P<0.0001.

[0054] Figure 7

[0055] Figure 7 shows cell-killing effect of anti-megalin ADC on human luminal invasive breast carcinoma cell lines. (A-B) Dose-response curves of IgGl-duocarmycin (IgGl-duo) control or anti-megalin-duocarmycin (aMegalin-duo) in MDAMB-134VI (A) or T47D (B) cells. Relative cell numbers are calculated compared to untreated control cells. Means (colored squares) are shown (n = 3 technical replicates). (C) Comparison of cell-killing effect of 500 pg / ml anti-megalin ADC in MDAMB-134VI and T47D human luminal invasive breast carcinoma cells. Means (bars) + / - standard error of mean (error bars) and individual values (colored squares) are shown (n = 3 technical replicates, unpaired t test). ****P<0.0001.

[0056] The present invention will now be described in more detail in the following.

[0057] Detailed description of the invention

[0058] Definitions

[0059] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

[0060] LRP2 / Meaalin

[0061] LRP2 / Megalin is a large endocytic receptor (600 kDa) highly expressed in specialized absorptive epithelia, such as the proximal tubules of the kidney, the thyroid gland, and glandular epithelium of the breast. LRP2 / megalin has more than 40 identified ligands, including lipoproteins, albumin, vitamin carrier proteins, hormones and signalling molecules, enzymes and enzyme inhibitors, and immunoglobulins.

[0062] In the present context, the terms "LRP2" and "megalin" are used interchangeably. Other well-known synonyms for LRP2 / megalin are "glycoprotein-330" and "gp330".

[0063] Mesothelioma

[0064] Mesothelioma is a type of cancer that develops from the thin layer of tissue that covers many of the internal organs (known as the mesothelium). The area most commonly affected is the lining of the lungs and chest wall. Symptoms or signs of mesothelioma may not appear until 20 to 50 years (or more) after exposure to asbestos. Shortness of breath, cough, and pain in the chest due to an accumulation of fluid in the pleural space (pleural effusion) are often symptoms of pleural mesothelioma.

[0065] Types of mesothelioma include epithelioid mesothelioma, biphasic mesothelioma and sarcomatiod mesothelioma. Preferably mesothelioma is epithelioid mesothelioma. Even more preferably the mesothelioma is pleural mesothelioma, such as pleural epithelioid mesothelioma. In the present context the terms "mesothelioma", "mesothelial cancer" and "mesotheliom" may be used interchangeably.

[0066] Pleural cavitv / space

[0067] The "pleural cavity" or "pleural space" is the space between the visceral and parietal pleura lining the outside of the lungs. The space contains a tiny amount of serous fluid, which has two key functions. The serous fluid continuously lubricates the pleural surface and makes it easy for them to slide over each other during the changes in the lung volume owing to the ongoing air inflation and deflation. Accumulation of fluid in the pleural cavity / space (pleural effusion) is most often seen in mesothelioma. Such a cancer is called a "fluid-secreting mesothelioma". This constitutes a threat to the patient because it causes pressure on the lungs which inhibits inflation and respiration. Treatment or alleviation of fluid accumulation will often require repeated drainage by a permanently inserted drainage tube connecting the pleural cavity with the outside.

[0068] AO Pl

[0069] AQP1 is a water channel protein found in cell membranes throughout the body and facilitates transcellular water transport.

[0070] In the present context, the immunohistochemical overexpression of AQP1 is defined as >50% of tumor cells showing membranous staining for AQP1.

[0071] Antibody

[0072] Antibodies of the invention include polyclonal, monospecific polyclonal, monoclonal, recombinant, chimeric, humanized, fully human, single chain and / or bispecific antibodies. Antibody fragments include those portions of an anti-megalin antibody which bind to an epitope on a megalin polypeptide. Examples of such fragments include nanobodies, Fab F(ab'), F(ab)', Fv, and sFv fragments. The antibodies may be generated by enzymatic cleavage of full-length antibodies or by recombinant DNA techniques, such as expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions.

[0073] Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen. An antigen is a molecule or a portion of a molecule capable of being bound by an antibody, which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen can have one or more epitopes. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies, which can be evoked by other antigens.

[0074] Polyclonal antibodies directed toward megalin polypeptide generally are raised in animals (e.g., rabbits or mice) by multiple subcutaneous or intraperitoneal injections of megalin and an adjuvant.

[0075] Monoclonal antibodies (mAbs) contain a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. A hybridoma producing a monoclonal antibody of the present invention may be cultivated in vitro, in situ, or in vivo. Production of high titers in vivo or in situ is a preferred method of production.

[0076] Monoclonal antibodies directed toward megalin are produced using any method which provides for the production of antibody molecules by continuous cell lines in culture. Examples of suitable methods for preparing monoclonal antibodies include hybridoma methods of Kohler et al., Nature 256, 495-497 (1975), and the human B-cell hybridoma method, Kozbor, J. Immunol. 133, 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988); the contents of which references are incorporated entirely herein by reference.

[0077] Preferred anti-megalin antibodies include monoclonal antibodies. Preferred methods for determining monoclonal antibody specificity and affinity by competitive inhibition can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol., 92:589- 601 (1983). Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.

[0078] The term "chimeric antibody", as used herein, includes monovalent, divalent or polyvalent immunoglobulins. A monovalent chimeric antibody is a dimer (HL) formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain. A divalent chimeric antibody is tetramer (H2L2) formed by two HL dimers associated through at least one disulfide bridge. A polyvalent chimeric antibody can also be produced, for example, by employing a CH region that aggregates (e.g., from an IgM H chain, or [micro] chain).

[0079] Murine and chimeric antibodies, fragments and regions of the present invention may comprise individual heavy (H) and / or light (L) immunoglobulin chains. A chimeric H chain comprises an antigen binding region derived from the H chain of a non-human antibody specific for megalin, which is linked to at least a portion of a human H chain C region (CR), such as CHI or CH2.

