Anti-megalin antibody-drug conjugate, for use in the treatment of melanoma in a subject, wherein said subject has previously been treated with IFN(GAMMA) and / or an IFN(GAMMA)-inducing compound

Abstract

The present invention relates to an anti-megalin antibody-drug conjugate (ADC), for use in the treatment and / or alleviation of cancer, preferably melanoma, in a subject, wherein said subject has previously been or is being treated with IFN(γ) and / or an IFN(γ)-inducing compound or therapy, such as immunotherapy. The present invention also relates to IFNγ and / or an IFNγ-inducing therapy for use as a pretreatment of a subject suffering from cancer, preferably melanoma, wherein said subject is scheduled for subsequent treatment with an anti-megalin antibody-drug conjugate (ADC) for use in the treatment and / or alleviation of cancer in the subject.

Description

[0001] PV ref: 85386PC01

[0002] 1

[0003] ANTI-MEGALIN ANTIBODY-DRUG CONJUGATE, FOR USE IN THE TREATMENT OF CANCER IN A SUBJECT, WHEREIN SAID SUBJECT HAS PREVIOUSLY BEEN TREATED WITH IFN(y) AND / OR AN IFN(Y)-INDUCING COMPOUND

[0004] Technical field of the invention

[0005] The present invention relates to an anti-megalin antibody-drug conjugate (ADC), for use in the treatment and / or alleviation of cancer, preferably melanoma, in a subject, wherein said subject has previously been or is being treated with IFN(y) and / or an IFN(y)-inducing compound or therapy, such as immunotherapy. The present invention also relates to IFNy and / or an IFNy-inducing therapy for use as a pretreatment of a subject suffering from cancer, preferably melanoma, wherein said subject is scheduled for subsequent treatment with an anti-megalin antibodydrug conjugate (ADC) for use in the treatment and / or alleviation of cancer in the subject.

[0006] Background of the invention

[0007] Megalin (gene name LRP2) is a 600 kilodalton multi-ligand endocytic membrane receptor expressed in several cell types during fetal development, including neuroepithelial cells, and in select absorptive epithelial cells in the adult.

[0008] In epithelial cancers, megalin expression is associated with a differentiated tumor cell state and better prognosis.

[0009] Andersen et al. (Melanoma tumors frequently acquire LRP2 / megalin expression, which modulates melanoma cell proliferation and survival rates. Pigment Cell Melanoma Res. 28; 267-280) discloses that the multiligand receptor megalin is more frequently expressed among malignant melanoma samples compared to benign counterparts.

[0010] Hence, an improved treatment of melanoma would be advantageous.

[0011] Summary of the invention

[0012] The present invention is based on the realization that inflammation, in particular IFNy, can increase megalin expression in melanoma cells. IFNy is a proinflammatory cytokine produced by activated T cells and NK cells. IFNy is therefore produced in the tumor microenvironment, and its levels dramaticallyPV ref: 85386PC01

[0013] 2

[0014] increased in response to cancer immunotherapy (e.g. immune checkpoint therapy, adoptive T cell transfer, CAR-T cell therapy, CAR-NK cell therapy).

[0015] Thus, the concept of the present invention is to combine megalin-targeting cancer therapies with immunotherapies in order to enhance megalin expression on cancer cells and thereby improve megalin-targeted drug delivery.

[0016] Thus, an object of the present invention relates to the provision of an improved treatment of melanoma.

[0017] In particular, it is an object of the present invention to provide an improved combinatorial treatment of melanoma.

[0018] Example 1 shows that IFNy exposure increases megalin expression in cancer cells both short-term and long-term.

[0019] Example 2 shows that megalin is positively correlated with T cell inflammation and IFNy signaling in melanoma.

[0020] Example 3 shows that megalin is upregulated in melanoma metastases from patients with disease control following immune checkpoint blockade - making megalin an interesting target for treating metastatic melanoma.

[0021] Example 4 shows that IFNy exposure increased the uptake of megalin-targeting antibodies by cancer cells.

[0022] Example 5 shows synthesis of antibody-drug conjugates.

[0023] Example 6 shows an increased cell-killing effect of anti-megalin ADCs towards megalin-expressing cancer cells following IFNy treatment.

[0024] Example 7 provides the sequences of the murine monoclonal IgG antibody "Megalin-41" including CDR sequences.

[0025] Thus, one aspect of the invention relates to an anti-megalin antibody-drug conjugate (ADC), for use in the treatment and / or alleviation of cancer, preferably melanoma, in a subject;

[0026] wherein said subject has previously been or is being treated with IFN(y) and / or an IFN(y)-inducing compound or therapy, such as immunotherapy.PV ref: 85386PC01

[0027] 3

[0028] Another aspect of the present invention relates to IFNy and / or an IFNy-inducing therapy for use as a pretreatment of a subject suffering from cancer, preferably melanoma;

[0029] wherein said subject is scheduled for subsequent treatment with an anti-megalin antibody-drug conjugate (ADC) for use in the treatment and / or alleviation of the cancer in the subject.

[0030] Yet another aspect of the present invention is to provide a kit or combination comprising

[0031] - IFNy and / or an IFNy-inducing compound or therapy, such as an IFNy- inducing immunotherapy; and

[0032] - an anti-megalin antibody-drug conjugate (ADC).

[0033] Brief description of the figures

[0034] Figure 1

[0035] Figure 1 shows IFNy exposure increases megalin expression in cancer cells. A) Bar plot of relative megalin mRNA expression determined by RT-qPCR in the indicated melanoma cell lines treated with vehicle control (circles) or IFNy (squares) for 48 hours (multiple unpaired t-tests). Mean (bars) ± SEM (error bars) and individual values (circles or squares) are shown (n = 3 technical replicates). Megalin mRNA relative quantities for A375 in the IFNy treated condition are set to 1 as megalin mRNA was not detected in the vehicle control. B-C) Whole-cell lysate immunoblots of megalin (upper panel) and clathrin loading control (lower panel) for the indicated melanoma cell lines treated with vehicle control or IFNy for 48 hours. D) Relative megalin mRNA expression) determined by RT-qPCR during a 24-hour time course in cells treated with vehicle control (circles) or IFNy (squares). Mean (circles or squares) ± SEM (error bars) are shown (n = 3 technical replicates). No error bars are displayed when the error is smaller than the size of the bar. E) Bar plot of megalin mRNA expression (relative quantities) determined by RT-qPCR during a 5-week time course (n = 3 technical replicates) of IFNy exposure. Mean (bars) ± SEM (error bars) shown (n = 3 technical replicates). **P < 0.01, ***p < 0.001. ND = not detected.PV ref: 85386PC01

[0036] 4

[0037] Figure 2

[0038] Figure 2 shows Megalin expression correlates with T cell inflammation and IFNy signalling in melanoma.

