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

Abstract

In tumor cells expressing the axon guiding factor 1, interference of the axon guiding factor 1 can trigger Mcl-1 degradation limited to the tumor cells expressing the axon guiding factor 1. An axon-directed factor 1 interference that triggers the degradation of tumor-specific Mcl-1 is presented. In addition, axon steering factor 1 interference may be combined with a BH3 mimetic, particularly a Bcl-2 inhibitor. An anti-axon director 1 antibody is used to interfere with the axon director 1. The present invention proposes an anti-axon guiding factor 1 antibody which is useful as a tumor-specific Mcl-1 inhibitor for the treatment of Mcl-1 + tumors, more particularly Mcl-1 +, axon guiding factor 1 + and axon guiding factor 1 receptor +, in particular UNC5B + tumors. Also provided are combinations of an anti-axon director 1 antibody and a Bcl-2 inhibitor (e.g., Vilentog).

Description

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

[0002] background

[0003] The balance between cell survival and death is essential for the homeostasis of an organism. This balance is controlled, at least in part, by the interaction of Bcl-2 family proteins, which consist of pro-apoptotic effectors Bax, Bak, and Bok, pro-apoptotic BH3-specific proteins (including these members: Bad, Bid, Puma, Bim, and Noxa), and anti-apoptotic proteins of the Bcl-2 family (including Bcl-2, Bcl-XL, Mcl1, Bcl-W, Bfl-1 / A1) (Singh et al., 2019). In cancer, the balance of this balance shifts toward pro-survival Bcl-2 members, which are often overexpressed and stabilized (Delbridge et al., 2016). Thus, Bcl-2 members enable cancer cells to evade cell death, a key step in acquiring a malignant phenotype (Hanahan and Weinberg, 2000; Juin et al., 2013). Cells protected by anti-apoptotic Bcl-2 members are often “sensitized” for apoptosis due to oncogenic stress and stabilization of BH3-specific proteins (Adams and Cory, 2018; Merino et al., 2012). Therefore, therapeutic strategies targeting anti-apoptotic Bcl-2 proteins are relevant, and selective and potent inhibitors against their members have been developed over the past decades (Merino et al., 2018). Such inhibitors, known as “BH3 mimetics,” are mainly effective in the treatment of hematopoietic cancers that express high levels of Bcl-2 family proteins (Ewald et al., 2019; Schefffold et al., 2018). However, combinations of different BH3 mimetics may be effective in the treatment of a wider range of tumors, including solid tumors, which rely on more than one Bcl-2 protein for survival (Soderquist et al., 2018). To date, only the Bcl-2-specific inhibitor venetoclax / ABT199 has received clinical approval (Souers et al., 2013).

[0004] Some molecules targeting other members of the Bcl-2 family have entered the clinic, such as the Mcl-1 inhibitor S63845 (Kotschy et al., 2016). Therefore, BH3 mimetics targeting Mcl-1 have been developed (S63845, AMG176 and AMG397). However, the clinical development of these compounds, especially those targeting Mcl-1, has been delayed due to their hit effects on healthy cells (Merino et al., 2018). In fact, the presence of this anti-apoptotic protein is essential for many tissues and cell types, including hematopoietic stem cells, thymic epithelium, mature and immature B and T lymphocytes, activated T cells, cardiomyocytes, neurons and hepatocytes (Merino et al., 2018). Preliminary testing of S63845 in humanized Mcl-1 mice showed a decrease in the B cell population during treatment, but overall good tolerance (Brennan et al., 2018). Nevertheless, the recent suspension of clinical studies of two Mcl-1 inhibitors, AMG 176 (NCT02675452) and AMG 397 (NCT03465540), evaluating the possible cardiotoxicity of these compounds, still raises some safety concerns (Wei et al., 2020). Several reports of biochemical restriction and relative toxicity of these Mcl-1 inhibitors on healthy hematopoietic cells have hampered the development of these therapies (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.

[0005] UNC5B / UNC5H2 (UNCoordinated Homolog 5) is a type I transmembrane receptor discovered for its effects on neuronal navigation during embryonic development. It is a receptor that can actively induce apoptosis when not bound to its ligand, axon guidance factor 1 (netrin-1) (Llambi et al., 2001). This property defines all members of the dependency receptor family, which activate dual signaling pathways: in the presence of their respective ligands, they induce "positive" signaling pathways, such as proliferation or migration signals, while in the absence of specific ligands, they activate "negative" signaling pathways, leading to programmed cell death (Mehlen and Bredesen, 2004, 2011; Negulescu and Mehlen, 2018). Therefore, the expression of these receptors makes cell survival dependent on the presence of their respective ligands (Bredesen et al., 2005; Mehlen and Puisieux, 2006). The dependency receptor family consists of more than 20 members ( Goldschneider and Mehlen, 2010 ), including the axon guidance factor 1 receptor UNC5H (i.e., UNC5H1 / A, UNC5H2 / B, UNC5H3 / C, and UNC5H4 / D) and DCC ( Llambi et al., 2001 ; Mehlen et al., 1998 ).

[0006] Together with AGF1, UNC5B exerts 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 is widely described as a pro-death receptor, a death activity that is blocked by multimerization after binding to AGF1. Therefore, UNC5B is defined as a conditional tumor suppressor, while AGF1 is defined as a tumor promoter (Llambi et al., 2001; Thiebault et al., 2003). Consistent with this concept, AGF1 is a protein that is primarily expressed during embryonic development and has been shown to be re-expressed by cancer cells and the tumor microenvironment in a large proportion of human tumors (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 NG1 and its receptor was sufficient to trigger cancer cell death and induce tumor growth inhibition. Based on these findings, a monoclonal antibody that neutralizes NG1 and blocks the NG1 / UNC5B interaction was developed, termed NP137 (Grandin et al., 2016), and is currently being evaluated in several Phase I and II clinical trials (ClinicalTrials.gov identifiers NCT02977195 and NCT04652076).

[0007] Therefore, there is a need for novel Mcl-1 inhibitors that are both effective and have a satisfactory safety profile. There is also a need for novel combination cancer therapies based on combination partners that exhibit synergistic effects, offer the advantages of significantly improved long-term efficacy and improved safety profiles. More interestingly, the combination allows the reduction of the therapeutic dose of one or both of the combined drugs (especially BH3 mimetics or Bcl-2 inhibitors), especially for safety reasons. In order to limit side effects on healthy tissues, it is of great interest to develop tumor-specific agents, especially Mcl-1 inhibitors. SUMMARY OF THE INVENTION

[0009] The mechanism by which UNC5B triggers apoptotic signaling is poorly understood. When deprived of AGF1, UNC5B assumes an open conformation, allowing the phosphatase PP2A to catalyze the dephosphorylation of DAPK (serine / threonine death-associated protein kinase), which in turn induces cell death through a still unclear mechanism (Guenebeaud et al., 2010; Llambi et al., 2005; Wang et al., 2009). In the current study, the CRL3-COMMD2 complex has been identified as a key mediator of UNC5B-induced apoptosis. Completely unexpectedly, in the presence of detached UNC5B (not bound to AGF1), this CRL3-COMMD2 complex ubiquitinates the anti-apoptotic Bcl-2 member Mcl-1, targeting it for proteasomal degradation. As a result, reduced levels of Mcl-1 trigger mitochondrial depolarization and activation of the intrinsic apoptotic pathway. "AGF1 interference" involves preventing AGF1 from binding to UNC5B. This can be achieved by providing anti-AGF1 specific antibodies. Prior knowledge was that dependency receptors, such as the AGF1 receptor, trigger cell death independently of the mitochondrial-dependent pathway. It was therefore completely unexpected that in tumor cells expressing AGF1, AGF1 interference would trigger Mcl-1 degradation. It was further completely unexpected to discover a method capable of inducing Mcl-1 degradation confined to these tumor cells expressing AGF1, thus enabling the proposal of AGF1 interference to trigger tumor-specific Mcl-1 degradation in Mcl-1+ tumors. This study provides preclinical evidence that NP137 can be used as a specific Mcl-1 inhibitor in tumor patients and in combination with a BH3 mimetic. This allows overcoming the toxicity originally associated with compounds of this type. The data provided herein indicate that other AGF1 receptors, particularly UNC5A and UNC5C, are present in Mcl1+ tumor cells and may also be involved in the present invention.

[0010] Therefore, the object of the present invention is an anti-axon guidance factor 1 antibody, especially an anti-axon guidance factor 1 monoclonal antibody, which is used as a tumor-specific Mcl-1 inhibitor for the treatment of Mcl-1+ tumors. Another object of the present invention is the use of an anti-axon guidance factor 1 antibody, especially an anti-axon guidance factor 1 monoclonal antibody, for the preparation of a medicament used as a tumor-specific Mcl-1 inhibitor for the treatment of Mcl-1+ tumors.

[0011] Another object of the present invention is a method for specifically inhibiting Mcl-1 in Mcl-1+ tumors in patients suffering from such tumors, comprising administering to the patient an effective amount of an anti-axon guidance factor 1 antibody, especially an anti-axon guidance factor 1 monoclonal antibody, and exerting a tumor-specific Mcl-1 inhibition effect. In one object, the present invention is a method for treating a patient suffering from Mcl-1+ tumors, comprising administering to the patient an effective amount of an anti-axon guidance factor 1 antibody, especially an anti-axon guidance factor 1 monoclonal antibody, and exerting a tumor-specific Mcl-1 inhibition or degradation effect.

[0012] As disclosed herein, a Mcl-1+ tumor or cancer can also be a tumor or cancer that expresses UNC5B (UNC5B+ tumor or cancer) and AxonGuidanceFactor1 (AxonGuidanceFactor1+ tumor or cancer).

[0013] As disclosed herein, Mcl-1+ tumors or cancers can also be tumors or cancers that express UNC5B (UNC5B+ tumors or cancers) and / or another axon guidance factor 1 receptor, such as UNC5A and / or UNC5C. The tumor or cancer can also express axon guidance factor 1 (axon guidance factor 1+ tumors or cancers).

[0014] "Tumor specificity" herein means that the antibody specifically induces Mcl-1 inhibition or degradation in tumor cells ("restricted to Mcl-1+ tumor cells"). The antibody does not induce this inhibition in healthy cells containing or expressing Mcl-1 ("healthy Mcl-1+ cells"). In fact, it has been documented above that, for example, the presence of this anti-apoptotic protein Mcl-1 is essential in many tissues and cell types, including hematopoietic stem cells, thymic epithelium, mature and immature B and T lymphocytes, activated T cells, cardiomyocytes, neurons and hepatocytes. Therefore, the antibody does not induce this inhibition in these cells.

[0015] In one aspect, anti-Agf1 antibodies, particularly anti-Agf1 monoclonal antibodies, are used as tumor-specific Mcl-1 inhibitors in tumor cells expressing Mcl-1, Agf1 and UNC5B.

[0016] In one aspect, the result of the binding of anti-axon guidance factor 1 antibody, especially anti-axon guidance factor 1 monoclonal antibody to axon guidance factor 1 is that UNC5B is disengaged from axon guidance factor 1 or cannot bind to axon guidance factor 1, which leads to Mcl-1 inhibition or degradation. In another aspect, the disengaged or unbound UNC5B causes the CRL3-COMMD2 complex to ubiquitinate the anti-apoptotic Bcl-2 member Mcl-1, targeting it for proteasomal degradation. In another aspect, the result of the binding of anti-axon guidance factor 1 antibody, especially anti-axon guidance factor 1 monoclonal antibody to axon guidance factor 1 is that UNC5B triggers Mcl-1 degradation.

[0017] In another aspect, all of these occur in tumors that express AGF 1, i.e., tumor cells and / or the tumor environment express AGF 1. In another aspect, all of these occur in tumors that express AGF 1 and AGF 1 receptors, particularly UNC5B.

[0018] Therefore, the present invention provides an anti-axon guidance factor 1 antibody, particularly an anti-axon guidance factor 1 monoclonal antibody, which is used as a tumor-specific Mcl-1 inhibitor for treating Mcl-1+ tumors, more particularly tumors expressing Mcl-1, axon guidance factor 1 and axon guidance factor 1 receptor, particularly Mcl-1+, axon guidance factor 1+ and UNC5B+ tumors, as disclosed herein.

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

[0020] Another object of the present invention is a combination of an anti-AGF1 antibody or an antigen-binding fragment thereof with an inhibitor of an anti-apoptotic protein of the Bcl-2 family, preferably a Bcl-2 inhibitor, for use in the treatment of Mcl1+ cancer or Mcl1+ and UNC5B+ cancer, or Mcl1+ and AGF1 receptor+ cancer (particularly a receptor selected from UNC5A, B and C). As disclosed herein, Mcl-1+ tumors or cancers may also be tumors or cancers expressing UNC5B (UNC5B+ tumors or cancers) and AGF1 (AGF1+ tumors or cancers).

[0021] Another object of the invention is a combination, pharmaceutical combination or anticancer combination comprising for simultaneous, sequential or separate use

[0022] (a) an anti-axon guidance factor 1 antibody, in particular an anti-axon guidance factor 1 monoclonal antibody, and

[0023] (b) Inhibitors of anti-apoptotic proteins of the Bcl-2 family, preferably Bcl-2 inhibitors.