[0080] A chimeric L chain according to the present invention comprises an antigen binding region derived from the L chain of a non-human antibody specific for megalin, linked to at least a portion of a human L chain C region (CL).

[0081] Selective binding agents, such as antibodies, fragments, or derivatives, having chimeric H chains and L chains of the same or different variable region binding specificity, can also be prepared by appropriate association of the individual polypeptide chains, according to known method steps, e.g., according to Ausubel et al., eds. Current Protocols in Molecular Biology, Wiley Interscience, N.Y. (1993), and Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). The contents of these references are incorporated entirely herein by reference. With this approach, hosts expressing chimeric H chains (or their derivatives) are separately cultured from hosts expressing chimeric L chains (or their derivatives), and the immunoglobulin chains are separately recovered and then associated.

[0082] Alternatively, the hosts can be co-cultured and the chains allowed to associate spontaneously in the culture medium, followed by recovery of the assembled immunoglobulin, fragment or derivative.

[0083] Megalin antibodies may be raised against full-length megalin as shown in the example section (see example 1). In embodiments of the invention the antibody is humanized.

[0084] Antibody-drug conjugates (ADCs)

[0085] Antibody-drug conjugates or ADCs are a class of biopharmaceutical drugs designed as a targeted therapy for treating cancer. Unlike chemotherapy, ADCs are intended to target and kill tumor cells while sparing healthy cells.

[0086] ADCs are complex molecules composed of an antibody linked to a biologically active cytotoxic (anticancer) payload or drug.

[0087] ADCs combine the targeting properties of monoclonal antibodies with the cancerkilling capabilities of cytotoxic drugs, designed to discriminate between healthy and diseased tissue.

[0088] Megalin-targeting conjugate comprising a megalin-targeting moiety and a conjugated drug

[0089] As outlined above, the inventing team has identified megalin / LRP2 mRNA and protein expression in certain cancer types. Thus, an aspect of the invention relates to a megalin-targeting conjugate comprising a megalin-targeting moiety and a conjugated drug, preferably being an antibody-drug conjugate (ADC), for use in the treatment and / or alleviation of mesothelial cancer (mesotheliom) and / or breast cancer and / or melanoma in a subject. In example 5 expression of megalin protein in mesothelioma tumors from patient samples are demonstrated. In example 6, specific binding and uptake of anti-megalin antibodies by megalin- expressing cancer cell lines is shown, namely melanoma cells and invasive breast carcinoma cells. Example 7 shows specific killing of megalin-expressing cancer cells by antibody drug-conjugates targeting megalin.

[0090] In a general aspect, the invention relates to a megalin-targeting conjugate comprising a megalin-targeting moiety and a conjugated drug, preferably being an antibody-drug conjugate (ADC), for use in the treatment and / or alleviation of a cancer in a subject.

[0091] A preferred aspect of the invention relates to a megalin-targeting conjugate comprising a megalin-targeting moiety and a conjugated drug, preferably being an antibody-drug conjugate (ADC), for use in the treatment and / or alleviation of mesothelial cancer (mesotheliom) in a subject.

[0092] In an embodiment, the megalin-targeting conjugate, preferably an antibody drug conjugate (ADC), is for use in the treatment and / or alleviation of mesothelial cancer in a subject.

[0093] In another embodiment, the megalin-targeting conjugate is an antibody-drug conjugate. In the example section, this format of conjugate has been tested.

[0094] In an embodiment, the mesothelial cancer is a pleural mesothelial cancer. As realized by the inventing team, a pleural mesothelial cancer may be treatable via the pleural cavity.

[0095] In a preferred embodiment, the megalin-targeting conjugate is an antibody-drug conjugate (ADC) and the cancer is pleural mesothelial cancer.

[0096] In yet an embodiment, the megalin-targeting conjugate, preferably an ADC, is administered to the pleural cavity.

[0097] In a related embodiment, the conjugate is administered to the pleural cavity via a (previously inserted) pleural cavity drainage. Treatment or alleviation of fluid accumulation in mesothelial cancer patients will often require drainage by a pleural cavity drainage. Thus, an administration route for the conjugate may be readily available.

[0098] In another embodiment, the cancer presents megalin in cell membranes, such as being exposed on the cell surface. For targeted drugs, the target must be available for the drug to reach its target.

[0099] In yet an embodiment, the mesothelial cancer presents megalin in cell membranes, such as being exposed on the cell surface, more preferably being present in the apical / luminal membrane facing the pleural cavity. In yet another embodiment, the cancer is polarized. As shown e.g. in figure 2, mesothelioma cells appear polarized with megalin localized to the apical membrane of tumor cells facing the pleural space. In a more specific embodiment, polarization refers to the localization of megalin. In a preferred embodiment, the mesothelioma cancer is polarized or comprises polarized cells with megalin localized to the apical membrane of tumor cells facing the pleural space.

[0100] In an embodiment, the pleural mesothelial cancer overexpresses AQP1 in cell membranes, such as being exposed on the cell surface.

[0101] In yet an embodiment, the mesothelial cancer presents megalin in cell membranes and wherein the pleural mesothelial cancer overexpresses AQP1 in cell membranes, such as being exposed on the cell surface.

[0102] In an embodiment the megalin targeting moiety, preferably an antibody, and the drug is conjugated via a linker, such as a cathepsin degradable linker. In the example section a cathepsin degradable linker has been used, namely the PEG4- vc-PAB linker. The skilled can easily identify other cathepsin degradable linkers, or other linkers, which a readily available from several suppliers.

[0103] In a related embodiment, the linker is selected from the group consisting of OSu- PEG4-vc-PAB linker, MA-PEG4-VC-PAB-DMEA linker, valine-citrulline-p- aminobenzyl carbamate (ValCitPABC), and GGFG Tetrapeptidyl-Aminomethoxy Linker.

