Novel Mcl-1 inhibitors and combinations of BH3 mimetics such as Mcl-1 and Bcl-2 inhibitors

Abstract

In netrin-1 expressing tumor cells, netrin-1 interference causes Mcl-1 degradation limited to these netrin-1 expressing tumor cells. It is proposed that netrin-1 interference causes tumor-specific Mcl-1 degradation. In addition, netrin-1 interference may be combined with a BH3-mimetic, in particular a Bcl-2 inhibitor. Netrin-1 interference is performed using an anti-netrin-1 antibody. The present invention proposes an anti-netrin-1 antibody for use as a tumor-specific Mcl-1 inhibitor in the treatment of Mcl-1+ tumors, more particularly Mcl-1+, netrin-1+ and netrin-1 receptor+, in particular UNC5B+ tumors. Also provided is a combination of anti-netrin-1 antibody and a Bcl-2 inhibitor, such as venetoclax.

Description

[Technical field]

[0001] The present invention relates to novel Mcl-1 inhibitors and combinations of such Mcl-1 inhibitors and BH3 mimetics such as Bcl-2 inhibitors. The present invention also relates to pharmaceutical compositions, combined compositions or pharmaceutical combinations for use in treating cancer, and methods for treating cancer. [Background technology]

[0002] The balance between cell survival and death is important for the homeostasis of an organism. This balance is controlled, at least in part, by the interplay of Bcl-2 family proteins, which consist of the proapoptotic effectors Bax, Bak, and Bok, the proapoptotic BH3-only proteins (including their members Bad, Bid, Puma, Bim, and Noxa), and the antiapoptotic proteins of the Bcl-2 family (including Bcl-2, Bcl-XL, Mcl1, Bcl-W, and Bfl-1 / A1) (Singh et al., 2019). In cancer, the equilibrium of this balance is shifted to prosurvival Bcl-2 members, which are often overexpressed and stabilized (Delbridge et al., 2016). Thus, Bcl-2 members allow cancer cells to escape cell death, which is a key step in the acquisition of a malignant phenotype (Hanahan and Weinberg, 2000; Juin et al., 2013). Cells protected by anti-apoptotic Bcl-2 members are often “primed” for apoptosis due to oncogenic stress and stabilization of BH3-only proteins (Adams and Cory, 2018; Merino et al., 2012). Hence, therapeutic strategies targeting anti-apoptotic Bcl-2 proteins are plausible, and selective and potent inhibitors of their members have been developed over the past decades (Merino et al., 2018). Such inhibitors, termed “BH3 mimetics”, are primarily effective in treating hematopoietic cancers that exhibit high levels of Bcl-2 family proteins (Ewald et al., 2019; Scheffold et al., 2018). However, combinations of different BH3 mimetics may be effective in treating a broader range of tumors, including solid tumors, that depend on more than one Bcl-2 protein for their survival (Soderquist et al., 2018). To date, only the Bcl-2 specific inhibitor venetoclax / ABT199 has been clinically approved ( Souers et al., 2013 ).

[0003] Some molecules targeting other members of the Bcl-2 family have reached the clinic, for example the Mcl-1 inhibitor S63845 (Kotschy et al., 2016). BH3-mimetics targeting Mcl-1 have thus been developed (S63845, AMG176, and AMG397). However, the clinical development of these compounds, especially those targeting Mcl-1, has been delayed due to on-target effects on healthy cells (Merino et al., 2018). In fact, the presence of this anti-apoptotic protein is important in many tissues and cell types, including hematopoietic stem cells, thymic epithelium, mature and immature B and T lymphoid cells, activated T cells, cardiomyocytes, neurons, and hepatocytes (Merino et al., 2018). Preliminary testing of S63845 on humanized Mcl-1 mice revealed a reduction in B cell populations during treatment, but also an overall good tolerability (Brennan et al., 2018). Nevertheless, the recent cessation of clinical trials evaluating the possible cardiotoxicity of two Mcl-1 inhibitors, AMG176 (NCT02675452) and AMG397 (NCT03465540), still raises some safety concerns (Wei et al., 2020). There are several reports on the biochemical limitations of these Mcl-1 inhibitors and their relative toxicity to healthy hematopoietic cells, which hinders the development of these therapeutics (Ashkenazi et al., 2017; Opferman et al., 2005; Zhang et al., 2007). Therefore, new leads that allow direct targeting of specific members of the Bcl-2 family in tumors are of great interest.

[0004] UNC5B / UNC5H2 (UNCoordinated Homolog 5), a type I transmembrane receptor discovered due to its effect on neuronal navigation during embryonic development, is a receptor that can actively induce apoptosis in the absence of its ligand, netrin-1 (Llambi et al., 2001). This feature defines all members of the dependence receptor family, which activates dual signaling pathways: in the presence of the respective ligand, they induce "positive" signaling pathways, e.g. proliferation or migration signals, and in the absence of the specific ligand, they activate "negative" signaling pathways that lead to programmed cell death (Mehlen and Bredesen, 2004, 2011; Negulescu and Mehlen, 2018). Thus, the expression of these receptors makes the cell dependent on the presence of the respective ligand for its survival (Bredesen et al., 2005; Mehlen and Puisieux, 2006). The dependence receptor family consists of more than 20 members (Goldschneider and Mehlen, 2010), including the netrin-1 receptors UNC5H (i.e., UNC5H1 / A, UNC5H2 / B, UNC5H3 / C and UNC5H4 / D) and DCC (Llambi et al., 2001; Mehlen et al., 1998).

[0005] UNC5B, together with netrin-1, triggers pleiotropic effects ranging from axon guidance and migration to angiogenesis (Castets et al., 2009; Lai Wing Sun et al., 2011). However, due to its death domain, UNC5B has been widely described as a death-promoting receptor, and upon netrin-1 binding, its death activity is blocked by multimerization. Thus, UNC5B has been defined as a conditional tumor suppressor, and netrin-1 as a carcinogenesis promoter (Llambi et al., 2001; Thiebault et al., 2003). Consistent with this idea, netrin-1, a protein expressed mainly during embryonic development, has been shown to be re-expressed by cancer cells and even the tumor microenvironment in the majority of human neoplasms (Mehlen et al., 2011; Sung et al., 2019). This has been shown to occur in inflammation-associated colorectal cancer (Paradisi et al., 2008; Paradisi et al., 2009), metastatic breast cancer (Fitamant et al., 2008), lung cancer (Delloye-Bourgeois et al., 2009a), neuroblastoma (Delloye-Bourgeois et al., 2009b), lymphoma (Broutier et al., 2016), and melanoma (Boussouar et al., 2020). In these models, interference between netrin-1 and its receptor was sufficient to cause cancer cell death and induce tumor growth inhibition. Based on these findings, a monoclonal antibody called NP137 that neutralizes netrin-1 and blocks the netrin-1 / UNC5B interaction has been developed ( Grandin et al., 2016 ) and is currently being evaluated in several phase I and II clinical trials (ClinicalTrials.gov identifiers NCT02977195 and NCT04652076).

[0006] Thus, there is a need for new Mcl-1 inhibitors that are effective and exhibit a satisfactory safety profile.There is also a need for new combination cancer therapies based on combination partners that show synergistic effects that bring the advantage of substantial increase in long-term efficacy and improved safety profile.It is also interesting that combinations reduce the effective dose of one agent or two combined agents, especially BH3 mimetics or Bcl-2 inhibitors, especially for safety reasons.It is also most interesting to have tumor-specific agents, especially Mcl-1 inhibitors, to limit secondary effects on healthy tissue. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] EP3092003 [Patent Document 2] WO2015 / 104360 [Patent Document 3] US10,494,427 [Patent Document 4] U.S. Patent No. 8,546,399 [Patent Document 5] EP0239400 [Patent Document 6] WO91 / 09967 [Patent Document 7] U.S. Patent No. 5,530,101 [Patent Document 8] U.S. Patent No. 5,585,089 [Patent Document 9] EP0592106 [Patent Document 10] EP0519596 [Patent Document 11] U.S. Patent No. 5,565,332 [Patent Document 12] U.S. Patent No. 4,444,887 [Patent Document 13] U.S. Patent No. 4,716,111 [Patent Document 14] U.S. Patent No. 5,545,806 [Patent Document 15] U.S. Patent No. 5,814,318 [Patent Document 16] International Patent Application No. WO98 / 46645 [Patent Document 17] International Patent Application No. WO98 / 50433 [Patent Document 18] International Patent Application No. WO98 / 24893 [Patent Document 19] International Patent Application No. WO98 / 16654 [Patent Document 20] International Patent Application No. WO96 / 34096 [Patent Document 21] International Patent Application No. WO96 / 33735 [Patent Document 22] International Patent Application No. WO91 / 10741 [Non-patent literature]

[0008] [Non-Patent Document 1] ClinicalTrials.gov [Non-Patent Document 2] Krajewska et al. (AJP 1996 May, Vol. 148, No. 5: 1567-1576) [Non-Patent Document 3] Erinna F. Lee et al. (CellDeath & Disease 2019, 10, 342) [Non-Patent Document 4] Archer T et al., Cancer Cell. 2018 Sep 10;34(3):396-410 (PMID: 30205044) [Non-Patent Document 5] Link et al., Annals of Chir. Onco. 2007 [Non-Patent Document 6] Dumartin et al., Gastro 2010 [Non-Patent Document 7] Kaufmann et al., Cellular Oncology 2009 Non-Patent Document 8 Panastasiou et al., Oncotarget 2011 Non-Patent Document 9 Mehlen et al., Nature Cancer Rev 2011 Non-Patent Document 10 Forcet et al. 2001, PNAS Non-Patent Document 11 Kohler and Milstein, Nature, 1975, 256(5517): 495-497 Non-Patent Document 12 Harlow et al., eds., 1988 "Antibodies: a laboratory manual" Non-Patent Document 13 Scott J.K., Smith G.P. Science 1990; 249: 386-390 Non-Patent Document 14 J.D. Marks et al., J. Mol. Biol., 222(1991), 581 Non-Patent Document 15 "Remington's Pharmaceutical Sciences" 15th Edition, pp. 1035-1038 and 1570-1580 Non-Patent Document 16 Needleman and Wunsch (1970) J. Mol. Biol. 48: 443 Non-Patent Document 17 Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA Non-Patent Document 18 (http: / / ) www.bioinf.org.uk / abs / #cdrdef Non-Patent Document 19 Lefranc M.-P., "Immunology Today, 18, 509 (1997) Non-Patent Document 20 Lefranc M.-P., The Immunologist, 7, pp. 132 - 136 (1999)

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[0009] Little is known about the mechanism by which apoptotic signaling is triggered by UNC5B. Upon netrin-1 depletion, UNC5B adopts an open conformation, allowing the phosphatase PP2A to catalyze the dephosphorylation of DAPK (serine / threonine death-associated protein kinase), which induces cell death by a still unknown mechanism (Guenebeaud et al., 2010; Llambi et al., 2005; Wang et al., 2009). Current studies identify the CRL3-COMMD2 complex as a key mediator of UNC5B-induced apoptosis. It was completely unexpected that in the presence of free UNC5B (not bound to netrin-1), this CRL3-COMMD2 complex ubiquitinates the anti-apoptotic Bcl-2-member Mcl-1, targeting it for proteasomal degradation. As a result, the reduced levels of Mcl-1 triggered mitochondrial depolarization and activation of the intrinsic apoptotic pathway. "Netrin-1 interference" includes blocking netrin-1 binding to UNC5B. This can occur by providing anti-netrin-1 specific antibodies. The prior knowledge was that dependency receptors such as netrin-1 receptors cause cell death independent of the mitochondria-dependent pathway. Therefore, it was completely unexpected that netrin-1 interference could cause Mcl-1 degradation in netrin-1 expressing tumor cells. It was even more completely unexpected to find a way to induce limited Mcl-1 degradation in these netrin-1 expressing tumor cells, and thus to propose netrin-1 interference to cause tumor-specific Mcl-1 degradation in Mcl-1+ tumors. The current study provides preclinical evidence that NP137 can be used as a specific Mcl-1 inhibitor in tumor patients, and in combination with BH3-mimetics. This makes it possible to overcome the toxicity otherwise associated with this type of compound. Data are presented herein that other netrin-1 receptors, in particular UNC5A and UNC5C, are present in Mcl1+ tumor cells and may also be involved in the present invention.

[0010] Thus, one object of the present invention is an anti-netrin-1 antibody, in particular an anti-netrin-1 monoclonal antibody, for use as a tumor-specific Mcl-1 inhibitor in the treatment of Mcl-1+ tumors. Another object of the present invention is the use of an anti-netrin-1 antibody, in particular an anti-netrin-1 monoclonal antibody, for the manufacture of a medicament for use as a tumor-specific Mcl-1 inhibitor in the treatment of Mcl-1+ tumors.

[0011] Another object of the invention is a method for specifically inhibiting Mcl-1 in Mcl-1+ tumors in a patient having such tumor, comprising administering to said patient an effective amount of an anti-netrin-1 antibody, in particular an anti-netrin-1 monoclonal antibody, which causes tumor-specific Mcl-1 inhibition.In one object, the invention is a method of treatment of a patient suffering from an Mcl-1+ tumor, comprising administering to said patient an effective amount of an anti-netrin-1 antibody, in particular an anti-netrin-1 monoclonal antibody, which causes tumor-specific Mcl-1 inhibition or degradation. [Means for solving the problem]

[0012] As disclosed herein, an Mcl-1+ tumor or cancer may be one that expresses UNC5B (UNC5B+ tumor or cancer) and netrin-1 (netrin-1+ tumor or cancer).

[0013] As disclosed herein, a Mcl-1+ tumor or cancer may express UNC5B (UNC5B+ tumor or cancer) and / or another netrin-1 receptor, such as UNC5A and / or UNC5C. The tumor or cancer may also express netrin-1 (netrin-1+ tumor or cancer).

[0014] "Tumor-specific" in this document means that the antibody induces Mcl-1 inhibition or degradation specifically in tumor cells ("restricted to Mcl-1+ tumor cells"). The antibody does not induce this inhibition in healthy cells that contain or express Mcl-1 ("healthy Mcl-1+ cells"). Indeed, it has been previously documented that the presence of this anti-apoptotic protein Mcl-1 is important in many tissues and cell types, including, for example, hematopoietic stem cells, thymic epithelium, mature and immature B and T lymphocyte cells, activated T cells, cardiomyocytes, neuronal cells and hepatocytes. Thus, the antibody does not induce this inhibition in these cells.

[0015] In one aspect, the anti-netrin-1 antibody, in particular the anti-netrin-1 monoclonal antibody, is for use as a tumor-specific Mcl-1 inhibitor in tumor cells expressing Mcl-1, netrin-1 and UNC5B.

[0016] In one embodiment, the significance of anti-netrin-1 antibodies, particularly anti-netrin-1 monoclonal antibodies, that bind to netrin-1 is that UNC5B is free from netrin-1 or cannot bind to netrin-1, which leads to Mcl-1 inhibition or degradation. In another embodiment, free or unbound UNC5B causes the CRL3-COMMD2 complex to ubiquitinate the anti-apoptotic Bcl-2-member Mcl-1, targeting Mcl-1 for proteasomal degradation. In another embodiment, the significance of anti-netrin-1 antibodies, particularly anti-netrin-1 monoclonal antibodies, that bind to netrin-1 is that UNC5B induces Mcl-1 degradation.

[0017] In yet another embodiment, all this occurs in a tumor expressing netrin-1, i.e. in tumor cells and / or in the tumor environment expressing netrin-1. In yet another embodiment, all this occurs in a tumor expressing netrin-1 and a netrin-1 receptor, in particular UNC5B.

[0018] The present invention therefore proposes anti-netrin-1 antibodies, in particular anti-netrin-1 monoclonal antibodies, for use as tumor-specific Mcl-1 inhibitors in the treatment of Mcl-1+ tumors, more particularly tumors expressing Mcl-1, netrin-1 and netrin-1 receptors, in particular Mcl-1+, netrin-1+ and UNC5B+ tumors, as disclosed herein.

[0019] In one aspect, the antibody is used or is for use to cause tumor-specific Mcl1 degradation and induce tumor-specific cell death.

[0020] Another object of the present invention is a combination of an anti-netrin-1 antibody, or an antigen-binding fragment thereof, and an inhibitor of an anti-apoptotic protein of the Bcl-2 family, preferably a Bcl-2 inhibitor, for use in the treatment of Mcl1+ cancers or Mcl1+ and UNC5B+ cancers or Mcl1+ and netrin-1 receptor+ cancers, particularly receptors selected from UNC5A, B and C. As disclosed herein, an Mcl-1+ tumor or cancer may be one that expresses UNC5B (UNC5B+ tumor or cancer) and netrin-1 (netrin-1+ tumor or cancer).

[0021] Another object of the present invention is to provide a method for the preparation of a medicament for simultaneous, sequential or separate use, (a) an anti-netrin-1 antibody, in particular an anti-netrin-1 monoclonal antibody, and (b) an inhibitor of an anti-apoptotic protein of the Bcl-2 family, preferably a Bcl-2 inhibitor;

[0033] The combination, pharmaceutical combination, or anti-cancer combination comprises

[0022] As used herein, (b) relates to BH3 mimetics as inhibitors of the Bcl-2 family of anti-apoptotic proteins, including inhibitors of the Bcl-2 protein, a member of the Bcl-2 family of anti-apoptotic proteins.

[0023] In one embodiment, the combination is for use in the treatment of cancer and (a) and (b) are intended for simultaneous, sequential or separate administration to a patient.

[0024] Another object of the present invention is to provide (a) an anti-netrin-1 antibody, in particular an anti-netrin-1 monoclonal antibody; (b) an inhibitor of an anti-apoptotic protein of the Bcl-2 family, preferably a Bcl-2 inhibitor; and (c) a pharma- ceutically acceptable vehicle or excipient; The present invention relates to a pharmaceutical composition or an anti-cancer composition comprising:

[0025] Another object of the present invention is to provide (a) a first vial or container containing an anti-netrin-1 antibody, in particular an anti-netrin-1 monoclonal antibody; (c) a second vial or container comprising an inhibitor of an anti-apoptotic protein of the Bcl-2 family, preferably a Bcl-2 inhibitor, and (b) Optionally, a notice with instructions regarding the administration regimen, e.g., instructions for proceeding with the simultaneous, sequential or separate administration to the patient of all or a portion of the contents of both vials or containers. A pharmaceutical composition kit comprising:

[0026] Another object of the present invention is the use of (a) an anti-netrin-1 antibody, in particular an anti-netrin-1 monoclonal antibody, and (b) an inhibitor of an anti-apoptotic protein of the Bcl-2 family, preferably a Bcl-2 inhibitor, for the manufacture of a medicament or pharmaceutical combination for use in the treatment of cancer. In one embodiment, (a) and (b) are intended for simultaneous, sequential or separate administration to a patient.

[0027] Another object of the present invention is a method for treating cancer in a patient in need thereof, comprising administering to said patient an effective amount of (a) an anti-netrin-1 antibody, in particular an anti-netrin-1 monoclonal antibody, and (b) an inhibitor of an anti-apoptotic protein of the Bcl-2 family, preferably a Bcl-2 inhibitor. This administration may be in the form of various administration regimens. This may include simultaneous, sequential or separate administration of (a) and (b) to said patient. The patient in need thereof is a patient with a tumor as disclosed herein.