[0024] Herein, (b) relates to BH3 mimetics as inhibitors of anti-apoptotic proteins of the Bcl-2 family. This includes inhibitors of Bcl-2 proteins, which are members of the Bcl-2 family of anti-apoptotic proteins.

[0025] In one aspect, the combination is for use in treating cancer, wherein (a) and (b) are intended for simultaneous, sequential or separate administration to a patient.

[0026] Another object of the present invention is a pharmaceutical composition or anticancer composition comprising

[0027] (a) an anti-axon guidance factor 1 antibody, in particular an anti-axon guidance factor 1 monoclonal antibody,

[0028] (b) an inhibitor of an anti-apoptotic protein of the Bcl-2 family, preferably a Bcl-2 inhibitor,

[0029] and

[0030] (c) a pharmaceutically acceptable carrier or excipient.

[0031] Another object of the present invention is a pharmaceutical composition kit comprising

[0032] (a) a first vial or container comprising an anti-axon guidance factor 1 antibody, in particular an anti-axon guidance factor 1 monoclonal antibody,

[0033] (c) a second vial or container comprising an inhibitor of an anti-apoptotic protein of the Bcl-2 family, preferably a Bcl-2 inhibitor,

[0034] and

[0035] (b) A notice that may carry instructions regarding the regimen of administration (e.g., instructions for administering all or part of the contents of two vials or containers to the patient simultaneously, sequentially, or separately).

[0036] Another object of the present invention is the use of (a) an anti-axon guidance factor 1 antibody, in particular an anti-axon guidance factor 1 monoclonal antibody and (b) an inhibitor of an anti-apoptotic protein of the Bcl-2 family, preferably a Bcl-2 inhibitor, for the preparation of a medicament or a drug combination for the treatment of cancer. In one aspect, (a) and (b) are intended to be administered to a patient simultaneously, sequentially or separately.

[0037] Another object of the present invention is a method for treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of (a) an anti-axon guidance factor 1 antibody, particularly an anti-axon guidance factor 1 monoclonal antibody, and (b) an inhibitor of an anti-apoptotic protein of the Bcl-2 family, preferably a Bcl-2 inhibitor. This administration can be carried out in the form of different administration regimens. It can include administering (a) and (b) to the patient simultaneously, sequentially or separately. The patient in need thereof is a patient with a tumor disclosed herein.

[0038] Among these different purposes related to the combined use of (a) and (b), the anti-axon guidance factor 1 antibody is a tumor-specific Mcl-1 inhibitor. The antibody provides the function of Mcl-1 inhibition or degradation in a tumor-specific manner, as described herein. Therefore, this function is now combined with the inhibition of anti-apoptotic Bcl-2 proteins known to those skilled in the art. However, unlike previous anti-Mcl-1 / anti-Bcl-2 combinations, the combination of the present invention benefits from Mcl-1 inhibition confined to tumor cells, as disclosed herein. In other words, the Bcl-2 inhibitor or BH3 mimetic in the combination can be any effective Bcl-2 inhibitor (excluding Mcl-1 inhibitors), and it can 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 generally referred to as BH3 mimetics and can include Bcl-2, Bcl-XL, Bcl-W, Bfl-1 / A1 proteins. It can be an inhibitor of Bcl-2 itself (Bcl-2 inhibitor) or an inhibitor of any other member of the Bcl-2 family. It may also be an inhibitor of at least two of these proteins, such as an inhibitor of Bcl-2 and Bcl-XL, or an inhibitor of Bcl-2, Bcl-W and Bfl-1 / A1.

[0039] Currently, the combination of two BH3 mimetics or Bcl-2 anti-apoptotic protein family inhibitors is thought to prevent compensation by Bcl-2 family members and enhance cancer cell death. This article demonstrates that combined treatment with anti-axon guidance factor 1 neutralizing antibodies and Bcl-2 inhibitor Venetoclax, which are tumor-specific Mcl-1 inhibitors, significantly reduces tumor growth in mouse models and triggers apoptosis in human breast cancer ex vivo slices. The combination of axon guidance factor 1 silencing and Navitoclax also triggers apoptosis quickly and strongly. This is the first time such a combination has been proposed, which combines a tumor-specific inhibitor, and current data support that combining axon guidance factor 1 interference with BH3 mimetics can effectively trigger cancer cell death and has reduced side effects, thereby providing potential therapeutic benefits.

[0040] Venetoclax is a clinically accepted BH3 mimetic. In one embodiment, venetoclax is used in the combination of the present invention. In another embodiment, nervitoclax is used as a BH3 mimetic in the combination of the present invention.

[0041] In one aspect, a Mcl-1+ tumor or cancer is particularly a tumor or cancer having a percentage of Mcl-1 positive tumor cells equal to or higher than grade 3, or equal to or higher than grade 4, or equal to grade 5 (see below for methods of determining Mcl1+ tumors).

[0042] Detailed Description

[0043] Cancers related to the present invention

[0044] RSSoderquist et al. showed in 2018 that different cancers have various dependencies or co-dependencies on the BCL-2 gene, and most cell lines rely on Bcl-2, Bcl-X L The authors propose an idea for a roadmap for rationally targeting Bcl-2 family dependencies in different human cancers.

[0045] The present invention is primarily directed to a specific group of cancers characterized by Mcl-1 acting as a pro-survival Bcl-2 member and unexpectedly associated with the presence and action of axon guidance factor 1. Typically, Mcl-1 is overexpressed and / or stabilized in these cancers. "Mcl-1+ tumors" refers to tumors that include tumor cells that escape programmed cell death or apoptosis through a mechanism involving Mcl-1 and axon guidance factor 1. In these tumors, Mcl-1 is expressed at a certain level that can be assessed and is sufficient to shift the balance between cell survival and death to the former. In addition, methods for assessing the presence or level of axon guidance factor 1 are available to technicians. These tumors (or cancers) can be identified as "Mcl-1+ / axon guidance factor 1+ tumors" or "Mcl-1+ / axon guidance factor 1+ / axon guidance factor 1 receptor+ tumors" or "Mcl-1+ / Unc5B+ tumors". "Mcl-1+ / axon guidance factor 1+ / Unc5B+ tumors". In one aspect, the tumor is Mcl1+ and axon guidance factor 1 receptor+ tumor (particularly a receptor selected from UNC5A, B and C). In one aspect, the cancer or tumor also includes the participation of other anti-apoptotic Bcl-2 members (e.g., Bcl-2), which can be designated herein as "Bcl-2+".

[0046] 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 May 1996, vol 148, No. 5: 1567-1576) showed that the production and characterization of anti-peptide antisera specific for human Bcl-2, Bax, Bcl-X, and Mcl-1 proteins and the methods for applying these antibodies to paraffin immunohistochemistry are known and described in detail in the article. Reference may also be made to Erinna F. Lee et al. (Cell Death & Disease 2019, 10, 342). According to these authors and others, Mcl-1 levels can be expressed in different ways: the percentage of Mcl-1-positive tumor cells or the intensity of immunostaining. Any suitable method used by hospital clinicians is also useful.

[0047] For example, according to Erinna F. Lee et al., the evaluation is performed by antibody staining, the positively stained cancer cells in the cytoplasm are counted, and the percentage of positive tumor cells is graded (grade): 0: none; 1: 1-5%; 2: 6-25%; 3: 26-50%; 4: 51-75% and 5: 76-100%. The staining intensity can be assessed as: 0: none; 1: weak; 2: moderate; 3: strong. The H score (ranging from 0-15) for each sample can be calculated by multiplying the grade and intensity scores, and then classified as high (9-15) or low (0-8). In the present invention, when the percentage of positive tumor cells is equal to or higher than grade 3, the tumor can preferably be identified as a "Mcl-1+ tumor". In some embodiments, when the percentage of positive tumor cells is equal to or higher than grade 3, or equal to or higher than grade 4, or equal to grade 5, the tumor can be identified as a "Mcl-1+ tumor".

[0048] In addition, recent methods allow the use of mass spectrometry to measure the amount of protein in a biopsy, such as using the method described in Archer T et al., Cancer Cell. 2018 Sep 10; 34(3): 396-410 (PMID: 30205044). This allows technicians to obtain the amount of Mcl-1 in a tumor biopsy, determine the minimum amount corresponding to a "Mcl-1+ tumor", and use this method to classify and select tumors for treatment.

[0049] In another aspect, these cancers express or overexpress Mcl-1, and the tumor cells present Mcl-1 receptors, such as, in particular, UNC5B (UNC5B+, or "Mcl-1+ / Mcl-1+ / UNC5B+ tumors").

[0050] In a given aspect, the patient is first assayed to determine whether he has a "Mcl-1+ tumor", "Mcl-1+ / axon guidance factor 1+ tumor" or "Mcl-1+ / axon guidance factor 1+ / axon guidance factor 1 receptor+ tumor" or "Mcl-1+ / axon guidance factor 1+ / Unc5B+ tumor". If he has such a tumor, he is treated with an anti-axon guidance factor 1 antibody. Therefore, different purposes (i.e., uses, methods of treatment) may include such an assay and / or determination of such a patient's status. In one embodiment, the assay is performed by antibody staining using any available antibody that binds to Mcl-1 (see Erinna F. Lee et al.), the cancer cells that stain positively in the cytoplasm are counted, and the percentage of positive tumor cells is graded (classified) as follows: 0: none; 1: 1-5%; 2: 6-25%; 3: 26-50%; 4: 51-75% and 5: 76-100%. In the present invention, preferably, when the percentage of positive tumor cells is equal to or higher than grade 3, the tumor can be identified as a "Mcl-1+ tumor". In some embodiments, when the percentage of (Mcl-1) positive tumor cells is equal to or higher than grade 3, or equal to or higher than grade 4, or equal to grade 5, the tumor is identified as a "Mcl-1+ tumor".

[0051] Some embodiments of cancers contemplated herein include metastatic breast cancer, non-small cell lung cancer, aggressive neuroblastoma, pancreatic adenocarcinoma, primary melanoma, melanoma metastasis, ovarian cancer, glioblastoma, acute myeloid leukemia, chronic lymphocytic leukemia, aggressive B-cell lymphoma, sarcoma, renal adenocarcinoma, head and neck cancer, testicular cancer (e.g., embryonal carcinoma, teratoma, yolk sac tumor), kidney cancer, gastric cancer, uterine cancer, colorectal cancer, lung adenocarcinoma.

[0052] Some specific embodiments of cancers that exhibit increased expression of axon guidance factor 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).

[0053] EP 3 092 003 provides an indication of the prevalence of cancers overexpressing axon guidance factor 1 in a variety of cancers: 60% of metastatic breast cancer (Fitamant et al., 2008), 47% of non-small cell lung cancer (Delloye-Bourgeois et al., 2009a), 38% of invasive neuroblastoma (Delloye-Bourgeois et al., 2009b), 61% of pancreatic adenocarcinoma (Link et al., Annals of Chir. Onco. 2007; Dumartin et al., Gastro 2010), 100% of primary melanomas (n=7), melanoma metastases (n=6) (Kaufmann et al., Cellular Oncology 2009), 76% of ovarian cancer (Panastasiou et al., Oncotarget 2009). 2011), 65% of glioblastomas, 60% of acute myeloid leukemia and chronic lymphocytic leukemia, 50% of aggressive B-cell lymphomas, 30% of sarcomas, 40% of renal adenocarcinomas, 22% of head and neck cancers, testicular cancer (36% of embryonal carcinomas, 50% of teratomas, 100% of yolk sac tumors), 50% of renal cancers, 26% of gastric cancers, and 19% of uterine cancers.

[0054] In cancers that overexpress AGF1, AGF1 has been shown to be a survival factor for cancer cells (Mehlen et al., Nature Cancer Rev 2011), but this survival was thought to be independent of Bcl-2 proteins (Forcet et al., 2001, PNAS), which is consistent with the idea that the cell death mechanism nomenclature proposes that dependency receptors (such as AGF1 receptor) trigger cell death independently of mitochondrial-dependent pathways (Galluzzi et al., 2018).

[0055] Anti-NTN1 antibody (anti-NTN1 antibody or antibody that binds to NTN1)

[0056] The antibody can be a polyclonal or monoclonal antibody (anti-axon guidance factor 1 antibody or an antibody that binds to axon guidance factor 1) that specifically binds to axon guidance factor 1 (NTN1), especially human axon guidance factor 1. In one aspect, the antibodies used and disclosed herein are neutralizing. In one aspect, the anti-axon guidance factor 1 antibody or its axon guidance factor 1 binding fragment or derivative of the antibody is neutralizing. The term "neutralizing antibody" as used herein refers to an antibody that captures axon guidance factor 1, blocks or reduces at least one activity of axon guidance factor 1, especially its binding to its receptor (mainly UNC5B).

[0057] Anti-axon guidance factor 1 polyclonal antibodies can be obtained, in particular, by immunizing animals (e.g., rabbits, mice, etc.) with the selected amino acid sequence, collecting and subsequently depleting the antiserum obtained, for example, on an immunosorbent containing a receptor (according to methods known to those skilled in the art). The amino acid sequence of axon guidance factor 1 is shown in SEQ ID NO: 1, and axon guidance factor 1 can be used in whole or in part to generate polyclonal or monoclonal antibodies.