[0104] In a preferred embodiment, the pleural mesothelial cancer is an epithelioid pleural mesothelioma.

[0105] In yet a preferred embodiment, the pleural mesothelial cancer is a malignant pleural mesothelioma (MPM).

[0106] In yet another preferred embodiment the pleural mesothelial cancer is a fluidsecreting mesothelial cancer. In an embodiment, the megalin-targeting moiety binds to an extracellular epitope of megalin.

[0107] In an embodiment, the antibody is selected from the group consisting of, monoclonal, polyclonal, monospecific polyclonal, recombinant, chimeric, humanized, fully human, single chain and bispecific antibodies.

[0108] In a further embodiment, the antibody has been raised against full-length megalin.

[0109] In yet a further embodiment, the antibody is humanized, preferably a humanized monoclonal antibody.

[0110] In an embodiment, the megalin-targeting conjugate is specific for megalin. Preferably the conjugate is an ADC.

[0111] In an embodiment, the megalin targeting moiety is selected from the group consisting of the megalin ligands receptor-associated protein (RAP), transcobalamin, retinol-binding protein, folate-binding protein, vitamin D binding protein, alpha-amylase, activated coagulation factor Vila and VIII, albumin, hemoglobin, myoglobin, lactoferrin, Clq, apolipoprotein B, E, H, J and M, alphal- microglobin, beta2-microglobulin, polybasic drugs such as aminoglycosides, and cytochrome C. The skilled person may identify other megalin ligands to which a drug could be conjugated using standard techniques. Megalin ligands are e.g. reviewed in Christensen et al. PHYSIOLOGY 27: 223-236, 201.

[0112] In an embodiment, the drug is selected from the group consisting of a cytotoxic agent, a therapeutic agent, a chemotherapeutic agent and a radioisotope.

[0113] In a more specific embodiment, the cytotoxic agent is selected from the group consisting of duocarmycin SA, taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicin; doxorubicin; daunorubicin; dihydroxy anthracin dione; a tubulin- inhibitor such as maytansine or an analogue or derivative thereof; an antimitotic agent such as monomethyl auristatin E or F or an analogue or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; a glucocorticoid; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analogue or derivative thereof; an antimetabolite such as methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabin, 5 fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; an alkylating agent such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; a platinum derivative such as cisplatin or carboplatin; duocarmycin A, , rachelmycin (CC-1065), or an analogue or derivative thereof; an antibiotic such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)); pyrrolo[2,l-c] [1,4]- benzodiazepines (PDB); diphtheria toxin and related molecules such as diphtheria A chain and active fragments thereof and hybrid molecules, ricin toxin such as ricin A or a deglycosylated ricin A chain toxin, cholera toxin, a Shiga-like toxin such as SLT I, SLT II, SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins such as PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins; ribonuclease (RNase); DNase I, Staphylococcal enterotoxin A; pokeweed antiviral protein; diphthera toxin; and Pseudomonas endotoxin.

[0114] In an embodiment, the cytotoxic agent is a duocarmycin, such as selected the group consisting of from duocarmycin SA, A, Bl, B2, Cl, C2, D, and CC-1065 or an analogue or derivative thereof such as selected from the group consisting of adozelesin, bizelesin, and carzelesin. In the example section duocarmycin SA has been tested.

[0115] In an embodiment, the subject is a mammal, preferably a human, and even more preferably an adult human, such as above 18 years of age. A method for determining if a mesothelial cancer or a breast cancer from a subject is susceptible to treatment with a megalin-targeting drug Another aspect of the invention relates to a method for determining if a mesothelial cancer or a breast cancer from a subject is susceptible to treatment with a megalin-targeting drug, such as an ADC, the method comprising a) determining in a (previously obtained) mesothelial cancer sample or breast cancer sample or melanoma sample in a subject from the subject the level of megalin protein, preferably membrane bound megalin; b) comparing said determined level of megalin protein to a reference level; and c)

[0116] - determining that said cancer from a subject is susceptible to treatment with a megalin targeting drug; when said determined level is equal to or above said reference level, or

[0117] - determining that said cancer from a subject is not susceptible to treatment with a megalin targeting drug; when said determined level is below said reference level.

[0118] As outlined in the example section, certain cancers have been identified to have high expression of megalin on the surface, making them excellent targets for drug conjugates targeting megalin. The skilled doctor such as a pathologist will be able to identify at set relevant reference levels for the cancer in question. In an embodiment, the reference level is based on (average) megalin levels from one or more corresponding samples from healthy subjects, or from neighbouring corresponding healthy samples from the same subject. Again, it is standard practice to establish relevant reference levels.

[0119] In a preferred embodiment, the cancer is mesothelial cancer.

[0120] In an embodiment, the level of megalin protein determined in step a) is the level in the apical / luminal membrane facing the pleural cavity.

[0121] In yet an embodiment, the cancer is mesothelial cancer, and the treatment is a treatment to be administered to the pleural cavity. In an embodiment, the level of megalin is determined using an antibody targeting megalin.

[0122] In another embodiment, the cancer sample in step a) is a biopsy or a pleural effusion, preferably a biopsy. Biopsies are often required for the doctor to establish a diagnosis, in particular for mesothelial cancers.

[0123] In a further embodiment, the level of megalin protein is determined using immunohistochemical staining such as on an FFPE sample.

[0124] In a related embodiment, the level of megalin protein in the apical / luminal membrane facing the pleural cavity is determined using immunohistochemical staining, such as on a FFPE sample of the cancer.

[0125] Other aspects of the invention

[0126] Yet an aspect of the invention relates to a method for the treatment and / or alleviation of mesothelial cancer (mesotheliom) and / or breast cancer and / or melanoma in a subject, the method comprising administering to said subject a megalin-targeting conjugate comprising a megalin-targeting moiety and a conjugated drug, preferably being an antibody-drug conjugate (ADC).