[0028] In these various purposes for the combined use of (a) and (b), the anti-netrin-1 antibody is a tumor-specific Mcl-1 inhibitor. This antibody provides the function of Mcl-1 inhibition or degradation in a tumor-specific manner, as described herein. Hence, this function is now combined with the inhibition of anti-apoptotic Bcl-2 proteins, known to those skilled in the art. However, in contrast to previous anti-Mcl-1 / anti-Bcl-2 combinations, this combination benefits from Mcl-1 inhibition limited to tumor cells, as disclosed herein. In other words, the Bcl-2 inhibitor or BH3 mimetic in the combination may be any efficient Bcl-2 inhibitor (with the exception of Mcl-1 inhibitors), which may be a currently clinically approved Bcl-2 inhibitor or BH3 mimetic, or a future clinically approved Bcl-2 inhibitor or BH3 mimetic. Inhibitors of the Bcl-2 anti-apoptotic protein family are usually called BH3 mimetics and may include Bcl-2, Bcl-XL, Bcl-W, Bfl-1 / A1 proteins. It may be an inhibitor of Bcl-2 itself (Bcl-2 inhibitor) or an inhibitor of any other member of the Bcl-2 family. It may be an inhibitor of at least two of these proteins, for example Bcl-2 and Bcl-XL, or Bcl-2, Bcl-W and Bfl-1 / A1.

[0029] This combination of two BH3 mimetics or Bcl-2 anti-apoptotic protein family inhibitors is believed to prevent recruitment of Bcl-2-family members and enhance cancer cell death. Here, we show that combined treatment with anti-netrin-1 neutralizing antibodies, acting as tumor-specific Mcl-1 inhibitors, and the Bcl-2 inhibitor venetoclax significantly reduced tumor growth in mouse models and triggered apoptosis in human breast cancer ex vivo sections. The combination of netrin-1 silencing and navitoclax also rapidly and significantly triggered apoptosis. This is the first time such a combination incorporating one tumor-specific inhibitor has been proposed, and the data support that combining netrin-1 interference with a BH3 mimetic can trigger cancer cell death efficiently and with reduced side effects, thus providing potential therapeutic benefits.

[0030] Venetoclax is a clinically approved BH3 mimetic. In one embodiment, venetoclax is used in the combination of the present invention. In another embodiment, navitoclax is used as the BH3 mimetic in the combination of the present invention.

[0031] In one embodiment, an Mcl-1+ tumor or cancer is particularly one having a percentage of Mcl-1 positive tumor cells of grade 3 or higher, or grade 4 or higher, or equal to grade 5 (see below for methods of determining Mcl1+ tumors). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Cancer related to the present invention RS Soderquist et al. (2018) reported that various cancers have various dependencies or co-dependencies on the BCL-2 gene, and that most cell lines have a role in determining whether Bcl-2, Bcl-X, or both are involved in survival. L We show that the combination of Bcl-2 family inhibitors and at least one Mcl-1 inhibitor is dependent on the induction of Bcl-2-dependent gliomas in various human cancers. The authors develop a conceptual roadmap for rational targeting of Bcl-2 family dependencies in various human cancers.

[0033] The present invention is primarily concerned with a particular group of cancers characterized by Mcl-1 acting as a pro-survival Bcl-2 member, which is unexpectedly associated with the presence and action of netrin-1. Generally, Mcl-1 is overexpressed and / or stabilized in these cancers. By "Mcl-1+ tumors" is meant that the tumor comprises tumor cells that escape programmed cell death or apoptosis by a mechanism involving Mcl-1 and netrin-1. In these tumors, Mcl-1 is expressed at a certain level that can be assessed, sufficient to shift the balance between cell survival and death in favor of the former. Also, methods are available to the skilled artisan to assess the presence or level of netrin-1. These tumors (or cancers) may be identified as "Mcl-1+ / netrin-1+ tumors" or "Mcl-1+ / netrin-1+ / netrin-1 receptor+ tumors" or "Mcl-1+ / Unc5B+ tumors". "Mcl-1+ / Netrin-1+ / Unc5B+ Tumors." In one embodiment, the tumor is a Mcl1+ and Netrin-1 receptor+ tumor (particularly a receptor selected from UNC5A, B and C). In one embodiment, the cancer or tumor further comprises the involvement of other anti-apoptotic Bcl-2 members (e.g., Bcl-2), which may be referred to herein as "Bcl-2+."

[0034] Mcl-1 levels, as well as other anti-apoptotic Bcl-2 members (e.g. Bcl-2), can be measured by classical immunohistochemical assays. Krajewska et al. (AJP 1996 May, Vol. 148, No. 5: pp. 1567-1576) show that the generation and characterization of anti-peptide antisera specific for human Bcl-2, Bax, Bcl-X, and Mcl-1 proteins, as well as methods for applying these antibodies in paraffin immunohistochemistry, are known and described in detail in the literature. Reference may also be made to Erinna F. Lee et al. (CellDeath & Disease 2019, 10, pp. 342). Mcl-1 levels, according to these and other authors, may be expressed in various ways: percentage of Mcl-1 positive tumor cells, or immunostaining intensity. Any suitable method used by clinicians in hospitals is also useful.

[0035] For example, according to Erinna F. Lee et al., the assessment is done by antibody staining, the cancer cells with positive staining in the cytoplasm are counted and the percentage of positive tumor cells is graded (grade) as: 0: none; 1: 1-5%; 2: 6-25%; 3: 26-50%; 4: 51-75% and 5: 76-100%. The intensity of the staining can be graded as: 0: none; 1: weak; 2: moderate; 3: strong. The H-score for each sample can be calculated by multiplying the grade and the intensity score (range 0-15) and can then be classified as high (9-15) or low (0-8). In the present invention, a tumor can be qualified as "Mcl-1+ tumor", preferably when the percentage of positive tumor cells is grade 3 or higher. In some embodiments, a tumor is qualified as an "Mcl-1+ tumor" if the percentage of positive tumor cells is grade 3 or greater, or grade 4 or greater, or equal to grade 5.

[0036] Recent methods also allow the use of mass spectrometry to measure protein amounts in biopsies, for example using the methods described in Archer T et al., Cancer Cell. 2018 Sep 10;34(3):396-410 (PMID: 30205044). This may allow the skilled artisan to obtain Mcl-1 amounts in tumor biopsies, determine the minimal amount that corresponds to an "Mcl-1+ tumor", and use methods to classify and select tumors for treatment.

[0037] In further embodiments, these cancers express or overexpress netrin-1 and the tumor cells exhibit netrin-1 receptors, such as, in particular, UNC5B (UNC5B+, or "Mcl-1+ / netrin-1+ / UNC5B+ tumors").

[0038] In a given embodiment, the patient is first assayed to determine whether he has an "Mcl-1+ tumor", "Mcl-1+ / Netrin-1+ tumor" or "Mcl-1+ / Netrin-1+ / Netrin-1 receptor+ tumor" or "Mcl-1+ / Netrin-1+ / Unc5B+ tumor". If he has such a tumor, then the patient is treated with an anti-Netrin-1 antibody. Thus, various purposes, i.e., uses, methods of treatment for use, may include such assays and / or determinations of the patient's condition. In one embodiment, the assay is done by antibody staining using any available antibody binding to Mcl-1 (see Erinna F. Lee et al.), cancer cells with positive staining in the cytoplasm are counted and the percentage of positive tumor cells is graded as follows: 0: none; 1: 1-5%; 2: 6-25%; 3: 26-50%; 4: 51-75% and 5: 76-100%. In the present invention, a tumor may be qualified as an "Mcl-1+ tumor" preferably if the percentage of positive tumor cells is equal to or greater than grade 3. In some embodiments, a tumor is qualified as an "Mcl-1+ tumor" if the percentage of (Mcl-1) positive tumor cells is equal to or greater than grade 3, or equal to or equal to grade 4, or equal to grade 5.

[0039] Some embodiments of cancers relevant herein include metastatic breast cancer, non-small cell lung cancer, invasive neuroblastoma, pancreatic adenocarcinoma, primary melanoma, melanoma metastasis, ovarian cancer, glioblastoma, acute myeloid leukemia, chronic lymphocytic leukemia, intermediate grade B-cell lymphoma, sarcoma, renal adenocarcinoma, head and neck cancer, testicular cancer (e.g., embryonal carcinoma, teratoma, yolk sac tumor), renal cancer, gastric cancer, uterine cancer, colorectal cancer, and lung adenocarcinoma.

[0040] Some specific embodiments of cancers that exhibit increased expression of netrin-1 include breast tumors, particularly metastatic breast cancer (Fitamant, 2008), lung cancer (Delloye-Bourgeois et al., 2009a), neuroblastoma (Delloye-Bourgeois et al., 2009b), lymphoma (Broutier et al., 2016), melanoma (Boussouar et al., 2020), and inflammation-associated colorectal cancer (Paradisi et al., 2008; Paradisi et al., 2009).

[0041] EP3092003 provides some indications of the prevalence of overexpressing netrin-1 in several cancers: 60% in metastatic breast cancer (Fitamant et al. 2008), 47% in non-small cell lung cancer (Delloye-Bourgeois et al. 2009a), 38% in invasive neuroblastoma (Delloye-Bourgeois et al. 2009b), 61% in pancreatic adenocarcinoma (Link et al., Annals of Chir. Onco. 2007; Dumartin et al., Gastro 2010), 100% in primary melanoma (n=7) and melanoma metastases (n=6) (Kaufmann et al., Cellular Oncology 2009), and 76% in ovarian cancer (Panastasiou et al., Oncotarget 2010). 2011), 65% for glioblastoma, 60% for acute myeloid leukemia and chronic lymphocytic leukemia, 50% for intermediate-grade B-cell lymphoma, 30% for sarcoma, 40% for renal adenocarcinoma, 22% for head and neck cancer, testicular cancer (36% for embryonal carcinoma, 50% for teratoma, 100% for yolk sac tumor), 50% for renal cancer, 26% for gastric cancer, and 19% for uterine cancer.

[0042] In such cancers that overexpress netrin-1, it has been shown that netrin-1 behaves as a survival factor for cancer cells (Mehlen et al., Nature Cancer Rev 2011), but this survival has been proposed to be independent of Bcl-2 proteins (Forcet et al. 2001, PNAS), in line with the view that nomenclature of cell death mechanisms suggests that dependence receptors, e.g. the netrin-1 receptor, trigger cell death independent of the mitochondria-dependent pathway (Galluzzi et al. 2018).

[0043] Anti-NTN1 antibody (anti-netrin-1 antibody or antibody that binds to netrin-1) The antibody may be a polyclonal or monoclonal antibody (anti-netrin-1 antibody or antibody that binds to netrin-1) that specifically binds to netrin-1 (NTN1), particularly human netrin-1. In one embodiment, the antibody used and disclosed herein is neutralizing. In one embodiment, the anti-netrin-1 antibody or its netrin-1 binding fragment or derivative of this antibody is neutralizing. As used herein, the term "neutralizing antibody" refers to an antibody that captures netrin-1 and blocks or reduces at least one activity of netrin-1, particularly its binding to its receptor, primarily UNC5B.

[0044] Anti-Netrin-1 polyclonal antibodies can be obtained, inter alia, by immunizing animals, such as rabbits, mice, and the like, with a selected amino acid sequence according to methods previously known to those skilled in the art, collecting the resulting antisera, for example on an immunoadsorbent containing the receptor, and then depleting it. Netrin-1 amino acid sequence is as shown in SEQ ID NO: 1, and Netrin-1 can be used in whole or in part to generate polyclonal or monoclonal antibodies.

[0045] Generally, monoclonal antibodies are obtained according to the conventional method of lymphocyte fusion and hybridoma culture described by Kohler and Milstein (Nature, 1975, 256(5517):495-7). Other methods for preparing monoclonal antibodies are also known (Harlow et al., eds., 1988 "Antibodies: a laboratory manual"). Monoclonal antibodies can be prepared by immunizing a mammal (e.g. mouse, rat, rabbit or even human, and the like) and resulting in a hybridoma using the lymphocyte fusion technique (Kohler and Milstein, 1975). Alternative techniques to this conventional technique exist. For example, it is possible to produce monoclonal antibodies by expressing a nucleic acid cloned from a hybridoma. Antibodies can also be produced by phage display techniques by introducing cDNA for an antibody into a vector, usually a filamentous phage that displays a gene library V on its surface (e.g., fUSE5 for E. coli, Scott JK, Smith GP Science 1990; 249:386-390). Protocols for constructing these antibody libraries are described in JD Marks et al., J. Mol. Biol., 222 (1991), 581. cDNA corresponding to full-length netrin-1 (SEQ ID NO: 2) or a suitable fragment thereof with a signal sequence may be used to produce monoclonal antibodies according to these methods.

[0046] The anti-Netrin-1 monoclonal antibody (mAb) may be a murine, chimeric, humanized or fully human monoclonal antibody. The fragment may be any kind of mAb fragment that substantially maintains the ability of the whole antibody to bind to Netrin-1, for example, Fab or F(ab')2. In particular, the monoclonal antibodies are those disclosed in WO2015 / 104360 or US10,494,427, which documents are incorporated herein by reference and disclose useful murine, chimeric and humanized monoclonal antibodies. These are antibodies or fragments thereof that specifically bind to NTN1 epitopes or polypeptides having the amino acid sequences SEQ ID NO: 3 or 33, or variants thereof.

[0047] Monoclonal antibodies useful in the present invention may be defined by their CDRs. In particular, these CDRs are derived from the murine antibody 4C11 disclosed in WO2015 / 104360, which specifically binds to a polypeptide having the amino acid sequence SEQ ID NO: 3 or 33. Preferably, the antibody comprises: - H-CDR1 having the sequence shown as SEQ ID NO:5, - H-CDR2 having the sequence shown as SEQ ID NO:6, - H-CDR3 having the sequence shown as SEQ ID NO: 7 A variable domain VH comprising - L-CDR1 having the sequence shown as SEQ ID NO: 8, - L-CDR2 with the sequence YAS, - L-CDR3 having the sequence shown as SEQ ID NO: 9 A variable domain VL comprising or - H-CDR1 having the sequence shown as SEQ ID NO: 28, - H-CDR2 having the sequence shown as SEQ ID NO: 29, - H-CDR3 having the sequence shown as SEQ ID NO: 30 A variable domain VH comprising - L-CDR1 having the sequence shown as SEQ ID NO: 31, - L-CDR2 having the sequence shown as SEQ ID NO: 32, - L-CDR3 having the sequence shown as SEQ ID NO: 9 A variable domain VL comprising The monoclonal antibody or antigen-binding fragment thereof comprises:

[0048] In a first set of embodiments, the antibody of the invention comprises the amino acid sequence of SEQ ID NO: 10, 11, 12 or 13. Usually it comprises a VH of sequence SEQ ID NO: 10 and a VL of sequence SEQ ID NO: 11, or a heavy chain of sequence SEQ ID NO: 12 and a light chain of sequence SEQ ID NO: 13.

[0049] In a second series of embodiments, the antibody is chimeric. Preferably, it comprises a VH of sequence SEQ ID NO: 27 and a VL of sequence SEQ ID NO: 19.

[0050] In a third series of embodiments, the antibody is humanized. Preferably, it comprises an amino acid sequence selected from the group of SEQ ID NOs: 14 to 18 (VL) and / or the group of SEQ ID NOs: 20 to 26 (VH). Usually, the antibody is humanized and comprises a VH having an amino acid sequence selected from the group of SEQ ID NOs: 14 to 18 and a VL having an amino acid sequence selected from the group of SEQ ID NOs: 20 to 26.

[0051] The antibody preferably comprises a monoclonal antibody (mAb) or an antigen-binding fragment thereof, which mAb or fragment thereof specifically binds to netrin-1. The mAb may be a murine, chimeric, humanized or fully human monoclonal antibody. The fragment may be any kind of mAb fragment that substantially retains the ability of the whole antibody to bind to netrin-1, for example Fab or F(ab')2.

[0052] Particular embodiments disclosed in this previous document and usable herein are the following antibodies listed in Table 1. The first antibody listed is a chimeric 4C11 antibody comprising the mouse VH and VL of the mouse 4C11 antibody. HUM00 listed in Table 1 corresponds to the grafting of mouse 4C11 CDRs onto human IgG1. The ten humanized mAbs HUM01-HUM10 correspond to humanized mAbs derived from HUM00 with the same CDRs but with specific modifications in the FR regions of human IgG. HUM03 is also known as NP137 and is currently in clinical trials. The sequence of the human IgG1 CH is derived from Genbank AEL33691.1 modified R97K. The sequence of the human IgG1 CL (kappa) is derived from Genbank CAC20459.1. Other allotypes are also usable. The specific binding of all these mAbs, murine, chimeric and humanized HUM01 to HUM10, Fab fragments and F(ab')2 fragments to netrin-1 and their ability to inhibit the binding of netrin-1 to its receptor UNC5B is shown in US 10,494,427 (Example 3).

[0053] In particular, these monoclonal antibodies specifically bind to a polypeptide having the amino acid sequence of SEQ ID NO:33.

[0054] [Table 1]

[0055] Preferably, the antibody is a monoclonal antibody, or an antigen-binding fragment thereof, comprising a pair of VH and VL sequences selected from the following pairs: SEQ ID NO:27 and 19, SEQ ID NO:20 and 14, SEQ ID NO:21 and 15, SEQ ID NO:22 and 16, SEQ ID NO:23 and 17, SEQ ID NO:24 and 17, SEQ ID NO:25 and 16, SEQ ID NO:26 and 17, SEQ ID NO:22 and 17, SEQ ID NO:25 and 18, SEQ ID NO:21 and 16. More preferably, the antibody is a monoclonal antibody, or an antigen-binding fragment thereof, comprising the pair of VH and VL sequences SEQ ID NO:22 and 16.

[0056] The anti-netrin-1 monoclonal antibody may further comprise a human IgG1 constant heavy chain (CH) and / or a human IgG1 constant light chain (CL), in particular a human kappa constant domain.

[0057] In one embodiment, the sequence of human IgG1 CH is from Genbank AEL33691.1 modified R97K. The sequence of human IgG1 CL (kappa) is from Genbank CAC20459.1. In one embodiment, the mAb is NP137 (AB_2811180 in Antibody Registry) and contains SEQ ID NOs: 22 and 16 as VH, VL sequences, respectively, and their unique IgG1 CH and CL.

[0058] The term "antigen-binding fragment" (or "antibody-binding portion") of a monoclonal antibody, as used herein, refers to one or more fragments of an antibody that maintain the ability to specifically bind to netrin-1, in particular to the same epitope as the whole antibody or to a peptide netrin-1 sequence (e.g., SEQ ID NO: 3 or 33) that is shorter than the whole antibody. In particular, the fragment comprises the same VH and VL regions as the whole antibody. The antibody fragment may comprise, for example, a Fab fragment, a F(ab')2 fragment, a Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. In one embodiment, the fragment comprises the VH and VL sequences of an antibody selected from HUM00 to HUM10.

[0059] Anti-netrin-1 monoclonal antibodies that can be used include other monoclonal antibodies developed against human netrin-1 or animal netrin-1, or antigen-binding fragments thereof, where netrin-1 is highly homologous between species. Abcam antibodies ab126729, ab122903, ab201324, ab39370; AF1109, AF6419, AF128.

[0060] BH3 mimetics and B-cell lymphoma 2 (Bcl-2) inhibitors: "Bcl-2 inhibitors" are a class of drugs that function by inhibiting the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein, resulting in programmed cell death of cells.

[0061] Venetoclax, also known in the art as GDC-0199, ABT-199, or RG7601, is a BCL-2 inhibitor. Venetoclax is chemically known as 4-[4-[[2-(4-chlorophenyl)-4,4-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[3-nitro-4-(oxan-4-ylmethylamino)phenyl]sulfonyl-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide. Its developed structure is well known. Venetoclax and methods for its preparation are disclosed in U.S. Patent No. 8,546,399. Venetoclax is marketed in the United States under the trade name VENCLEXTA™ by AbbVie, Inc. and is indicated for the treatment of chronic lymphocytic leukemia.

[0062] The BH3 mimetic may be an inhibitor of Bcl-2 itself (Bcl-2 inhibitor) or of any other anti-apoptotic member of the Bcl-2 family, as navitoclax is, an inhibitor of at least two of these proteins, for example Bcl-2 and Bcl-XL, or Bcl-2, Bcl-W and Bfl-1 / A1.