[0058] Typically, monoclonal antibodies are The conventional lymphocyte fusion and hybridoma culture method described by Milstein (Nature, 1975, 256 (5517): 495-7) is obtained. Other methods for preparing monoclonal antibodies are also known (Harlow et al., ed., 1988 "Antibodies: a laboratory manual"). Monoclonal antibodies can be obtained by immunizing mammals (such as mice, rats, rabbits or even humans) and using the lymphocyte fusion technique ( and Milstein, 1975) to prepare monoclonal antibodies. There are alternatives to this conventional technology. For example, monoclonal antibodies can be produced by expressing nucleic acids cloned from hybridomas. Antibodies can also be produced by phage display technology, that is, the cDNA of the antibody is introduced into a vector, which is usually a filamentous phage, which displays a gene library V on the surface of the phage (for example, fUSE5 for Escherichia coli, Scott JK, Smith GPScience 1990; 249: 386-390). The scheme for constructing these antibody libraries is described in JD Marks et al., J. Mol. Biol., 222 (1991), p. 581). According to these methods, monoclonal antibodies can be produced using cDNA corresponding to the full-length axon guidance factor 1 with a signal sequence (SEQ ID NO: 2) or a suitable fragment thereof.

[0059] The anti-axon guidance factor 1 monoclonal antibody (mAb) can be a mouse, chimeric, humanized or fully human monoclonal antibody. The fragment can be any type of mAb fragment that substantially retains the ability of the intact antibody to bind to the axon guidance factor 1, which can be, for example, Fab or F(ab')2. Specifically, the monoclonal antibody is an antibody disclosed in WO2015 / 104360 or US10,494,427, which are incorporated herein by reference and disclose useful mouse, chimeric and humanized monoclonal antibodies. These are antibodies or fragments thereof that specifically bind to an NTN1 epitope or polypeptide or a variant thereof having an amino acid sequence of SEQ ID NO: 3 or 33.

[0060] The monoclonal antibodies useful in the present invention can 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 an amino acid sequence of SEQ ID NO: 3 or 33. Preferably, the antibody is a monoclonal antibody or an antigen-binding fragment thereof, comprising:

[0061] A variable domain VH comprising:

[0062] - H-CDR1 having the sequence shown in SEQ ID NO: 5;

[0063] - H-CDR2 having the sequence shown in SEQ ID NO: 6;

[0064] - H-CDR3 having the sequence shown in SEQ ID NO: 7;

[0065] A variable domain VL comprising:

[0066] - L-CDR1 having the sequence shown in SEQ ID NO: 8;

[0067] - L-CDR2 having the sequence YAS;

[0068] - L-CDR3 having the sequence shown in SEQ ID NO: 9;

[0069] or

[0070] A variable domain VH comprising:

[0071] - H-CDR1 having the sequence shown in SEQ ID NO: 28;

[0072] - H-CDR2 having the sequence shown in SEQ ID NO: 29;

[0073] - H-CDR3 having the sequence shown in SEQ ID NO: 30;

[0074] A variable domain VL comprising:

[0075] - L-CDR1 having the sequence shown in SEQ ID NO: 31;

[0076] - L-CDR2 having the sequence shown in SEQ ID NO: 32;

[0077] - L-CDR3 having the sequence shown in SEQ ID NO:9.

[0078] In a first series of embodiments, the antibody of the invention comprises the amino acid sequence of SEQ ID NO: 10, 11, 12 or 13. Typically, 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.

[0079] 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.

[0080] 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 selected from the group of SEQ ID NOs: 20 to 26 (VH). Typically, 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.

[0081] The antibody preferably comprises a monoclonal antibody (mAb) or an antigen-binding fragment thereof, wherein the mAb or its fragment specifically binds to axon guidance factor 1. The mAb can be a mouse, chimeric, humanized or fully human monoclonal antibody. The fragment can be any type of mAb fragment that substantially retains the ability of the intact antibody to bind to axon guidance factor 1, for example, it can be a Fab or F(ab')2.

[0082] Specific embodiments disclosed in the previous literature and useful 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 transplantation of the mouse 4C11 CDR into human IgG1. The ten humanized mAbs HUM01 to 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 publicly known as NP137 and is currently undergoing clinical trials. The sequence of human IgG1 CH is from Genbank AEL33691.1 modified R97K. The sequence of human IgG1 CL (κ) is from Genbank CAC20459.1. Other isotypes may also be used. The specific binding of all these mAbs, murine, chimeric and humanized HUM01-HUM10, Fab fragments and F(ab')2 fragments to Axon Guidance Factor 1 and their ability to inhibit the binding of Axon Guidance Factor 1 to its receptor UNC5B were demonstrated in US Pat. No. 10,494,427 (Example 3).

[0083] In particular, these monoclonal antibodies specifically bind to a polypeptide having the amino acid sequence of SEQ ID NO:33.

[0084] Table 1 VH SEQ ID NO: Constant heavy chain VL SEQ ID NO: Constant light chain 4C11 10 Human IgG1 11 Human IgG1 HUM00 27 Human IgG1 19 Human IgG1 HUM01 20 Human IgG1 14 Human IgG1 HUM02 21 Human IgG1 15 Human IgG1 HUM03 22 Human IgG1 16 Human IgG1 HUM04 23 Human IgG1 17 Human IgG1 HUM05 24 Human IgG1 17 Human IgG1 HUM06 25 Human IgG1 16 Human IgG1 HUM07 26 Human IgG1 17 Human IgG1 HUM08 22 Human IgG1 17 Human IgG1 HUM09 25 Human IgG1 18 Human IgG1 HUM10 21 Human IgG1 16 Human IgG1

[0085] Preferably, the antibody is a monoclonal antibody or an antigen-binding fragment thereof, comprising a VH and VL sequence pair 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 a VH and VL sequence pair SEQ ID NO: 22 and 16.

[0086] The anti-AGF-1 monoclonal antibody may also comprise a human IgG1 constant heavy chain (CH) and / or a human IgG1 constant light chain (CL), in particular a human kappa constant domain.

[0087] In one embodiment, the sequence of human IgG1 CH is from Genbank AEL33691.1 modified R97K. The sequence of human IgG1 CL (κ) is from Genbank CAC20459.1. In one embodiment, the mAb is NP137 (AB_2811180 in the Antibody Registry) and comprises SEQ ID NOs: 22 and 16 as VH, VL sequences, respectively, and those specific IgG1 CH and CL.

[0088] As used herein, the term "antigen-binding fragment" (or "antibody binding portion") of a monoclonal antibody refers to one or more fragments of an antibody that retains the ability to specifically bind to axon guidance factor 1, particularly to the same epitope as the intact antibody or to a short peptide axon guidance factor 1 sequence (e.g., SEQ ID NO: 3 or 33). In particular, the fragment comprises the same VH and VL regions as the intact antibody. Antibody fragments may include, for example, Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, CDR-containing fragments, or isolated CDRs. In one aspect, the fragment comprises the VH and VL sequences of an antibody selected from HUM00 to HUM10.

[0089] As the anti-AGF1 monoclonal antibody that can be used, other monoclonal antibodies or antigen-binding fragments thereof developed against human AGF1 or against animal AGF1 can be cited, because AGF1 is highly homologous among species. The following can be cited: Abcam antibodies ab126729, ab122903, ab201324, ab39370; AF1109, AF6419, AF128.

[0090] BH3 mimetics and B-cell lymphoma 2 (Bcl-2) inhibitors:

[0091] "Bcl-2 inhibitors" are a class of drugs that work by inhibiting the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein, leading to programmed cell death of cells.

[0092] Venetoclax, also known in the art as GDC-0199, ABT-199 or RG7601, is a BCL-2 inhibitor. The chemical name of venetoclax is 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 its preparation method are disclosed in U.S. Patent No. 8,546,399. Venetoclax is marketed in the United States under the brand name VENCLEXTA TM Marketed by AbbVie, Inc. for the treatment of chronic lymphocytic leukemia.

[0093] The BH3 mimetic can be an inhibitor of Bcl-2 itself (Bcl-2 inhibitor) or an inhibitor of any other anti-apoptotic member of the Bcl-2 family. It can also be an inhibitor of at least two of these proteins, such as an inhibitor of Bcl-2 and Bcl-XL, or an inhibitor of Bcl-2, Bcl-W and Bfl-1 / A1, as in the case of navitoc.

[0094] In some embodiments, the inhibitor is venetoclax (ABT-199 or GDC-0199), ABT-737, BP 1002, SPC2996, APG-1252, obatocla mesylate (GX15-070MS) or PNT2258, navitoc.

[0095] Definitions and further embodiments, variants and alternatives of the invention:

[0096] In the context of the present invention, the terms "treat" or "treatment" as used herein means to reverse, alleviate, inhibit the progression of, or prevent the disorder or condition to which the term applies, or one or more symptoms of the disorder or condition.

[0097] As used herein, the term "treating cancer" refers specifically to inhibiting the growth of malignant cells of a tumor and / or the progression of metastases from said tumor. Such treatment may also result in regression of tumor growth, i.e., a reduction in the size of a measurable tumor. In a specific embodiment, such treatment results in partial regression of a tumor or metastasis. In another specific embodiment, such treatment results in complete regression of a tumor or metastasis. In some aspects, treatment prevents metastasis. In the present invention, such treatment results in apoptosis or programmed cell death of cancer cells.

[0098] According to the present invention, the term "patient" or "patient in need thereof" refers to a human or non-human mammal suffering from or likely to suffer from a malignant tumor.

[0099] "Therapeutically effective amount" refers to an amount of an active agent sufficient to treat the cancer disease and has a reasonable benefit / risk ratio applicable to any medical treatment. However, it should be understood that the total daily dosage of the active agent will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the specific polypeptide or antibody used; the specific composition used, the patient's age, weight, general health, sex and diet; the administration time, administration route and excretion rate of the specific active agent used; the duration of treatment; drugs used in combination or concomitantly with the specific active agent used; and similar factors well known in the medical field.

[0100] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when properly administered to mammals, especially humans. Pharmaceutically acceptable carriers or excipients refer to any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0101] Pharmaceutical composition:

[0102] The form and administration route of the pharmaceutical composition comprising the polypeptide or antibody of the present invention will naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, and the like.

[0103] The active agents of the invention may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration, etc. In a specific embodiment, the active agents of the invention are administered intravenously.

[0104] In particular, the pharmaceutical composition comprising the active agent of the invention may contain a pharmaceutically acceptable vehicle for an injectable formulation. These may be in particular isotonic, sterile, saline solutions (monosodium phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of these salts), or dried, in particular lyophilized compositions which, as the case may be, allow the constitution of injectable solutions after the addition of sterile water or physiological saline.

[0105] To prepare a pharmaceutical composition, an effective amount of the active agent of the present invention may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0106] Pharmaceutical forms suitable for injection 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 it is easy to inject. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating effects of microorganisms (e.g., bacteria and fungi).

[0107] The carrier can be a solvent or dispersion medium, containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating (e.g., lecithin), by maintaining the desired particle size in the case of dispersion, and by using a surfactant, stabilizer, cryoprotectant, or antioxidant. The action of microorganisms can be prevented by antibacterial and antifungal agents. In many cases, isotonic agents, such as sugars or sodium chloride, will preferably be included.

[0108] Sterile injections are prepared by incorporating the desired amount of active agent in a suitable solvent along with several other ingredients listed above (as needed), followed by filtration sterilization. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle comprising a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injections, preferred preparation methods are vacuum drying and freeze drying techniques, which produce powders of the active ingredient and any other desired ingredients from their previously sterile filtered solutions.

[0109] After formulation, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. These preparations are easily administered in a variety of dosage forms, such as injections of the type described above, but drug release capsules and the like may also be used.

[0110] For example, for parenteral administration in aqueous solution, if necessary, appropriate buffer solution should be used, and first the liquid diluent should be isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, those skilled in the art will understand the sterile aqueous medium that can be used according to the present disclosure. For example, a dose can be dissolved in 1mL isotonic NaCl solution, then added to 1000mL subcutaneous injection or injected into the intended infusion site (for example, referring to " Remington's Pharmaceutical Sciences " the 15th edition, pages 1035-1038 and pages 1570-1580). According to the condition of the subject being treated, some changes will inevitably occur in the dosage. The person responsible for administration will determine the dosage suitable for individual subjects.

[0111] Drug administration and method of use:

[0112] As used herein, "simultaneous" is used to refer to the simultaneous administration of two agents, while the term "combination" is used to refer to their "sequential" administration within a certain time, if not simultaneous, so that both agents can exert their therapeutic effects within the same time frame. Thus, "sequential" administration can allow one agent to be administered 5 minutes, 10 minutes, or hours after the administration of the other agent, provided that the circulating half-life of the first administered agent is such that both agents are present in therapeutically effective amounts at the same time. The time delay between the administration of the components will vary depending on the exact nature of the components, the interactions between them, and their respective half-lives.

[0113] In contrast to "in combination" or "sequentially," "separately" is used herein to mean that the interval between administration of one agent and administration of the other agent is substantial, i.e., several hours, which may include situations where the first administered agent is no longer present in the bloodstream in therapeutically effective amounts when the second agent is administered. However, the first administered agent may still be present and active in cancer cells.

[0114] In one embodiment of the present invention, the anti-AGF1 antibody or antigen-binding fragment thereof is administered sequentially or separately before the Bcl-2 inhibitor.