[0039] A) Volcano plot of gene set enrichment analysis results for megalin mRNA correlated genes in TCGA SKCM. NES = Normalized Enrichment Score. Gene sets of interest are highlighted with text. B) Mountain plot of the IFNy response gene set for megalin mRNA correlated genes in TCGA SKCM. NES = Normalized Enrichment Score. C) Boxplot of megalin mRNA expression (log2(RSEM norm_count+l) in T cell inflammation-high and T cell inflammation-low groups in TCGA SKCM (unpaired t-test). D) Boxplot of megalin protein expression in melanoma cells (SOX10+ area fraction) in T cell inflammation-high and T cell inflammation-low groups from multiplex immunohistochemistry analysis of primary melanomas (Mann-Whitney test). The boxplot lines represent the lower quartile, median, and upper quartile. Whiskers extend to 1.5 times above or below the interquartile range. *P < 0.05, ****P < 0.0001.

[0040] Figure 3

[0041] Figure 3 shows transcriptomic data indicating that megalin is upregulated in melanoma metastases from patients with disease control following immune checkpoint blockade. Dot plots of megalin mRNA expression determined by bulk RNA sequencing in paired pre-therapy and on-therapy melanoma metastases samples from patients with disease progression (panel A) or disease control (panel B) following immune checkpoint blockade. Statistics from paired t tests are shown. PD = progressive disease. SD = stable disease. PR = partial response. CR = complete response. *P < 0.05. ns = not significant.

[0042] Figure 4

[0043] Figure 4 shows IFNy exposure increases the uptake of megalin-targeting antibodies by cancer cells. Bar plot of median fluorescent signal (MFI) for FM3 melanoma cell lines treated with vehicle control or IFNy and then incubated for 2 hours with AF647-labeled anti-megalin antibody to measure antibody binding and uptake. MFI = Median fluorescent signal. AF647 = AlexaFluor 647.

[0044] Figure 5

[0045] Figure 5 shows that IFNy increases killing of megalin-expressing cancer cells by antibody-drug conjugates targeting megalin.PV ref: 85386PC01

[0046] 5

[0047] Bar plot of cell number (relative) for FM3 melanoma cells (panel A) or the corresponding megalin-knockout cells, FM3 sgLRP2 #3, (panel B) that have been pre-treated with IFNy or vehicle for 24 hours, followed by treatment with indicated ADC for 96 hours, while IFNy treatment was maintained. Statistical analysis was performed using multiple unpaired t-tests with Holm-Sidak's multiple comparison correction, ns = not significant, * P < 0.05.

[0048] The present invention will now be described in more detail in the following.

[0049] Detailed description of the invention

[0050] Definitions

[0051] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

[0052] LRP2 / Meaalin

[0053] 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.

[0054] In the present context, the terms "LRP2" and "megalin" are used interchangeably. Other well-known synonyms for LRP2 / megalin are "glycoprotein-330" and "gp330".

[0055] Antibody

[0056] 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.

[0057] Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen. An antigen is aPV ref: 85386PC01

[0058] 6

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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).PV ref: 85386PC01

[0064] 7

[0065] 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.

[0066] 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).

[0067] 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.

[0068] 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).

[0069] 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.

[0070] 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.

[0071] Megalin antibodies may be raised against full-length megalin as shown in the example section (see example 4).PV ref: 85386PC01

[0072] 8

[0073] In embodiments of the invention the antibody is humanized.

[0074] Antibody-drug conjugates (ADCs)

[0075] 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.

[0076] ADCs are complex molecules composed of an antibody linked to a biologically active cytotoxic (anticancer) payload or drug.

[0077] ADCs combine the targeting properties of monoclonal antibodies with the cancerkilling capabilities of cytotoxic drugs, designed to discriminate between healthy and diseased tissue.

[0078] Melanoma

[0079] As used herein, "melanoma" refers to a condition characterized by the growth of a tumor arising from the melanocytic system of the skin and other organs. Most melanocytes occur in the skin, but are also found in the meninges, digestive tract, lymph nodes and eyes. When melanoma occurs in the skin, it is referred to as cutaneous melanoma. Melanoma can also occur in the eyes and is called ocular or intraocular melanoma. Melanoma occurs rarely in the meninges, the digestive tract, lymph nodes or other areas where melanocytes are found. 40-60 % of melanomas carry an activating mutation in the gene encoding the serinethreonine protein kinase B-RAF (BRAF). Among the BRAF mutations observed in melanoma, over 90 % are at codon 600, and among these, over 90 % are a single nucleotide mutation resulting in substitution of glutamic acid for valine (BRAFV600E).

[0080] In some embodiments, the subject suffers from melanoma.

[0081] Treatment

[0082] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The term "treatment" as used herein also includes administration to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolongPV ref: 85386PC01

[0083] 9

[0084] the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0085] Immune checkpoint inhibitor

[0086] As used herein, the term "immune checkpoint inhibitor" has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein. As used herein the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480-489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. Inhibition includes reduction of function and full blockade. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint inhibitors are known and in analogy of thesePV ref: 85386PC01

[0087] 10

[0088] known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future. The immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. Examples of immune checkpoint inhibitor includes PD-1 antagonist, PD-L1 antagonist, PD-L2 antagonist CTLA-4 antagonist, VISTA antagonist, TIM-3 antagonist, LAG-3 antagonist, IDO antagonist, KIR2D antagonist, A2AR antagonist, B7-H3 antagonist, B7-H4 antagonist, and BTLA antagonist.

[0089] Thus, in an embodiment, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 antagonist, a PD-L1 antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a VISTA antagonist, a TIM-3 antagonist, a LAG-3 antagonist, an IDO antagonist, a KIR2D antagonist, an A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist and combinations thereof.

[0090] Combination

[0091] As used the terms "combination" and "combination therapy" are interchangeable and refer to treatments comprising the administration of at least two compounds or therapies administered simultaneously, separately or sequentially. As used herein the term "co-administering" as used herein means a process whereby the combination of at least two compounds is administered to the same patient. The at least two compounds may be administered simultaneously, at essentially the same time, or sequentially. The at least two compounds can be administered separately by means of different vehicles or composition. The at least two compounds can also be administered in the same vehicle or composition (e.g. pharmaceutical composition). The at least two compounds may be administered one or more times and the number of administrations of each component of the combination may be the same or different.

[0092] Such combination therapies, in which administration of one or more antibodies of the invention is suitably (meaning, in suitable doses according to a suitable dosage regimen) combined with administration of one or more other active principles, depending on the disease or disorder to be treated. A combination therapy will usually at least provide an additive effect. It is also within the scope of the invention that the use of a combination or combination therapy may result in a synergistic effect.PV ref: 85386PC01

[0093] 11

[0094] Anti-megalin antibody-drug conjugates (ADCs), for use in the treatment and / or alleviation of cancer

[0095] As outlined in the example section, the inventing team has realized that inflammation, in particular IFNy, can increase megalin expression in melanoma cells. IFNy is a proinflammatory cytokine produced by activated T cells and NK cells. IFNy is therefore produced in the tumor microenvironment, and its levels dramatically increased in response to cancer immunotherapy (e.g. immune checkpoint therapy, adoptive T cell transfer, CAR-T cell therapy, CAR-NK cell therapy). Thus, the concept of the present invention is to combine megalin-targeting cancer therapies with immunotherapies in order to enhance megalin expression on cancer cells and thereby improve megalin-targeted drug delivery. Thus, an aspect of the invention relates to an anti-megalin antibody-drug conjugate (ADC), for use in the treatment and / or alleviation of cancer, preferably melanoma, in a subject;

[0096] wherein said subject has previously been or is being treated with IFN(y) and / or an IFN(y)-inducing compound or therapy, such as immunotherapy.