[0063] In some embodiments, the inhibitor is venetoclax (ABT-199 or GDC-0199), ABT-737, BP1002, SPC2996, APG-1252, obatoclax mesylate (GX15-070MS), or PNT2258, navitoclax.

[0064] Definition of the invention and further embodiments, variations and alternatives: In the context of the present invention, the term "treating" or "treatment" as used herein means reversing, alleviating or inhibiting the progression of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

[0065] By the term "treating cancer", as used herein, it is specifically intended to inhibit the proliferation of malignant cells of a tumor and / or the progression of metastases from said tumor. Such treatment may also lead to regression of tumor growth, i.e., a reduction in the size of a measurable tumor. In certain embodiments, such treatment leads to partial regression of tumors or metastases. In another particular embodiment, such treatment leads to complete regression of tumors or metastases. In some aspects, the treatment prevents metastasis. In the present invention, such treatment leads to cancer cell apoptosis or programmed cell death.

[0066] According to the present invention, the term "patient" or "patient in need thereof" is intended to mean a human or non-human mammal suffering from or likely to suffer from a malignant tumor.

[0067] By "therapeutically effective amount" is intended a sufficient amount of active agent to treat said cancer disease with a reasonable benefit / risk ratio applicable to any medical treatment. However, it should be understood that the total daily amount of active agent is determined by the attending physician within the scope of sound medical judgment. The therapeutically effective dose level specific to any particular patient depends on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific polypeptide or antibody being used; the specific composition being used, the age, weight, general health, sex and diet of the patient; the administration time, route of administration and excretion rate of the specific active agent being used; the duration of treatment; the drugs used in combination with or in the presence of the specific active agent being used; and similar factors well known in the medical art.

[0068] "Pharmaceutically" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to a mammal, particularly a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating substance or formulation auxiliary of any type.

[0069] Pharmaceutical Compositions: The form of a pharmaceutical composition comprising a polypeptide or antibody of the invention, and the route of administration will necessarily depend on the condition to be treated, the severity of the disease, the age, weight, and sex of the patient, and the like.

[0070] The active agents of the present invention may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like. In one particular embodiment, the active agents of the present invention are administered intravenously.

[0071] In particular, pharmaceutical compositions containing the active agents of the present invention may contain pharma- ceutically acceptable vehicles for injectable preparations, which may in particular be isotonic and sterile saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dried, in particular lyophilized, compositions that, optionally, upon addition of sterile water or saline, allow the constitution of an injectable solution.

[0072] To prepare pharmaceutical compositions, an effective amount of the active agents of the present invention may be dissolved or dispersed in a pharma- ceutically acceptable carrier or aqueous medium.

[0073] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0074] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants, stabilizers, cryoprotectants, or antioxidants. Prevention of the action of microorganisms can be brought about by antibacterial and antifungal agents. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride.

[0075] Sterile injectable solution is prepared by incorporating the required amount of active agent in a suitable solvent with some of the other ingredients listed above as necessary, and then sterilizing by filtration.In general, dispersion is prepared by incorporating various sterilized active ingredients into a sterile medium that contains a basic dispersion medium and other necessary ingredients from the ingredients listed above.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technique, which produces powder of active ingredient and any other desired ingredients from its solution that has been previously sterile-filtered.

[0076] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, although drug release capsules and the like are also available.

[0077] For parenteral administration as an aqueous solution, for example, the solution should be suitably buffered if necessary, and the liquid diluent is first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, available sterile aqueous media will be known to those skilled in the art in light of this disclosure. For example, a dosage can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous infusion therapy liquid, or injected at the proposed injection site (see, for example, "Remington's Pharmaceutical Sciences", 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will determine the appropriate dose for the individual subject.

[0078] Method of drug administration and use: As used herein, "simultaneously" is used to mean that two agents are administered in parallel, and the term "in combination" is used to mean that two agents are administered "sequentially" within the same time frame, if not simultaneously, that is, both available to act therapeutically within the same time frame. Thus, "sequential" administration allows one agent to be administered within 5 minutes, 10 minutes, or just a few hours after the other has exhibited the circulating half-life of the agent administered first, such that the agents are both present in parallel in therapeutically effective amounts. The time delay between administration of the components varies depending on the exact nature of the components, the interaction between the components, and their respective half-lives.

[0079] "Separately," as opposed to "in combination" or "sequentially," is used herein to mean a large time lag between administration of one agent and the other, i.e., hours, which may include cases where the first administered agent is no longer present in the bloodstream in therapeutically effective amounts when the second agent is administered, but the first administered agent may still be present in cancer cells and may still be active.

[0080] In one embodiment of the invention, the anti-netrin-1 antibody or antigen-binding fragment thereof is administered sequentially or separately prior to the Bcl-2 inhibitor.

[0081] In a particularly preferred embodiment, the Bcl-2 inhibitor is administered sequentially or separately before the anti-netrin-1 antibody or antigen-binding fragment thereof.

[0082] In one embodiment, both the anti-netrin-1 antibody or antigen-binding fragment thereof and the Bcl-2 inhibitor are present in the same composition, together with pharma- ceutically acceptable carriers, excipients and / or diluents.

[0083] In another embodiment, they are provided as separate pharmaceutical forms or kit of parts. This forms a composition or set or kit of parts that includes anti-netrin-1 antibody or its antigen-binding fragment and Bcl-2 inhibitor for simultaneous, separate or sequential administration to a patient. Thus, the present invention may include (i) a composition that includes two active ingredients as a mixture, or (ii) a composition that includes these active ingredients that are kept separately in the same state or in separate states, in the case of (ii), each of the active ingredients is present in its own vial or container, which is usually called the kit of parts idea.

[0084] In one embodiment, the antibody or antigen-binding fragment, or the simultaneous combination of an anti-netrin-1 antibody or antigen-binding fragment thereof and a Bcl-2 inhibitor thereof, is administered as a single dose, or an initial dose followed by administration of a second or multiple subsequent doses of the same, wherein the subsequent doses are spaced at least 1 day; at least 1 week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks apart.

[0085] In one embodiment of the methods of treatment, uses and compositions for use, the administration of the anti-netrin-1 antibody or antigen-binding fragment thereof and the Bcl-2 inhibitor is sequential or separate. The interval between the administration of both may be at least 5, 10, 15, 20 or 24 hours, preferably 24 to 96 hours, more preferably 24 to 72 hours or more, especially 24 to 48 hours, for example 24 hours. In one embodiment, one agent or drug is simply administered the day after the administration of the other agent or drug. Also, the first administered active ingredient (anti-netrin-1 antibody or antigen-binding fragment thereof, or Bcl-2 inhibitor) may be administered as a single dose, or an initial dose followed by administration of a second or multiple subsequent doses of the same, with subsequent doses spaced at least 1 day; at least 1 week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks apart, depending on which of the above time intervals are calculated.

[0086] A variety of pharmaceutical forms may be used in sufficient amounts in the methods of treatment of the present invention.

[0087] The present invention does not imply or may not imply a change in the dosage regimen of the Bcl-2 inhibitor, such as venetoclax. However, the synergistic effect generated by the anti-netrin-1 antibody or antigen-binding fragment thereof may allow the use of a smaller dosage regimen of the Bcl-2 inhibitor, such as venetoclax, in the patient. Those skilled in the art can determine the optimal dosage regimen in the context of the combination therapy provided by the present invention.

[0088] The pharmaceutical composition may be administered to the subject at a suitable dose, i.e., at least 1 mg / kg body weight, for example about 1 mg / kg body weight to about 100 mg / kg body weight, in particular about 10 mg / kg body weight to about 60 mg / kg body weight, of the subject whose cancer is to be treated, for the anti-netrin-1 antibody or antigen-binding fragment thereof. The Bcl-2 inhibitor, e.g., venetoclax, may be administered at a normal dose or at a reduced dose relative to the normal dose to the extent that the combination has synergistic efficacy. For example, the dose of the Bcl-2 inhibitor, e.g., venetoclax, may be reduced by 10, 20, 30, 40, 50%, or more.

[0089] As used herein, the term "synergistic" means that when an active ingredient, such as an antibody, is used in combination, it produces an effect that is greater than would be expected from adding the individual effects of the two ingredients. Advantageously, synergistic interactions allow for lower doses of each ingredient to be administered to a patient, thereby reducing the toxicity of chemotherapy while producing and / or maintaining the same therapeutic effect. Thus, in a particularly preferred embodiment, each ingredient may be administered in a sub-therapeutic amount.

[0090] [Table 2A]

[0091] [Table 2B]

[0092] [Table 2C]

[0093] [Table 2D]

[0094] [Table 2E]

[0095] [Table 2F]

[0096] As used herein, a sequence that is "at least 85% identical to a reference sequence" is a sequence that has 85% or more, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity over its entire length to the reference sequence.

[0097] The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, where a portion of the polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which contains no additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical amino acid residues appear in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences for comparison is performed by global pairwise alignment, for example, using the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443. The percentage of sequence identity can be readily determined, for example, using the program Needle, using the BLOSUM62 matrix and the following parameters: gap open=10, gap extend=0.5.

[0098] In the context of the present invention, a "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain group with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. Examples of groups of amino acids with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine-tryptophan, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.

[0099] Throughout this application, the term "comprising" should be interpreted as embracing all features specifically mentioned, as well as optional further unspecified features. As used herein, use of the term "comprising" also discloses embodiments in which no features are present other than those specifically mentioned (i.e., "consisting of").

[0100] An "antibody" may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains a unique sequence domain. The light chain contains two domains or regions, the variable domain (VL) and the constant domain (CL). The heavy chain contains four domains, the variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable parts of one light chain and one heavy chain. The specificity of an antibody resides in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is composed mainly of residues from the hypervariable or complementarity determining regions (CDRs). In some cases, residues from non-hypervariable or framework regions (FRs) influence the overall domain structure and hence the binding site.

[0101] "Complementarity determining region" or "CDR" refers to an amino acid sequence that together defines the binding affinity and specificity of the natural Fv region of a natural immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, the antigen-binding site of a conventional antibody contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions.

[0102] "Framework region" (FR) refers to the amino acid sequences interposed between the CDRs, i.e., portions of the immunoglobulin light and heavy chain variable regions that are relatively conserved among various immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively.

[0103] As used herein, a "human framework region" is a framework region that is substantially identical (about 85% or more, particularly 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

[0104] In the context of the present invention, the CDR / FR definitions in an immunoglobulin light or heavy chain will be determined according to the Kabat or IMGT definitions.

[0105] Residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al., which is set out in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, United States Department of Health and Human Services, NIH, USA (hereinafter "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not always correspond directly to the linear numbering of amino acid residues in the SEQ ID NO: sequence. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, which corresponds to truncation of, or insertion into, structural elements, whether framework or complementarity determining regions (CDRs) of the basic variable domain structure. The exact Kabat numbering of residues can be determined for a given antibody by alignment of homologous residues in the antibody sequence with the "standard" Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35B (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system. ((http: / / ) www.bioinf.org.uk / abs / #cdrdef)

[0106] In the context of the present invention, the amino acid residues of the antibodies of the present invention may be numbered according to the IMGT numbering system. The IMGT unique numbering has been defined to compare variable domains of any antigen receptor, chain type, or species (Lefranc M.-P., "Immunology Today, 18, 509 (1997); Lefranc M.-P., The Immunologist, 7, pp. 132-136 (1999); Lefranc, M.-P. et al. Dev. Comp. Immunol., 27, pp. 55-77 (2003). In the unique numbering of IMGT, conserved amino acids always have the same positions, e.g., cysteine ​​23, tryptophan 41, hydrophobic amino acid 89, cysteine ​​104, phenylalanine or tryptophan 118. The unique numbering of IMGT is divided into the framework regions (FR1-IMGT: 1-26, FR2-IMGT: 39-55, FR3-IMGT: 66-104 and FR4-IMGT: 118-128 positions) and the complementarity determining regions: CDR1-IMGT: 27-38, CDR2-IMGT: 28-39, CDR3-IMGT: 36-41, CDR4-IMGT: 38-42, CDR5-IMGT: 39-55, CDR6-IMGT: 39-55, CDR7-IMGT: 39-55, CDR8-IMGT: 39-61, CDR9-IMGT: 39-70, CDR10-IMGT: 39-82, CDR11-IMGT: 39-90, CDR12-IMGT: 39-102, CDR13-IMGT: 39-114, CDR14-IMGT: 39-126, CDR15-IMGT: 39-136, CDR16-IMGT: 39-140, CDR17-IMGT: 39-152, CDR18-IMGT: 39-162, CDR19-IMGT: 39-202, CDR20-IMGT: 39-216, CDR21-IMGT: 39-32, CDR22-IMGT: 39-55, CDR This results in standardized delimitations of CDR3-IMGT: 56-65 and CDR3-IMGT: 105-117. If the CDR3-IMGT length is less than 13 amino acids, gaps are created from the tip of the loop in the following order: 111, 112, 110, 113, 109, 114, etc. If the CDR3-IMGT length is more than 13 amino acids, additional positions are created at the tip of the CDR3-IMGT loop between positions 111 and 112 in the following order: 112.1, 111.1, 112.2, 111.2, 112.3, 111.3, etc. ((http: / / )www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html).

[0107] As used herein, the term "antibody" refers to conventional antibodies and fragments thereof, as well as to single domain antibodies and fragments thereof, in particular the variable heavy chains of single domain antibodies, and chimeric, humanized, bispecific or multispecific antibodies.

[0108] As used herein, antibody or immunoglobulin also includes the more recently described "single domain antibodies", which are antibodies whose complementarity determining regions are part of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from traditional four-chain antibodies, engineered single domain antibodies. Single domain antibodies may be derived from any species, including but not limited to mouse, human, camel, llama, goat, rabbit and cow. Single domain antibodies may be naturally occurring single domain antibodies, known as heavy chain antibodies devoid of light chains. In particular, species of the Camelidae family, such as camels, dromedaries, llamas, alpacas and guanacos, produce heavy chain antibodies naturally devoid of light chains. Camelid heavy chain antibodies also lack the CH1 domain.

[0109] The variable heavy chains of these single domain antibodies, devoid of light chains, are known in the art as "VHH" or "nanobodies". Similar to conventional VH domains, VHH contain four FRs and three CDRs. Nanobodies have advantages over conventional antibodies: they are about 10 times smaller than IgG molecules, and as a result properly folded and functional nanobodies can be produced by in vitro expression while achieving high yields. Furthermore, nanobodies are highly stable and resistant to the action of proteases. The properties and production of nanobodies have been reviewed by Harmsen and De Haard (2007) Appl. Microbiol. Biotechnol. 77:13-22.

[0110] The terms "monoclonal antibody" or "mAb," as used herein, refer to an antibody molecule of a single amino acid composition directed against a particular antigen and should not be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be produced by a single clone of B cells or hybridoma, but may also be recombinant, i.e., produced by protein engineering.

[0111] A "fragment" of a (conventional) antibody comprises a portion of an intact antibody, in particular the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may be a single domain antibody, such as a heavy chain antibody or a VHH.

[0112] The term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 Da and antigen-binding activity, in which approximately half of the N-terminal side of the H chain and the entire L chain are bound together via disulfide bonds, among fragments obtained by treating IgG with the protease papain.

[0113] The term "F(ab')2" refers to an antibody fragment obtained by treating IgG with the protease pepsin, which is bound by disulfide bonds in the hinge region, is slightly larger than Fab, has a molecular weight of about 100,000 Da, and has antigen-binding activity.

[0114] Single-chain Fv ("scFv") polypeptides are covalently linked VH::VL heterodimers that are usually expressed from gene fusions containing genes encoding VH and VL linked by a peptide-encoding linker. Human scFv fragments of the invention contain the CDRs maintained in the proper conformation, particularly by using recombinant gene techniques. Bivalent and multivalent antibody fragments may form spontaneously by association of monovalent scFvs or may be produced by linking monovalent scFvs by peptide linkers, such as bivalent sc(Fv)2.

[0115] A "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond.

[0116] "(dsFv)2" refers to two dsFvs linked by a peptide linker.

[0117] The term "bispecific antibody" refers to small antibody fragments having two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain, creating two antigen-binding sites.

[0118] In certain embodiments, the epitope-binding fragment is selected from the group consisting of an Fv, a Fab, a F(ab')2, a Fab', a dsFv, a (dsFv)2, a scFv, a sc(Fv)2, a bispecific antibody, and a VHH.

[0119] A "chimeric antibody," as used herein, is an antibody in which the constant region, or a portion thereof, has been altered, replaced or exchanged, so that the variable region is linked to a constant region belonging to a different species or to another antibody class or subclass. "Chimeric antibody" also refers to an antibody in which the variable region, or a portion thereof, has been altered, replaced or exchanged, so that the constant region is linked to a variable region belonging to a different species or to another antibody class or subclass.

[0120] The term "humanized antibody" refers to an antibody that is originally of fully or partially non-human origin and has been modified to replace certain amino acids, particularly in the framework regions of the heavy and light chains, to avoid or minimize immune responses in humans. The constant domains of a humanized antibody are most often human CH and CL domains. In one embodiment, a humanized antibody has constant domains of human origin. As used herein, the term "humanized antibody" refers to a chimeric antibody that contains minimal sequences derived from non-human immunoglobulin, such as CDRs.

[0121] The term "antibody" is used to encompass all these types of antibodies, fragments or combinations thereof.

[0122] The goal of humanization is to reduce the immunogenicity of xenogeneic antibodies, e.g., murine antibodies, for introduction into humans while maintaining the full antigen-binding affinity and specificity of the antibody. Humanized antibodies, or antibodies adapted to not be rejected by other mammals, may be generated using several techniques, e.g., resurfacing and CDR grafting. As used herein, resurfacing techniques use a combination of molecular modeling, statistical analysis, and mutagenesis to alter the non-CDR surfaces of antibody variable regions to resemble the surfaces of known antibodies of the target host.

[0123] Antibodies may be humanized using a variety of other techniques, including CDR grafting (EP 0239400; WO 91 / 09967; U.S. Pat. Nos. 5,530,101 and 5,585,089), veneering or resurfacing (EP 0592106; EP 0519596; Padlan (1991) Molecular Immunology 28(4 / 5):489-498; Studnicka et al. (1994) Protein Engineering 7(6):805-814; Roguska et al. (1994) Proc. Natl. Acad. Sci USA 91:969-973), and chain shuffling (U.S. Pat. No. 5,565,332). Human antibodies can be produced by a variety of methods known in the art, including phage display methods. See also U.S. Patent No. 4,444,887, U.S. Patent No. 4,716,111, U.S. Patent No. 5,545,806, and U.S. Patent No. 5,814,318; and International Patent Application No. WO 98 / 46645, International Patent Application No. WO 98 / 50433, International Patent Application No. WO 98 / 24893, International Patent Application No. WO 98 / 16654, International Patent Application No. WO 96 / 34096, International Patent Application No. WO 96 / 33735, and International Patent Application No. WO 91 / 10741.

[0124] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to an antigen, such as an epitope presented on netrin-1, but have relatively little detectable reactivity with non-netrin-1 proteins or structures (e.g., other proteins presented on cancer cells or other cell types). Specificity can be relatively determined by binding or competitive binding assays, for example, using Biacore instruments, as described elsewhere herein. Specificity can be shown by an affinity / avidity ratio of, for example, about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1 or more, in binding to a specific antigen versus nonspecific binding to other unrelated molecules (in this case, the specific antigen is netrin-1).

[0125] The term "affinity" as used herein refers to the strength of binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab]x[Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988, Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are incorporated herein by reference in their entireties. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of a Biacore instrument.