[0115] In a particularly preferred embodiment, the Bcl-2 inhibitor is administered sequentially or separately prior to the anti-AGF1 antibody or antigen-binding fragment thereof.

[0116] In one embodiment, the anti-AGF1 antibody or antigen-binding fragment thereof and the Bcl-2 inhibitor are in the same composition with a pharmaceutically acceptable carrier, excipient and / or diluent.

[0117] In another embodiment, they are present in the form of separate drugs or a kit of parts. This forms a composition or a kit or a kit of parts comprising an anti-axon guidance factor 1 antibody or its antigen-binding fragment and a Bcl-2 inhibitor for simultaneous, separate or sequential administration to a patient. Thus, the present invention may include (i) a composition comprising two active ingredients as a mixture, or (ii) a composition comprising those active ingredients stored separately in the same condition or in different conditions, each active ingredient being in its own vial or container, and in case (ii) the concept of a kit of parts is generally mentioned.

[0118] In one embodiment, the antibody or antigen-binding fragment or a combination of an anti-axon guidance factor 1 antibody or antigen-binding fragment thereof and a Bcl-2 inhibitor is administered in a single dose, or in an initial dose, followed by a second dose or multiple subsequent doses thereof, wherein the subsequent doses are separated by at least one day; at least one 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.

[0119] In one embodiment of the method of treatment, use and composition used, the administration of the anti-axon guidance factor 1 antibody or its antigen binding fragment and the Bcl-2 inhibitor is continuous or separate. The interval between two administrations can be at least 5, 10, 15, 20 or 24 hours, preferably 24 to 96 hours, more preferably 24 to 72 hours, or more particularly 24 to 48 hours, for example 24 hours. In one embodiment, one agent or drug is simply administered the next day after the administration of another agent or drug. In addition, the first administered active ingredient (anti-axon guidance factor 1 antibody or its antigen binding fragment, or Bcl-2 inhibitor) relative to which the time interval is calculated can be administered as a single dose, or as an initial dose, followed by a second dose or multiple subsequent doses thereof, wherein the subsequent doses are separated by at least one day; at least one 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.

[0120] Various pharmaceutical forms may be used in sufficient amounts in the therapeutic methods of the present invention.

[0121] The present invention does or may not imply a change in the dosage regimen of the Bcl-2 inhibitor (e.g., venetoclax). However, the synergistic effect with the anti-axon guidance factor 1 antibody or its antigen-binding fragment may allow the use of a lower dosage regimen of the Bcl-2 inhibitor (e.g., venetoclax) in patients. The skilled person is able to determine the optimal dosage regimen in the context of the combination therapy provided by the present invention.

[0122] The pharmaceutical composition can be administered to the subject at a suitable dose, i.e., for anti-axon guidance factor 1 antibodies or antigen-binding fragments thereof, at least 1 mg / kg body weight, such as about 1 mg / kg body weight to about 100 mg / kg body weight, particularly about 10 mg / kg body weight to about 60 mg / kg body weight, the body weight of the subject in which cancer is to be treated. Bcl-2 inhibitors (e.g., venetoclax) can be administered at conventional doses, or at doses reduced relative to conventional doses, as long as the combination has a synergistic effect. For example, the dose of a Bcl-2 inhibitor (e.g., venetoclax) is reduced by 10%, 20%, 30%, 40%, 50% or more.

[0123] As used herein, the term "synergistic" refers to the effect of the active ingredients (e.g., antibodies) when used in combination, which is greater than the effect expected by adding the individual effects of the two ingredients. Advantageously, the synergistic interaction can allow each ingredient to be administered to the patient at a lower dose, thereby reducing the toxicity of the chemotherapy while producing and / or maintaining the same therapeutic effect. Therefore, in particularly preferred embodiments, each ingredient can be administered in a subtherapeutic amount.

[0124] Table 2: Sequence description:

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133] The CDRs under IMGT are highlighted in bold in Table 1 where appropriate.

[0134] As used herein, a sequence that is "at least 85% identical to a reference sequence" is a sequence that has 85% or more, specifically 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 entire length of the reference sequence.

[0135] The percentage of "sequence identity" can be determined by comparing two sequences that are optimally aligned over a comparison window, wherein the portion of the polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) to achieve optimal alignment of the two sequences. The percentage is calculated as follows: the number of positions where the identical amino acid residues occur in the two sequences is determined to give the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window, and the result is multiplied by 100 to give the percentage of sequence identity. The sequences are optimally aligned for comparison 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 easily determined, for example using the Needle program, using the BLOSUM62 matrix, and the following parameters gap-open=10, gap-extended=0.5.

[0136] In the context of the present invention, "conservative amino acid substitution" refers to the substitution of an amino acid residue by another amino acid residue having a side chain group of similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not significantly change the functional properties of proteins. Examples of amino acid groups with side chains of 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 acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine-tryptophan, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0137] Throughout this application, the term "comprising" should be interpreted as covering all specifically mentioned features as well as optional, additional, unspecified features. As used herein, the use of the term "comprising" also discloses embodiments in which there are no features other than the specifically mentioned features (i.e., "consisting of ...").

[0138] "Antibodies" can be natural or conventional antibodies, in which two heavy chains are interconnected by disulfide bonds, and each heavy chain is connected to a light chain by a disulfide bond. There are two types of light chains, λ (lambda) and κ (kappa). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA and IgE. Each chain contains different sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties, such as antibody chain binding, secretion, transplacental mobility, complement binding and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and is composed of one light chain and the variable portion of one heavy chain. The specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site consists of residues primarily from the hypervariable regions or complementarity determining regions (CDRs). Sometimes, residues from non-hypervariable regions or framework regions (FRs) affect the overall domain structure and thus the binding site.

[0139] "Complementarity determining region" or "CDR" refers to the amino acid sequence that together determines the binding affinity and specificity of the native Fv region of a natural immunoglobulin binding site. The light chain and heavy chain of an immunoglobulin each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, a conventional antibody antigen binding site includes six CDRs, comprising each of the heavy and light chain V regions.

[0140] "Framework region" (FR) refers to the amino acid sequence between CDRs, i.e., the relatively conserved portion of the variable region of the immunoglobulin light chain and heavy chain between different immunoglobulins of a single species. The light chain and heavy chain of immunoglobulins each have four FRs, designated as FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively.

[0141] 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.

[0142] In the context of the present invention, the CDR / FR definition in an immunoglobulin light or heavy chain should be determined based on the Kabat or IMGT definition.

[0143] The residues in the variable domain of an antibody are usually numbered according to the system designed by Kabat et al. This system is described in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, NIH, USA (hereinafter referred to as "Kabat et al."). This numbering system is used in this specification. The Kabat residue names do not always directly correspond to the linear numbering of the amino acid residues in the SEQ ID sequence. The actual linear amino acid sequence may contain fewer or more amino acids than the strict Kabat numbering, which corresponds to the shortening of the structural components of the basic variable domain structure (whether framework or complementary determining region (CDR)) or insertion therein. The correct Kabat residue numbering of a given antibody can be determined by comparing the homologous residues in the antibody sequence with the "standard" Kabat numbering 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).

[0144] 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. IMGT unique numbering has been defined to compare variable domains regardless of antigen receptor, chain type or species (Lefranc M.-P., "Immunology Today, 18, 509 (1997); Lefranc M.-P., The Immunologist, 7, 132-136 (1999).; Lefranc, M.-P. et al. Dev. Comp. Immunol., 27, 55-77 (2003).). In the IMGT unique numbering, the conserved amino acids always have the same position, such as cysteine ​​23, tryptophan 41, hydrophobic amino acid 89, cysteine ​​104, phenylalanine or tryptophan 118. The IMGT unique numbering provides standardized boundaries for the framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and the complementary determining regions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 119 to 29, CDR3-IMGT: 28 to 30, CDR4-IMGT: 31 to 31, CDR5-IMGT: 33 to 32, CDR6-IMGT: 34 to 35, CDR7-IMGT: 36 to 37, CDR8-IMGT: 38 to 39, CDR9-IMGT: 40 to 41, CDR10-IMGT: 41 to 42, CDR11-IMGT: 42 to 43, CDR12-IMGT: 43 to 44, CDR13-IMGT: 44 to 45, CDR14-IMGT: 45 to 46, CDR15-IMGT: 46 to 47, CDR16-IMGT: 47 to 48, CDR17-IMGT: 48 to 49, CDR20-40 IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. If the CDR3-IMGT is less than 13 amino acids long, a gap is created from the top of the loop in the following order 111, 112, 110, 113, 109, 114, etc. If the CDR3-IMGT is longer than 13 amino acids, an additional position is created between positions 111 and 112 at the top of the CDR3-IMGT loop 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).

[0145] As used herein, the term "antibody" refers to conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular the variable heavy chain of a single domain antibody, as well as chimeric, humanized, bispecific or multispecific antibodies.

[0146] As used herein, antibodies or immunoglobulins also include the recently described "single domain antibodies", which are antibodies in which the complementary determining region is a part of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, antibodies naturally lacking light chains, single domain antibodies derived from conventional four-chain antibodies, and engineered single domain antibodies. Single domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. Single domain antibodies can be naturally occurring single domain antibodies, referred to as heavy chain antibodies lacking light chains. In particular, camelid species, such as camels, dromedaries, llamas, alpacas, and guanacos, produce heavy chain antibodies naturally lacking light chains. Camelid heavy chain antibodies also lack the CH1 domain.

[0147] The variable heavy chains of these single-domain antibodies lacking light chains are referred to in the art as "VHH" or "nanobodies". Similar to conventional VH domains, VHHs contain four FRs and three CDRs. Nanobodies have advantages over traditional antibodies: they are about ten times smaller than IgG molecules, so correctly folded functional nanobodies can be produced by in vitro expression while achieving high yields. In addition, nanobodies are very stable and resistant to the action of proteases. Harmsen and De Haard (2007) Appl. Microbiol. Biotechnol. 77: 13-22 have reviewed the properties and production of nanobodies.

[0148] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule of a single amino acid composition directed against a specific antigen, and should not be construed as requiring the antibody to be produced by any particular method. Monoclonal antibodies can be produced by a single B cell clone or hybridoma, but can also be recombinant, i.e., produced by protein engineering.

[0149] A "fragment" of a (conventional) antibody includes a portion of an intact antibody, in particular the antigen binding region or variable region of an intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific antibodies and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a single domain antibody, such as a heavy chain antibody or VHH.

[0150] The term "Fab" means an antibody fragment having a molecular weight of about 50,000 Da and having antigen-binding activity, in which about half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond in a fragment obtained by treating IgG with protease papain.

[0151] The term "F(ab')2" refers to an antibody fragment obtained by treating IgG with the protease pepsin, having a molecular weight of about 100,000 Da and having slightly greater antigen-binding activity than Fab bound by a disulfide bond in the hinge region.

[0152] Single-chain Fv ("scFv") polypeptides are covalently linked VH::VL heterodimers, typically expressed by gene fusions comprising VH and VL encoding genes linked by a peptide encoding linker. The human scFv fragments of the present invention include CDRs that retain an appropriate conformation, particularly by using genetic recombination techniques. Divalent and multivalent antibody fragments can be formed spontaneously by the association of monovalent scFvs, or can be produced by coupling monovalent scFvs by peptide linkers, such as divalent sc(Fv)2.

[0153] "dsFv" is a VH::VL heterodimer stabilized by disulfide bonds.

[0154] "(dsFv)2" indicates two dsFvs connected by a peptide linker.

[0155] The term "diabody" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to pair between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0156] In a specific embodiment, the epitope binding fragment is selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabody and VHH.

[0157] As used herein, "chimeric antibody" refers to an antibody whose constant region or a portion thereof is altered, replaced or exchanged so that the variable region is linked to a constant region of a different species or to a constant region belonging to another antibody class or subclass. "Chimeric antibody" also refers to an antibody whose variable region or a portion thereof is altered, replaced or exchanged so that the constant region is linked to a variable region of a different species or to a variable region belonging to another antibody class or subclass.

[0158] The term "humanized antibody" refers to an antibody originally derived in whole or in part from a non-human source that has been modified to replace certain amino acids, particularly in the framework regions of the heavy and light chains, to avoid or minimize human immune responses. The constant domains of a humanized antibody are most often human CH and CL domains. In one embodiment, a humanized antibody has a constant domain of human origin. As used herein, the term "humanized antibody" refers to a chimeric antibody comprising a minimal sequence derived from a non-human immunoglobulin, such as CDR.

[0159] The term "antibody" is used to encompass all these types of antibodies, fragments or combinations thereof.

[0160] The goal of humanization is to reduce the immunogenicity of xenoantibodies (e.g., mouse antibodies) so as to introduce humans while maintaining the complete antigen binding affinity and specificity of the antibody. Humanized antibodies or antibodies suitable for other mammals that are not rejected can be produced using a variety of techniques (e.g., surface remodeling and CDR transplantation). As used herein, surface remodeling technology combines molecular modeling, statistical analysis, and mutagenesis techniques to change the non-CDR surface of the antibody variable region to make it similar to the surface of the known antibodies of the target host.