[0097] The treatment described may find use for different types of cancers. Thus, in an embodiment, the cancer is selected from the group consisting of melanoma, mesothelial cancer, breast cancer, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, liver cancer, kidney cancer, thyroid cancer and ovarian cancer.

[0098] In the example section (examples 1-4), data is provided in relation to melanoma. Thus, in an embodiment, the cancer is melanoma.

[0099] In another embodiment, the cancer presents megalin in cell membranes, such as being exposed on the cell surface, at a higher level than before the treatment with IFN(y) and / or an IFN(y)-inducing compound or therapy.

[0100] In yet an embodiment, the anti-megalin antibody and the drug is conjugated via a linker, such as a cathepsin degradable linker.

[0101] 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-PV ref: 85386PC01

[0102] 12

[0103] aminobenzyl carbamate (ValCitPABC), and GGFG Tetrapeptidyl-Aminomethoxy Linker.

[0104] In a further embodiment, the anti-megalin antibody binds to an extracellular epitope of megalin.

[0105] Different kinds of antibodies may be used. Thus, in an embodiment, the anti-megalin antibody is selected from the group consisting of, monoclonal, polyclonal, monospecific polyclonal, recombinant, chimeric, humanized, fully human, single chain and bispecific antibodies.

[0106] In a further embodiment, anti-megalin antibody has been raised against full-length megalin.

[0107] In yet an embodiment, the anti-megalin antibody is humanized, preferably a humanized monoclonal antibody.

[0108] In yet a further embodiment, the anti-megalin antibody-drug conjugate (ADC) is specific for megalin.

[0109] In yet another embodiment, the antibody is an antibody according to the present invention.

[0110] Different kind of drugs may form part of the conjugate. Thus, in an embodiment, the drug is selected from the group consisting of a cytotoxic agent, a therapeutic agent, a chemotherapeutic agent and a radioisotope.

[0111] In a related 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; aPV ref: 85386PC01

[0112] 13

[0113] 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; diphtherin toxin; and Pseudomonas endotoxin.

[0114] In yet a related 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.

[0115] In an embodiment, the subject is a mammal, preferably a human.

[0116] IFNy and / or an IFNy-inducing therapy for use as a pretreatment of a subject suffering from cancer

[0117] IFNy and / or an IFNy-inducing therapy may be used as a pretreatment of a subject suffering from cancer, when the subject is scheduled for subsequent treatment with an anti-megalin antibody-drug conjugate (ADC). Thus, another aspect of thePV ref: 85386PC01

[0118] 14

[0119] invention relates to IFNy and / or an IFNy-inducing therapy for use as a pretreatment of a subject suffering from cancer, preferably melanoma; wherein said subject is scheduled for subsequent treatment with an anti-megalin antibody-drug conjugate (ADC) for use in the treatment and / or alleviation of the cancer in the subject.

[0120] In an embodiment, the IFNy and / or an IFNy-inducing therapy is administered to the subject simultaneously with the ADC or at the most 7 days before the ADC is administered to the subject, such as being administered 1 hour to 3 days before the ADC is administered to the subject.

[0121] In another embodiment, IFNy and / or an IFNy-inducing therapy is administered to the subject at the most 90 days before the ADC is administered to the subject, such as being administered at the most 60 days before the ADC is administered to the subject, preferably at the most 30 days, such as at the most 14 days. For example, "time to response" in case of immunotherapy is typically a couple of months. A median time to response of 2.8 months is described in Hamid et al. (Ann Oncol. 2019 Jan 31;30(4):582-588).

[0122] In an embodiment, the cancer is selected from the group consisting of melanoma, mesothelial cancer, breast cancer, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, liver cancer, kidney cancer, thyroid cancer and ovarian cancer.

[0123] In yet an embodiment, the cancer is melanoma.

[0124] Kit and combination

[0125] Since the present invention relates to a treatment protocol involving two independent components, such invention may also relate a kit or a combination. Thus, an aspect of the invention relates to a kit or combination comprising

[0126] - IFNy and / or an IFNy-inducing compound or therapy, such as an IFNy- inducing immunotherapy; and

[0127] - an anti-megalin antibody-drug conjugate (ADC).

[0128] In an embodiment, the kit or combination is for use in the treatment or alleviation of cancer in the subject, preferably melanoma.PV ref: 85386PC01

[0129] 15

[0130] In an embodiment, IFNy and / or an IFNy-inducing compound is to be administered to the subject simultaneously with the anti-megalin antibody-drug conjugate (ADC) or before the anti-megalin antibody-drug conjugate (ADC).

[0131] In yet another embodiment relating to:

[0132] - The anti-megalin antibody-drug conjugate (ADC), for use according to the invention,

[0133] - the IFNy and / or an IFNy-inducing compound or therapy for use according to the invention; and / or

[0134] - the kit or combination according to the invention.

[0135] In an embodiment, the IFNy-inducing therapy is selected from the group consisting of immune checkpoint inhibitors (such as anti-PD-(L)l, anti-CLTA-4, anti-LAG-3), cellular immunotherapy (such as tumor infiltrating lymphocyte therapy, T cell receptor (TCR)-engineered T cell therapy, CAR-T cell therapy, CAR-NK cell therapy), cytokine-based therapies (such as interleukin-2 therapies), bispecific T-cell engagers (such as targeting CD3 and tumor antigens), cancer vaccines (such as neoantigen-based personalized vaccines), oncolytic viral therapies with immunostimulatory effects (such as talimogene laherparepvec (T-VEC)).

[0136] In a further embodiment relating to:

[0137] - The anti-megalin antibody-drug conjugate (ADC), for use according to the invention,

[0138] - the IFNy and / or an IFNy-inducing compound or therapy for use according to the invention; and / or

[0139] - the kit or combination according to the invention.

[0140] In a preferred embodiment, the cancer is melanoma and the melanoma is a malignant melanoma or a benign melanoma

[0141] and / or

[0142] the melanoma is selected from the group consisting of cutaneous melanoma, acral melanoma, mucosal melanoma, uveal melanoma, and metastatic melanoma.PV ref: 85386PC01

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[0144] Antibodies

[0145] An aspect of the invention relates to an antibody targeting megalin comprising o a light chain variable region comprising

[0146] ■ a CDR 1 region according to SEQ ID NO: 3;

[0147] ■ a CDR 2 region according to SEQ ID NO: 4; and

[0148] ■ a CDR 3 region according to SEQ ID NO: 5;

[0149] o a heavy chain variable region comprising

[0150] ■ a CDR 1 region according to SEQ ID NO: 6;

[0151] ■ a CDR 2 region according to SEQ ID NO: 7; and

[0152] ■ a CDR 3 region according to SEQ ID NO: 8.