[0126] The invention will now be described by way of non-limiting examples with reference to the drawings. [Brief description of the drawings]

[0127] [Figure 1] CRISPR-Cas9 screening identifies genes and molecular pathways involved in UNC5B-induced cell death. (A): UNC5B overexpression causes cell death. Stable HeLa cells inducible for UNC5B (HeLa UNC5B) were treated with the indicated amounts of doxycycline (Dox). To assess cell death, propidium iodide (PI) incorporation was followed in real time for up to 48 hours by Incucyte Zoom technology (n=3, ***p<0.01, Kolmogorov-Smirnov test). Values ​​are given as mean ± SEM. (B, C): UNC5B-induced cell death is associated with DNA fragmentation and caspase-3 activation and is inhibited by netrin-1. HeLa UNC5B cells constitutively expressing netrin-1 (UNC5B+NTN1) were treated with the indicated concentrations of Dox for 18 h and apoptosis was assessed by DNA fragmentation assay via flow cytometry (n=3, **p<0.01, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test) (B). Caspase-3 activity was measured in real time for up to 24 h using an Incucyte Zoom instrument (n=3, ****p<0.0001, Kolmogorov-Smirnov test) (C). Values ​​are given as mean ± SEM. [Figure 2-1]CRISPR-Cas9 screening identifies genes and molecular pathways involved in UNC5B-induced cell death. Related to Figure 1. (A): UNC5B induction upon treatment with doxycycline. Stable HCT116 cells inducible for UNC5B were treated with the indicated concentrations of doxycycline (Dox). After 24 hours, the protein levels of FLAG-UNC5B and GAPDH, used as a housekeeping gene, were assessed by Western blot. (B): UNC5B induction upon treatment with doxycycline on HeLa cells. Stable HeLa cells inducible for UNC5B (HeLa UNC5B) were treated with the indicated doses of Dox for 18 hours. The protein levels of His-UNC5B and β-actin, used as a housekeeping gene, were measured by Western blot. (C, D, E): Induction of UNC5B causes apoptotic cell death in HCT116 cells. Stable HCT116 cells inducible for human UNC5B were treated with Dox. To assess cell death, propidium iodide (PI) incorporation was followed in real time for up to 36 hours using Incucyte Zoom technology (n=3, **p<0.01, Kolmogorov-Smirnov test) (C). Apoptosis was assessed 18 hours after Dox treatment by DNA fragmentation via flow cytometry (assessed 18 hours after Dox treatment by flow cytometry (D) and caspase-3 activity (E) quantified by a fluorometric detection kit) (n=3, *p<0.05, **p<0.01, ***p<0.001, ordinary two-way ANOVA). Values ​​are given as mean ± SEM. (F): Caspase inhibition rescues apoptosis caused by UNC5B. HeLa UNC5B cells were treated with Dox and the pan-caspase inhibitor Q-VC-OPh (QVD). After 18 hours, DNA fragmentation was assessed by flow cytometry (n=3, ****p<0.0001, ordinary two-way ANOVA with Sidak's multiple comparison test). Values ​​are given as mean±SEM. (G): Candidate genes identified in the CRISPR-Cas9-based screen.The number of guides showing increased reads compared to the control condition is shown for each gene. [Figure 2-2]CRISPR-Cas9 screening identifies genes and molecular pathways involved in UNC5B-induced cell death. Related to Figure 1. (A): UNC5B induction upon treatment with doxycycline. Stable HCT116 cells inducible for UNC5B were treated with the indicated concentrations of doxycycline (Dox). After 24 hours, the protein levels of FLAG-UNC5B and GAPDH, used as a housekeeping gene, were assessed by Western blot. (B): UNC5B induction upon treatment with doxycycline on HeLa cells. Stable HeLa cells inducible for UNC5B (HeLa UNC5B) were treated with the indicated doses of Dox for 18 hours. The protein levels of His-UNC5B and β-actin, used as a housekeeping gene, were measured by Western blot. (C, D, E): Induction of UNC5B causes apoptotic cell death in HCT116 cells. Stable HCT116 cells inducible for human UNC5B were treated with Dox. To assess cell death, propidium iodide (PI) incorporation was followed in real time for up to 36 hours using Incucyte Zoom technology (n=3, **p<0.01, Kolmogorov-Smirnov test) (C). Apoptosis was assessed 18 hours after Dox treatment by DNA fragmentation via flow cytometry (assessed 18 hours after Dox treatment by flow cytometry (D) and caspase-3 activity (E) quantified by a fluorometric detection kit) (n=3, *p<0.05, **p<0.01, ***p<0.001, ordinary two-way ANOVA). Values ​​are given as mean ± SEM. (F): Caspase inhibition rescues apoptosis caused by UNC5B. HeLa UNC5B cells were treated with Dox and the pan-caspase inhibitor Q-VC-OPh (QVD). After 18 hours, DNA fragmentation was assessed by flow cytometry (n=3, ****p<0.0001, ordinary two-way ANOVA with Sidak's multiple comparison test). Values ​​are given as mean±SEM. (G): Candidate genes identified in the CRISPR-Cas9-based screen.The number of guides showing increased reads compared to the control condition is shown for each gene. [Figure 3-1]COMMD2 is required for UNC5B-induced cell death and tumor suppression. (A): Silencing COMMD2 inhibits UNC5B-induced DNA fragmentation. Stable HeLa cells inducible for UNC5B were transfected with two separate siRNAs scrambled (siCTRL) or siRNAs targeting COMMD2 (siCOMMD2-1 and -2). Upon treatment with the indicated concentrations of doxycycline (Dox) for 18 h, DNA fragmentation was assessed by FACS analysis (n=3, ****p<0.0001, ordinary two-way ANOVA). Values ​​are given as mean±SEM. (B,C,D): Tumor growth inhibition driven by netrin-1 interference requires UNC5B and COMMD2. NMRI nude mice were implanted with stable H322 cells knocked down for luciferase (sgLUC, used as a non-targeting control), UNC5B (sgUNC5B), or COMMD2 (sgCOMMD2) generated by the CRISPR-Cas-9-KRAB strategy. Tumors obtained from nude mice implanted with stable H322 cells were lysed and gene expression was assessed by quantitative RT-PCR (n=2-3, *p<0.05, Kruskal-Wallis test with Dunn's multiple comparison test) (B). After tumor volumes reached 50 mm3, mice were treated intravenously with anti-Netrin-1 (NP137) or control (Iso-mAb) antibodies three times a week and tumor growth was assessed (n=10 mice / group, nsp>0.05, *p<0.05, Mann-Whitney test, two-tailed) (C). Histograms showing tumor volumes 35 days after the first treatment are shown in (D) (n=10 mice / group, nsp>0.05, *p<0.05, ***p<0.001, Kruskal-Wallis test with Dunn's test uncorrected). (E) Silencing of UNC5B and COMMD2 suppresses NP137-induced apoptosis in vivo. After sacrificing mice, tumors were excised and apoptosis was quantified by immunostaining for active post-cleavage caspase-3. Positive cells were then indexed in the excised tumors in mice treated with Iso-mAb in each stable cell line (n=10 mice / group, nsp>0.05, *p<0.05, ***p<0.001, Kruskal-Wallis test with Dunn's test uncorrected).Histograms represent the mean±SEM. [Figure 3-2]COMMD2 is required for UNC5B-induced cell death and tumor suppression. (A): Silencing COMMD2 inhibits UNC5B-induced DNA fragmentation. Stable HeLa cells inducible for UNC5B were transfected with two separate siRNAs scrambled (siCTRL) or siRNAs targeting COMMD2 (siCOMMD2-1 and -2). Upon treatment with the indicated concentrations of doxycycline (Dox) for 18 h, DNA fragmentation was assessed by FACS analysis (n=3, ****p<0.0001, ordinary two-way ANOVA). Values ​​are given as mean±SEM. (B,C,D): Tumor growth inhibition driven by netrin-1 interference requires UNC5B and COMMD2. NMRI nude mice were implanted with stable H322 cells knocked down for luciferase (sgLUC, used as a non-targeting control), UNC5B (sgUNC5B), or COMMD2 (sgCOMMD2) generated by the CRISPR-Cas-9-KRAB strategy. Tumors obtained from nude mice implanted with stable H322 cells were lysed and gene expression was assessed by quantitative RT-PCR (n=2-3, *p<0.05, Kruskal-Wallis test with Dunn's multiple comparison test) (B). After tumor volumes reached 50 mm3, mice were treated intravenously with anti-Netrin-1 (NP137) or control (Iso-mAb) antibodies three times a week and tumor growth was assessed (n=10 mice / group, nsp>0.05, *p<0.05, Mann-Whitney test, two-tailed) (C). Histograms showing tumor volumes 35 days after the first treatment are shown in (D) (n=10 mice / group, nsp>0.05, *p<0.05, ***p<0.001, Kruskal-Wallis test with Dunn's test uncorrected). (E) Silencing of UNC5B and COMMD2 suppresses NP137-induced apoptosis in vivo. After sacrificing mice, tumors were excised and apoptosis was quantified by immunostaining for active post-cleavage caspase-3. Positive cells were then indexed in the excised tumors in mice treated with Iso-mAb in each stable cell line (n=10 mice / group, nsp>0.05, *p<0.05, ***p<0.001, Kruskal-Wallis test with Dunn's test uncorrected).Histograms represent the mean±SEM. [Figure 4-1]UNC5B triggers proteasomal degradation of Mcl-1. (A, B): Proteasome activity is required for UNC5B-induced apoptosis. Stable HeLa cells inducible for UNC5B (HeLa UNC5B) were treated with 2 μg / mL doxycycline (Dox) in combination with the indicated doses of proteasome inhibitors MG132 and bortezomib (BTZ). 18 h after treatment, apoptosis was measured by DNA fragmentation (A) and caspase-3 activity (B) (n=3-4, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. Treatment with proteasome inhibitors was sufficient to significantly reduce apoptosis induction upon UNC5B overexpression. (C): UNC5B triggers Mcl-1 degradation. HeLa UNC5B cells were treated with 2 μg / mL Dox in combination with bortezomib (BTZ, 1 μM) and the pan-caspase inhibitor Q-VD-OPh (QVD, 10 μM). After 16 h of treatment, cells were lysed by detachment and sonication in SDS-containing buffer. Protein levels of Mcl-1, Bcl-2 and Bcl-XL, as well as UNC5B and β-actin, used as a housekeeping gene, were assessed by Western blot. The ratio of protein levels between untreated (0) and doxycycline-treated (2), normalized to β-actin, is shown below each Western blot. UNC5B overexpression specifically promoted Mcl-1 protein reduction. Proteasome inhibition by treatment with bortezomib rescued this degradation, whereas caspase inhibition by Q-VD-OPh, although it inhibited cell death, had no effect on Mcl-1 degradation. (D): UNC5B-induced Mcl-1 degradation requires the COMMD2 / CRL3 complex. HeLa UNC5B cells were transfected with scrambled (siCTRL), COMMD2 (siCOMMD2) or RBX1 (siRBX1) siRNA. 24 h after transfection, cells were treated with 2 μg / mL Dox for an additional 18 h.Protein levels of Mcl-1, UNC5B, and Ku80, used as a housekeeping protein, were assessed by Western blot. Silencing COMMD2 or RBX1 was sufficient to block Mcl-1 degradation induced by UNC5B overexpression. (E): Mcl-1 overexpression is not sufficient to rescue UNC5B-induced apoptosis. Stable HeLa UNC5B cells constitutively expressing Mcl-1 or empty vector were treated with doxycycline and apoptosis was assessed 18 h later by DNA fragmentation (n=3, nsp>0.05, ordinary two-way ANOVA with Sidak's multiple comparison test). Values ​​are given as mean ± SEM. (F): Overexpressed Mcl-1 protein, but not Bcl-2 or Bcl-XL, is significantly degraded upon UNC5B induction. Stable HeLa UNC5B cells constitutively expressing Mcl-1, Bcl-2 or Bcl-XL were treated with Dox and protein levels were assessed as described in (C). (G): Proteasome activity is required for overexpressed Mcl-1 degradation upon UNC5B induction. Stable HeLa UNC5B cells constitutively expressing Mcl-1 were treated with 2 μg / mL Dox in combination with the proteasome inhibitor bortezomib (BTZ, 1 μM). After 18 h, protein levels of Mcl-1, UNC5B and β-actin, used as a housekeeping gene, were assessed by Western blot. (H): UNC5B accelerates the degradation and decreases the half-life of Mcl-1. HeLa UNC5B cells constitutively expressing Mcl-1 were treated with Dox for 6 h and then cycloheximide (CHX) was added. At the indicated time points after CHX treatment, cells were harvested and protein levels were assessed by Western blot. Mcl-1 expression was normalized by β-actin. In the presence of UNC5B, Mcl-1 half-life was decreased compared to control conditions. [Figure 4-2]UNC5B triggers proteasomal degradation of Mcl-1. (A, B): Proteasome activity is required for UNC5B-induced apoptosis. Stable HeLa cells inducible for UNC5B (HeLa UNC5B) were treated with 2 μg / mL doxycycline (Dox) in combination with the indicated doses of proteasome inhibitors MG132 and bortezomib (BTZ). 18 h after treatment, apoptosis was measured by DNA fragmentation (A) and caspase-3 activity (B) (n=3-4, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. Treatment with proteasome inhibitors was sufficient to significantly reduce apoptosis induction upon UNC5B overexpression. (C): UNC5B triggers Mcl-1 degradation. HeLa UNC5B cells were treated with 2 μg / mL Dox in combination with bortezomib (BTZ, 1 μM) and the pan-caspase inhibitor Q-VD-OPh (QVD, 10 μM). After 16 h of treatment, cells were lysed by detachment and sonication in SDS-containing buffer. Protein levels of Mcl-1, Bcl-2 and Bcl-XL, as well as UNC5B and β-actin, used as a housekeeping gene, were assessed by Western blot. The ratio of protein levels between untreated (0) and doxycycline-treated (2), normalized to β-actin, is shown below each Western blot. UNC5B overexpression specifically promoted Mcl-1 protein reduction. Proteasome inhibition by treatment with bortezomib rescued this degradation, whereas caspase inhibition by Q-VD-OPh, although it inhibited cell death, had no effect on Mcl-1 degradation. (D): UNC5B-induced Mcl-1 degradation requires the COMMD2 / CRL3 complex. HeLa UNC5B cells were transfected with scrambled (siCTRL), COMMD2 (siCOMMD2) or RBX1 (siRBX1) siRNA. 24 h after transfection, cells were treated with 2 μg / mL Dox for an additional 18 h.Protein levels of Mcl-1, UNC5B, and Ku80, used as a housekeeping protein, were assessed by Western blot. Silencing COMMD2 or RBX1 was sufficient to block Mcl-1 degradation induced by UNC5B overexpression. (E): Mcl-1 overexpression is not sufficient to rescue UNC5B-induced apoptosis. Stable HeLa UNC5B cells constitutively expressing Mcl-1 or empty vector were treated with doxycycline and apoptosis was assessed 18 h later by DNA fragmentation (n=3, nsp>0.05, ordinary two-way ANOVA with Sidak's multiple comparison test). Values ​​are given as mean ± SEM. (F): Overexpressed Mcl-1 protein, but not Bcl-2 or Bcl-XL, is significantly degraded upon UNC5B induction. Stable HeLa UNC5B cells constitutively expressing Mcl-1, Bcl-2 or Bcl-XL were treated with Dox and protein levels were assessed as described in (C). (G): Proteasome activity is required for overexpressed Mcl-1 degradation upon UNC5B induction. Stable HeLa UNC5B cells constitutively expressing Mcl-1 were treated with 2 μg / mL Dox in combination with the proteasome inhibitor bortezomib (BTZ, 1 μM). After 18 h, protein levels of Mcl-1, UNC5B and β-actin, used as a housekeeping gene, were assessed by Western blot. (H): UNC5B accelerates the degradation and decreases the half-life of Mcl-1. HeLa UNC5B cells constitutively expressing Mcl-1 were treated with Dox for 6 h and then cycloheximide (CHX) was added. At the indicated time points after CHX treatment, cells were harvested and protein levels were assessed by Western blot. Mcl-1 expression was normalized by β-actin. In the presence of UNC5B, Mcl-1 half-life was decreased compared to control conditions. [Figure 4-3]UNC5B triggers proteasomal degradation of Mcl-1. (A, B): Proteasome activity is required for UNC5B-induced apoptosis. Stable HeLa cells inducible for UNC5B (HeLa UNC5B) were treated with 2 μg / mL doxycycline (Dox) in combination with the indicated doses of proteasome inhibitors MG132 and bortezomib (BTZ). 18 h after treatment, apoptosis was measured by DNA fragmentation (A) and caspase-3 activity (B) (n=3-4, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. Treatment with proteasome inhibitors was sufficient to significantly reduce apoptosis induction upon UNC5B overexpression. (C): UNC5B triggers Mcl-1 degradation. HeLa UNC5B cells were treated with 2 μg / mL Dox in combination with bortezomib (BTZ, 1 μM) and the pan-caspase inhibitor Q-VD-OPh (QVD, 10 μM). After 16 h of treatment, cells were lysed by detachment and sonication in SDS-containing buffer. Protein levels of Mcl-1, Bcl-2 and Bcl-XL, as well as UNC5B and β-actin, used as a housekeeping gene, were assessed by Western blot. The ratio of protein levels between untreated (0) and doxycycline-treated (2), normalized to β-actin, is shown below each Western blot. UNC5B overexpression specifically promoted Mcl-1 protein reduction. Proteasome inhibition by treatment with bortezomib rescued this degradation, whereas caspase inhibition by Q-VD-OPh, although it inhibited cell death, had no effect on Mcl-1 degradation. (D): UNC5B-induced Mcl-1 degradation requires the COMMD2 / CRL3 complex. HeLa UNC5B cells were transfected with scrambled (siCTRL), COMMD2 (siCOMMD2) or RBX1 (siRBX1) siRNA. 24 h after transfection, cells were treated with 2 μg / mL Dox for an additional 18 h.Protein levels of Mcl-1, UNC5B, and Ku80, used as a housekeeping protein, were assessed by Western blot. Silencing COMMD2 or RBX1 was sufficient to block Mcl-1 degradation induced by UNC5B overexpression. (E): Mcl-1 overexpression is not sufficient to rescue UNC5B-induced apoptosis. Stable HeLa UNC5B cells constitutively expressing Mcl-1 or empty vector were treated with doxycycline and apoptosis was assessed 18 h later by DNA fragmentation (n=3, nsp>0.05, ordinary two-way ANOVA with Sidak's multiple comparison test). Values ​​are given as mean ± SEM. (F): Overexpressed Mcl-1 protein, but not Bcl-2 or Bcl-XL, is significantly degraded upon UNC5B induction. Stable HeLa UNC5B cells constitutively expressing Mcl-1, Bcl-2 or Bcl-XL were treated with Dox and protein levels were assessed as described in (C). (G): Proteasome activity is required for overexpressed Mcl-1 degradation upon UNC5B induction. Stable HeLa UNC5B cells constitutively expressing Mcl-1 were treated with 2 μg / mL Dox in combination with the proteasome inhibitor bortezomib (BTZ, 1 μM). After 18 h, protein levels of Mcl-1, UNC5B and β-actin, used as a housekeeping gene, were assessed by Western blot. (H): UNC5B accelerates the degradation and decreases the half-life of Mcl-1. HeLa UNC5B cells constitutively expressing Mcl-1 were treated with Dox for 6 h and then cycloheximide (CHX) was added. At the indicated time points after CHX treatment, cells were harvested and protein levels were assessed by Western blot. Mcl-1 expression was normalized by β-actin. In the presence of UNC5B, Mcl-1 half-life was decreased compared to control conditions. [Figure 5-1]UNC5B-mediated Mcl-1 degradation induces MOMP and caspase-9 activation. (A): UNC5B overexpression causes mitochondrial outer membrane permeabilization (MOMP) in a COMMD2 / CRL3 complex-dependent manner. HeLa cells were transfected with scrambled (siCTRL), COMMD2 (siCOMMD2-1) or RBX1 (siRBX1-1) siRNA. 24 h after transfection, cells were treated with Dox, and after a further 18 h, mitochondrial depolarization rates were quantified by 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye staining using a NucleoCounter NC3000 (n=3, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. (B, C, D): Caspase-9 is required for apoptotic cell death induced by UNC5B overexpression. HeLa UNC5B cells were transfected with scrambled (siCTRL) or caspase-9 (siCASP9) siRNA and treated with the indicated concentrations of Dox after 24 h. Propidium iodide (PI) positive cells were assessed in real time for up to 48 h using an Incucyte Zoom instrument (C) (n=3, ***p<0.01, Kolmogorov-Smirnov test). DNA fragmentation was assessed by FACS analysis (D) and caspase-3 was measured using a caspase-3 fluorescence quantification kit (E) (n=3, *p>0.05, ****p<0.0001, ordinary two-way ANOVA with Sidak's multiple comparison test). Values ​​are given as mean ± SEM. Inhibition of caspase-9 gene expression reduced cell death caused by UNC5B upon Dox treatment. (E): UNC5B overexpression activates caspase-9 but not caspase-8. HeLa UNC5B cells were treated with Dox and caspase-9 and -8 activities were assessed 18 h later by fluorometric kits using specific substrates (n=3, nsp>0.05, *p<0.05, Mann-Whitney test, one-tailed). Values ​​are given as mean ± SEM.UNC5B caused a marked induction of caspase-9 activity, whereas caspase-8 activity was not increased after Dox treatment. (F): UNC5B-induced caspase-9 activity requires the COMMD2 / CRL3 complex. HeLa UNC5B cells were transfected with scrambled siRNA (siCTRL) or siRNA targeting COMMD2 (siCOMMD2-1 and -2), Cullin-3 (siCUL3-1 and -2), or RBX1 (siRBX1-1 and -2). After 24 h, cells were treated with Dox and caspase-9 activity was measured by a fluorometric kit (n=3, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. Silencing of at least one member of the COMMD2 / CRL3 complex was sufficient to suppress UNC5B-induced caspase-9 activity. (G): Proteasome or NEDD8 inhibition limits UNC5B-triggered caspase-9 activity. HeLa UNC5B cells were treated with the indicated concentrations of Dox and the proteasome inhibitors MG132 and bortezomib (BTZ), the NEDD8 inhibitor pevonedistat or the pan-caspase inhibitor Q-VD-OPh (QVD). 