[0127] In an embodiment, the cancer is (pleural) mesothelial cancer and the megalin- targeting conjugate (preferably an ADC) is administered to the pleural cavity, such as via a pleural cavity drainage.

[0128] In yet an embodiment, the subject has been determined to have a pleural mesothelial cancer expressing megalin before said treatment is initiated.

[0129] Items of the invention

[0130] 1. A megalin-targeting conjugate comprising a megalin-targeting moiety and a conjugated drug, preferably being an antibody-drug conjugate (ADC), for use in the treatment and / or alleviation of mesothelial cancer and / or breast cancer and / or melanoma in a subject. 2. The megalin-targeting conjugate, preferably an antibody drug conjugate (ADC), according to item 1, for use in the treatment and / or alleviation of mesothelial cancer in a subject.

[0131] 3. The megalin-targeting conjugate for use according to item 1 or 2, being an antibody-drug conjugate.

[0132] 4. The megalin-targeting conjugate for use according to any of the preceding items, wherein the mesothelial cancer is a pleural mesothelial cancer.

[0133] 5. The megalin-targeting conjugate for use according to any of the preceding items, being an antibody-drug conjugate (ADC) for use in the treatment and / or alleviation of pleural mesothelial cancer in a subject.

[0134] 6. The megalin-targeting conjugate for use according to any of the preceding items, being administered to the pleural cavity.

[0135] 7. The megalin-targeting conjugate for use according to any of the preceding items, wherein the conjugate is administered to the pleural cavity via a (previously inserted) pleural cavity drainage.

[0136] 8. The megalin-targeting conjugate for use according to any of the preceding items, wherein the cancer presents megalin in cell membranes, such as being exposed on the cell surface.

[0137] 9. The megalin-targeting conjugate for use according to any of the preceding items, wherein the mesothelial cancer presents megalin in cell membranes, such as being exposed on the cell surface, more preferably being present in the apical / luminal membrane facing the pleural cavity.

[0138] 10. The megalin-targeting conjugate for use according to any of the preceding items, wherein the cancer is polarized. 11. The megalin-targeting conjugate for use according to any of the preceding items, wherein the pleural mesothelial cancer overexpresses AQP1 in cell membranes, such as being exposed on the cell surface.

[0139] 12. The megalin-targeting conjugate for use according to any of the preceding items, wherein the megalin targeting moiety, preferably an antibody, and the drug is conjugated via a linker, such as a cathepsin degradable linker.

[0140] 13. The megalin-targeting conjugate for use according to item 12, wherein the linker is selected from the group consisting of OSu-PEG4-vc-PAB linker, MA-PEG4- vc-PAB-DMEA linker, valine-citrulline-p-aminobenzyl carbamate (ValCitPABC), and GGFG Tetrapeptidyl-Aminomethoxy Linker.

[0141] 14. The megalin-targeting conjugate for use according to any of the preceding items, wherein the mesothelial cancer presents megalin in cell membranes and wherein the pleural mesothelial cancer overexpresses AQP1 in cell membranes, such as being exposed on the cell surface.

[0142] 15. The megalin-targeting conjugate for use according to any of the preceding items, wherein the pleural mesothelial cancer is an epithelioid pleural mesothelioma.

[0143] 16. The megalin-conjugate for use according to any of the preceding items, wherein the pleural mesothelial cancer is a malignant pleural mesothelioma (MPM).

[0144] 17. The megalin-targeting conjugate for use according to any of the preceding items, wherein the pleural mesothelial cancer is a fluid-secreting mesothelial cancer.

[0145] 18. The megalin-targeting conjugate for use according to any of the preceding items, wherein the megalin-targeting moiety binds to an extracellular epitope of megalin. 19. The megalin-targeting conjugate for use according to any of the preceding items, being an antibody, wherein the antibody is selected from the group consisting of, monoclonal, polyclonal, monospecific polyclonal, recombinant, chimeric, humanized, fully human, single chain and bispecific antibodies.

[0146] 20. The megalin-targeting conjugate for use according to item 19, wherein the antibody has been raised against full-length megalin.

[0147] 21. The megalin-targeting conjugate according to any of the proceeding items 19 or 20, wherein the antibody is humanized, preferably a humanized monoclonal antibody.

[0148] 22. The megalin-targeting conjugate according to any of the proceeding items, being specific for megalin.

[0149] 23. The megalin-targeting drug conjugate, for use according to any of item 1-18, wherein the megalin targeting moiety is selected from the group consisting of the megalin ligands, receptor-associated protein (RAP), transcobalamin, retinol- binding protein, folate-binding protein, vitamin D binding protein, alpha-amylase, activated coagulation factor Vila and VIII, albumin, hemoglobin, myoglobin, lactoferrin, Clq, apolipoprotein B, E, H, J and M, alphal-microglobin, beta2- microglobulin, polybasic drugs such as aminoglycosides, and cytochrome C.

[0150] 24. The megalin-targeting conjugate for use according to any of the preceding items, wherein the drug is selected from the group consisting of a cytotoxic agent, a therapeutic agent, a chemotherapeutic agent and a radioisotope.

[0151] 25. The megalin-targeting conjugate for use according to item 24, wherein the cytotoxic agent is selected from the group consisting of duocarmycin SA, taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicin; doxorubicin; daunorubicin; dihydroxy anthracin dione; a tubulin- inhibitor such as maytansine or an analogue or derivative thereof; an antimitotic agent such as monomethyl auristatin E or F or an analogue or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1- dehydrotestosterone; a glucocorticoid; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analogue or derivative thereof; an antimetabolite such as methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabin, 5 fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; an alkylating agent such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; a platinum derivative such as cisplatin or carboplatin; duocarmycin A, , rachelmycin (CC-1065), or an analogue or derivative thereof; an antibiotic such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)); pyrrolo[2,l-c][l,4]-benzodiazepines (PDB); diphtheria toxin and related molecules such as diphtheria A chain and active fragments thereof and hybrid molecules, ricin toxin such as ricin A or a deglycosylated ricin A chain toxin, cholera toxin, a Shiga-like toxin such as SLT I, SLT II, SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins such as PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins; ribonuclease (RNase); DNase I, Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtherin toxin; and Pseudomonas endotoxin.