[0153] An aspect of the invention relates to an antibody targeting megalin comprising • a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1, or sequences having at least 90% sequence identity, such as at least 95% sequence identity to SEQ ID NO: 1;

[0154] o the light chain variable region comprising

[0155] ■ a CDR 1 region according to SEQ ID NO: 3;

[0156] ■ a CDR 2 region according to SEQ ID NO: 4; and

[0157] ■ a CDR 3 region according to SEQ ID NO: 5;

[0158] • a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2, or sequences having at least 90% sequence identity, such as at least 95% sequence identity to SEQ ID NO: 2;

[0159] o the heavy chain variable region comprising

[0160] ■ a CDR 1 region according to SEQ ID NO: 6;

[0161] ■ a CDR 2 region according to SEQ ID NO: 7; and

[0162] ■ a CDR 3 region according to SEQ ID NO: 8.

[0163] In an embodiment, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2.

[0164] In an embodiment, the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, an antibody, wherein the heavy chain and the light chain are connected by a flexible linker, an Fv molecule, an antigenPV ref: 85386PC01

[0165] 17

[0166] binding fragment, a Fab fragment, a Fab' fragment, a F(ab')? molecule, a fully human antibody, a humanized antibody, and a chimeric antibody.

[0167] In an embodiment, is a F(ab')2 molecule.

[0168] In a preferred embodiment, the antibody is humanized, preferably a humanized monoclonal antibody.

[0169] In another preferred embodiment, the antibody is coupled to a detectable label or a substance having toxic or therapeutic activity.

[0170] Vector

[0171] An aspect of the invention relates to a vector or vectors encoding the antibody targeting megalin according to the invention. In one embodiment, LC and HC are encoded on the same vector. In another embodiment, the LC is expressed from one vector and the HC is expressed from another vector.

[0172] Cell

[0173] Yet an aspect of the invention relates to a cell expressing the antibody targeting megalin according to the invention, and / or a cell comprising the vector or vectors according to the invention.

[0174] Composition

[0175] The antibodies according to the invention may form part of a composition. Thus, a further aspect relates to a composition comprising the antibody targeting megalin according to the invention, and one or more physiologically acceptable carriers, excipients and / or diluents.

[0176] In an embodiment, the composition is a pharmaceutical composition.

[0177] Other aspects

[0178] Yet another aspect relates to a method for the treatment and / or alleviation of cancer, preferably melanoma, in a subject, the method comprising administering to said subject a IFNy and / or an IFNy-inducing compound or therapy, such as anPV ref: 85386PC01

[0179] 18

[0180] IFNy-inducing immunotherapy, before or simultaneously with an anti-megalin antibody-drug conjugate (ADC).

[0181] Items of the invention

[0182] 1. An anti-megalin antibody-drug conjugate (ADC), for use in the treatment and / or alleviation of cancer, preferably melanoma, in a subject;

[0183] wherein said subject has previously been or is being treated with IFN(y) and / or an IFN(y)-inducing compound or therapy, such as immunotherapy.

[0184] 2. The anti-megalin antibody-drug conjugate (ADC), for use according to item 1, wherein the cancer is selected from the group consisting of melanoma, mesothelial cancer, breast cancer, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, liver cancer, kidney cancer, thyroid cancer and ovarian cancer.

[0185] 3. The anti-megalin antibody-drug conjugate (ADC), for use according to item 1 or 2, wherein the cancer is melanoma.

[0186] 4. The anti-megalin antibody-drug conjugate (ADC), 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, at a higher level than before the treatment with IFN(y) and / or an IFN(y)-inducing compound or therapy.

[0187] 5. The anti-megalin antibody-drug conjugate (ADC) for use according to any of the preceding items, wherein the anti-megalin antibody and the drug is conjugated via a linker, such as a cathepsin degradable linker.

[0188] 6. The anti-megalin antibody-drug conjugate (ADC) for use according to item 5, 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.

[0189] 7. The anti-megalin antibody-drug conjugate (ADC) for use according to any of the preceding items, wherein the anti-megalin antibody binds to an extracellular epitope of megalin.PV ref: 85386PC01

[0190] 19

[0191] 8. The anti-megalin antibody-drug conjugate (ADC) for use according to any of the preceding items, wherein the anti-megalin antibody is selected from the group consisting of, monoclonal, polyclonal, monospecific polyclonal, recombinant, chimeric, humanized, fully human, single chain and bispecific antibodies.

[0192] 9. The anti-megalin antibody-drug conjugate (ADC) for use according to any of the preceding items, wherein anti-megalin antibody has been raised against full-length megalin.

[0193] 10. The anti-megalin antibody-drug conjugate (ADC) for use according to any of the preceding items, wherein the anti-megalin antibody is humanized, preferably a humanized monoclonal antibody.

[0194] 11. The anti-megalin antibody-drug conjugate (ADC) for use according to any of the preceding items, being specific for megalin.

[0195] 12. The anti-megalin antibody-drug conjugate (ADC) 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.

[0196] 13. The anti-megalin antibody-drug conjugate (ADC) for use according to item 12, 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),PV ref: 85386PC01

[0197] 20

[0198] 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.

[0199] 14. The anti-megalin antibody-drug conjugate (ADC) for use according to any of the preceding items, 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.

[0200] 15. The anti-megalin antibody-drug conjugate (ADC) for use according to any of the preceding items, wherein the subject is a mammal, preferably a human.

[0201] 16. IFNy and / or an IFNy-inducing therapy for use as a pretreatment of a subject suffering from cancer, preferably melanoma;

[0202] wherein said subject is scheduled for subsequent treatment with an anti-megalin antibody-drug conjugate (ADC) for use in the treatment and / or alleviation of the cancer in the subject.

[0203] 17. The IFNy and / or an IFNy-inducing therapy for use according to item 16, wherein the IFNy and / or an IFNy-inducing therapy is administered to the subjectPV ref: 85386PC01

[0204] 21

[0205] simultaneously with the ADC or at the most 7 days before the ADC is administered to the subject, such as being administered 1 hour to 3 days before the ADC is administered to the subject.

[0206] 18. The IFNy and / or an IFNy-inducing therapy for use according to item 16 or 17, wherein the cancer is selected from the group consisting of melanoma, mesothelial cancer, breast cancer, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, liver cancer, kidney cancer, thyroid cancer and ovarian cancer.

[0207] 19. The IFNy and / or an IFNy-inducing therapy for use according to any of items 16-18, wherein the cancer is melanoma.