[0161] Antibodies can be humanized using a variety of other techniques, including CDR transplantation (EP0239400; WO91 / 09967; U.S. Pat. Nos. 5,530,101 and 5,585,089), veneering or surface remodeling (EP0592106; EP0519596; 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 prepared by a variety of methods known in the art, including phage display methods. See also U.S. Patent Nos. 4,444,887, 4,716,111, 5,545,806 and 5,814,318; and International Patent Applications WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735 and WO 91 / 10741.

[0162] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to an epitope present on an antigen (e.g., axon guidance factor 1) while having relatively little detectable reactivity with non-axon guidance factor 1 proteins or structures (e.g., other proteins present on cancer cells or other cell types). Specificity can be relatively determined by binding or competitive binding assays, such as using a Biacore instrument as described elsewhere herein. Specificity can be demonstrated, for example, by an affinity / avidity ratio of about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1, or greater for binding to a specific antigen relative to nonspecific binding to other unrelated molecules (in this case, the specific antigen is axon guidance factor 1).

[0163] The term "affinity" as used herein refers to the binding strength 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 as 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 are incorporated herein by reference in their entirety. A preferred standard method for determining the affinity of a mAb, well known in the art, is by use of a Biacore instrument.

[0164] The invention will now be described using non-limiting examples with reference to the accompanying drawings.

[0165] Figure 1.CRISPR-Cas9 screening identifies genes and molecular pathways associated with UNC5B-induced cell death. (A): UNC5B overexpression triggers cell death. Stable HeLa cells (HeLa UNC5B) in which UNC5B is induced were treated with the indicated amounts of doxycycline (Dox). To assess cell death, propidium iodide (PI) incorporation was tracked in real time for up to 48 h by Incucyte Zoom technology (n=3, ***p<0.01, Kolmogorov-Smirnov test). Values ​​are expressed as mean ± SEM. (B, C): UNC5B-induced cell death is associated with DNA fragmentation and caspase-3 activation and is inhibited by axon guidance factor 1. HeLa UNC5B cells constitutively expressing axon guidance factor 1 (UNC5B+NTN1) were treated with the indicated concentrations of Dox for 18 hours and DNA fragmentation assay was performed by flow cytometry to assess apoptosis (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 up to 24 hours using an Incucyte Zoom instrument (n=3, ****p<0.0001, Kolmogorov-Smirnov test) (C). Values ​​are expressed as mean ± SEM.

[0166] Figure 2. CRISPR-Cas9 screening identifies genes and molecular pathways involved in UNC5B-induced cell death. Figure 1Related. (A): UNC5B induction after treatment with doxycycline. Stable HCT116 cells in which UNC5B is induced were treated with the indicated concentrations of doxycycline (Dox). After 24 hours, the protein levels of FLAG-UNC5B and GAPDH, which are used as housekeeping genes, were assessed by Western blotting. (B): UNC5B induction after treatment of HeLa cells with doxycycline. Stable HeLa cells in which UNC5B is induced (HeLa UNC5B) were treated with the indicated doses of Dox for 18 hours. The protein levels of His-UNC5B and β-actin, which are used as housekeeping genes, were measured by Western blotting. (C, D, E): Induction of UNC5B triggers apoptosis in HCT116 cells. Stable HCT116 cells in which human UNC5B is induced were treated with Dox. To assess cell death, propidium iodide (PI) incorporation was followed in real time for up to 36 h using IncucyteZoom technology (n=3, **p<0.01, Kolmogorov Smirnov test) (C). Apoptosis was assessed by flow cytometry via DNA fragmentation 18 h after Dox treatment (D) and caspase-3 activity quantified by a fluorescence detection kit (E) was assessed 18 h after Dox treatment (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 UNC5B-induced apoptosis. HeLa UNC5B cells were treated with Dox and the pan-caspase inhibitor Q-VC-OPh (QVD). After 18 h, DNA fragmentation was assessed by flow cytometry (n=3, ****p<0.0001, using (ANOVA with multiple comparisons). Values ​​are given as mean ± SEM. (G): Candidate genes identified in CRISPR-Cas9-based screening. The number of guides showing an increase in reads compared to the control condition is shown for each gene.

[0167] Figure 3. COMMD2 is required for UNC5B-induced cell death and tumor suppression. (A): Silencing COMMD2 inhibits UNC5B-induced DNA fragmentation. Stable HeLa cells inducing UNC5B were transfected with two different scrambled siRNAs (siCTRL) or siRNAs targeting COMMD2 (siCOMMD2-1 and -2). DNA fragmentation was assessed by FACS analysis after 18 h of treatment with the indicated concentrations of doxycycline (Dox) (n=3, ****p<0.0001, ordinary two-way ANOVA). Values ​​are given as mean ± SEM. (B, C, D): Tumor growth inhibition driven by axon guidance factor 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), which were 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). 3 After that, mice were treated intravenously with anti-axon guidance factor 1 (NP137) or control (Iso-mAb) antibodies three times a week, and tumor growth was assessed (n = 10 mice / group, ns p>0.05, *p<0.05, Mann-Whitney test, two-tailed) (C). (D) shows the histogram of tumor volume 35 days after the first treatment (n=10 mice / group, ns p>0.05, *p<0.05, ***p<0.001, Kruskal-Wallis test using uncorrected Dunn's test). (E). Silencing of UNC5B and COMMD2 inhibits NP137-induced apoptosis in vivo. After mice were sacrificed, tumors were excised and apoptosis was quantified by immunostaining for active cleaved caspase-3. Positive cells on excised tumors in Iso-mAb-treated mice were then indexed for each stable cell line (n=10 mice / group, ns p>0.05, *p<0.05, ***p<0.001, Kruskal-Wallis test using uncorrected Dunn's test). Histograms represent mean±SEM.

[0168] Figure 4. UNC5B triggers proteasomal degradation of Mcl-1. (A, B): UNC5B-induced apoptosis requires proteasome activity. Stable HeLa cells (HeLa UNC5B) in which UNC5B is induced were treated with 2 μg / mL of doxycycline (Dox) in combination with the indicated doses of the proteasome inhibitors MG132 and bortezomib (BTZ). 18 hours 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 expressed as mean ± SEM. Treatment with proteasome inhibitors is sufficient to significantly reduce apoptosis induction caused by 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). 16 h after treatment, cells were lysed in a buffer containing SDS by scraping and sonication. Protein levels of Mcl-1, Bcl-2, and Bcl-XL, as well as UNC5B and β-actin used as housekeeping genes, were assessed by Western blotting. The bottom of each Western blot shows the ratio of protein levels between untreated (0) and doxycycline-treated (2), normalized to β-actin. UNC5B overexpression specifically promoted the reduction of Mcl-1 protein. Proteasome inhibition by treatment with bortezomib rescued this degradation, while caspase inhibition by Q-VD-OPh, although inhibiting cell death, did not affect 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 further treated with 2 μg / mL Dox for 18 h. Protein levels of Mcl-1, UNC5B, and Ku80, which serves as a housekeeping protein, were assessed by western blotting. Silencing COMMD2 or RBX1 was sufficient to block Mcl-1 degradation caused 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 by DNA fragmentation 18 h later (n=3, ns p>0.05, use Ordinary two-way ANOVA with multiple comparison test). Values ​​are expressed as mean ± SEM. (F): Overexpressed Mcl-1 protein, but not Bcl-2 or Bcl-XL, is strongly 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 degradation of overexpressed Mcl-1 upon UNC5B induction. Stable HeLa UNC5B cells (constitutively expressing Mcl-1) were treated with 2 μg / mL Dox and 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 blotting. (H): UNC5B accelerates the degradation of Mcl-1 and reduces the half-life. HeLa UNC5B cells constitutively expressing Mcl-1 were treated with Dox for six hours, followed by the addition of cycloheximide (CHX). Cells were harvested at the indicated times after CHX treatment, and protein levels were assessed by Western blotting. Mcl-1 expression was normalized to β-actin. In the presence of UNC5B, Mcl-1 half-life was shortened compared to control conditions.

[0169] Figure 5. UNC5B-mediated Mcl-1 degradation induces MOMP and caspase-9 activation. (A): UNC5B overexpression triggers 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 hours after transfection, cells were treated with Dox, and 18 hours later, mitochondrial depolarization rates were quantified by 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye staining using 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): Apoptotic cell death induced by UNC5B overexpression requires caspase-9. HeLaUNC5B cells were transfected with scrambled (siCTRL) or caspase-9 (siCASP9) siRNA and treated with the indicated concentrations of Dox 24 hours later. Propidium iodide (PI)-positive cells were assessed in real time using an Incucyte Zoom instrument for up to 48 hours (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 kit (E), both performed 18 hours after treatment (n=3, *p>0.05, ****p<0.0001, using (ANOVA with multiple comparisons test). Values ​​are given as mean ± SEM. Inhibition of caspase-9 gene expression reduces UNC5B-induced cell death after 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 a fluorescent kit using specific substrates, (n = 3, nsp>0.05, *p<0.05, Mann-Whitney test, one-sided). Values ​​are given as mean ± SEM. UNC5B triggers a strong induction of caspase-9 activity, while caspase-8 activity is 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 hours, cells were treated with Dox and caspase-9 activity was measured by a fluorescence 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 is sufficient to inhibit UNC5B-induced caspase-9 activity. (G): Proteasome or NEDD8 inhibition limits UNC5B-triggered caspase-9 activity. HeLa UNC5B cells were treated with Dox and the proteasome inhibitors MG132 and bortezomib (BTZ), the NEDD8 inhibitor pevonedistat, or the pan-caspase inhibitor Q-VD-OPh (QVD) at the indicated concentrations. 18 hours after treatment, caspase-9 activity was assessed using a fluorescent 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): UNC5B-triggered MOMP does not require caspase-9. HeLa UNC5B cells were transfected with scrambled or caspase-9-specific siRNA. After 24 hours, cells were treated with Dox, and after another 18 hours, the mitochondrial depolarization rate was 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. The cells were then treated with Dox and Mcl-1 protein was assessed by western blotting. Silencing of caspase-9 was not sufficient to reverse the Mcl-1 degradation observed after UNC5B induction. (J): UNC5B-induced apoptosis involves the pro-apoptotic proteins BAX and BAK1. HeLa UNC5B cells were transfected with siRNA targeting BAK1 and BAX (alone or in combination). After 24 hours, the cells were treated with Dox and DNA fragmentation was assessed by flow cytometry after an additional 18 hours (n=4, ****p<0.0001, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are given as mean ± SEM.

[0170] 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 hours after transfection, total RNA was extracted and caspase-9 (A) and -8 (B) gene expression was evaluated by quantitative RT-PCR. Relative gene expression was normalized to siCTRL conditions using TATA binding protein (TBP) and β-glucuronidase (GUSB) genes as housekeeping genes (n=3-4, ****p<0.0001, ordinary two-way ANOVA using Dunnett's multiple comparison test). Values ​​are given as mean ± SEM. (C): Caspase-8 is not required for UNC5B to induce cell death. HeLa UNC5B cells were transfected with scrambled (siCTRL) or caspase-8 (siCASP8) siRNA. 24 hours after transfection, cells were treated with doxycycline (Dox) and cell death was assessed by real-time measurement of propidium iodide (PI)-positive cells for up to 26 hours using the Incucyte Zoom instrument (n=3, nsp>0.05, Kolmogorov-Smirnov test). Values ​​are given as mean ± SEM. (D): Validation of BAX and BAK1 silencing. HeLaUNC5B 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.

[0171] Figure 7. Axon guidance factor 1 interference triggers Mcl-1 degradation in vitro and in vivo. (A, B): Axon guidance factor 1 silencing triggers Mcl-1 degradation. Breast cancer MCF7 cells were transfected with scrambled (siCTRL) or Axon guidance factor 1 (siNet) targeting siRNA. 48 hours after transfection, cells were lysed by scraping and sonication in SDS-containing buffer. The protein levels of Mcl-1, Axon guidance factor 1 and UNC5B, as well as β-actin and Ku80 used as housekeeping genes, were assessed by western blotting (A). (B) Relative Mcl-1 protein levels are shown, normalized to β-actin or Ku80 (n=2, ***p<0.001, ****p<0.0001, using Ordinary two-way ANOVA with multiple comparisons test). Activation of endogenous UNC5B after NGF1 silencing reduces Mcl-1 protein levels. Values ​​are given as mean ± SEM. (C, D): NGF1 interference degrades Mcl-1 in a syngeneic breast cancer model. Stable EMT6 cells (constitutively overexpressing human Mcl-1) were implanted into BALB / c mice. When tumors reached 100 mm 3Mice were treated with Iso-mAb or NP137 antibody at a volume of , and were killed 8 hours after treatment. The protein level of Mcl-1 was then assessed by Western blotting (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 middle of the box is drawn at the median. (E, F): NP137 antibody triggers Mcl-1 degradation in a group of cancer patients. According to the design of the Phase I clinical trial, patients with advanced solid cancer participating in the Phase I clinical trial (NCT02977195) received 14 mg / kg NP137 treatment every two weeks. Biopsy samples were collected at the time of 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 of paired C1D1 and C3D1 biopsy samples and compared with clinical benefit (stable disease at 6 weeks, SD or progressive disease, PD). Mcl-1 intensity in all, PD or SD biopsy samples was assessed (E) (n=6, Wilcoxon test). The dotted line indicates Mcl-1 expression in the same patient before and after NP137 infusion. Representative IHC images of three SD patients are shown in (F).