[0152] 26. The megalin-targeting conjugate for use according to item 16, wherein the cytotoxic agent is a duocarmycin, such as selected the group consisting of from duocarmycin SA, A, Bl, B2, Cl, C2, D, and CC-1065 or an analogue or derivative thereof such as selected from the group consisting of adozelesin, bizelesin, and carzelesin.

[0153] 27. The megalin-targeting conjugate for use according to any of the preceding items, wherein the subject is a mammal, preferably a human, and even more preferably an adult human, such as above 18 years of age. 28. A method for determining if a mesothelial cancer or a breast cancer from a subject is susceptible to treatment with a megalin-targeting drug, such as an ADC, the method comprising a) determining in a (previously obtained) mesothelial cancer sample or breast cancer sample or melanoma cancer sample in a subject from the subject the level of megalin protein, preferably membrane bound megalin; b) comparing said determined level of megalin protein to a reference level; and c)

[0154] - determining that said cancer from a subject is susceptible to treatment with a megalin targeting drug; when said determined level is equal to or above said reference level, or

[0155] - determining that said cancer from a subject is not susceptible to treatment with a megalin targeting drug; when said determined level is below said reference level.

[0156] 29. The method according to item 28, wherein the cancer is mesothelial cancer.

[0157] 30. The method according to item 29, wherein the level of megalin protein determined in step a) is the level in the apical / luminal membrane facing the pleural cavity.

[0158] 31. The method according to any of items 29-30, wherein the cancer is mesothelial cancer, and the treatment is a treatment to be administered to the pleural cavity.

[0159] 32. The method according to any of items 28-31, wherein the level of megalin is determined using an antibody targeting megalin.

[0160] 33. The method according to any of the preceding items 28-32, wherein the cancer sample in step a) is a biopsy or a pleural effusion, preferably a biopsy.

[0161] 34. The method according to any of the preceding items 28-33, wherein the level of megalin protein is determined using immunohistochemical staining, FISH, such as on a FFPE sample. 35. The method according to any of the preceding items 30-34, wherein the level of megalin protein in the apical / luminal membrane facing the pleural cavity is determined using immunohistochemical staining, such as on a FFPE sample of the cancer.

[0162] 36. A method for the treatment and / or alleviation of mesothelial cancer and / or breast cancer and / or melanoma in a subject, the method comprising administering to said subject a megalin-targeting conjugate comprising a megalin- targeting moiety and a conjugated drug, preferably being an antibody-drug conjugate (ADC).

[0163] 37. The method according to item 36, wherein the megalin-targeting conjugate is administered to the pleural cavity.

[0164] 38. The method according to item 19 or 20, wherein the subject has been determined to have a pleural mesothelial cancer expressing megalin before said treatment is initiated.

[0165] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0166] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0167] The invention will now be described in further details in the following non-limiting examples.

[0168] Examples

[0169] Example 1 - Generation of antibody binding human megalin

[0170] Aim of study

[0171] To generate an antibody that can be used to demonstrate expression of megalin on cells and for initial targeting experiments of megalin expressing cells. Materials and methods

[0172] Full length megalin was purified by Receptor-Associated Protein (RAP) - affinity chromatography from human renal cotex as described for purification megalin (previous name gp330) from rabbit renal cortex (Moestrup et al. 1993).

[0173] Polyclonal antibodies against human megalin were raised in rabbits using Davids Biotechnologie as service provider (https : / / www. davids-bio.com / Dacjes / Dolvclonal- rabbit-antisera.html).

[0174] MAb was produced by hybridoma technique described by Kohler and Milstein. Briefly, two NMRI mice (Taconic, Ejby, Denmark) were immunized twice subcutaneously with 10 pg of purified megalin with two-week intervals using Gerbu adjuvant P (Gerbu Biotechnik GmbH, Heidelberg, Germany). Three days before splenectomy, mice were boosted i.v. with 10 pg purified megalin. HGPRT negative Sp2 / 0-AG14 myeloma cells and splenocytes were fused and hybridomas were screened by direct ELISA for megalin binding. Finally, the hybridomas were cloned by limiting dilution to obtain mAbs. MAbs were purified by affinity chromatography using a HiTrap protein G HP column® (GE Healthcare) according to the manufacturer's instructions using 0.1M sodium-citrate buffer (pH 3.0) for elution.

[0175] Direct ELISA:

[0176] A direct sandwich ELISA was established to identify hybridomas expressing antibodies binding megalin. Maxisorp® plates (Nunc, Roskilde, Denmark) were coated with human megalin (2 pg / ml in PBS, overnight, 4°C). Wells were blocked in 3% bovine serum albumin (BSA) for 90 minutes and washed with PBS + 0.5% Tween-20. Hybridoma supernatants diluted 1: 10 in PBS + 0.5% Tween 20 was added to wells and plates were incubated for two hours. Plates were washed four times with PBS + 0.5% Tween 20. HRP conjugated polyclonal goat anti-mouse diluted 1:2000 in PBS + 0.5% Tween 20 was added to each well and incubated for 1 hour, followed by four times washing with PBS + 0.5% Tween 20. Plates were developed using TMB one® (Kem-En-Tec) and stopped after 10 minutes by adding 0.2 M sulfuric acid. Polyclonal megalin antibody was used as positive control. Results

[0177] Several mAbs were generated for human megalin. One of these was selected and the antibody purified.

[0178] Conclusion

[0179] One hybridoma was identified that expressed an antibody positive in both ELISA, western blot (see example 2).