18 hours after treatment, caspase-9 activity was assessed by a fluorometric quantification kit (n=5, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. (H): Caspase-9 is not required for UNC5B-triggered MOMP. Hela UNC5B cells were transfected with scrambled or caspase-9-specific siRNA. After 24 h, cells were treated with Dox and after a further 18 h, mitochondrial depolarization rates were quantified as described in (B) (n=3, nsp<0.05, **p<0.01, ***p<0.001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. (I): Caspase-9 is not required for UNC5B-dependent Mcl-1 degradation. HeLa UNC5B cells were transfected with scrambled or caspase-9 siRNA.Cells were then treated with Dox and Mcl-1 protein was assessed by Western blot. Silencing of caspase-9 was not sufficient to reverse the Mcl-1 degradation observed upon UNC5B induction. (J): UNC5B-induced apoptosis involves the proapoptotic proteins BAX and BAK1. HeLa UNC5B cells were transfected with siRNAs targeting BAK1 and BAX, either alone or in combination. After 24 h, cells were treated with Dox and after a further 18 h, DNA fragmentation was assessed by flow cytometry (n=4, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. [Figure 5-2]UNC5B-mediated Mcl-1 degradation induces MOMP and caspase-9 activation. (A): UNC5B overexpression causes mitochondrial outer membrane permeabilization (MOMP) in a COMMD2 / CRL3 complex-dependent manner. HeLa cells were transfected with scrambled (siCTRL), COMMD2 (siCOMMD2-1) or RBX1 (siRBX1-1) siRNA. 24 h after transfection, cells were treated with Dox, and after a further 18 h, mitochondrial depolarization rates were quantified by 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye staining using a NucleoCounter NC3000 (n=3, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. (B, C, D): Caspase-9 is required for apoptotic cell death induced by UNC5B overexpression. HeLa UNC5B cells were transfected with scrambled (siCTRL) or caspase-9 (siCASP9) siRNA and treated with the indicated concentrations of Dox 24 h later. Propidium iodide (PI) positive cells were assessed in real time for up to 48 h using an Incucyte Zoom instrument (B) (n=3, ***p<0.01, Kolmogorov-Smirnov test). DNA fragmentation was assessed by FACS analysis (C) and caspase-3 was measured using a caspase-3 fluorescence quantification kit (D) (n=3, *p>0.05, ****p<0.0001, ordinary two-way ANOVA with Sidak's multiple comparison test) after both treatments. Values ​​are given as mean ± SEM. Inhibition of caspase-9 gene expression reduced cell death caused by UNC5B upon Dox treatment. (E): UNC5B overexpression activates caspase-9 but not caspase-8. HeLa UNC5B cells were treated with Dox and caspase-9 and -8 activities were assessed 18 h later by fluorometric kits using specific substrates (n=3, nsp>0.05, *p<0.05, Mann-Whitney test, one-tailed). Values ​​are given as mean ± SEM.UNC5B caused a marked induction of caspase-9 activity, whereas caspase-8 activity was not increased after Dox treatment. (F): UNC5B-induced caspase-9 activity requires the COMMD2 / CRL3 complex. HeLa UNC5B cells were transfected with scrambled siRNA (siCTRL) or siRNA targeting COMMD2 (siCOMMD2-1 and -2), Cullin-3 (siCUL3-1 and -2), or RBX1 (siRBX1-1 and -2). After 24 h, cells were treated with Dox and caspase-9 activity was measured by a fluorometric kit (n=3, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. Silencing of at least one member of the COMMD2 / CRL3 complex was sufficient to suppress UNC5B-induced caspase-9 activity. (G): Proteasome or NEDD8 inhibition limits UNC5B-triggered caspase-9 activity. HeLa UNC5B cells were treated with the indicated concentrations of Dox and the proteasome inhibitors MG132 and bortezomib (BTZ), the NEDD8 inhibitor pevonedistat or the pan-caspase inhibitor Q-VD-OPh (QVD). 18 hours after treatment, caspase-9 activity was assessed by a fluorometric quantification kit (n=5, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. (H): Caspase-9 is not required for UNC5B-triggered MOMP. Hela UNC5B cells were transfected with scrambled or caspase-9-specific siRNA. After 24 h, cells were treated with Dox and after a further 18 h, mitochondrial depolarization rates were quantified as described in (B) (n=3, nsp<0.05, **p<0.01, ***p<0.001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. (I): Caspase-9 is not required for UNC5B-dependent Mcl-1 degradation. HeLa UNC5B cells were transfected with scrambled or caspase-9 siRNA.Cells were then treated with Dox and Mcl-1 protein was assessed by Western blot. Silencing of caspase-9 was not sufficient to reverse the Mcl-1 degradation observed upon UNC5B induction. (J): UNC5B-induced apoptosis involves the proapoptotic proteins BAX and BAK1. HeLa UNC5B cells were transfected with siRNAs targeting BAK1 and BAX, either alone or in combination. After 24 h, cells were treated with Dox and after a further 18 h, DNA fragmentation was assessed by flow cytometry (n=4, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. [Figure 5-3]UNC5B-mediated Mcl-1 degradation induces MOMP and caspase-9 activation. (A): UNC5B overexpression causes mitochondrial outer membrane permeabilization (MOMP) in a COMMD2 / CRL3 complex-dependent manner. HeLa cells were transfected with scrambled (siCTRL), COMMD2 (siCOMMD2-1) or RBX1 (siRBX1-1) siRNA. 24 h after transfection, cells were treated with Dox, and after a further 18 h, mitochondrial depolarization rates were quantified by 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye staining using a NucleoCounter NC3000 (n=3, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. (B, C, D): Caspase-9 is required for apoptotic cell death induced by UNC5B overexpression. HeLa UNC5B cells were transfected with scrambled (siCTRL) or caspase-9 (siCASP9) siRNA and treated with the indicated concentrations of Dox 24 h later. Propidium iodide (PI) positive cells were assessed in real time for up to 48 h using an Incucyte Zoom instrument (B) (n=3, ***p<0.01, Kolmogorov-Smirnov test). DNA fragmentation was assessed by FACS analysis (C) and caspase-3 was measured using a caspase-3 fluorescence quantification kit (D) (n=3, *p>0.05, ****p<0.0001, ordinary two-way ANOVA with Sidak's multiple comparison test) after both treatments. Values ​​are given as mean ± SEM. Inhibition of caspase-9 gene expression reduced cell death caused by UNC5B upon Dox treatment. (E): UNC5B overexpression activates caspase-9 but not caspase-8. HeLa UNC5B cells were treated with Dox and caspase-9 and -8 activities were assessed 18 h later by fluorometric kits using specific substrates (n=3, nsp>0.05, *p<0.05, Mann-Whitney test, one-tailed). Values ​​are given as mean ± SEM.UNC5B caused a marked induction of caspase-9 activity, whereas caspase-8 activity was not increased after Dox treatment. (F): UNC5B-induced caspase-9 activity requires the COMMD2 / CRL3 complex. HeLa UNC5B cells were transfected with scrambled siRNA (siCTRL) or siRNA targeting COMMD2 (siCOMMD2-1 and -2), Cullin-3 (siCUL3-1 and -2), or RBX1 (siRBX1-1 and -2). After 24 h, cells were treated with Dox and caspase-9 activity was measured by a fluorometric kit (n=3, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. Silencing of at least one member of the COMMD2 / CRL3 complex was sufficient to suppress UNC5B-induced caspase-9 activity. (G): Proteasome or NEDD8 inhibition limits UNC5B-triggered caspase-9 activity. HeLa UNC5B cells were treated with the indicated concentrations of Dox and the proteasome inhibitors MG132 and bortezomib (BTZ), the NEDD8 inhibitor pevonedistat or the pan-caspase inhibitor Q-VD-OPh (QVD). 18 hours after treatment, caspase-9 activity was assessed by a fluorometric quantification kit (n=5, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. (H): Caspase-9 is not required for UNC5B-triggered MOMP. Hela UNC5B cells were transfected with scrambled or caspase-9-specific siRNA. After 24 h, cells were treated with Dox and after a further 18 h, mitochondrial depolarization rates were quantified as described in (B) (n=3, nsp<0.05, **p<0.01, ***p<0.001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. (I): Caspase-9 is not required for UNC5B-dependent Mcl-1 degradation. HeLa UNC5B cells were transfected with scrambled or caspase-9 siRNA.Cells were then treated with Dox and Mcl-1 protein was assessed by Western blot. Silencing of caspase-9 was not sufficient to reverse the Mcl-1 degradation observed upon UNC5B induction. (J): UNC5B-induced apoptosis involves the proapoptotic proteins BAX and BAK1. HeLa UNC5B cells were transfected with siRNAs targeting BAK1 and BAX, either alone or in combination. After 24 h, cells were treated with Dox and after a further 18 h, DNA fragmentation was assessed by flow cytometry (n=4, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. [Figure 6]UNC5B-mediated Mcl-1 degradation induces MOMP and caspase-9 activation. (A, B): Validation of caspase-9 and -8 silencing. HeLa UNC5B cells were transfected with scrambled (siCTRL), caspase-9 (siCASP9-1 and -2) or caspase-8 (siCASP8-1 and -2) siRNA. 24 h after transfection, total RNA was extracted and caspase-9 (A) and -8 (B) gene expression was assessed by quantitative RT-PCR. Relative gene expression was normalized to the siCTRL condition using TATA-binding protein (TBP) and β-glucuronidase (GUSB) genes as housekeeping genes (n=3-4, ****p<0.0001, ordinary two-way ANOVA with Dunnett's multiple comparison test). Values ​​are given as mean ± SEM. (C): Caspase-8 is not required for UNC5B-induced cell death. HeLa UNC5B cells were transfected with scrambled (siCTRL) or caspase-8 (siCASP8) siRNA. 24 h after transfection, cells were treated with doxycycline (Dox) and cell death was assessed by measuring propidium iodide (PI) positive cells in real time for up to 26 h using an Incucyte Zoom instrument (n=3, nsp>0.05, Kolmogorov-Smirnov test). Values ​​are given as mean ± SEM. (D): Validation of BAX and BAK1 silencing. HeLa UNC5B cells were transfected with scrambled (siCTRL), BAX (siBAX) or BAK1 (siBAK1) siRNA. Quantitative RT-PCR was performed as in (A) (n=3, ****p<0.0001, ordinary two-way ANOVA with Dunnett's multiple comparison test). Values ​​are given as mean±SEM. [Figure 7-1]Netrin-1 interference causes Mcl-1 degradation in vitro and in vivo. (A, B): Netrin-1 silencing causes Mcl-1 degradation. Breast cancer MCF7 cells were transfected with siRNA targeting scrambled (siCTRL) or netrin-1 (siNet). 48 h after transfection, cells were lysed by detachment and sonication in SDS-containing buffer. Protein levels of Mcl-1, netrin-1, and UNC5B, as well as β-actin and Ku80, used as housekeeping genes, were assessed by Western blot (A). Relative Mcl-1 protein levels normalized to β-actin or Ku80 are shown in (B) (n=2, ***p<0.001, ****p<0.0001, ordinary two-way ANOVA with Sidak's multiple comparison test). Activation of endogenous UNC5B upon netrin-1 silencing reduced Mcl-1 protein levels. Values ​​are given as mean ± SEM. (C, D): Netrin-1 interference degrades Mcl-1 in a syngeneic breast cancer model. Stable EMT6 cells constitutively overexpressing human Mcl-1 were implanted in BALB / c mice. When tumors reached a volume of 100 mm3, mice were treated with Iso-mAb or NP137 antibody and sacrificed 8 h after treatment. Protein levels of Mcl-1 were then assessed by Western blot (C). Relative Mcl-1 levels normalized to β-actin are shown in (D) (n=4-5, *p<0.01, Mann-Whitney test). The line in the center of the box is plotted at the median. (E, F): NP137 antibody causes Mcl-1 degradation in a cohort of cancer patients. Patients with advanced solid tumors enrolled in a Phase I trial (NCT02977195) were treated with NP137 at 14 mg / kg once every 2 weeks according to the Phase I trial design. Biopsies were collected at patient enrollment (before the first dose of NP137) (C1D1) and before the third infusion of NP137 (C3D1). Mcl-1 protein was quantified in paraffin-embedded sections for paired C1D1 and C3D1 biopsies and compared by clinical benefit (stable, SD, or progressive, PD, week 6).Mcl-1 intensity was assessed in all PD or SD biopsies (E) (n=6, Wilcoxon test). The dashed line represents Mcl-1 expression in the same patients before and after NP137 infusion. Representative IHC images for three SD patients are shown in (F). [Figure 7-2]Netrin-1 interference causes Mcl-1 degradation in vitro and in vivo. (A, B): Netrin-1 silencing causes Mcl-1 degradation. Breast cancer MCF7 cells were transfected with siRNA targeting scrambled (siCTRL) or netrin-1 (siNet). 48 h after transfection, cells were lysed by detachment and sonication in SDS-containing buffer. Protein levels of Mcl-1, netrin-1, and UNC5B, as well as β-actin and Ku80, used as housekeeping genes, were assessed by Western blot (A). Relative Mcl-1 protein levels normalized to β-actin or Ku80 are shown in (B) (n=2, ***p<0.001, ****p<0.0001, ordinary two-way ANOVA with Sidak's multiple comparison test). Activation of endogenous UNC5B upon netrin-1 silencing reduced Mcl-1 protein levels. Values ​​are given as mean ± SEM. (C, D): Netrin-1 interference degrades Mcl-1 in a syngeneic breast cancer model. Stable EMT6 cells constitutively overexpressing human Mcl-1 were implanted in BALB / c mice. When tumors reached a volume of 100 mm3, mice were treated with Iso-mAb or NP137 antibody and sacrificed 8 h after treatment. Protein levels of Mcl-1 were then assessed by Western blot (C). Relative Mcl-1 levels normalized to β-actin are shown in (D) (n=4-5, *p<0.01, Mann-Whitney test). The line in the center of the box is plotted at the median. (E, F): NP137 antibody causes Mcl-1 degradation in a cohort of cancer patients. Patients with advanced solid tumors enrolled in a Phase I trial (NCT02977195) were treated with NP137 at 14 mg / kg once every 2 weeks according to the Phase I trial design. Biopsies were collected at patient enrollment (before the first dose of NP137) (C1D1) and before the third infusion of NP137 (C3D1). Mcl-1 protein was quantified in paraffin-embedded sections for paired C1D1 and C3D1 biopsies and compared by clinical benefit (stable, SD, or progressive, PD, week 6).Mcl-1 intensity was assessed in all PD or SD biopsies (E) (n=6, Wilcoxon test). The dashed line represents Mcl-1 expression in the same patients before and after NP137 infusion. Representative IHC images for three SD patients are shown in (F). [Figure 8-1] Combining Netrin-1 interference and BH3 mimetics inhibits tumor growth in preclinical in vivo and ex vivo models. (A, B, C): Combining Netrin-1 interference and Bcl-2 inhibition suppresses tumor growth in a syngeneic breast cancer model. EMT6 cells were implanted in BALB / c mice and, when tumors reached a volume of 50 mm3, were treated with isotype (Iso-mAb) or NP137 antibody and the Bcl-2 inhibitor venetoclax, either as single agents or in combination. Tumor volumes were measured the indicated days after the first treatment (n=9, **p<0.01, ordinary two-way ANOVA with Tukey's multiple comparison test) (A, B). Survival rates were assessed at the time of sacrifice of mice bearing tumors >500 mm3 (C). (D, E, F): Combination treatment is efficient to induce apoptosis in human ex vivo sections. Cells positive for active caspase-3 after cleavage were assessed by IHC staining in fresh ex vivo breast tumor tissue sections. Combination treatment with NP137 antibody and venetoclax increased the apoptosis rate compared to single agent treatment (n=8, **p<0.01, ordinary two-way ANOVA with Fisher's LSD uncorrected). [Figure 8-2]Combining Netrin-1 interference and BH3 mimetics inhibits tumor growth in preclinical in vivo and ex vivo models. (A, B, C): Combining Netrin-1 interference and Bcl-2 inhibition suppresses tumor growth in a syngeneic breast cancer model. EMT6 cells were implanted in BALB / c mice and, when tumors reached a volume of 50 mm3, were treated with isotype (Iso-mAb) or NP137 antibody and the Bcl-2 inhibitor venetoclax, either as single agents or in combination. Tumor volumes were measured the indicated days after the first treatment (n=9, **p<0.01, ordinary two-way ANOVA with Tukey's multiple comparison test) (A, B). Survival rates were assessed at the time of sacrifice of mice bearing tumors >500 mm3 (C). (D, E, F): Combination treatment is efficient to induce apoptosis in human ex vivo sections. Cells positive for active caspase-3 after cleavage were assessed by IHC staining in fresh ex vivo breast tumor tissue sections. Combination treatment with NP137 antibody and venetoclax increased the apoptosis rate compared to single agent treatment (n=8, **p<0.01, ordinary two-way ANOVA with Fisher's LSD uncorrected). [Figure 9-1]Combining netrin-1 interference and BH3 mimetics inhibits tumor growth in preclinical in vivo and ex vivo models. Related to Figure 8. (A): UNC5B overexpression sensitizes tumor cells to Bcl-2 inhibition. Stable breast cancer T47D cells inducible for UNC5B were treated with 2 μg / mL doxycycline (Dox) and the Bcl-2 inhibitor venetoclax at the indicated concentrations. After 24 hours, apoptosis was quantified by Annexin V / propidium iodide (PI) staining. EC50 values ​​were calculated using nonlinear regression of dose-response curves with GraphPad Prism software. (B): Silencing netrin-1 gene expression enhances cell death induced by Bcl-2 inhibition. Breast cancer MCF7 cells were transfected with siRNA targeting scrambled (siCTRL) or netrin-1 (siNet). 24 hours after transfection, cells were treated with venetoclax for another 48 hours. Apoptosis was then assessed by 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye staining using a NucleoCounter NC3000 (n=2, *p<0.05, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. (C): Tumor growth in a syngeneic EMT6 breast cancer model is unresponsive to both Netrin-1 interference and Bcl-2 inhibition. EMT6 cells were implanted in BALB / c mice and, when tumors reached a volume of 50 mm3, were treated with isotype (Iso-mAb) or NP137 antibodies as single agents and the Bcl-2 inhibitor venetoclax. Tumor volumes were measured the indicated days after the first treatment (n=9, nsp>0.05, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean±SEM. (D): Combination treatments are efficient in inducing apoptosis in human ex vivo slices. RNA from breast tumor biopsies (n=8) was extracted and gene expression of netrin-1 (NTN1), UNC5B, Bcl-2 (BCL2) and Bcl-XL (BCL2L1) was assessed by quantitative RT-PCR.The heatmap represents basal mRNA levels for each patient, shown as a percentage of gene expression relative to the patient expressing the highest transcript level. Patients in red responded to combination treatment with NP137 and venetoclax compared to single agent treatment. [Figure 9-2]Combining netrin-1 interference and BH3 mimetics inhibits tumor growth in preclinical in vivo and ex vivo models. Related to Figure 8. (A): UNC5B overexpression sensitizes tumor cells to Bcl-2 inhibition. Stable breast cancer T47D cells inducible for UNC5B were treated with 2 μg / mL doxycycline (Dox) and the Bcl-2 inhibitor venetoclax at the indicated concentrations. After 24 hours, apoptosis was quantified by Annexin V / propidium iodide (PI) staining. EC50 values ​​were calculated using nonlinear regression of dose-response curves with GraphPad Prism software. (B): Silencing netrin-1 gene expression enhances cell death induced by Bcl-2 inhibition. Breast cancer MCF7 cells were transfected with siRNA targeting scrambled (siCTRL) or netrin-1 (siNet). 24 hours after transfection, cells were treated with venetoclax for another 48 hours. Apoptosis was then assessed by 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye staining using a NucleoCounter NC3000 (n=2, *p<0.05, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM. (C): Tumor growth in a syngeneic EMT6 breast cancer model is unresponsive to both Netrin-1 interference and Bcl-2 inhibition. EMT6 cells were implanted in BALB / c mice and, when tumors reached a volume of 50 mm3, were treated with isotype (Iso-mAb) or NP137 antibodies as single agents and the Bcl-2 inhibitor venetoclax. Tumor volumes were measured the indicated days after the first treatment (n=9, nsp>0.05, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean±SEM. (D): Combination treatments are efficient in inducing apoptosis in human ex vivo slices. RNA from breast tumor biopsies (n=8) was extracted and gene expression of netrin-1 (NTN1), UNC5B, Bcl-2 (BCL2) and Bcl-XL (BCL2L1) was assessed by quantitative RT-PCR.The heatmap represents basal mRNA levels for each patient, shown as a percentage of gene expression relative to the patient expressing the highest transcript level. Patients in red responded to combination treatment with NP137 and venetoclax compared to single agent treatment. [Figure 10] Cell death assessment by combining netrin-1 silencing and navitoclax. To assess cell death, propidium iodide (PI) incorporation normalized to cell confluence was tracked in real time for up to 24 hours by Incucyte Zoom technology for scrambled (siCTRL), netrin-1 (siNet), scrambled (siCTRL) and navitoclax, and netrin-1 (siNet) and navitoclax.