[0172] Figure 8. Combination of Axon Guidance Factor 1 Interference and BH3 Mimetics Inhibit Tumor Growth in Preclinical In Vivo and Ex Vivo Models. (A, B, C): Combination of Axon Guidance Factor 1 Interference and Bcl-2 Inhibition Suppresses Tumor Growth in Syngeneic Breast Cancer Model. EMT6 cells were implanted into BALB / c mice and once tumors reached 50 mm 3 If the tumor volume was greater than 500 mm, the isotype (Iso-mAb) or NP137 antibody and Bcl-2 inhibitor venetoclax were used as single agents or in combination. Tumor volume was measured on the indicated days after the first treatment (n=9, **p<0.01, ordinary two-way ANOVA with Tukey's multiple comparison test) (A, B). 3 Survival was assessed when mice were treated with NP 137 (C). (D, E, F): Combination treatment effectively induces apoptosis in human ex vivo sections. Cleaved active caspase-3 positive cells were assessed by IHC staining in fresh ex vivo breast tumor tissue sections. Combination treatment with NP 137 antibody and venetoclax increased apoptosis rate compared to single agent treatment (n=8, **p<0.01, ordinary two-way ANOVA with uncorrected Fisher's LSD).

[0173] FIG9. Combined NGF1 perturbation and BH3 mimetics inhibit tumor growth in preclinical in vivo and in vitro models. Related to FIG8. (A): UNC5B overexpression sensitizes tumor cells to Bcl-2 inhibition. UNC5B-stable breast cancer T47D cells were treated with 2 μg / mL doxycycline (Dox) and the indicated concentrations of the Bcl-2 inhibitor venetoclax. After 24 hours, apoptosis was quantified by Annexin V / propidium iodide (PI) staining. EC50 values ​​were calculated using nonlinear regression of dose-response curves using GraphPad Prism software. (B): Silencing of NGF1 gene expression increases cell death induced by Bcl-2 inhibition. Breast cancer MCF7 cells were transfected with scrambled (siCTRL) or NGF1 (siNet) targeting siRNA. 24 hours after transfection, cells were treated with venetoclax for 48 hours. Apoptosis was then assessed by staining with 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye using a NucleoCounter NC3000 (n=2, *p<0.05, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are expressed as mean ± SEM. (C): Tumor growth in the syngeneic EMT6 breast cancer model is unresponsive to axon guidance factor 1 interference or Bcl-2 inhibition. EMT6 cells were implanted into BALB / c mice and once tumors reached 50 mm 3 Tumor volumes were measured at designated days after the first treatment (n=9, ns p>0.05, ordinary two-way ANOVA with Tukey's multiple comparison test). Values ​​are expressed as mean ± SEM. (D): Combination treatment effectively induces apoptosis in human ex vivo sections. RNA from breast tumor biopsy samples (n=8) was extracted and gene expression of axon guidance factor 1 (NTN1), UNC5B, Bcl-2 (BCL2), and Bcl-XL (BCL2L1) was evaluated by quantitative RT-PCR. The heat map represents the basal mRNA level of each patient, shown as the 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 monotherapy.

[0174] Fig.10Cell death assessment by combined NAGN1 silencing and Navitoclax To assess cell death, propidium iodide (PI) incorporation was tracked in real time (normalized to cell confluence) for up to 24 hours for scrambled (siCTRL), NAGN1 (siNet), scrambled (siCTRL) and Navitoclax, and NAGN1 (siNet) and Navitoclax using Incucyte Zoom technology.

[0175] result

[0176] Working model for UNC5B-mediated Mcl-1 degradation and activation of the intrinsic apoptotic pathway

[0177] Induction of UNC5B activates 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 apoptotic bodies, and ultimately activation of caspase-9, leading to apoptosis.

[0178] COMMD2 is required for UNC5B-induced cell death and tumor suppression

[0179] To identify the pro-apoptotic pathways triggered by UNC5B, we generated stable HCT116 and HeLa cancer cell lines that could induce UNC5B using Sleeping Beauty-based vectors. Forced UNC5B expression following doxycycline treatment (confirmed by Western blotting) (Figures 2A and 2B) strongly induced apoptosis in both models compared with cells stably transfected with empty vector, as shown by PI staining ( Figure 1 A and 2C), DNA fragmentation ( Figure 1 B and 2D) and caspase-3 activity ( Figure 1 C and 2E). This strong sensitivity to UNC5B induction suggests the presence of functional pro-apoptotic UNC5B signaling in HCT116 and HeLa cells. To confirm that UNC5B-induced apoptosis was not simply due to protein overexpression, we stably expressed the ligand axon guidance factor 1 in cells in which UNC5B can be inducible. Constitutive expression of axon guidance factor 1 was sufficient to suppress the apoptosis that would otherwise be observed after UNC5B induction ( Figure 1 B). Furthermore, inhibition of caspases by the pan-caspase inhibitor Q-VD-OPh completely blocked UNC5B-induced apoptosis (Figure 2F).

[0180] We decided to perform a CRISPR-Cas9-based screen using a stable HCT116 cell line that can induce UNC5B to identify UNC5B pro-apoptotic effectors. To this end, we transduced Sleeping Beauty-modified HCT116 cells with a whole-genome lentiviral library (GeCKO library, (Sanjana et al., 2014; Shalem et al., 2014)). After doxycycline treatment and UNC5B induction, only cell clones with knockout of genes required for UNC5B pro-apoptotic signaling survived. Using next-generation sequencing (NGS), we identified inactive genes in cell clones. Considering the enrichment of all genes presenting at least three small guide RNAs (sgRNAs) compared to control cells and identified in more than 100 reads, we identified 75 genes that are potentially involved in UNC5B-induced apoptosis.

[0181] After 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 different siRNAs targeting COMMD2. Compared with cells transfected with control siRNA, the reduction of COMMD2 gene expression was sufficient to reduce DNA fragmentation after UNC5B challenge with doxycycline (Figure 3A). In addition, COMMD2 silencing suppressed the severe increase in PI-positive HeLa cells triggered by UNC5B overexpression, as well as the caspase-3 activation observed in control cells (not shown). Therefore, COMMD2 has a specific role in the UNC5B pro-apoptotic pathway.

[0182] Next, we assessed the requirement for COMMD2 and UNC5B for tumor suppression associated with NG1 inhibition. To this end, we investigated the effects of NP137, a neutralizing anti-NG1 antibody that is able to induce 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 strongly inhibited tumor growth in control cells, but tumors with knockout of COMMD2 or UNC5B were less sensitive to anti-NG1 antibodies, as shown by tumor growth analysis and percentage of tumor growth inhibition (Figure 3C, 3D). We confirmed that NP137 antitumor activity was associated with apoptosis induction, as shown by immunohistochemical staining of cleaved active caspase-3 (Figure 3E). In contrast, we failed to detect a similar increase in the percentage of caspase-3-positive cells after treatment with NP137 antibody in tumors with COMMD2 or UNC5B knockdown ( Figure 3E ), further supporting the idea that interference with the interaction of NF1 / UNC5B with NP137 triggers COMMD2-dependent cancer cell death associated with tumor growth inhibition.

[0183] UNC5B triggers proteasomal degradation of Mcl-1

[0184] Considering our demonstration that UNC5B-induced cell death requires COMMD2, we hypothesized that UNC5B might lead to the ubiquitination and subsequent proteasomal degradation of unknown target proteins. Along these lines, treatment of stable HeLa cells with the proteasome inhibitors MG132 and bortezomib inhibited DNA fragmentation and caspase-3 activation triggered by UNC5B induction (Figures 4A and 4B). In the search for UNC5B target proteins that are degraded during apoptosis induction, we focused on members of the Bcl-2 family, which are anti-apoptotic proteins whose cellular abundance is regulated by the ubiquitin-proteasome system (Kale et al., 2018; Singh et al., 2019). Western blot analysis revealed a specific decrease in Mcl-1 protein after 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 untreated and doxycycline-treated cells, supporting the notion that the decrease in Mcl-1 levels associated with UNC5B induction is dependent on proteasome activity and is therefore associated with protein degradation. Interestingly, inhibition of caspase activity by Q-VD-OPh was not sufficient to prevent UNC5B-triggered Mcl-1 degradation (Fig. 4C), although it effectively prevented UNC5B-induced apoptosis (see Fig. 2F), suggesting that caspase activation is downstream of Mcl-1 degradation. Furthermore, inactivation of the COMMD2 / CRL3 complex by silencing COMMD2 or RBX1 gene expression inhibited Mcl-1 degradation in doxycycline-treated HeLa cells (Fig. 4D).

[0185] To more formally demonstrate that Mcl-1 degradation is specifically induced by UNC5B, we generated stable HeLa cells that can induce UNC5B and constitutively express high levels of Mcl-1. Interestingly, Mcl-1 overexpression was not sufficient to inhibit UNC5B-induced apoptotic cell death (Figure 4E), and Western blot analysis showed that even ectopically overexpressed Mcl-1 was significantly degraded after UNC5B induction, while overexpressed Bcl-2 and Bcl-XL proteins were not degraded under the same circumstances (Figure 4F). In addition, treatment with bortezomib inhibited the degradation of ectopically expressed Mcl-1 (Figure 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, and UNC5B-induced HeLa cells with CHX showed an increased rate of Mcl-1 degradation in the presence of UNC5B, resulting in a shortened Mcl-1 half-life (108.5 min in control cells versus 37.2 min upon UNC5B induction, monophasic decay equation) (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.

[0186] UNC5B-mediated Mcl-1 degradation induces MOMP and caspase-9 activation

[0187] Mcl-1, as well as other Bcl-2 family members, act as anti-apoptotic proteins by inhibiting the pro-apoptotic proteins BCL2-related X, regulator of apoptosis (BAX) and BCL2 antagonist / killer 1 (BAK1) and the ensuing mitochondrial outer membrane permeabilization (MOMP) (Galluzzi et al., 2018). After the release of apoptotic factors residing in the mitochondrial inner membrane space into the cytoplasm, a pro-apoptotic complex, known as the apoptosome, is formed, leading to caspase-9 activation and the subsequent execution of the intrinsic apoptotic program. Since we have shown that UNC5B triggers Mcl-1 degradation, we hypothesized that UNC5B-induced apoptosis requires MOMP and caspase-9 activation. Indeed, UNC5B overexpression induced MOMP in stable HeLa cells (Figure 5A). Interestingly, silencing of COMMD2 and RBX1 suppressed UNC5B-triggered MOMP (Figure 5A), suggesting that the ubiquitin ligase activity of the COMMD2 / CRL3 complex may be involved in the induction of UNC5B-triggered MOMP.

[0188] Next, we determined whether caspase-9, activated after MOMP induction, is a mediator of UNC5B-triggered apoptosis. Stable HeLa cells inducible for UNC5B were transfected with siRNA targeting caspase-9, and cell death rate was assessed after treatment with doxycycline. Silencing caspase-9 rescued the strong increase in PI-positive cells after 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 apoptotic pathway, failed to affect UNC5B-induced cell death (Figures 5B and 6A). Figure 6 B and 6C).

[0189] The dependence of UNC5B-induced death on caspase-9 suggests that UNC5B-induced MOMP triggers apoptosis via caspase-9 activation. Indeed, we showed that in stable HeLa cells, UNC5B triggers caspase-9 but not caspase-8 activation, as indicated by caspase activity using specific fluorescent substrates (Figure 5E). Since we demonstrated that Mcl-1 degradation requires UNC5B to activate the COMMD2 / CRL3 complex, we determined whether UNC5B-dependent caspase-9 activation is a consequence of this event. Stable HeLa cells were transfected with two different siRNAs targeting COMMD2, CUL3, and RBX1. Silencing the expression of the COMMD2 / CRL3 complex members completely suppressed the caspase-9 activation observed upon UNC5B induction in cells transfected with scrambled siRNA (Figure 5F). Furthermore, treatment of stable HeLa cells with NEDD8 (Pevonedistat) or proteasome inhibitors (MG132 and bortezomib) was sufficient to prevent UNC5B-induced caspase-9 activation (Figure 5G).

[0190] To confirm that UNC5B-induced Mcl-1 degradation and MOMP were not simply an effect of cellular perturbations caused by UNC5B overexpression, we determined whether caspase-9 knockdown affected MOMP or Mcl-1 degradation associated with UNC5B induction. Figure 6 A) Even though UNC5B-induced apoptosis was inhibited (Figures 5B, 5C, and 5D), it was not sufficient to inhibit UNC5B-induced mitochondrial depolarization (Figure 5H). As a control, caspase 8 silencing ( Figure 6 B) Unable to inhibit UNC5B-induced cell death ( Figure 6 C). Furthermore, transfection of stable HeLa cells with siRNA targeting caspase-9 did not prevent UNC5B-induced Mcl-1 degradation (Figure 5I). Similarly, silencing of BAX and BAK1 ( Figure 6D), a molecular mediator that drives apoptosis through Mcl-1 inhibition of MOMP and caspase-9 activation, reduced UNC5B-dependent apoptosis induction ( FIGS. 5J and 8D ).