[0208] 20. A kit or combination comprising

[0209] - IFNy and / or an IFNy-inducing compound or therapy, such as an IFNy- inducing immunotherapy; and

[0210] - an anti-megalin antibody-drug conjugate (ADC).

[0211] 21. The kit or combination according to item 20, for use in the treatment or alleviation of cancer in the subject, preferably melanoma.

[0212] 22. The kit or combination for use according to item 20 or 21, wherein IFNy and / or an IFNy-inducing compound is to be administered to the subject simultaneously with the anti-megalin antibody-drug conjugate (ADC) or before the anti-megalin antibody-drug conjugate (ADC).

[0213] 23.

[0214] - The anti-megalin antibody-drug conjugate (ADC), for use according to any of items 1-15,

[0215] - the IFNy and / or an IFNy-inducing compound or therapy for use according to any of items 16-19; and / or

[0216] - the kit or combination according to any of items 20-22;

[0217] wherein the IFNy-inducing therapy is selected from the group consisting of immune checkpoint inhibitors, such as anti-PD-(L)l, anti-CLTA-4, anti-LAG-3, cellular immunotherapy, such as tumor infiltrating lymphocyte therapy, T cellPV ref: 85386PC01

[0218] 22

[0219] receptor (TCR)-engineered T cell therapy, CAR-T cell therapy, CAR-NK cell therapy), cytokine-based therapies, such as interleukin-2 therapies, bispecific T-cell engagers such as targeting CD3 and tumor antigens, cancer vaccines, such as neoantigen-based personalized vaccines, oncolytic viral therapies with immunostimulatory effects, such as talimogene laherparepvec (T-VEC).

[0220] 24.

[0221] - The anti-megalin antibody-drug conjugate (ADC), for use according to any of items 1-15,

[0222] - the IFNy and / or an IFNy-inducing compound or therapy for use according to any of items 16-19; and / or

[0223] - the kit or combination according to any of items 20-22;

[0224] wherein the cancer is melanoma and the melanoma is a malignant melanoma or a benign melanoma

[0225] and / or

[0226] wherein the melanoma is selected from the group consisting of cutaneous melanoma, acral melanoma, mucosal melanoma, uveal melanoma, and metastatic melanoma.

[0227] 25. A method for the treatment and / or alleviation of cancer, preferably melanoma, in a subject, the method comprising administering to said subject a IFNy and / or an IFNy-inducing compound or therapy, such as an IFNy-inducing immunotherapy, before or simultaneously with an anti-megalin antibody-drug conjugate (ADC).

[0228] 26. An antibody targeting megalin comprising

[0229] o the light chain variable region comprising

[0230] ■ a CDR 1 region according to SEQ ID NO: 3;

[0231] ■ a CDR 2 region according to SEQ ID NO: 4; and

[0232] ■ a CDR 3 region according to SEQ ID NO: 5;

[0233] o the heavy chain variable region comprising

[0234] ■ a CDR 1 region according to SEQ ID NO: 6;

[0235] ■ a CDR 2 region according to SEQ ID NO: 7; and

[0236] ■ a CDR 3 region according to SEQ ID NO: 8.PV ref: 85386PC01

[0237] 23

[0238] 27. The antibody targeting megalin according to item 26 comprising

[0239] • a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1, or sequences having at least 90% sequence identity, such as at least 95% sequence identity to SEQ ID NO: 1;

[0240] o the light chain variable region comprising

[0241] ■ a CDR 1 region according to SEQ ID NO: 3;

[0242] ■ a CDR 2 region according to SEQ ID NO: 4; and

[0243] ■ a CDR 3 region according to SEQ ID NO: 5;

[0244] • a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2, or sequences having at least 90% sequence identity, such as at least 95% sequence identity to SEQ ID NO: 2;

[0245] o the heavy chain variable region comprising

[0246] ■ a CDR 1 region according to SEQ ID NO: 6;

[0247] ■ a CDR 2 region according to SEQ ID NO: 7; and

[0248] ■ a CDR 3 region according to SEQ ID NO: 8.

[0249] 28. The antibody targeting megalin according to item 26 or 27, wherein the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2.

[0250] 29. The antibody targeting megalin according to any of items 26-28, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, an antibody, wherein the heavy chain and the light chain are connected by a flexible linker, an Fv molecule, an antigen binding fragment, a Fab fragment, a Fab' fragment, a F(ab')2 molecule, a fully human antibody, a humanized antibody, and a chimeric antibody.

[0251] 30. The antibody targeting megalin according to any of items 26-29, wherein the antibody is a F(ab')2 molecule.

[0252] 31. The antibody targeting megalin according to any of items 26-30, the antibody is humanized, preferably a humanized monoclonal antibody.PV ref: 85386PC01

[0253] 24

[0254] 32. The antibody targeting megalin according to any of items 26-31, wherein the antibody is coupled to a detectable label or a substance having toxic or therapeutic activity.

[0255] 33. A vector or vectors encoding the antibody targeting megalin according to any of items 26-32, such as wherein the light chain (LC) and the heavy chain (HC) are encoded on the same vector or such as wherein the LC is expressed from one vector and the HC is expressed from another vector.

[0256] 34. A cell expressing the antibody targeting megalin according to any of items 26-32, and / or a cell comprising the vector or vectors according to item 33.

[0257] 35. A composition comprising the antibody targeting megalin according to any of items 26-32, and one or more physiologically acceptable carriers, excipients and / or diluents.

[0258] 36. The composition according to item 35, wherein the composition is a pharmaceutical composition.

[0259] 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.

[0260] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0261] The invention will now be described in further details in the following non-limiting examples.

[0262] Examples

[0263] Example 1 - IFNy exposure increases megalin expression in cancer cells.

[0264] Aim of study

[0265] To assess the impact of IFNy exposure on megalin expression in cancer cells.

[0266] Materials and methodsPV ref: 85386PC01

[0267] 25

[0268] Cell culture

[0269] Human melanoma cell lines were cultured under standard conditions in a humidified incubator with 5% CO2 at 37 °C. FM3, FM88, SKMEL28, WM2664 and WM115 were cultured in RPMI1640 (Sigma Aldrich) and A375 and RPMI7951 were cultured in DMEM (Gibco). Cell culture media was supplemented with 10% FBS (Gibco) and 100 units / mL penicillin and streptomycin (Sigma Aldrich).

[0270] IFNy stimulation

[0271] Cells were plated and cultured for 24 hours prior to IFNy stimulation. Recombinant human IFNy (PeproTech) was used at 50 ng / mL for 24-hour time course and 48-hour stimulation experiments and at 25 ng / mL for 5-week time course experiments. Cell culture medium was replaced with fresh medium containing IFNy every 2-3 days for the 5-week time course experiments.