[0128] result A model of action for UNC5B-mediated Mcl-1 degradation and activation of the intrinsic apoptotic pathway Induction of UNC5B can activate the cullin-RING-ligase (CRL) complex 3 (CRL3) via COMMD2. Through an unknown mechanism involving NEDDylation of CRL3 and activation of the E2 ubiquitin-conjugating enzyme UBE2L6, UNC5B promotes ubiquitination and proteasomal degradation of Mcl-1, allowing mitochondrial outer membrane permeabilization (MOMP), release of cytochrome C, formation of the apoptosome, and ultimately activation of caspase-9, thus leading to apoptosis.

[0129] COMMD2 is required for UNC5B-induced cell death and tumor suppression To identify the pro-apoptotic pathway triggered by UNC5B, we used Sleeping Beauty-based vectors to generate stable HCT116 and HeLa cancer cell lines inducible for UNC5B. Forced UNC5B expression upon doxycycline treatment, confirmed by Western blot (Figures 2A and 2B), significantly induced apoptotic cell death in both models compared to cells stably transfected with empty vector, as revealed by PI staining (Figures 1A and 2C), DNA fragmentation (Figures 1B and 2D), and caspase-3 activity (Figures 1C and 2E). This strong sensitivity to UNC5B induction indicates the presence of functional pro-apoptotic UNC5B signaling in HCT116 and HeLa cells. To confirm that UNC5B-induced apoptosis was not due to protein overexpression alone, we stably expressed the ligand Netrin-1 in cells inducible for UNC5B. Constitutive expression of netrin-1 was sufficient to inhibit the apoptosis otherwise observed upon UNC5B induction (Fig. 1B), and furthermore, caspase inhibition with the pan-caspase inhibitor Q-VD-OPh completely blocked UNC5B-induced apoptosis (Fig. 2F).

[0130] We decided to take advantage of a stable HCT116 cell line inducible for UNC5B to perform a CRISPR-Cas9-based screen to allow the identification of UNC5B pro-apoptotic effectors. For this purpose, we transduced a genome-wide lentiviral library (GeCKO library, (Sanjana et al., 2014; Shalem et al., 2014)) into Sleeping Beauty modified HCT116 cells. Upon doxycycline treatment and UNC5B induction, only cell clones that have knocked out genes required for UNC5B pro-apoptotic signaling should survive. Using next generation sequencing (NGS), we identified inactivated genes in cell clones. Considering all genes presenting at least three small guide RNAs (sgRNAs) that were enriched compared to control cells and identified with more than 100 reads, we identified 75 genes potentially involved in UNC5B-induced apoptosis.

[0131] Following literature-based selection, we highlighted eight genes listed in Figure 2G. To confirm whether COMMD2 is required for UNC5B-induced cell death, we transfected stable HeLa and HCT116 cells with two separate siRNAs targeting COMMD2. Reduction of COMMD2 gene expression was sufficient to reduce DNA fragmentation upon UNC5B challenge with doxycycline compared to cells transfected with control siRNA (Figure 3A). Furthermore, COMMD2 silencing inhibited the significant increase in PI-positive HeLa cells caused by UNC5B overexpression, as well as the caspase-3 activation observed in control cells (not shown). Thus, there is a unique role for COMMD2 in the UNC5B proapoptotic pathway.

[0132] Next, we evaluated the requirement of COMMD2 and UNC5B for tumor suppression associated with netrin-1 inhibition. To this end, we investigated the effect of NP137, a neutralizing anti-netrin-1 antibody capable of inducing cancer cell death and tumor growth inhibition in several preclinical models (Boussouar et al., 2020; Grandin et al., 2016; Sun et al., 2021), on stable knockout H322 cells implanted in nude mice (Figure 3B). Treatment of mice with NP137 antibody significantly inhibited tumor growth in control cells, whereas tumors with COMMD2 or UNC5B knockout were less sensitive to anti-netrin-1 antibody, as shown by both tumor growth analysis and percentage of tumor growth inhibition (Figure 3C, Figure 3D). We confirmed that NP137 anti-tumor activity was associated with apoptosis induction, as shown by immunohistochemical staining of active caspase-3 after cleavage (Figure 3E). In contrast, in tumors with COMMD2 or UNC5B knockout, we were unable to detect a similar increase in the percentage of caspase-3 positive cells upon treatment with NP137 antibody (Figure 3E), further supporting the notion that interference with netrin-1 / UNC5B interaction by NP137 induces COMMD2-dependent cancer cell death associated with tumor growth inhibition.

[0133] UNC5B triggers proteasomal degradation of Mcl-1 Given that we have shown that UNC5B-induced cell death requires COMMD2, we hypothesized that UNC5B may lead to ubiquitination of unknown target proteins and their subsequent proteasomal degradation. In this light, treatment of stable HeLa cells with proteasome inhibitors MG132 and bortezomib suppressed DNA fragmentation and caspase-3 activation caused by UNC5B induction (Figures 4A and 4B). In search of UNC5B target proteins that are degraded during apoptosis induction, we focused on Bcl-2 family members, anti-apoptotic proteins whose cellular abundance is regulated via the ubiquitin-proteasome system (Kale et al., 2018; Singh et al., 2019). Western blot analysis revealed a specific decrease in Mcl-1 protein upon UNC5B induction in stable HeLa cells, whereas we did not observe such a decrease in Bcl-2 and Bcl-XL protein levels (Figure 4C). Furthermore, treatment with the proteasome inhibitor bortezomib resulted in increased but similar Mcl-1 protein levels in both untreated and doxycycline-treated cells, supporting the view that the decrease in the levels of Mcl-1 associated with UNC5B induction is proteasome activity dependent and therefore protein degradation-related. Interestingly, inhibition of caspase activity by Q-VD-OPh, although it efficiently blocked UNC5B-induced apoptosis (see Figure 2F), was not sufficient to prevent Mcl-1 degradation caused by UNC5B (Figure 4C), indicating that caspase activation is downstream of Mcl-1 degradation. Moreover, inactivation of the COMMD2 / CRL3 complex by silencing COMMD2 or RBX1 gene expression prevented Mcl-1 degradation in doxycycline-treated HeLa cells (FIG. 4D).

[0134] To more formally demonstrate that Mcl-1 degradation is specifically induced by UNC5B, we generated stable HeLa cells inducible for UNC5B and constitutively expressing high levels of Mcl-1. Interestingly, Mcl-1 overexpression was not sufficient to inhibit UNC5B-induced apoptotic cell death (Fig. 4E), and Western blot analysis revealed that even ectopically overexpressed Mcl-1 was substantially degraded upon UNC5B induction, whereas overexpressed Bcl-2 and Bcl-XL proteins were not degraded in the same situation (Fig. 4F). Furthermore, treatment with bortezomib suppressed the degradation of ectopically expressed Mcl-1 (Fig. 4G). To address the question of whether UNC5B affects Mcl-1 degradation, we performed cycloheximide (CHX) chase experiments. Time-dependent treatment of stable, Mcl-1-overexpressing, UNC5B-induced HeLa cells with CHX revealed an increased rate of Mcl-1 degradation in the presence of UNC5B, resulting in a decreased Mcl-1 half-life (108.5 min in control cells vs. 37.2 min upon UNC5B induction, one-phase decay) (Figure 4H). Together, these results suggest that UNC5B drives the proteasomal degradation of Mcl-1 via the ubiquitin-ligase activity of the COMMD2 / CRL3 complex.

[0135] UNC5B-mediated Mcl-1 degradation induces MOMP and caspase-9 activation Mcl-1, as well as other Bcl-2 family members, act as anti-apoptotic proteins by inhibiting the pro-apoptotic proteins BCL2-associated X, regulator of apoptosis (BAX) and BCL2 antagonist / killer 1 (BAK1) and the resulting mitochondrial outer membrane permeabilization (MOMP) (Galluzzi et al., 2018). After cytoplasmic release of apoptotic factors present in the mitochondrial intermembrane space, a pro-apoptotic complex called the apoptosome is formed, leading to caspase-9 activation and the resulting execution of the intrinsic apoptotic program. Since we have shown that UNC5B causes Mcl-1 degradation, we hypothesized that MOMP and caspase-9 activation are required for UNC5B-induced apoptosis. Indeed, UNC5B overexpression induced MOMP in stable HeLa cells (Figure 5A). Interestingly, silencing of COMMD2 and RBX1 inhibited UNC5B-induced MOMP (Fig. 5A ), suggesting that the ubiquitin-ligase activity of the COMMD2 / CRL3 complex may be involved in UNC5B-induced MOMP induction.

[0136] We next determined whether caspase-9, activated upon MOMP induction, is a mediator of apoptosis triggered by UNC5B. Stable HeLa cells inducible for UNC5B were transfected with siRNA targeting caspase-9, and upon treatment with doxycycline, we assessed the cell death rate. Silencing of caspase-9 rescued the significant increase in PI-positive cells upon UNC5B induction (Figures 5B and 6A), as well as DNA fragmentation and caspase-3 activation (Figures 5C and 5D). In contrast, transfection of siRNA targeting caspase-8, an effector caspase involved in the extrinsic apoptosis pathway, did not affect UNC5B-induced cell death (Figures 6B and 6C).

[0137] The dependency of UNC5B-induced death on caspase-9 suggests that UNC5B-induced MOMP triggers apoptosis via caspase-9 activation. Indeed, we showed that UNC5B triggers caspase-9 but not caspase-8 activation in stable HeLa cells, as shown by caspase activity performed using a specific fluorescent substrate (Figure 5E). Since we showed that activation of the COMMD2 / CRL3 complex by UNC5B is required for Mcl-1 degradation, we determined whether UNC5B-dependent caspase-9 activation is a consequence of this phenomenon. Stable HeLa cells were transfected with two separate siRNAs targeting COMMD2, CUL3 and RBX1. Silencing the expression of the COMMD2 / CRL3 member complex completely suppressed the caspase-9 activation observed upon UNC5B induction in scramble-transfected cells (Figure 5F). Furthermore, treatment of stable HeLa cells with NEDD8 (pevonedistat) or proteasome inhibitors (MG132 and bortezomib) was sufficient to block UNC5B-induced caspase-9 activation (Figure 5G).

[0138] To confirm that UNC5B-induced Mcl-1 degradation and MOMP were not the only effects of cellular perturbation as a result of UNC5B overexpression, we determined whether caspase-9 knockdown affected MOMP or Mcl-1 degradation associated with UNC5B induction. Although caspase-9 silencing (Fig. 6A) suppressed UNC5B-induced apoptosis (Fig. 5B, 5C, and 5D), it was not sufficient to suppress UNC5B-induced mitochondrial depolarization (Fig. 5H). As a control, caspase-8 silencing (Fig. 6B) did not inhibit UNC5B-induced cell death (Fig. 6C). Furthermore, transfection of siRNA targeting caspase-9 in stable HeLa cells did not prevent Mcl-1 degradation as a result of UNC5B induction (Fig. 5I). Similarly, silencing of BAX and BAK1, molecular mediators that lead to apoptosis via Mcl-1 inhibition leading to MOMP and caspase-9 activation (Figure 6D), reduced UNC5B-dependent apoptosis induction (Figures 5J and S8D).

[0139] Netrin-1 interference causes Mcl-1 degradation To confirm that Mcl-1 degradation was associated with endogenously expressed, free UNC5B, we hypothesized that in cancer cells expressing netrin-1 and UNC5B, netrin-1 silencing should be associated with Mcl-1 degradation. Indeed, transfection of breast cancer MCF7 cells with siRNA targeting netrin-1 induced Mcl-1 degradation (Figure 7A and Figure 7B).

[0140] To move to a more relevant therapeutic context, we used the EMT6 syngeneic breast cancer model to evaluate Mcl-1 degradation in vivo. Indeed, EMT6 cells endogenously express netrin-1 and UNC5B. Stable EMT6 cells constitutively overexpressing human Mcl-1 were implanted in BALB / c mice, and tumor-bearing animals were treated iv with a therapeutic anti-netrin-1 antibody (NP137). Mcl-1 was measured by immunoblot from mouse tumors, and interestingly, netrin-1 blockade significantly reduced Mcl-1 protein levels compared to control mice (Figure 7C and Figure 7D).

[0141] The NP137 antibody is currently being evaluated in several phase I and II clinical trials in patients with advanced solid tumors. Paired biopsies from 12 patients treated with NP137 in the phase I trial were collected before the first injection of NP137 (C1D1) and after two cycles of NP137 (1 month, C3D1). Thus, the presence of Mcl-1 was analyzed by immunohistochemistry. NP137 administration reduced Mcl-1 levels in 9 of 12 patients tumors tested (Figure 7E and Figure F). Furthermore, when comparing clinical benefit for 12 patients in whom Mcl-1 detection was monitored (who experienced either disease stabilization (SD) or disease progression (PD) in the 6-week RESIST analysis), 6 patients with SD showed Mcl-1 reduction upon NP137 treatment (Figure 7E and Figure F), whereas we observed a reduction in Mcl-1 in only 3 of 6 patients with PD. Collectively, these data indicate that netrin-1 blockade causes tumor-specific Mcl-1 loss in both animal models and patients.

[0142] Combining netrin-1 interference and BH3 mimetics (venetoclax and navitoclax) inhibits tumor growth in preclinical in vivo and ex vivo models BH3 mimetics are a new class of therapeutic molecules that target Bcl-2 family proteins (Cerella et al., 2020). The Bcl-2 inhibitor ABT-199 (also known as venetoclax) is currently the only BH3 mimetic approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), or acute myeloid leukemia (AML) (Souers et al., 2013). Several BH3 mimetics targeting Mcl-1 have been developed. Despite initial enthusiasm for Mcl-1 targeting, early clinical trials of Mcl-1 inhibitors have been halted in recent years due to potential cardiac toxicity (Wei et al., 2020). BH3 mimetics used as monotherapy in solid tumors are less effective, possibly because solid tumors are less "primed" for apoptosis than hematopoietic tumors (Sarosiek et al., 2017) or because of compensation (van Delft et al., 2006). For this reason, the current strategy in solid tumors is considered to be combinations of selective BH3 mimetics, particularly combination treatments with Mcl-1 and Bcl-XL or Bcl-2 inhibitors (Soderquist et al., 2018). As we have previously shown that the UNC5B receptor causes Mcl-1 degradation in cancer cells, we hypothesized that causing Mcl-1 degradation by UNC5B could enhance the efficacy of Bcl-2 inhibitors. To confirm this hypothesis, we first generated stable breast cancer T47D cells inducible for UNC5B and evaluated venetoclax efficiency upon UNC5B induction. Indeed, UNC5B induction was sufficient to significantly reduce the venetoclax EC50 (Figure 9A).

[0143] Since we showed that in MCF7 cells expressing both netrin-1 and UNC5B, netrin-1 silencing is associated with Mcl-1 degradation, we hypothesized that interference with netrin-1 expression may have an enhanced effect of Bcl-2 inhibitors in these cells. Indeed, in contrast to netrin-1 interference or Bcl-2 inhibition alone, the combination of netrin-1 silencing and venetoclax treatment caused mitochondrial depolarization, suggesting an enhanced cell death induced by Bcl-2 inhibition (Figure 9B).

[0144] Subsequently, to evaluate the antitumor effect of the combination treatment in vivo in a conventional preclinical model, we utilized the EMT6 syngeneic breast cancer model. Indeed, these cells express high levels of both netrin-1 and UNC5B. However, interfering with netrin-1 via anti-netrin-1 NP137 antibody has no effect on tumor growth in this model (not shown). EMT6 cells were implanted in BALB / c mice and tumor-bearing animals were treated with NP137 and venetoclax. Treatment with both drugs in monotherapy had no effect on tumor growth, whereas the combination treatment significantly reduced tumor growth compared to isotype control antibody treatment (Figure 8A, Figure 8B and Figure 9C). This reduced tumor growth was associated with the overall survival of mice treated with the combination (Figure 8C).