[0191] Axon guidance factor 1 disruption triggers Mcl-1 degradation

[0192] To confirm that Mcl-1 degradation is associated with endogenously expressed detached UNC5B, we hypothesized that in cancer cells expressing both NGF1 and UNC5B, NGF1 silencing would be associated with Mcl-1 degradation. Indeed, transfection of breast cancer MCF7 cells with siRNA targeting NGF1 induced Mcl-1 degradation (Figures 7A and 7B).

[0193] To move to a more relevant therapeutic setting, we evaluated Mcl-1 degradation in vivo using the EMT6 syngeneic breast cancer model. Indeed, EMT6 cells express endogenous NGF1 and UNC5B. Stable EMT6 cells (constitutively overexpressing human Mcl-1) were implanted into BALB / c mice, and tumor-bearing animals were treated intravenously with a therapeutic anti-NGF1 antibody (NP137). Mcl-1 in tumors of mice was measured by immunoblotting, and interestingly, NGF1 blockade significantly reduced Mcl-1 protein levels compared to control mice (Figure 7C and 7D).

[0194] The NP137 antibody is currently being evaluated in patients with advanced solid tumors in several Phase I and Phase II clinical trials. In the Phase I trial, paired biopsy samples from 12 patients treated with NP137 were collected before the first infusion of NP137 (C1D1) and after two cycles of NP137 (1 month, C3D1). Therefore, the presence of Mcl-1 was analyzed by immunohistochemistry. NP137 administration reduced Mcl-1 levels in 9 of the 12 patient tumors tested (Figures 7E and F). In addition, when comparing the clinical benefits of 12 patients who monitored Mcl-1 testing (experienced disease stabilization (SD: stable disease) or disease progression (PD: progressive disease) in the 6-week RESIST analysis), 6 SD patients showed a decrease in Mcl-1 after NP137 treatment (Figures 7E and F), while we observed a decrease in Mcl-1 in only 3 of the 6 PD patients. These data together indicate that axon guidance factor 1 blockade triggers tumor-specific Mcl-1 loss, both in animal models and in patients.

[0195] Combination of axon guidance factor 1 disruption and BH3 mimetics (venetoclax and navitoc) inhibits tumor growth in preclinical in vivo and ex vivo models

[0196] BH3 mimetics represent 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 U.S. 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 have been developed that target Mcl-1. Despite initial enthusiasm for Mcl-1 targeting, clinical trials of early Mcl-1 inhibitors were recently suspended due to potential cardiotoxicity (Wei et al., 2020). BH3 mimetics used as monotherapy in solid tumors have been less effective, perhaps because solid tumors are less “sensitized” to apoptosis than hematopoietic tumors (Sarosiek et al., 2017) or perhaps because of compensatory effects (van Delft et al., 2006). This is why the current strategy for solid tumors seems to be a combination of selective BH3 mimetics, especially combined treatment with Mcl-1 and Bcl-XL or Bcl-2 inhibitors (Soderquist et al., 2018). As we showed above that the UNC5B receptor triggers Mcl-1 degradation in cancer cells, we hypothesized that UNC5B-triggered Mcl-1 degradation could enhance the efficacy of Bcl-2 inhibitors. To confirm this hypothesis, we first generated stable breast cancer T47D cells in which UNC5B can be induced and evaluated the efficacy of venetoclax in the presence of UNC5B induction. Indeed, UNC5B induction was sufficient to significantly reduce the venetoclax EC50 (Figure 9A).

[0197] As we have demonstrated that in MCF7 cells expressing AGF1 and UNC5B, AGF1 silencing is associated with Mcl-1 degradation, we hypothesized that, in these cells, interference with AGF1 expression might tolerate enhanced efficacy of Bcl-2 inhibitors. In fact, combined AGF1 silencing and venetoclax treatment triggered mitochondrial depolarization compared with AGF1 interference or Bcl-2 inhibition alone, indicating enhanced induction of cell death by inhibition of Bcl-2 (Fig. 9B).

[0198] Subsequently, to evaluate the anti-tumor effect of the combination therapy in vivo in a traditional preclinical model, we took advantage of the EMT6 syngeneic breast cancer model. Indeed, these cells express high levels of neurite 1 and UNC5B. However, interference with neurite 1 by the anti-neurite 1 NP137 antibody did not affect tumor growth in this model (not shown). EMT6 cells were implanted into BALB / c mice, and animals bearing tumors were treated with NP137 and venetoclax. Although the drugs in the monotherapy had no effect on tumor growth, the combination treatment significantly reduced tumor growth compared to the isotype control antibody treatment (Figures 8A, 8B, and 9C). This reduced tumor growth was associated with overall survival of mice treated with the combination (Figure 8C).

[0199] Finally, to confirm the effectiveness of the combination treatment in human patient tissues, we evaluated the effects of NG1 disruption and Bcl-2 inhibition in an ex vivo breast cancer organotypic culture technique, allowing the study of tumors maintained in their natural microenvironment (de Graaf et al., 2010). Fresh tumor sections were treated with NP137 antibody and venetoclax as single agents or in combination, and apoptosis was quantified by immunohistochemical analysis of cleaved caspase-3. Treatment with NP137 antibody and venetoclax significantly increased the percentage of cleaved caspase-3-positive cells compared to NG1 disruption or Bcl-2 inhibition alone (Figure 8D). Notably, the combination treatment increased the rate of apoptosis in five out of eight tumor biopsies (Figures 8E and 8F). Of note, of the three patients who did not respond to the combination treatment, patient B21.015 expressed lower levels of NG1 and UNC5B compared to the other patients, leading to resistance to the NP137 antibody. Furthermore, although patient B21.030 exhibited elevated AGF1 and UNC5B transcript levels, resistance to the combined treatment was likely due to relatively low Bcl-2 levels (i.e., poor sensitivity to venetoclax) and elevated Bcl-XL expression, indicating resistance to Mcl-1 inhibition (Soderquist et al., 2018) (Figure 9D). These results confirm that inhibition of Bcl-2 alone is not sufficient to trigger apoptosis in solid tumors (i.e., human breast cancer patients) and that simultaneous interference with the AGF1 / UNC5B interaction by the NP137 antibody is required to induce Mcl-1 degradation.

[0200] In addition, breast cancer HCC70 was transfected with scrambled siRNA (siCTRL) or siRNA targeting axon guidance factor 1 (siNet). 24 hours after transfection, cells were isolated, seeded again into 24-well plates, and treated with 0.5 μM of the Bcl-2 / Bcl-XL / Bcl-w inhibitor navitoc (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. Although axon guidance factor 1 silencing or Bcl-2 / Bcl-XL / Bcl-w inhibition alone had a slight effect on cell death induction, the combined treatment rapidly and strongly triggered apoptosis ( Fig.10 ).

[0201] Expression of UNC5 Receptor in Selected Cell Lines:

[0202] We have shown that the reduction of AGF1 presence in some tumor cell lines such as MCF7 and H322 is associated with Mcl-1 degradation. We have also demonstrated the combined effect of AGF1 depletion and BH3 mimetics on cell death induction in the tumor cell line HCC70. We then demonstrated that UNC5B as well as another AGF1 receptor such as UNC5A and UNC5C are well expressed in these cell lines.

[0203] The mRNA expression of the receptor UNC5A-C was assessed using the Cancer Cell Line Encyclopedia (CCLE) database. The transcript abundance of the receptor was assessed in breast cancer cells (MCF7 and HCC70) and lung cancer cells (H322) and expressed as reads per million kilobases (RPKM).

[0204] Table: Gene expression (RPKM)

[0205] MCF7 HCC70 H322

[0206] UNC5A 0,854 0,000 0,157

[0207] UNC5B 0,540 1,297 0,575

[0208] UNC5C 0,095 1,917 0,305

[0209] In addition to UNC5B, which was expressed at high and stable levels in all three cell lines analyzed, at least one receptor between UNC5A and UNC5C was expressed. Therefore, it is considered that other UNC5 receptors, especially UNC5A and UNC5C, may be involved in cell death caused by axon guidance factor 1 perturbation or silencing (alone or in combination with BH3 mimetics).

[0210] discuss

[0211] The dependence receptor UNC5B conditionally induces apoptosis in cancer cells. Using CRISPR-Cas9 screening, we identified the protein COMMD2 as a pro-apoptotic mediator of UNC5B-induced cell death. In the absence of axon guidance factor 1, UNC5B triggers COMMD2 activation through an unknown mechanism. Once activated, COMMD2 interacts with the CRL3 complex, then ubiquitinates Mcl-1 and causes its degradation by the proteasome. This results in a shift in the pro-apoptotic balance, leading to mitochondrial permeabilization and sequential activation of caspase-9 and caspase-3, ultimately leading to apoptosis in cancer cells. To date, several ubiquitin ligases are known to degrade Mcl-1, including Mule, SCF β-TrCP 、SCF FBW7 、TRIM17、APC / C Cdc20 , MARCH5, and Parkin (Mojsa et al., 2014; Senichkin et al., 2020). However, the CRL3 complex has never been described in this context before. Here, we show for the first time that this E3 ligase is involved in the degradation of Mcl-1.

[0212] The CRL E3 ligase family is primarily regulated by the pseudomimetic cycle, which allows for its activation and inactivation (Duda et al., 2011). CRL E3 ligases are activated by covalent binding to 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-pseudomimetic complex and inhibit its activity (Duda et al., 2011). Interestingly, COMMD1 inhibits the binding of CAND1 by associating with Cullin-2, thereby activating the CRL2 complex (Mao et al., 2011). In addition, COMMD2 specifically interacts with Cullin-3, but not Cullin-2 (Mao et al., 2011). Our data suggest that COMMD2, as well as members of the CRL3 complex, are required for UNC5B-induced cell death, whereas COMMD1 and other CRLs are not involved in the UNC5B pro-apoptotic pathway. Furthermore, we were able to show that inhibition of pseudomimetic remodeling by treatment with pevonedistat, a specific inhibitor of NEDD8, prevented UNC5B-induced cell death. Since CAND1 binds and inhibits nearly all Cullins (Duda et al., 2011), we can assume that COMMD2, by specifically interacting with CRL3, inhibits the interaction of CAND1 with Cullin-3, thereby allowing its activation by pseudomimetic remodeling. However, how UNC5B supports the interaction of COMMD2 with CRLs and activates these complexes remains unclear.

[0213] From a therapeutic point of view, our results suggest that interfering with the binding between AGF1 and UNC5B using the neutralizing anti-AGF1 antibody NP137 is an original approach to degrade Mcl-1, thereby providing an interesting alternative to the BH3 mimetics currently under development against Mcl-1. The NP137 antibody degrades Mcl-1 in an indirect but tumor-specific manner and is an attractive alternative to BH3 mimetic compounds. In fact, using NP137, Mcl-1 degradation is thought to occur only in cells expressing AGF1, and preclinical development of NP137 showed the absence of tissue cross-reactivity in healthy adult individuals. In addition, the safety of this antibody has been tested in clinical phase I and showed an excellent safety profile (Cassier et al., 2019). By preferentially targeting tumor cells overexpressing AGF1, NP137 offers considerable advantages over chemical inhibitors that indiscriminately target tumors and healthy cells. The good tolerability of the antibody by patients offers the possibility of combining it with other molecules.

[0214] Another interesting approach is to target other Bcl-2 family members in addition to Mcl-1. While the vast majority of solid tumors are resistant to the use of single inhibitors against Bcl-2, Bcl-XL, or Mcl-1, combinations that inhibit two or even all three proteins are very effective (Soderquist et al., 2018). In particular, the combination of Mcl-1 and Bcl-XL inhibitors appears to be effective against a large proportion of tumors and therefore offers promise. However, this combination has been reported to be lethal in mouse models due to acute liver toxicity (Weeden et al., 2018). The use of antibodies against axon guidance factor 1 represents a more selective BH3 mimetic against Mcl-1, thus reducing the toxicity of the combination therapy and allowing for combination with other BH3 mimetics, as we have shown in several preclinical and ex vivo tumor models treated with NP137 and the Bcl-2 inhibitor venetoclax.

[0215] Another BH3 mimetic has been tested. Navitoclax (ABT-263) is known to induce Bcl-2 / Bcl-XL / Bcl-w inhibition. The combination of ANF1 silencing and Navitoclax rapidly and strongly induces apoptosis (Figure 9).

[0216] method:

[0217] Cell lines, transfection procedures, reagents

[0218] Human colorectal cancer HCT116 and cervical cancer HeLa cell lines were cultured in DMEM medium (Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum. HEK 293T 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 cancer H322 cells were cultured in RPMI 1640+Glutamax medium (Life Technologies) containing 10% fetal bovine serum. For RNA interference experiments, cell lines were transfected using lipofectamineRNAiMAX reagent (Life Technologies). For plasmid transfection, HCT116 cell line was transfected using lipofectamine Plus reagent (Life Technologies); HeLa cell line was transfected using jet prime (PolyPlus-Transfection). All transfections were performed according to the manufacturer's protocol. BH3 mimetic venetoclax (ABT-199) and NEDD8 inhibitor pevonedistat were purchased from Selleck Chemicals GmbH (Munich, Germany). Proteasome inhibitor MG132 and bortezomib were purchased from Sigma-Aldrich (St. Louis, MO, USA). Caspase inhibitor QVD was purchased from Adooq Bioscience (Irvine, CA, USA).