[0180] Example 2 - Demonstration of megalin specificity of antibody using western blotting

[0181] Aim of study

[0182] As also described further above, it has previously been demonstrated in Rasmussen et al. that megalin encoding RNA is upregulated in certain cancers. However, what has not been clarified is whether megalin protein is present in cell surfaces in mesothelioma tumors in clinically relevant amounts, and if the protein is present on the cell surface allowing for using megalin as a gateway for antibody-drug conjugates.

[0183] To clarify the above, antibodies targeting megalin are needed. Furthermore, megalin targeting antibodies exhibiting specific binding to megalin in a complex human sample, and that targeting and staining of specimens hence will be megalin specific is also needed.

[0184] Materials and Methods

[0185] Antibody

[0186] See example 1.

[0187] Western blot

[0188] 0.025 pg purified human megalin from kidney was loaded per well of an 4-16% Bis-Tris SDS-PAGE gel with a MOPS running buffer (Invitrogen). PageRuler Plus pre-stained protein ladder (Invitrogen) was also loaded. After electrophoresis, the gel was wet-blotted onto a PVDF membrane, running for 16 hours at 100 mA in blotting buffer (25 mM Tris-Base, 192 mM glycine). The membrane was blocked for 2 hours at room temperature in 50 mM Tris, 250 mM NaCI, 2% Tween-20, pH 9.0, followed by 3 times 5 minutes wash in PBS, 0.05% Tween-20 pH 7.4. The membrane was incubated overnight with hybridoma clone supernatant diluted 1:2 and added skimmed milk powder to 2.5%. This was followed by 3 times 5 minutes wash in PBS, 0.05% Tween-20 pH 7.4 and incubation for 1 hour with secondary antibody, rabbit anti-mouse IgG AP conjugated (Sigma) diluted 1: 10000 in PBS, 0.05% Tween-20 pH 7.4 + 5% skimmed milk powder, followed by 3 times 5 minutes wash in PBS, 0.05% Tween- 20 pH 7.4.

[0189] The membrane was developed using BCIP-NBT Blue (Sigma).

[0190] Results

[0191] The megalin mAb exhibited specific binding to megalin, as reflected in binding of one band, with intensity at around the predicted size of 600 kDa as well as a degradation products of megalin (data not shown).

[0192] Conclusion

[0193] The antibody can be used for specific staining and specific targeting of megalin.

[0194] Example 3 - Synthesis of antibody drug conjugate (ADC) Aim of study

[0195] To produce antibody drug conjugates (ADCs) targeting the highly toxic drug duocarmycin to cells expressing megalin. The example describes the conjugation of duocarmycin to primary amino groups of the antibody through a cathepsin degradable linker. Cathepsin is contained in lysosomes where the megalin-binding ADC ends up owing to endocytosis mediated by megalin. The drug may then be cleaved of and then be able to exert its function in the cell.

[0196] Materials and Methods

[0197] Antibody

[0198] See example 1.

[0199] Conjugation Antibody was dissolved in PBS pH 7.4 at 3 mg / ml. Osu-PEG4-vc-PAB-DMEA Duocarmycin SA (cat. No. ADC-S-011, Creative Biolabs), to conjugate duocarmycin SA to amino groups using the linker Osu-PEG4-vc-PAB-DMEA, was dissolved in DMSO at a concentration of 5 mM and 6 pl added per mg antibody. Conjugation was allowed to take place by incubation overnight at 4 C followed by buffer exchange not PBS pH 7.4 using a PD-10 column (Cytiva). Drug-antibody ratio was calculated by measuring protein and duocarmycin concentrations based on absorption of light at 280 nm and 337 nm, respectively.

[0200] Samples were sterile filtered and snap frozen.

[0201] Results

[0202] The drug-antibody ration was generally found to be between 4 and 5, there was generally a yield of above 80% in terms of recovered antibody.

[0203] Conclusion

[0204] Synthesis of ADC was successful.

[0205] Example 4 - Demonstration of human megalin affinity of the ADCs using surface plasmon resonance

[0206] Aim of study

[0207] To show that the mAb binds megalin both before and after conjugation with toxin.

[0208] Material and Methods

[0209] Antibodv-druo conjugate See example 3.

[0210] Surface plasmon resonance (SPR) analysis

[0211] Surface plasmon resonance (SPR) analysis of the binding of antibody to megalin was carried out using a Biacore 3000 instrument (Cytiva). The CM5 sensor chip (Cytiva) was activated with a 1 : 1 mixture of 0.2 M N-ethyl-N'-(3- dimethylaminopropyl) carbodiimide and 0.05 M N-hydroxysuccimide in H2O. Human and murine megalin was immobilized on the chip in 10 mM sodium phosphate, pH 5.0, and the remaining binding sites blocked with 1 M ethanolamine, pH 8.5. The SPR signal generated from immobilized megalin generally corresponded to 20-40 fmol of protein / mm2. Sensorgrams were generated using the following running buffer: lOmM Hepes, 150mM NaCI, 3mM CaCI2, +0.05% Tween 20, pH 7.4. An antibody concentration of 5 pg / ml was used. The flow cells were regenerated with 100 mM phosphoric acid, which lead to a more than 98% functional regeneration of the chip in terms of antibody binding. The FXII constructs were dissolved in running buffer. All binding experiments were at least done in triplicate and data were analyzed using the Biomolecular Interaction Analysis evaluation program version 3.1 (Cytiva).

[0212] Results

[0213] The mAb binds human megalin (Figure 1A), whereas it did not bind murine megalin (figure IB). Thus, binding was specific for human megalin and conjugation with toxin did not lower this binding.

[0214] Conclusion:

[0215] The megalin targeting ADC is suitable for targeting megalin expressing cells.