[0272] Quantification of meqalin mRNA expression by real-time quantitative polymerase chain reaction (RT-qPCR)

[0273] Cells were washed in PBS and lysed with RLT buffer (Qiagen). RNA was purified using the RNeasy Mini Kit (Qiagen) following the manufacturers protocol with on-column DNase digestion and eluted in nuclease-free water. Purified RNA concentration was measured on a Nanodrop 100 Spectrophotometer (Thermo Fisher Scientific). For all samples, a total of 1440 ng RNA in a 20 pl reaction volume was used for cDNA synthesis. cDNA synthesis mixtures were prepared using the High Capacity RNA-to-cDNA kit (Applied Biosystems) and cDNA synthesis (37 °C for 1 hour) and denaturation (95 °C for 5 minutes) steps were run on a Verity Thermal Cycler (Applied Biosystems). RT-qPCR was performed using TaqMan Fast Advanced Master Mix and assays (Life Technologies) according to the manufacturers protocol. RT-qPCR was run on a QuantStudio Real-Time PCR system (Thermo Fisher Scientific). Cycle threshold (Ct) values were determined using a baseline-subtracted normalized reporter value (ARn) of 0.4 for all genes. Ct values above 36 were considered as not detected. ACt values were calculated as the difference between the geometric mean of Ct values between the gene of interest and the reference gene CASC3 (Christensen et al., 2020). Relative quantification (RQ) values were calculated using the formula 2AACt. Ct values for GAPDH measured on a stock preparation of cDNA from FM88 were used as an interplate control. Taqman probes used in this study: CASC3 (Hs00201226_ml), LRP2 (Hs00189742_ml) and GAPDH (Hs02758991_gl).PV ref: 85386PC01

[0274] 26

[0275] Quantification of mecjalin protein expression by immunoblottincj

[0276] Cell pellets were lysed in "membrane buffer" (20 mM CaCh, 10 mM MgCh, 100 mM HEPES, 1400 mM NaCI, pH 7.8) with 1% Triton X-100 (Merck) and complete Mini Protease Inhibitor Cocktail for 15 minutes on ice. Cell lysates were centrifuged at 13.000 rpm for 15 minutes at 4 °C and supernatant was collected. Total protein concentration was determined using the Pierce™ BCA Protein Assay kit (Thermo Scientific). 5% SDS sample buffer was added to samples which were then boiled for 5 minutes. Protein samples were loaded onto non-reducing NuPAGE™ 3-8% Tris-Acetate gels (Invitrogen) along with SeeBlue Plus Pre-stained Protein Standard and run at 200 V for 75 minutes. Protein blotting onto a polyvinylidene difluoride (PVDF) membrane was performed overnight at 4 °C using a constant current of 100 mA in a Mini Tank Transfer Unit (GE Healthcare Life Sciences). Following blotting, PVDF membranes were blocked in lx Tris- buffered saline with 2% Tween® 20 (Sigma Aldrich) (TBST) overnight at 4 °C. Primary antibodies were diluted in 5 % w / v nonfat dry milk, and incubation was performed for two hours at room temperature. Following washing, incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (SIGMA) was performed for one hour at room temperature. Pierce ECL Western Blotting Substrate (Thermo Scientific) was added and protein bands were visualized on a LAS-3000 Imaging System (FujiFilm). Primary antibodies were mouse monoclonal anti-human megalin (Rasmussen et al., 2023) and mouse monoclonal anti-clathrin (from Linton M. Traub) (Nathke et al., 1992).

[0277] Results

[0278] IFNy exposure increased megalin mRNA expression (Figure 1A) and megalin protein expression (Figure 1B-C) in melanoma cell lines. IFNy exposure triggered a rapid increase in megalin mRNA levels (Figure ID). Long-term IFNy exposure demonstrated that increased megalin mRNA levels were sustained for at least 5 weeks (Figure IE).

[0279] Conclusion

[0280] IFNy exposure increases megalin expression in cancer cells both short-term and long-term.

[0281] Example 2 - Megalin expression correlates with T cell inflammation and IFNy signalling in melanoma

[0282] Aim of studyPV ref: 85386PC01

[0283] 27

[0284] To assess the correlation between megalin expression and inflammatory signaling in cancer, exemplified by melanoma.

[0285] Materials and methods

[0286] The Cancer Genome Atlas

[0287] TCGA data was downloaded from the UCSC Xena data portal (https: / / xena.ucsc.edu). TCGA RIMA sequencing data were processed using the Toil pipeline (Vivian et al., 2017), which uses STAR (Dobin et al., 2013) to generate alignments (reference genome GRCh38) and performs quantification using RSEM (Li & Dewey, 2011) and Kallisto (Bray et al., 2016). RNA sequencing data were downloaded as RSEM-based normalized gene quantifications and then Iog2-transformed with an offset of 1. For all analyses, the TCGA SKCM dataset was filtered to include only metastatic samples (n = 366).

[0288] Gene Set Enrichment Analysis (GSEA)

[0289] Genes were ranked based on their correlation to megalin mRNA in TCGA SKCM metastases. Gene set enrichment analysis (GSEA) was performed on the ranked gene list using fgsea (https: / / www.biorxiv.orq / content / 10.1101 / 060012v3) in R. Gene sets included HALLMARKS and GO Biological Process (BP). Enrichment plots were based on running enrichment scores extracted from the ClusterProfiler (Yu et al., 2012) package in R.

[0290] T cell inflammation scores

[0291] T cell inflammation scores for TCGA SKCM were calculated as aggregated meancentered scores (z scores) of a previously defined T cell inflammation gene signature: CD8A, CCL2, CCL3, CCL4, CXCL9, CXCL10, ICOS, GZMK, IRF1, HLA-DMA, HLA-DMB, HLA-DOA and HLA-DOB (Spranger et al., 2015). T cell inflammation-low and T cell inflammation-high stratification was based on a median cutoff.

[0292] Patient samples for immunohistochemistry

[0293] Patient samples (n = 150) from a previously established cohort of Danish patients with primary cutaneous melanoma (n = 385) (Bonnelykke-Behrndtz et al., 2017) were included for immunohistochemical analysis. Patient samples were included in pairs matched according to ulceration, Breslow thickness, and age. The Regional Committee for Health Research in the Central Denmark Region approved the study.

[0294] Four-color multiplexed immunohistochemistry and digital image analysisPV ref: 85386PC01

[0295] 28

[0296] Formalin-fixed paraffin-embedded (FFPE) melanoma tumors were sectioned and stained at the Department of Pathology at Aarhus University Hospital. FFPE samples were sectioned at a thickness of 3 JJM and mounted on TOMO® Adhesion Microscope Slides (Matsunami Glass Ind., Ltd.). Slides were dried at 60 °C for one hour prior to staining. Sequential staining was performed on a DISCOVERY ULTRA (Roche) in the following order: CD8 (clone C8 / 144B, Dako, 1:150) detected with DISCOVERY ChromoMap DAB kit (RUO)(Roche), CD4 (clone SP35, Ventana, ready to use) detected with DISCOVERY Purple HRP (Roche), megalin (protein G-purified polyclonal rabbit anti-human megalin antibody (Andersen et al., 2015) (1:100) detected with DISCOVERY Teal HRP (Roche), and SOX10 (clone SP267, Ventana, ready to use) detected with DISCOVERY Yellow AP (Roche). Upon staining, tissue sections were scanned at 20x magnification on a NanoZoomer S60 Digital Slide Scanner (Hamamatsu Phototonics).