[0145] Finally, to confirm the efficiency of the combination treatment in tissues from human patients, we evaluated the effect of netrin-1 interference and Bcl-2 inhibition in an ex vivo breast cancer organ culture technique (de Graaf et al., 2010), which allows the study of tumors maintained in their natural microenvironment. Fresh tumor sections were treated with NP137 antibody and venetoclax as single agents or in combination, and apoptosis was quantified by immunohistochemical analysis of post-cleavage caspase-3. Treatment with NP137 antibody and venetoclax significantly increased the percentage of post-cleavage caspase-3 positive cells compared to netrin-1 interference or Bcl-2 inhibition alone (Figure 8D). Notably, the combination treatment increased the apoptosis rate in 5 out of 8 tumor biopsies (Figures 8E and 8F). Unexpectedly, among the three patients not responding to the combination treatment, patient B21.015 expressed low levels of both netrin-1 and UNC5B compared to the other patients, thus contributing to NP137 antibody resistance. Furthermore, although patient B21.030 presented high transcript levels of netrin-1 and UNC5B, its resistance to the combination treatment may be explained by relatively low levels of Bcl-2 (i.e., insufficient sensitivity to venetoclax) and high expression of Bcl-XL, indicating resistance to Mcl-1 inhibition (Soderquist et al., 2018) (Figure 9D). These results confirmed that Bcl-2 inhibition alone is not sufficient to trigger apoptosis in solid tumors (i.e., human breast cancer patients) and that simultaneous interference with netrin-1 / UNC5B interaction via NP137 antibody is required to induce Mcl-1 degradation.

[0146] In addition, breast cancer HCC70 cells were transfected with siRNA targeting scrambled (siCTRL) or netrin-1 (siNet). 24 hours after transfection, cells were detached, replated in 24-well plates, and treated with 0.5 μM Bcl-2 / Bcl-XL / Bcl-w inhibitor navitoclax (Tse et al., Cancer Res, 2008; Shoemaker et al., Clin Cancer Res, 2008). To assess cell death, propidium iodide (PI) incorporation normalized to cell confluence was tracked in real time for up to 24 hours by Incucyte Zoom technology. Netrin-1 silencing or Bcl-2 / Bcl-XL / Bcl-w inhibition alone had only a minor effect on cell death induction, whereas the combined treatment rapidly and significantly triggered apoptosis (Figure 10).

[0147] Expression of UNC5 receptor in selected cell lines: The inventors have shown that the reduced presence of netrin-1 in some tumor cell lines, such as MCF7 and H322, correlates with Mcl-1 degradation. The inventors have also shown the combined effect of netrin-1 deficiency and BH3 mimetics on cell death induction in the tumor cell line HCC70. The inventors then showed that UNC5B, as well as other netrin-1 receptors, such as UNC5A and UNC5C, are fully expressed in these cell lines.

[0148] The mRNA expression of receptors UNC5A-C was assessed using the Cancer Cell Line Encyclopedia (CCLE) database. The transcript abundance of the receptors, expressed as Reads Per Kilobase Million (RPKM), was evaluated in breast (MCF7 and HCC70) and lung (H322) cancer cells.

[0149] [Table 3]

[0150] Except for UNC5B, whose expression is high and stable in all three analyzed cell lines, at least one receptor, UNC5A-UNC5C, is also expressed. Therefore, other UNC5 receptors, especially UNC5A and UNC5C, are likely to be involved in cell death induced by netrin-1 interference or silencing alone or in combination with BH3 mimetics.

[0151] Consideration The dependence receptor UNC5B conditionally induces apoptosis in cancer cells. Using CRISPR-Cas9 screening, we identified the protein COMMD2 as a proapoptotic mediator of UNC5B-induced cell death. In the absence of netrin-1, UNC5B triggers COMMD2 activation by a still unknown mechanism. Upon activation, COMMD2 interacts with the CRL3 complex, which then ubiquitinates Mcl-1, triggering its degradation by the proteasome. This results in a shift in the proapoptotic balance, leading to mitochondrial permeabilization and subsequent activation of caspase-9 and caspase-3, and ultimately apoptosis of cancer cells. To date, Mule, SCF β-TrCP , SCF FBW7 , TRIM17, APC / C Cdc20 Several ubiquitin ligases, including MARCH5 and Parkin, are known to degrade Mcl-1 (Mojsa et al., 2014; Senichkin et al., 2020). However, the CRL3 complex has not been described previously in this context. Here, for the first time, we show the involvement of this E3 ligase in the degradation of Mcl-1.

[0152] The CRL E3 ligase family is primarily regulated by a NEDDylation cycle that allows their activation and inactivation (Duda et al., 2011). CRL E3 ligases are activated by the covalent binding of the NEDD8 protein (Enchev et al., 2015). Subsequently, NEDD8 can be dissociated from the complex by the action of the COP9 (constitutive photomorphogenesis 9) signalosome, allowing the CAND1 protein to interact with the non-NEDDylated complex and inhibit its activity (Duda et al., 2011). Interestingly, COMMD1 inhibits the binding of CAND1 by binding to cullin-2, thus activating the CRL2 complex (Mao et al., 2011). In addition, COMMD2 specifically interacts with cullin-3, but not with cullin-2 (Mao et al., 2011). Our data indicate that COMMD2, as well as members of the CRL3 complex, are required for UNC5B-induced cell death, whereas COMMD1 and other CRLs were not involved in the UNC5B proapoptotic pathway. Furthermore, we could show that inhibition of NEDDylation via treatment with pevonedistat, a specific inhibitor of NEDD8, prevents UNC5B-induced cell death. Since CAND1 binds and inhibits virtually all cullins (Duda et al., 2011), we can hypothesize that COMMD2, by specifically interacting with CRL3, inhibits CAND1's interaction with cullin-3 and thus allows its activation by NEDDylation. However, it remains unclear how UNC5B preferentially interacts with COMMD2 with CRLs and activates these complexes.

[0153] From a therapeutic point of view, our results suggest that interference with the binding between netrin-1 and UNC5B using the neutralizing anti-netrin-1 antibody NP137 constitutes the original means of degrading Mcl-1 and therefore provides an interesting alternative to the currently developed BH3-mimetics targeting Mcl-1. By indirectly but tumor-specifically degrading Mcl-1, the NP137 antibody represents an attractive alternative to BH3-mimetic compounds. Indeed, with NP137, Mcl-1 degradation appears to occur only in netrin-1 expressing cells, and preclinical development of NP137 revealed the absence of tissue cross-reactivity in healthy adult individuals. Moreover, the safety of this antibody has already been tested in phase I clinical trials, showing an extremely good safety profile (Cassier et al., 2019). By preferentially targeting tumor cells overexpressing netrin-1, NP137 has an important advantage over chemical inhibitors that indiscriminately target both tumor and healthy cells. The good tolerance of the antibody in patients offers the possibility of using it in combination with other molecules.

[0154] Another interesting approach is to target other Bcl-2 family members in addition to Mcl-1. The majority of solid tumors are resistant to the use of single inhibitors targeting Bcl-2, Bcl-XL, or Mcl-1, but the combination of inhibition of two or even all three proteins is highly effective (Soderquist et al., 2018). The combination of Mcl-1 and Bcl-XL inhibitors in particular seems to be effective in the majority of tumors and is therefore promising. However, this combination has been reported to be lethal in mouse models due to acute liver toxicity (Weeden et al., 2018). Using an antibody against netrin-1 would be a more selective BH3 mimetic targeting Mcl-1, thus reducing the toxicity of co-treatment and allowing combination with other BH3 mimetics, as we have shown here in several preclinical ex vivo tumor models treated with NP137 and the Bcl-2 inhibitor venetoclax.

[0155] Another BH3 mimetic was assayed. Navitoclax (ABT-263) is known to induce inhibition of Bcl-2 / Bcl-XL / Bcl-w. The combination of netrin-1 silencing and navitoclax rapidly and significantly induced apoptosis (Figure 9).

[0156] method: Cell lines, transfection procedures, and reagents Human colorectal HCT116 and cervical HeLa cancer cell lines were cultured in DMEM medium (Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum. HEK293T cells were cultured in DMEM medium (Life Technologies) containing 10% fetal bovine serum, non-essential amino acids (NEAA, Life Technologies) and 50 μg / ml Geneticin (Life Technologies). Human lung H322 cancer cells were cultured in RPMI1640+Glutamax medium (Life Technologies) containing 10% fetal bovine serum. For RNA interference experiments, cell lines were transfected using Lipofectamine RNAiMAX reagent (Life Technologies). For plasmid transfection, HCT116 cell lines were transfected using Lipofectamine Plus reagent (Life Technologies) and HeLa cell lines were transfected using jet prime (PolyPlus-Transfection). All transfections were performed according to the manufacturer's protocols. The BH3 mimetic venetoclax (ABT-199) and the NEDD8 inhibitor pevonedistat were purchased from Selleck Chemical GmbH (Munich, Germany). The proteasome inhibitors MG132 and bortezomib were purchased from Sigma-Aldrich (St. Louis, MO, USA). The caspase inhibitor QVD was obtained from Adooq Bioscience (Irvine, CA, USA).

[0157] Generation of stable cell lines For the generation of stable inducible cell lines, a histidine-tagged rat UNC5B coding sequence was cloned into the SfiI site of the pSBtet-GH plasmid, a Sleeping Beauty-based vector that allows doxycycline-inducible expression of UNC5B, as well as constitutive expression of the rtTA protein, the hygromycin resistance gene, and green fluorescent protein (GFP), expressed under the control of the synthetic RPBSA promoter. HCT116, HeLa, and T47D cell lines were cotransfected with UNC5B-pSBtet-GH and the Sleeping Beauty transposase expression vector (SB100X). The Sleeping Beauty transposase expression vector was a generous gift from Professor Rolf Marschalek (Goethe-University of Frankfurt, Germany). pSBtet-GH was a gift from Eric Kowarz (Addgene, Watertown, MA, plasmid #60498; http: / / n2t.net / addgene:60498; RRID:Addgene60498). Stable cell clones were selected in hygromycin-containing medium for 7 days. Stable UNC5B cell lines were then sorted by flow cytometry to select high GFP positive cells. All stable inducible cell lines were further tested for UNC5B expression in response to doxycycline.

[0158] Stable cell lines constitutively expressing netrin-1 were generated by lentiviral infection using a self-inactivating HIV-1 derived vector (pWPXLd) encoding netrin-1 under the control of the human elongation factor-1 alpha (EF-1 alpha) promoter. For the generation of HCT116 stable cell lines inducible for UNC5B used in the screening, the pCW57.1 vector was used and stable cell lines were generated by lentiviral infection. This lentiviral vector allows doxycycline-inducible expression of UNC5B and constitutive expression of the rtTA protein and the hygromycin resistance gene. pCW57.1 (Addgene, Plasmid #41393; http: / / n2t.net / addgene:41393; RRID:Addgene_41393).

[0159] For the generation of stable UNC5B or COMMD2 knockout H322 cell lines, various small guide RNAs (sgRNAs) were designed using the sgRNA designer tool CRISPick (CRISPRi) available at (https: / / )portals.broadinstitute.org / gppx / crispick / public. sgRNAs were designed near the UNC5B or COMMD2 promoter. BsmBI linkers were added to the sgRNAs, and the oligonucleotides were then annealed by incubation at 90°C for 3 min and 37°C for 15 min and inserted into the BsmBI-digested pLVhU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro vector (Thakore et al., 2015) to express a truncated Cas9 enzyme (dCas9) fused to a Kruppel-associated box (KRAB) transcriptional repressor (dCas9-KRAB). pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro (Addgene, plasmid #71236; (http: / / )n2t.net / addgene:71236; RRID:Addgene_71236).

[0160] Quantitative RT-PCR Total RNA was extracted using the NucleoSpin® RNA Plus kit (Macherey Nagel, Duren, Germany) according to the manufacturer's protocol. RT-PCR reactions were performed with the PrimeScript RT Reagent Kit (Takara Bio Europe, Saint-Germain-en-Laye, France). 500 ng of total RNA was reverse transcribed using the following program: 37°C for 15 min and 85°C for 5 s. For expression studies, target transcripts were amplified using the Premix Ex Taq (probe qPCR) kit (Takara Bio Europe) in a LightCycler® 2.0 instrument (Roche Applied Science, Penzberg, Germany) according to the manufacturer's instructions. Expression of target genes was normalized to TATA-binding protein (TBP) and beta-glucuronidase (GUSB) genes, which were used as housekeeping genes. The amount of target transcript normalized to the housekeeping genes was calculated using the comparative CT method. A validation experiment was performed to show that the efficiencies of target and housekeeping genes were approximately equal. Primer sequences are available upon request.

[0161] Western blot analysis For immunoblot analysis, cells were lysed by sonication in SDS buffer (10 mM Tris-HCl pH 7.4, 10% glycerol, 5% SDS, 1% TX-100, 100 mM DTT) in the presence of a protease inhibitor cocktail (Roche Applied Science). Protein extracts (20–50 μg per lane) were loaded onto 4–15% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and blotted onto nitrocellulose sheets using the Trans-Blot Turbo Transfer System (Bio-Rad). Nitrocellulose sheets were blocked with 10% nonfat dry milk in PBS / 0.1% Tween 20 (PBS-T) for 1 h and then incubated with the following antibodies: rabbit monoclonal antibody α-UNC5B (1:1000, clone D9M7Z, #13851, Cell Signaling, Danvers, MA); mouse monoclonal antibody α-β-actin, HRP-conjugated (1:10000, clone BA3R, #MA5-15739-HRP, Life Technologies); rabbit monoclonal antibody α-Mcl-1 (1:1000, clone D5V5L, #39224, Cell Signaling); rabbit monoclonal antibody α-Bcl-XL (1:1000, clone 54H6, #2764, Cell Signaling); rabbit polyclonal antibody α-Bcl-2 (1:1000, clone 21, #783, Santa Cruz, CA) rabbit polyclonal antibody α-COMMD2 (1:1000, HPA044190, Sigma-Aldrich); rabbit monoclonal antibody α-Ku80 (1:1000, clone C48E7, ​​#2180, Cell Signaling) overnight at 4°C. After three washes with PBS-T, the membranes were incubated with the appropriate HRP-conjugated secondary antibody (1:10000, Jackson ImmunoResearch, Suffolk, UK) for 1 h.Detection was performed using the West Dura Chemiluminescence System (Life Technologies). Membranes were imaged with a ChemiDoc Touch Imaging System (Bio-Rad). Membranes were imaged with a ChemiDoc Touch Imaging System (Bio-Rad). Protein band quantification was performed with Image Lab 6.1 software (Bio-Rad) taking into account adjusted volumetric intensities normalized to housekeeping genes.

[0162] Cell death assay For propidium iodide (PI) staining, stable inducible cell lines were cultured in 48-well plates at a density of 25 x 10 3 Cells were seeded at 1000 x g / well. The next day, cells were treated with doxycycline and 0.33 mg / ml PI in serum-free (HCT116) or 10% serum (HeLa) medium and plates were transferred into an Incucyte Zoom instrument (Essen Bioscience, Royston, UK). Images of live and PI-stained dead cells were taken every 2 or 3 hours and the number of PI-stained cells was calculated.

[0163] For DNA fragmentation analysis (SubG1), HCT116 and HeLa cell lines were cultured in 6-well plates at a density of 2*10 per well. 5The following day, cells were transfected with siRNA as described above and incubated in serum-free medium for 24 hours. The transfected cells were then treated with doxycycline in 10% serum medium and further incubated for 18 hours. The cells were then harvested, fixed in 70% ethanol, and incubated at -20°C for at least overnight. Afterwards, the cells were washed twice with PBS and incubated in PBS containing 2mg / ml RNase A and 40mg / ml PI for 15 minutes at room temperature. DNA content was assessed by fluorescence-activated cell sorting (FACS) analysis on a FACScalibur or FacsCantoII (BD Bioscience, Franklin Lakes, NJ, USA).

[0164] Caspase-3 and -9 activity assays were performed as previously described (Paradisi et al., 2013; Paradisi et al., 2009). Briefly, HCT116 and HeLa cells were seeded and treated similarly to the DNA fragmentation assay. Cells were then harvested and caspase-3 / caspase-9 activity assays were performed using a Caspase-3 / Caspase-9 Fluorometric Assay Kit (BioVision, Milpitas, CA, USA) according to the manufacturer's instructions. Caspase activity (activity / min / mg protein) was calculated from 1 h kinetic cycle readings on a spectrofluorometer (405 nm / 510 nm, Infinite F500, Tecan, Mannedorf, Switzerland).

[0165] Mitochondrial potential assay HCT116 and HeLa cells were seeded and treated in the same manner as in the DNA fragmentation assay. The cells were then harvested and washed with PBS. 1 × 10 cells were used for each sample. 6The cells were suspended in 1 mL of PBS containing JC-1 dye (2.5 μg / mL, Chemometec, Allerod, Denmark). After 15 min of incubation at 37°C, the stained cells were centrifuged at 800×g for 5 min at room temperature and the supernatant was completely removed without disturbing the cell pellet. The cell pellet was then resuspended in 1 ml of PBS for washing, then centrifuged at 800×g for 5 min at room temperature and the supernatant was removed again. The washing step was repeated twice. The cell pellet was then resuspended in 250 μl of PBS buffer containing 1 μg / ml DAPI by pipetting. The final stained cells were analyzed by using a NucleoCounter NC-3000 (Chemometec).

[0166] Conditioned medium generation HCT116 stable cells constitutively expressing netrin-1 were seeded in 100 mm dishes (3*10 per dish). 6 After 24 hours, the cell culture medium was collected and centrifuged at 1200×g for 5 minutes, and the supernatant was collected as netrin-1 conditioned medium.

[0167] CRISPR-Cas9 screening Gecko Library The human GeCKOv2 CRISPR knockout pooled library (targeting 19,050 genes and split into two half-libraries, each containing three sgRNAs for each gene) was a gift from Feng Zhang (Addgene).

[0168] Library DNA amplification Each lentiviral library (A and B) was amplified and prepared separately following the same protocol. 2 μL of each GeCKO library at 50 ng / μL was electroporated into Endura electrocompetent cells (Lucigen, Middleton, WI, USA). Electroporation into competent cells was performed four times per library. Cells were resuspended in recovery medium and placed in a shaking incubator at 37°C for 1 h. For each library, electroporated cells were transferred to two pre-heated 600 cm 2 1000-fold dilutions of the complete transformation were plated in parallel to estimate the transformation efficiency and ensure that complete library representation was maintained. The total number of colonies was at least 3,106, corresponding to approximately 50 colonies per construct in each GeCKO library. Colonies were picked by adding 10 mL of LB medium to each plate and detaching with a cell spreader. The liquid with the detached colonies was collected in a tube and the procedure was repeated a second time on the same plate with an additional 10 mL of medium. An appropriate number of plasmid preparations (NucleoBond Xtra, Macherey Nagel) were performed and the concentration of each library was determined by UV-VIS spectroscopy (NanoDrop 2000, Thermo Fischer Scientific, Waltham, MA, USA). Plasmid libraries A and B were pooled in equimolar proportions for lentivirus production.

[0169] Lentivirus production and titration HEK 293T cells were plated at 5.10 µl per 150 mm dish one day before transfection. 6Cells were seeded with 10 μg of phCMV-VSV-g, 15 μg of phCMV-Gag-Pol-HIV, and 20 μg of GeCKO plasmid library, using Xtreme Gene Reagent (Roche Applied Science). Six hours after transfection, the medium was changed. Two viral harvests were collected at 40 and 60 hours after transfection. The two harvests were pooled, centrifuged, and filtered through a 0.45 μm filter. Aliquots of the clarified supernatant were stored at -80°C. For lentivirus production, titration was performed by infecting HCT116 cells with several different volumes of lentivirus in 12-well plates. The day after infection, each well was split into two wells: one received 0.8 μg / mL puromycin and the other did not receive puromycin. After 72 hours of selection, cells were counted to calculate the percentage of transduction and the virus titer was determined.

[0170] Cell library creation The day before transduction, HCT116 cells were cultured at a concentration of 10 per well. 5 Cells were seeded into eight 12-well plates. Cells were then transduced with the GeCKO lentiviral library in a volume previously determined to achieve a multiplicity of infection (MOI) of 0.3-0.5. HCT116 cells were transduced by spin infection for 1 h 30 min at 37°C in complete DMEM medium supplemented with 8 μg polybrene. The next day, cells were trypsinized and seeded into eight 150 mm dishes and treated with 0.8 μg / mL puromycin. As a selection control, non-transduced cells were seeded and treated with 0.8 μg / mL puromycin.