[0216] Example 5 - Expression of megalin protein in mesothelioma tumors from patient samples

[0217] Aim of study

[0218] Megalin mRNA expression is detectable in bulk RIMA sequencing data from mesothelioma tumors (Rasmussen et al.) but there has been no previous assessment of megalin protein expression and localization in mesothelioma. In the present study we assessed the abundance and localization of megalin protein in mesothelioma tumors.

[0219] Materials and methods

[0220] Formalin-fixed paraffin-embedded pleural (FFPE)

[0221] Sections of 2.5 jim were cut from formalin-fixed paraffin-embedded pleural (FFPE) mesothelioma tumor samples using a Leica RM2165 rotary microtome (Leica, Ballerup, Denmark), mounted on positive charged Superfrost glass slides (Thermo Scientific, Slangerup, Denmark), and dried for 1 h at 60 °C.

[0222] Antibody

[0223] See example 1. Immunohistochemical staining

[0224] Immunohistochemical staining was performed using a Ventana XT Benchmark automated staining system (Ventana Medical Systems, Roche, Tucson, AZ, USA). Heat-induced epitope retrieval was performed at pH 9. Sections were incubated with primary antibody for 30 minutes. Visualization of megalin labeled with polyclonal rabbit anti-human megalin was performed using the UltraView DAB detection kit (#760-500, Ventana Medical Systems, Roche), including an additional Ultra-Wash step. Sections were dehydrated and mounted with xylene and film using a Tissue-Tek SCA film coverslipper (Sakura Finetek, Zoeterwoude, The Netherlands). Whole slides images were captured using a Nanozoomer 1.0-HT scanner (Hamamatsu Photonics K.K., Hamamatsu City, Japan) with a magnification of 20 x. Slides were analyzed using NDP viewer (Hamamatsu Photonics K.K.).

[0225] Results

[0226] FFPE sections of 6 epithelioid mesothelioma tumors were stained with a polyclonal rabbit anti-human megalin antibody. High levels of megalin were observed in mesothelioma cells but not in adjacent normal cells (Figure 2A and Figure 2E). Mesothelioma cells appear polarized with megalin localized to the apical membrane of tumor cells facing the pleural space (Figure 2B-D and Figure 2F-H). The morphology as seen by microscopic analysis further supports a high degree of differentiation of many of the mesothelioma cells which are organized like seen for instance the in the kidney proximal tubules cells, where megalin is highly expressed. This morphology is rather different from that of normal epithelium and this may explain the fluid-secretion properties of most mesothelioma.

[0227] FFPE sections of primary invasive breast carcinoma, primary cutaneous melanoma and a melanoma brain metastasis further also identified megalin positive cancer cells (Figure 2I-K).

[0228] FFPE sections of pleural effusion cytology specimens from two patients with epithelioid mesothelioma were stained for megalin. In both cases, megalin was observed at the plasma membrane of mesothelioma cells but was not present in normal cells (Figure 3A-D). Conclusion

[0229] High levels of megalin are present on the (apical) plasma membrane of mesothelioma cells in tumors and pleural effusions, while adjacent normal stromal and immune cells are devoid of megalin. In view of its endocytic capabilities, these observations make megalin an attractive target for drug delivery in mesothelioma. Polarization of megalin to the apical membrane of mesothelioma cells facing the pleural space suggests that efficient megalin targeting can be achieved with administration to the pleural space.

[0230] Example 6 - Specific binding and uptake of anti-megalin antibodies by megalin-expressing cancer cell lines

[0231] Aim of study

[0232] To assess the binding and uptake of mouse monoclonal anti-human megalin antibodies by human cancer cells.

[0233] Materials and methods

[0234] Antibodies

[0235] Mouse monoclonal antibodies raised against ligand-affinity purified full-length human megalin of renal origin were generated using hybridoma technology. See example 1.

[0236] Antibody labeling

[0237] Alexa Fluor 647 (AF647) was conjugated to primary amines (e.g. lysines) on anti- megalin antibodies using a Lightning-Link Conjugation Kit (Abeam) per manufacturer instructions.

[0238] Cell lines

[0239] FM3 sgLRP2 generation (megalin knockout) FM3 sgLRP2 melanoma cell lines were generated by nucleofection of FM3 cells with Streptococcus pyogenes SF370 Cas9 mRNA (TriLink Biotechnologies) and sgRNA targeting the exon 17 in the megalin (LRP2) gene (Synthego).

[0240] Antibody uptake assay

[0241] Human cancer cell lines were incubated at 37°C with 1 pg / ml anti-megalin antibodies in cell culture media for 2 hours. Cells were harvested, washed, and placed on ice. Cells were stained with LIVE / DEAD™ Fixable Violet (Thermo Fisher Scientific) prior to flow cytometric analysis on a Novocyte Quanteon. At least 5000 cells were recorded for each sample. FlowJo was used for data analysis.

[0242] Imaqinq-flow cytometry

[0243] FM3 cells were incubated at 37°C with 1 pg / ml AF647-labeled anti-megalin antibody in cell culture media for 2 hours. Cells were harvested, washed and placed on ice. Cells were stained with FITC-labeled HLA-A / B / C antibody (clone W6 / 32, Biolegend) and LIVE / DEAD™ Fixable Violet (Thermo Fisher Scientific) for 1 hour on ice prior to imaging-flow cytometric analysis on a ImageStream®xMark II Imaging Flow Cytometer (Amnis, Luminex Corporation). At least 5000 cells were recorded for each sample. IDEAS software (Amnis, Luminex Corporation) was used for data analysis.

[0244] Results

[0245] To assess the cell surface binding of anti-megalin antibodies to cancer cells, we incubated human cancer cell lines with AF647-labeled anti-megalin antibodies for 2 hours and measured fluorescence by flow cytometry, megalin-expressing FM3 human melanoma cells showed marked binding of anti-megalin antibodies.

[0246] Binding was megalin-specific, as it was abolished in the corresponding megalin knockout population, FM3 sgLRP2 #3 (90% knockout efficiency), and the non- megalin expressing FM92 human melanoma cell line showed negligible binding of the antibodies (Figure 4A).