[0297] Image analysis was performed using VisioPharm software (Visiopharm A / S). We applied a threshold-based color classification to label positive staining for the four markers: CD8 (DAB), CD4 (purple), megalin (teal) and SOX10 (yellow). First, a global tumor region of interest (ROI) was outlined manually, which included bulk tumor and surrounding lymphocytes, while excluding epidermis, large blood vessels, heavily pigmented regions, and artifacts from sample preparation.

[0298] Second, an automated image analysis pipeline was applied, which calculated the area fraction of CD8 and CD4 staining within the global tumor ROI, used the SOX10 label to define a SOX10+ melanoma cell ROI (with manual correction if needed), and calculated the area fraction of megalin staining within the SOX10 + melanoma cell ROI. Following quality control of sectioning and staining of the 150 sections, there were 132 sections available for measurement of both megalin (SOX10+ area fraction) and T cells (CD4 and CD8 tumor area fraction). T cell inflammation stratification was based on aggregated CD4 (tumor area fraction) and CD8 (tumor area fraction) scores using a median cutoff.

[0299] Results

[0300] To characterize megalin-expressing melanomas, we performed gene set enrichment analysis of megalin (gene name LRP2) correlated genes in The Cancer Genome Atlas (TCGA) Skin Cutaneous Melanoma (SKCM) cohort. The top enriched pathways were related to immune responses and immune signaling, such as adaptive immune response, lymphocyte-mediated immunity, and IFNy response (Fig. 2A-B), indicating a more inflamed tumor microenvironment in megalin-PV ref: 85386PC01

[0301] 29

[0302] expressing melanoma. We applied a previously defined T cell-inflammation signature for the classification of TCGA SKCM tumors (Spranger et al., 2015) and observed higher megalin expression in T cell-high compared to T cell-low melanomas (Fig. 2C).

[0303] To validate the positive correlation between megalin expression and T cell inflammation in melanoma, we performed a four-color multiplex immunohistochemistry analysis of SOXIO, megalin, CD4, and CD8 in sections of formalin-fixed paraffin-embedded (FFPE) primary melanomas using a cohort of 150 patients diagnosed with primary melanoma. One section of each tumor was scored for CD4 and CD8 T cells (fraction of bulk tumor region) and megalin (fraction of SOX10+ tumor region). We stratified primary melanomas into T cell-high and T cell-low and compared megalin expression in SOX10+ melanoma cells. Megalin expression in SOX10+ melanoma cells was higher in T cell-high versus T cell-low tumors (Fig. 2D), consistent with observations in the TCGA SKCM cohort. In combination, these results demonstrate increased expression of megalin in T cell-inflamed melanomas.

[0304] Conclusion

[0305] Megalin is positively correlated with T cell inflammation and IFNy signaling in melanoma.

[0306] Example 3 - Transcriptomic data indicating that megalin is upregulated in melanoma metastases from patients with disease control following immune checkpoint blockade.

[0307] Aim of study

[0308] To assess the impact of immune checkpoint blockade on megalin expression in melanoma metastases.

[0309] Materials and methods

[0310] Bulk RIMA sequencing data (normalized transcript per million (TPM) values) for melanoma metastases pre- or on-therapy with immune checkpoint blockade (nivolumab) were available from Riaz et al. (Tumor and microenvironment evolution during immunotherapy with nivolumab. Cell 2017, 171, 4). Patients were stratified based on disease control (stable disease, partial response, and complete response) or disease progression. Megalin mRNA expression was compared between pre- and on-therapy samples for each group.

[0311] ResultsPV ref: 85386PC01

[0312] 30

[0313] No significant difference in megalin mRNA expression was observed between pre-and on-therapy samples in patients with disease progression following immune checkpoint blockade (Figure 3A). In contrast, a significant increase in megalin mRNA expression was observed on-therapy in patients with disease control following immune checkpoint blockade (Figure 3B).

[0314] Conclusion

[0315] Megalin is upregulated in melanoma metastases from patients with disease control following immune checkpoint blockade - making megalin an interesting target for treating metastatic melanoma.

[0316] Example 4 - IFNy exposure increases the uptake of megalin-targeting antibodies by cancer cells

[0317] Aim of study

[0318] To assess the impact of IFNy exposure on the uptake of megalin-targeting antibodies by cancer cells.

[0319] Materials and methods

[0320] Cell culture

[0321] For cell culture conditions see example 1.

[0322] IFNy exposure

[0323] Cells were plated and cultured for 24 hours prior to IFNy stimulation. Cells were treated with vehicle control or 25 ng / mL recombinant human IFNy (PeproTech) for 48 hours prior to antibody uptake assays.

[0324] Antibodies

[0325] Mouse monoclonal antibodies raised against ligand-affinity purified full-length human megalin of renal origin were generated using hybridoma technology.

[0326] Briefly, 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). Polyclonal antibodies against human megalin were raised in rabbits using Davids Biotechnologie as service provider (https: / / www.davids-bio.com / paQes / polvclonal-rabbit-antisera.html).

[0327] 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 daysPV ref: 85386PC01

[0328] 31

[0329] 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.

[0330] Direct ELISA:

[0331] 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.

[0332] Several mAbs were generated for human megalin. One of these was selected and the antibody purified. One hybridoma was identified that expressed an antibody positive in both ELISA and western blot (Data not shown). The antibody also showed specific staining and specific targeting of megalin (Data not shown).

[0333] Antibody labeling

[0334] 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.

[0335] Antibody uptake assay

[0336] 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.

[0337] ResultsPV ref: 85386PC01

[0338] 32

[0339] FM3 metastatic melanoma cells were cultured for 48 hours under standard conditions or exposed to IFNy. Then, cells were incubated with AF647-labeled anti-megalin antibodies for 2 hours, and fluorescence was measured by flow cytometry. IFNy exposure resulted in an increased uptake of megalin-targeting antibodies by FM3 metastatic melanoma cells (Figure 4A).

[0340] Conclusion

[0341] IFNy exposure increased the uptake of megalin-targeting antibodies by cancer cells.

[0342] Example 5 - Synthesis of antibody-drug conjugates

[0343] The antibody "Megalin-41" or irrelevant murine monoclonal IgG control mAb were dissolved in PBS (pH 7.4) at 1 mg / ml. The toxin, OSu-PEG4-vc-PAB-DMEA-Duocarmycin SA (Levana Biopharma, cat. no. SET0103) was dissolved at a concentration of 8.33 mg / ml in DMSO.

[0344] 40 pl of dissolved toxin was added per ml of 1 mg / ml antibody solution and samples incubated for 16 hours at 4 C.