[0171] UNC5B loading in library-transduced cells After puromycin selection, a portion of the cell library was dissolved for genome extraction and sequencing. 24,106 cells of the two conditions were seeded in serum-free DMEM, respectively, and treated or not treated with 2 μg / ml doxycycline. In parallel, a stable HCT116 cell line inducible for UNC5B and not transduced by the CRISPR library in the control condition was induced with the same doxycycline concentration. When all cells died in the control condition, the surviving cells of the two conditions of the library were amplified for 3 days and then dissolved for genome extraction and sequencing.

[0172] Genome extraction and DNA amplification Genomic DNA (gDNA) was extracted from the cell library before UNC5B induction and from surviving cells after UNC5B induction using the NucleoSpin Tissue Kit (Macherey-Nagel). Amplification of the pooled sgRNA isolated from the cell samples was performed using Clone Amp HiFi enzyme (Takara Bio Europe). Primers were designed to amplify a 150 bp region around the sgRNA sequence and to add a barcode sequence specific for each sample and an adapter required to immobilize the PCR product in the NGS flow cell. Three separate PCRs with 250 ng of gDNA each were performed for each sample. The PCR products were purified with the PCR Clean-up / Gel extraction kit (Macherey Nagel) and then run on an agarose gel, followed by a new purification step with the same kit. A final step of purification (AMPure, Beckman Coulter, Brea, CA, USA) was performed before sequencing.

[0173] Next Generation Sequencing and Analysis Next generation sequencing (NGS) was performed on the Ion Torrent platform (Thermo Fischer Scientific) by the Institute of functional genomics of Lyon (IGFL, Lyon) sequencing platform using the Ion PI HiQ Chef Kit and Ion PI HiQ chip v3 (100pb-80M reads). Amplified samples were pooled together on a sequencing chip for running and differentiated by barcode identification. Raw data from sequencing was processed to eliminate incorrect sequences and to trim sequences adjacent to the sequence of the sgRNA. Trimmed sequences were BLASTed against the GeCKO library database with determined parameters (95% identity for 90% of the length) and only the first hit was kept. Finally, a list with the number of reads for each sgRNA per experimental condition was obtained. For a given condition, the number of reads for each sgRNA was normalized by the total number of reads in this condition. The reads were then normalized by subtracting the number of reads in the UNC5B-induced condition from the control condition. Finally, sgRNA sequences with more than 100 normalized reads were selected, and then genes with more than three sgRNA sequences were selected and confirmed.

[0174] Xenografts in nude mice Five-week-old female athymic NMRI nude mice were obtained from Janvier Labs (Saint Bertevin, France). Mice were maintained in a specific and opportunistic pathogen-free (SOPF) animal facility. Parental or stable H322 cells deficient in COMMD2 or UNC5B were injected at 5*10 ng / ml in 100 μl PBS into the right flank of nude mice. 6 The tumors were implanted by subcutaneous injection of 100 mm 3Upon reaching 10 mg / Kg, mice were injected intravenously with 10 mg / Kg of anti-Netrin-1 (NP137) or isotype control antibody, 3 times a week for 36 days. Tumor size was measured 3 times a week with a Vernier caliper. Tumor volume was calculated according to the formula v=0.5×(l×w2), where v is volume, l is length, and w is width. All experiments were performed in accordance with the relevant guidelines and regulations of the Animal Ethics Committee (approval APAFIS n°:28723; accreditation for laboratory animal care by CECCAP, ENS Lyon-PBES, France).

[0175] EMT6 syngeneic breast tumor model Five-week-old Balb / c females were purchased from Janvier Labs. EMT6 cells were implanted by subcutaneous injection of 5*105 cells suspended in 100 μl of PBS. Tumors were 50 mm 3 Once the tumor size reached 100 mg / kg, mice were intravenously injected with a loading dose of 20 mg / kg of NP137 or an isotype control antibody for three consecutive days, followed by a maintenance dose of 10 mg / kg, three times a week for 12 days. Venetoclax (5 mg / kg, Selleck Chemicals) or DMSO (resuspended in 50% PEG 300 + 5% Tween 80 in distilled water, Selleck Chemicals GmbH) was administered by oral gavage, five times a week for two weeks. Tumor size was measured three times a week with a caliper. Tumor volume was calculated according to the formula v = 0.5 x (l x w2), where v is volume, l is length, and w is width. All experiments were performed in accordance with the relevant guidelines and regulations of the Animal Ethics Committee (approval APAFIS n°:28780; accreditation for laboratory animal care by CECCAP, ENS Lyon-PBES, France).

[0176] For in vivo Mcl-1 degradation, EMT6 cells were cultured at 5*10 5 The tumors were implanted by subcutaneous injection of 100 mm 3 Upon reaching 100% CI, mice were injected intravenously with 20 mg / Kg of NP137 or isotype control antibody. Eight hours after injection, mice were sacrificed and tumors were harvested and immediately frozen at -80°C.

[0177] Ex vivo breast cancer organ culture A total of eight fresh human breast samples were obtained from chemotherapy-naïve patients with invasive cancer after surgical resection at the Centre Leon Berard (CLB, Lyon, France). All patients signed an informed consent document authorizing the use of their samples for research purposes, as required by French bioethics law, and the study was approved by the ethical committee of the CLB. After surgical resection and gross anatomopathological examination, tumor tissues were immediately kept in PBS at 4°C and transported to the laboratory. Thereafter, tumors were routinely examined and classified by a board-certified pathologist. After removal of excess necrotic tissue, 200 mm 3 Fresh tumor fragments were included in the agarose matrix. Automated slicing was performed using a MICROM HM650V Vibratome (Thermo Fisher Scientific, Walldorf, Germany) with a section thickness of 250 μm, an amplitude of 8 mm, and a slice speed of 0.6 mm / sec. The slices were cultured within 2 hours after the tumor was removed from the patient. Culture was performed at 5% CO2, 37°C, and atmospheric oxygen levels. The slices were individually incubated in DMEM medium supplemented with 5% fetal bovine serum, 1% GlutaMAX, 1% penicillin / streptomycin, 1% MEM, 1% HEPES (all from Life Technologies) for 24 hours using 24-well plates. Several consecutive sections of each fresh tumor were then treated with 10 μg / ml anti-netrin-1 (NP137) or isotype control (NP001) antibodies in DMSO and 1 μM venetoclax for 24 hours. Apoptosis in treated sections was assessed by immunohistochemical staining for active caspase-3.

[0178] Immunohistochemical staining For histological examination of tumors implanted in nude mice, ex vivo breast cancer sections, and in human clinical trials, tumors were fixed in 10% buffered formalin and embedded in paraffin. 4 μm thick tissue sections of formalin-fixed paraffin-embedded tissues were prepared according to conventional procedures. Sections were then stained with hematoxylin and eosin and examined under a light microscope. Immunohistochemistry was performed using the Omnimap DAB Kit in an automated immunostainer (Ventana Discovery XT, Roche, Meylan, France) according to the manufacturer's instructions. Sections were incubated with rabbit anti-post-cleavage caspase-3 (diluted 1:500, #9661S, Cell Signaling) or rabbit anti-Mcl-1 (diluted 1:500, #HPA031125, Sigma-Aldrich) antibodies. HRP-conjugated anti-rabbit antibodies were applied to the sections. Staining was visualized with DAB solution with 3,3'-diaminobenzidine as chromogenic substrate. Sections were then counterstained with Gill's hematoxylin. Finally, sections were scanned at 20X with a Panoramic Scan II (3D Histech, Budapest, Hungary). Quantification of caspase-3 and Mcl-1 positive cells was performed with HALO software and adapted algorithm cytonuclear v2.0.8 6 (Indica Labs, Albuquerque, NM, USA). MCL1 intensity was calculated as a percentage over the surface of the tumor cells. All immunohistochemical staining and analyses were performed by the Research Pathology Platform East (Plateforme Anatomopathologie Recherche, Universite de Lyon, Universite Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Leon Berard, Centre de recherche en cancerologie de Lyon (CRCL), Lyon, 69373, France).

[0179] statistical analysis Statistical tests are indicated in each figure and were performed using GraphPad Prism version 9.4.1 for Windows (GraphPad Software, La Jolla, CA, USA). Survival curves were generated according to the Kaplan-Meier method in GraphPad software. (References) TIFF2025513747000009.tif224164TIFF2025513747000010.tif209162

Claims

1. A combination of an anti-Netrin-1 antibody, or an antigen-binding fragment thereof, and an inhibitor of an anti-apoptotic protein of the Bcl-2 family for use in the treatment of Mcl1+ and Netrin-1 receptor+, particularly UNC5B+ cancers.

2. 2. The combination for use according to claim 1, which results in the simultaneous, separate or sequential administration to a patient of an anti-netrin-1 antibody, or an antigen-binding fragment thereof, and an inhibitor of an anti-apoptotic protein of the Bcl-2 family.

3. 3. The combination for use according to claim 1 or 2, wherein the inhibitor of an anti-apoptotic protein of the Bcl-2 family is an inhibitor of the Bcl-2 protein.

4. The anti-netrin-1 antibody or antigen-binding fragment thereof, (i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 3 or 35; (ii) or - H-CDR1 having the sequence shown as SEQ ID NO:5, - H-CDR2 having the sequence shown as SEQ ID NO:6, - H-CDR3 having the sequence shown as SEQ ID NO: 7 A variable domain VH comprising - L-CDR1 having the sequence shown as SEQ ID NO: 8, - L-CDR2 with the sequence YAS, - L-CDR3 having the sequence shown as SEQ ID NO: 9 A variable domain VL comprising Contains or (iii) or - H-CDR1 having the sequence shown as SEQ ID NO: 28, - H-CDR2 having the sequence shown as SEQ ID NO: 29, - H-CDR3 having the sequence shown as SEQ ID NO: 30 A variable domain VH comprising - L-CDR1 having the sequence shown as SEQ ID NO: 31, - L-CDR2 having the sequence shown as SEQ ID NO: 32, - L-CDR3 having the sequence shown as SEQ ID NO: 9 A variable domain VL comprising Including, A combination for use according to any one of claims 1 to 3.

5. The combination for use according to claim 4, wherein the anti-netrin-1 antibody or antigen-binding fragment thereof is selected from the group consisting of anti-netrin-1 antibodies and antigen-binding fragments thereof comprising the VH and VL amino acid sequence pairs SEQ ID NOs: 20 and 14, SEQ ID NOs: 21 and 15, SEQ ID NOs: 22 and 16, SEQ ID NOs: 23 and 17, SEQ ID NOs: 24 and 17, SEQ ID NOs: 25 and 16, SEQ ID NOs: 26 and 17, SEQ ID NOs: 22 and 17, SEQ ID NOs: 25 and 18, SEQ ID NOs: 21 and 16, or SEQ ID NOs: 27 and 19.

6. The combination for use according to claim 4 or 5, wherein the anti-netrin-1 antibody comprises a human IgG1 constant heavy chain (CH) and / or a human IgG1 constant light chain (CL), in particular a human kappa constant domain.

7. 7. A combination for use according to any one of claims 1 to 6, comprising a VH of sequence SEQ ID NO: 22 and a VL of sequence SEQ ID NO:

16.

8. The combination for use according to claim 7, further comprising human IgG1 CH, and human IgG1 CL kappa.

9. 8. The combination for use according to claim 7, further comprising human IgG1 CH Genbank AEL33691.1 modified R97K, and human IgG1 CL kappa Genbank CAC20459.

1.

10. 10. The combination for use according to any one of claims 1 to 9, wherein the inhibitor of anti-apoptotic proteins of the Bcl-2 family is venetoclax or navitoclax.

11. 11. The combination for use according to any one of claims 1 to 10, which induces Mcl-1 degradation limited to said Mcl-1+ and UNC5B+ cancer in said patient.

12. 12. The combination for use according to any one of claims 1 to 11, wherein the cancer is a cancer with a percentage of positive tumor cells of grade 3 or higher, or grade 4 or higher, or equal to grade 5, as measured by staining with an antibody binding to Mcl-1, counting the cancer cells in the cytoplasm, and assessing the percentage of stained positive cancer cells.

13. A pharmaceutical composition comprising an anti-netrin-1 antibody, or an antigen-binding fragment thereof, an inhibitor of an anti-apoptotic protein of the Bcl-2 family, and a pharma- ceutically acceptable vehicle or excipient.

14. The composition of claim 13, wherein the inhibitor of anti-apoptotic proteins of the Bcl-2 family is a Bcl-2 inhibitor.

15. The composition according to claim 13 or 14, wherein the anti-netrin-1 antibody, or antigen-binding fragment thereof, is as defined in any one of claims 4 to 9.

16. A composition according to any one of claims 13 to 15, wherein the inhibitor of anti-apoptotic proteins of the Bcl-2 family is venetoclax or navitoclax.

17. An anti-Netrin-1 antibody, or an antigen-binding fragment thereof, for use as a tumor-specific Mcl-1 inhibitor in the treatment of Mcl-1+ and Netrin-1 receptor+, particularly UNC5B+ tumors.

18. The anti-netrin-1 antibody, or an antigen-binding fragment thereof, for use according to claim 17, wherein the tumor is a Mcl-1+, netrin-1+ and UNC5B+ tumor.

19. 19. An anti-netrin-1 antibody, or an antigen-binding fragment thereof, for use according to claim 17 or 18, as defined in any one of claims 4 to 9.

20. 20. The anti-netrin-1 antibody, or antigen-binding fragment thereof, for use according to any one of claims 17 to 19, wherein the tumor is a tumor having a percentage of positive tumor cells of grade 3 or higher, or grade 4 or higher, or equal to grade 5, as measured by staining with an antibody binding to Mcl-1, counting tumor cells in the cytoplasm, and assessing the percentage of stained positive tumor cells.

21. An anti-Netrin-1 antibody, or an antigen-binding fragment thereof, for use according to any one of claims 17 to 20 in patients with Mcl1+ and Netrin-1 receptor+, in particular UNC5B+, tumors, wherein the patient is further treated with an inhibitor of an anti-apoptotic protein of the Bcl-2 family, preferably an inhibitor of a Bcl-2 protein.

22. A method for the treatment of Mcl1+ and netrin-1 receptor+, particularly UNC5B+ cancers, comprising administering to a patient having such cancer an effective amount of an anti-netrin-1 antibody, or an antigen-binding fragment thereof, and an inhibitor of an anti-apoptotic protein of the Bcl-2 family.

23. The method of claim 22, wherein the anti-netrin-1 antibodies and antigen-binding fragments thereof cause tumor-specific Mcl-1 inhibition or degradation in the patient.

24. The method described in claim 22 or 23, wherein the anti-netrin-1 antibody and its antigen-binding fragment inhibit the binding of netrin-1 to UNC5B, and the released UNC5B causes the CRL3-COMMD2 complex to ubiquitinate Mcl-1, targeting Mcl-1 for proteasomal degradation.

25. 25. The method of any one of claims 22 to 24, wherein the cancer has a percentage of Mcl-1 positive tumor cells of grade 3 or higher, or grade 4 or higher, or equal to grade 5, as measured by staining with an antibody bound to Mcl-1, counting the cancer cells in the cytoplasm, and assessing the percentage of stained positive cancer cells.

26. The anti-netrin-1 antibody and the antigen-binding fragment thereof are - H-CDR1 having the sequence shown as SEQ ID NO: 28, - H-CDR2 having the sequence shown as SEQ ID NO: 29, - H-CDR3 having the sequence shown as SEQ ID NO: 30 A variable domain VH comprising - L-CDR1 having the sequence shown as SEQ ID NO: 31, - L-CDR2 having the sequence shown as SEQ ID NO: 32, - L-CDR3 having the sequence shown as SEQ ID NO: 9 A variable domain VL comprising 26. The method of any one of claims 22 to 25, comprising:

27. The method of claim 26, wherein the anti-netrin-1 antibodies and antigen-binding fragments thereof are selected from the group consisting of anti-netrin-1 antibodies and antigen-binding fragments thereof comprising the VH and VL amino acid sequence pairs SEQ ID NOs: 20 and 14, SEQ ID NOs: 21 and 15, SEQ ID NOs: 22 and 16, SEQ ID NOs: 23 and 17, SEQ ID NOs: 24 and 17, SEQ ID NOs: 25 and 16, SEQ ID NOs: 26 and 17, SEQ ID NOs: 22 and 17, SEQ ID NOs: 25 and 18, SEQ ID NOs: 21 and 16, and SEQ ID NOs: 27 and 19.

28. The method according to claim 26 or 27, wherein the anti-netrin-1 antibody and its antigen-binding fragments comprise a VH of the sequence of SEQ ID NO: 22 and a VL of the sequence of SEQ ID NO:

16.

29. The method of claim 26 or 27, wherein the anti-netrin-1 antibody further comprises human IgG1 CH, and human IgG1 CL kappa.

30. 30. The method according to any one of claims 22 to 29, wherein the inhibitor of anti-apoptotic proteins of the Bcl-2 family is a Bcl-2 inhibitor.

31. 31. The method of claim 30, wherein the inhibitor is venetoclax or navitoclax.

32. A method for treating a patient suffering from an Mcl-1+ and netrin-1 receptor+, particularly UNC5B+, tumor, comprising administering to the patient an effective amount of an anti-netrin-1 monoclonal antibody or an antigen-binding fragment thereof, and causing tumor-specific Mcl-1 inhibition or degradation.

33. The method of claim 32, wherein the anti-netrin-1 antibody and its antigen-binding fragment inhibit the binding of netrin-1 to UNC5B, and the released UNC5B allows the CRL3-COMMD2 complex to ubiquitinate Mcl-1, targeting Mcl-1 for proteasomal degradation.

34. The anti-netrin-1 antibody and the antigen-binding fragment thereof are - H-CDR1 having the sequence shown as SEQ ID NO: 28, - H-CDR2 having the sequence shown as SEQ ID NO: 29, - H-CDR3 having the sequence shown as SEQ ID NO: 30 A variable domain VH comprising - L-CDR1 having the sequence shown as SEQ ID NO: 31, - L-CDR2 having the sequence shown as SEQ ID NO: 32, - L-CDR3 having the sequence shown as SEQ ID NO: 9 A variable domain VL comprising 34. The method of claim 32 or 33, comprising:

35. The method of claim 34, wherein the anti-netrin-1 antibodies and antigen-binding fragments thereof are selected from the group consisting of anti-netrin-1 antibodies and antigen-binding fragments thereof comprising the VH and VL amino acid sequence pairs SEQ ID NOs: 20 and 14, SEQ ID NOs: 21 and 15, SEQ ID NOs: 22 and 16, SEQ ID NOs: 23 and 17, SEQ ID NOs: 24 and 17, SEQ ID NOs: 25 and 16, SEQ ID NOs: 26 and 17, SEQ ID NOs: 22 and 17, SEQ ID NOs: 25 and 18, SEQ ID NOs: 21 and 16, and SEQ ID NOs: 27 and 19.

36. The method of claim 35, wherein the anti-netrin-1 antibody and its antigen-binding fragments comprise a VH of the sequence of SEQ ID NO: 22 and a VL of the sequence of SEQ ID NO:

16.

37. 37. The method according to any one of claims 32 to 36, wherein the patient is further treated with an inhibitor of an anti-apoptotic protein of the Bcl-2 family, preferably an inhibitor of the Bcl-2 protein.

38. 38. The method of any one of claims 32 to 37, wherein the tumor has a percentage of Mcl-1 positive tumor cells of grade 3 or higher, or grade 4 or higher, or equal to grade 5, as measured by staining with an antibody bound to Mcl-1, counting tumor cells in the cytoplasm, and assessing the percentage of stained positive cancer cells.

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