[0247] In similar experiments for human luminal invasive breast carcinoma cells, there was binding of anti-megalin antibodies to megalin-expressing MDAMB-134VI cells, but not non-megalin expressing T47D cells (Figure 4B). To demonstrate uptake of anti-megalin antibodies, we carried out similar antibody incubation experiments for FM3 and performed imaging flow cytometry. AF647- labeled anti-megalin antibodies were localized to intracellular compartments, likely endolysosomal vesicles, indicating rapid receptor-mediated endocytosis of the antibodies (Figure 5).

[0248] Conclusion

[0249] The data demonstrates the generation of specific mouse monoclonal anti-human megalin antibodies, which bind to megalin on the cell surface of cancer cells and are internalized to intracellular compartments.

[0250] Example 7 - Specific killing of megalin-expressing cancer cells by antibody drug-conjugates targeting megalin Aim of study

[0251] To assess the cell-killing ability of an anti-megalin antibody-drug conjugate towards megalin-expressing cancer cells in vitro.

[0252] Materials and methods

[0253] Antibodv-druo conjugate

[0254] To assess the cell-killing ability of anti-megalin ADCs, anti-megalin antibodies were coupled to duocarmycin, a highly potent DNA-alkylating agent, using a cathepsin-cleavable linker. Cells were cultured with a serial dilution of ADC for 6 days followed by cell viability assessment.

[0255] See also example 3.

[0256] Cell lines

[0257] FM3 human melanoma cells

[0258] - FM3 sgLRP2

[0259] - FM92

[0260] - MDAMB-134VI

[0261] See also example 5.

[0262] Cell viability assay

[0263] 1000 FM3 cells were plated in 96 well plates and allowed to adhere overnight.

[0264] Then cells were treated with a serial dilution (0, 0.03, 0.125, 0.5, 1 or 2 pg / ml) of anti-megalin-duocarmycin or an IgGl-duocarmycin control ADC for 6 days. Each condition was performed in triplicates. Cells were lysed in 100 pl CellTiterGlo 2.0 and incubated for 15 minutes prior to luminescence measurements on a Leica plate reader. Relative cell numbers were calculated compared to untreated control cells and ICso values were calculated by non-linear regression in GraphPad Prism.

[0265] Results

[0266] FM3 melanoma cell lines were sensitive to anti-megalin ADC (ICso = 0.37 pg / ml) compared to control IgGl-ADC (ICso > 2 pg / ml) (Figure 6A).

[0267] The cytotoxic effect of anti-megalin ADC on FM3 was megalin-mediated, as it was reduced in the corresponding megalin knockout cell line, FM3 sgLRP2 #3 (ICso = 1.04 pg / ml), as well as a non-megalin expressing melanoma cell line, FM92 (ICso > 2 pg / ml) (Figure 6B-D).

[0268] In similar experiments for luminal invasive breast carcinoma cell lines, there was high sensitivity of megalin-ADC in the megalin-expressing cell line MDAMB-134VI (ICso = 0.03 pg / ml) compared to the non-megalin expressing cell line T47D (ICso « 2 pg / ml) (Figure 7A-C).

[0269] Conclusion

[0270] A potent and selective killing activity of anti-megalin ADCs towards megalin- expressing cancer cell lines is demonstrated.

Claims

Claims1. A megalin-targeting antibody-drug conjugate (ADC), for use in the treatment and / or alleviation of mesothelial cancer and / or breast cancer in a subject.

2. The megalin-targeting antibody-drug conjugate (ADC) according to claim 1, for use in the treatment and / or alleviation of mesothelial cancer in a subject.

3. The megalin-targeting antibody-drug conjugate (ADC), for use according to any of the preceding claims, wherein the mesothelial cancer is a pleural mesothelial cancer.

4. The megalin-targeting antibody-drug conjugate (ADC), for use according to any of the preceding claims, being administered to the pleural cavity.

5. The megalin-targeting antibody-drug conjugate (ADC), for use according to any of the preceding claims, wherein the conjugate is administered to the pleural cavity via a pleural cavity drainage.

6. The megalin-targeting antibody-drug conjugate (ADC), for use according to any of the preceding claims, wherein the mesothelial cancer presents megalin in cell membranes, such as being exposed on the cell surface, more preferably being present in the apical / luminal membrane facing the pleural cavity.

7. The megalin-targeting antibody-drug conjugate (ADC), for use according to any of the preceding claims, wherein the pleural mesothelial cancer is an epithelioid pleural mesothelioma.

8. The megalin-targeting antibody-drug conjugate (ADC), for use according to any of the preceding claims, wherein the pleural mesothelial cancer is a fluid-secreting mesothelial cancer.

9. The megalin-targeting antibody-drug conjugate (ADC), for use according to any of the preceding claims, wherein the drug is selected from the group consisting ofa cytotoxic agent, a therapeutic agent, a chemotherapeutic agent and a radioisotope.

10. A method for determining if a mesothelial cancer from a subject is susceptible to treatment with a megalin-targeting antibody-drug conjugate (ADC), via administration to the pleural cavity, the method comprising a) determining in a mesothelial cancer sample from the subject the level of megalin protein, preferably membrane bound megalin; b) comparing said determined level of megalin protein to a reference level; and c)- determining that said cancer from a subject is susceptible to treatment with a megalin targeting antibody-drug conjugate (ADC), when said determined level is equal to or above said reference level, or- determining that said cancer from a subject is not susceptible to treatment with a megalin targeting antibody-drug conjugate (ADC), when said determined level is below said reference level; wherein the reference level is based on megalin levels from one or more corresponding samples from healthy subjects, or from neighboring corresponding healthy samples from the same subject; wherein the reference level is determined using immunohistochemical staining; and wherein the level of megalin protein determined in step a) is the level in the apical / luminal membrane facing the pleural cavity.

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