[0345] The antibody-drug-conjugates (ADCs) were buffer exchanged into PBS (pH 7.4) and sterile filtered. Protein concentrations were determined using a BCA assay (ThermoFisher, cat. no. 23225) while the amount of duocarmycin SA was determined by measuring absorbance at 337 nm. The drug-antibody ratio (DAR) calculated from these measurements was 2.1 for Megalin-41 ADC and 1.9 for IgG control ADC. Megalin-41 ADC retained binding to megalin as determined by surface plasmon resonance analysis (Biacore) (data not shown).

[0346] Example 6 - IFNy increases killing of megalin-expressing cancer cells by antibody-drug conjugates targeting megalin.

[0347] Aim of study

[0348] To determine if IFNy enhances the cytotoxic effect of megalin-targeting ADCs on cancer cells.

[0349] Materials and methods

[0350] FM3 human melanoma cells or the corresponding megalin-knockout cells, FM3 sgLRP2 #3, were seeded in 96-well plates and allowed to adhere overnight. Cells were then stimulated with 10 ng / ml recombinant human IFNy (PeproTech) or vehicle for 24 hours. Following stimulation, cells were treated with 500 ng / mlPV ref: 85386PC01

[0351] 33

[0352] Megalin-41 ADC or IgG control ADC for 96 hours, while IFNy exposure was maintained. Each condition was performed in duplicate.

[0353] Cells were lysed in 100 pL CellTiterGlo 2.0 reagent and incubated for 15 minutes prior to luminescence measurement on a Spectramax ID5 plate reader. Relative cell viability was calculated by normalizing luminescence signal to vehicle-treated cells for each ADC.

[0354] Results

[0355] In FM3 human melanoma cells, IFNy treatment increased the cytotoxic effect of Megalin-41 ADC, as demonstrated by a reduced relative cell number compared to vehicle-treated cells (Figure 5A). In contrast, IFNy did not increase the cytotoxic effect of the IgG control ADC (Figure 5A). The cell-killing effect of Megalin-41 ADC following IFNy stimulation was eliminated in the megalin-knockout cell line, FM3 sgLRP2 #3, demonstrating that this effect is mediated by megalin (Figure 5B). Conclusion

[0356] An increased cell-killing effect of anti-megalin ADCs towards megalin-expressing cancer cells following IFNy treatment is demonstrated.

[0357] Example 7 - Sequencing of the antibody Megalin-41

[0358] The sequence of the murine monoclonal IgG antibody Megalin-41 was determined by the company Yumab, Gottingen, Germany. Briefly, using PCR, cDNA was synthesized from isolated mRNA using murine IgG variable heavy- and light-chain specific primers. The PCR products were cloned into vectors and sequenced.

[0359] The sequence of Megalin-41 light chain variable region was as follows:

[0360] DIQMTQTPASLSASVGETVTITCRASGNIHIYLAWYQQRQGKSPQLLVYNAKTLADGVP SRFSGSGSRTQYSLKINSLQPEDFGTYYCQNFWSTPWTFGGGTKLEIK (SEQ ID NO: 1)

[0361] The sequence of Megalin-41 heavy chain variable region was as follows:

[0362] QVQLKQSGPGLVAPPQSLSITCTVSGFSLTSYVVHWIRQPPGKGLEWLGMIWAGGITNY NSALMSRLSISKDNSRNQVFLKMNSLQTDDTAIYYCARGDNFDYWGQGTTLTVSS (SEQ ID NO: 2)

[0363] Using Kabat numbering and annotation of CDR, the following CDRs were identified:

[0364]

[0365] PV ref: 85386PC01

[0366] 34

[0367]

Claims

PV ref: 85386PC0135Claims1. An anti-megalin antibody-drug conjugate (ADC), for use in the treatment and / or alleviation of melanoma in a subject;wherein said subject has previously been or is being treated with IFN(y) and / or an IFN(y)-inducing compound or therapy, such as immunotherapy.

2. The anti-megalin antibody-drug conjugate (ADC), for use according to any of the preceding claims, wherein the cancer presents megalin in cell membranes, such as being exposed on the cell surface, at a higher level than before the treatment with IFN(y) and / or an IFN(y)-inducing compound or therapy.

3. The anti-megalin antibody-drug conjugate (ADC) for use according to any of the preceding claims, wherein the anti-megalin antibody binds to an extracellular epitope of megalin.

4. The anti-megalin antibody-drug conjugate (ADC) for use according to any of the preceding claims, being specific for megalin.

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

6. The anti-megalin antibody-drug conjugate (ADC) for use according to any of the preceding claims, 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.

7. IFNy and / or an IFNy-inducing therapy for use as a pretreatment of a subject suffering from melanoma;wherein said subject is scheduled for subsequent treatment with an anti-megalin antibody-drug conjugate (ADC) for use in the treatment and / or alleviation of the melanoma in the subject.PV ref: 85386PC01368. A kit or combination comprising- IFNy and / or an IFNy-inducing compound or therapy, such as an IFNy- inducing immunotherapy; and- an anti-megalin antibody-drug conjugate (ADC).

9. The kit or combination according to claim 11, for use in the treatment or alleviation of cancer in a subject, preferably melanoma.

10. The kit or combination according to claim 8 or 9, for use in the treatment or alleviation of melanoma in a subject.

11. The kit or combination for use according to any of claims 8-10, wherein IFNy and / or an IFNy-inducing compound is to be administered to the subject simultaneously with the anti-megalin antibody-drug conjugate (ADC) or before the anti-megalin antibody-drug conjugate (ADC).12.- The anti-megalin antibody-drug conjugate (ADC), for use according to any of claims 1-6,- the IFNy and / or an IFNy-inducing compound or therapy for use according to claim 7; and / or- the kit or combination according to any of claims 8-11;wherein the IFNy-inducing therapy is selected from the group consisting of immune checkpoint inhibitors, such as anti-PD-(L)l, anti-CLTA-4, anti-LAG-3, cellular immunotherapy, such as tumor infiltrating lymphocyte therapy, T cell receptor (TCR)-engineered T cell therapy, CAR-T cell therapy, CAR-NK cell therapy), cytokine-based therapies, such as interleukin-2 therapies, bispecific T-cell engagers such as targeting CD3 and tumor antigens, cancer vaccines, such as neoantigen-based personalized vaccines, oncolytic viral therapies with immunostimulatory effects, such as talimogene laherparepvec (T-VEC).PV ref: 85386PC0137- The anti-megalin antibody-drug conjugate (ADC), for use according to any of claims 1-6 or 12,- the IFNy and / or an IFNy-inducing compound or therapy for use according to claim 7 or 12; and / or- the kit or combination for use according to claim 9 or 10;wherein the cancer is melanoma and the melanoma is a malignant melanoma or a benign melanomaand / orwherein the melanoma is selected from the group consisting of cutaneous melanoma, acral melanoma, mucosal melanoma, uveal melanoma, and metastatic melanoma.

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