[0219] Generation of stable cell lines

[0220] To generate stable inducible cell lines, the 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 expressed under the control of a synthetic RPBSA promoter, as well as constitutive expression of the rtTA protein, a hygromycin resistance gene, and green fluorescent protein (GFP). HCT116, HeLa, and T47D cell lines were co-transfected with UNC5B-pSBtet-GH and the Sleeping Beauty transposase expression vector (SB100X). The Sleeping Beauty transposase expression vector was a generous gift from Prof. 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:Addgene_60498). Stable cell clones were selected in medium containing hygromycin for 7 days. Stable UNC5B cell lines were then sorted by flow cytometry to select highly GFP-positive cells. All stable inducible cell lines were further tested for UNC5B expression in response to doxycycline.

[0221] Stable cell lines constitutively expressing axon guidance factor 1 were generated by lentiviral infection using a self-inactivating HIV-1-derived vector (pWPXLd) encoding axon guidance factor 1 under the control of the human elongation factor-1α (EF-1α) promoter. To generate HCT116 stable cell lines that can induce UNC5B for screening, the pCW57.1 vector was used and stable cell lines were generated by lentiviral infection. This lentiviral vector allows doxycycline-inducible UNC5B expression as well as constitutive expression of the rtTA protein and hygromycin resistance gene. pCW57.1 (Addgene, plasmid #41393; http: / / n2t.net / addgene:41393; RRID:Addgene_41393).

[0222] To generate stable UNC5B or COMMD2 knockout H322 cell lines, different small guide RNAs (sgRNAs) were designed using the sgRNA design tool CRISPick (CRISPRi), which can be accessed from (https: / / )portals.broadinstitute.org / gppx / crispick / public. The sgRNAs were designed close to the UNC5B or COMMD2 promoter. The BsmBI linker was added to the sgRNA, and the oligonucleotides were then annealed by incubation at 90°C for 3 minutes and at 37°C for 15 minutes and then inserted into the BsmBI-digested pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro vector (Thakore et al., 2015), allowing the expression of the defective Cas9 enzyme (dCas9) fused to the Krüppel-associated cassette (KRAB) transcriptional repressor (dCas9-KRAB). pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro (Addgene, plasmid #71236; (http: / / )n2t.net / addgene:71236; RRID: Addgene_71236).

[0223] Quantitative RT-PCR

[0224] According to the manufacturer's protocol, use Total RNA was extracted using the RNA Plus Kit (Macherey Nagel, Düren, Germany). RT-PCR reactions were performed using the PrimeScript RT 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, 85°C for 5 s. For expression studies, the Premix Ex Taq (probe qPCR) Kit (Takara Bio Europe) was used according to the manufacturer's instructions. Target transcripts were amplified in the 2.0 instrument (Roche Applied Science, Penzberg, Germany). The expression of the target gene was normalized to the TATA binding protein (TBP) and β-glucuronidase (GUSB) genes, which were used as housekeeping genes. The target transcript amount normalized to the housekeeping gene was calculated using the comparative CT method. Validation experiments were performed to demonstrate that the efficiency of the target gene and the housekeeping gene was approximately equal. Primer sequences are available upon request.

[0225] Western blot analysis

[0226] 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 overnight at 4°C 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 Biotechnology, Inc.); Biotechnology, Dallas, TX, USA); rabbit polyclonal antibody α-COMMD2 (1:1000, HPA044190, Sigma-Aldrich); rabbit monoclonal antibody α-Ku80 (1:1000, clone C48E7, ​​#2180, Cell Signaling). After washing three times with PBS-T, the membrane was incubated with the appropriate HRP-conjugated secondary antibody (1:10000, Jackson ImmunoResearch, Suffolk, UK) for 1 hour. Detection was performed using the WestDura chemiluminescence system (Life Technologies). The membrane was imaged on the ChemiDoc Touch imaging system (Bio-rad). The membrane was imaged on the ChemiDoc Touch imaging system (Bio-rad). The protein bands were quantified using Image Lab6.1 software (Bio-rad), taking into account the adjusted volume intensity normalized using housekeeping genes.

[0227] Cell death assay

[0228] For propidium iodide (PI) staining, stable inducible cell lines were seeded in 48-well plates at a density of 25 × 10 3 Cells / well. The next day, cells were treated with 0.33 mg / ml PI and doxycycline in serum-free (HCT116) or 10% serum (HeLa) medium, and the plates were transferred to an Incucyte Zoom instrument (Essen Bioscience, Royston, UK). Images of live cells and PI-stained dead cells were taken every two to three hours, and the number of PI-stained cells was counted.

[0229] For DNA fragmentation analysis (SubG1), HCT116 and HeLa cell lines were seeded in 6-well plates at a density of 2*10 5 cells. The next 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 collected, fixed in 70% ethanol, and incubated at -20°C for at least one night. Afterwards, the cells were washed twice with PBS and incubated at room temperature for 15 minutes in PBS containing 2mg / ml Rnase A and 40mg / ml PI. DNA content was assessed on FACScalibur or FacsCantoII (BD Bioscience, Franklin Lakes, NJ, USA) by fluorescence activated cell sorting (FACS) analysis.

[0230] 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 processed for DNA fragmentation assays. Cells were then collected and caspase-3 / caspase-9 activity assays were performed using a caspase-3 / caspase-9 fluorescence assay kit (BioVision, Milpitas, CA, USA) according to the manufacturer's instructions. The activity of caspase-3 / caspase-9 was measured using a fluorescence spectrophotometer (405 nm / 510 nm, Infinite F500, Tecan, Caspase activity (activity / min / mg protein) was calculated using a 1 h kinetic cycle readout on a PCR plate reader (Switzerland).

[0231] Mitochondrial potential measurement

[0232] HCT116 and HeLa cells were seeded and processed for DNA fragmentation assay. The cells were then collected and washed with PBS. 1x10 6Each cell was suspended in 1 mL PBS containing JC-1 dye (2.5 μg / mL, Chemometec, Allerod, Denmark). After incubation at 37 ° C for 15 minutes, the stained cells were centrifuged at 800 x g for 5 minutes at room temperature, and the supernatant was completely removed without disturbing the cell pellet. Afterwards, the cell pellet was suspended in 1 ml PBS and washed, then centrifuged at 800 x g for 5 minutes at room temperature, and the supernatant was removed again. The washing step was repeated twice. Afterwards, the cell pellet was suspended in 250 μl PBS buffer containing 1 μg / ml DAPI by pipetting. The final stained cells were analyzed using NucleoCounter NC-3000 (Chemometec).

[0233] Conditioned medium production

[0234] HCT116 stable cells constitutively expressing axon guidance factor 1 were seeded in 100 mm culture dishes (3*10 cells per dish). 6 After 24 hours, the cell culture medium was collected and centrifuged at 1200×g for 5 minutes, and the supernatant was recovered as the axon guidance factor 1 conditioned medium.

[0235] CRISPR-Cas9 screening

[0236] Gecko Library

[0237] The human GeCKOv2 CRISPR knockout pooled library (targeting 19.050 genes, divided into two half-libraries, each containing 3 sgRNAs for each gene) was gifted by Feng Zhang (Addgene).

[0238] DNA amplification of the library

[0239] Each lentiviral library (A and B) was amplified and prepared according to the same protocol. 2 μL of 50 ng / μL GeCKO library was electroporated into Endura electroporation competent cells (Lucigen, Middleton, WI, USA). Each library was electroporated four times in competent cells. The cells were suspended in recovery medium and placed in a shaking incubator at 37°C for 1 hour. For each library, the electroporated cells were inoculated in two preheated 600 cm 2Luria broth (LB) agar plates and grown at 37 ° C for 16 hours. At the same time, 30.000 times diluted complete transformants were inoculated to estimate the transformation efficiency and ensure that the complete library representation was retained. The total number of colonies was at least 3.106, equivalent to about 50 colonies per construct in each GeCKO library. Colonies were harvested by adding 10mL LB culture medium on each plate and scraped with a cell spreader. The liquid with the scraped colonies was collected in a test tube, and another 10mL culture medium was used to repeat the process on the same plate for the second time. 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 combined in equimolar ratios for lentiviral production.

[0240] Lentiviral production and titration

[0241] One day before transfection, HEK 293T cells were plated at 5.10 per 150 mm dish. 6 Cells were inoculated. Xtreme gene reagent (Roche Applied Science) was used to transfect cells with three expression vectors, namely 10 μg phCMV-VSV-g, 15 μg phCMV-Gag-Pol-HIV, and 20 μg GeCKO plasmid library. Six hours after transfection, the culture medium was changed. Two viral harvests were collected 40 hours and 60 hours after transfection. The two harvests were merged, centrifuged and filtered through a 0.45 μm filter. Aliquots of the clarified supernatant were stored at -80 ° C. The production of slow viruses was titrated by infecting HCT116 cells in 12-well plates with several different volumes of slow viruses. The next day after infection, each well was divided into two wells: one well received 0.8 μg / mL of puromycin, and the other well did not receive puromycin. After 72 hours of selection, cells were counted to calculate the transduction percentage and determine the viral titer.

[0242] Cell library generation

[0243] One day before transduction, HCT116 cells were seeded in eight 12-well plates at a concentration of 10 5cells. The cells were then transduced with a previously determined amount of GeCKO lentiviral library for achieving a multiplicity of infection (MOI) of 0.3 to 0.5. HCT116 cells were transduced by rotational infection during 1 hour and 30 minutes at 37°C in complete DMEM medium supplemented with 8 μg of polybrene. The next day, the cells were trypsinized, seeded into eight 150 mm culture dishes, and treated with 0.8 μg / mL of puromycin. As a selection control, non-transduced cells were seeded and treated with 0.8 μg / mL of puromycin.

[0244] UNC5B attack in library-transduced cells

[0245] After puromycin selection, a portion of the cell library was lysed for genome extraction and sequencing. 24.106 cells of the two conditions were inoculated in serum-free DMEM and treated or not treated with 2 μg / ml doxycycline. At the same time, the stable HCT116 cell line that can induce UNC5B in the control condition and is not transduced with the CRISPR library was induced with the same concentration of doxycycline. When all cells under the control condition died, the surviving cells of the libraries of the two conditions were expanded for three days and then lysed for genome extraction and sequencing.

[0246] Genomic extraction and DNA amplification

[0247] Genomic DNA (gDNA) was extracted from the cell library before UNC5B induction using the NucleoSpin tissue kit (Macherey-Nagel) and from the surviving cells after UNC5B induction. The merged sgRNA isolated from the cell samples was amplified using CloneAmp HiFi enzyme (Takara Bio Europe). Primers were designed to amplify the 150bp region around the sgRNA sequence and add barcode sequences specific to each sample and the adapters required to fix the PCR products in the NGS flow cell. Three separate PCRs were performed for each sample, using 250ng gDNA each time. The PCR products were purified with a PCR cleanup / gel extraction kit (Macherey Nagel) and then run on an agarose gel, followed by a new purification step with the same kit. A final purification step was performed before sequencing (AMPure, Beckman Coulter, Brea, CA, USA).

[0248] Next-generation sequencing and analysis

[0249] Next generation sequencing (NGS) was performed on the Ion Torrent platform (ThermoFischer Scientific) by the sequencing platform of the Lyon Institute of Functional Genomics (IGFL, Lyon) using the IonPI HiQ Chef kit and the Ion PI HiQ chip v3 (100pb-80M readings). The amplified samples were pooled together on the sequencing chip for running and distinguished by barcode identification. The raw data from sequencing were processed to eliminate erroneous sequences and to trim the sequences on both sides of the sgRNA sequence. The trimmed sequences were aligned against the GeCKO library database with determined parameters (95% identity at 90% length), retaining only the first hit. Finally, a list was obtained containing the number of reads for each sgRNA under each experimental condition. For a given condition, the number of reads for each sgRNA was standardized by the total number of reads under that condition. The reads were then standardized by subtracting the number of reads under the control condition from the number of reads under the UNC5B-induced condition. Finally, sgRNA sequences with more than 100 normalized reads were selected, and then genes with more than 3 sgRNA sequences were selected and confirmed.

[0250] Xenotransplantation in nude mice

[0251] Five-week-old female athymic NMRI nude mice were obtained from Janvier Labs (Saint Bertevin, France). The mice were housed in a specific and opportunistic pathogen-free (SOPF) animal facility. 5*10 6 Cells were injected subcutaneously into the right flank of nude mice implanted with parental or stable H322 cells (with COMMD2 or UNC5B deleted). 3 Mice were intravenously injected with 10 mg / kg anti-axon guidance factor 1 (NP137) or isotype control antibody three times a week for 36 days. Tumor size was measured with a caliper three times a week. Tumor volume was calculated according to the formula v = 0.5x (lx 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 (authorization APAFIS number: 28723; experimental animal care was certified by CECCAP, ENS Lyon-PBES, France).

[0252] EMT6 syngeneic breast tumor model

[0253] Five-week-old Balb / c female mice were purchased from Janvier Labs. EMT6 cells were implanted subcutaneously with 5*105 cells suspended in 100 μl PBS. When tumors reached 50 mm 3At 3 pm, mice were intravenously injected with a loading dose of 20 mg / Kg of NP137 or 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% PEG300+5% Tween80 in distilled water, Selleck Chemicals GmbH) was administered by oral gavage five times a week for two weeks. Tumor size was measured with a caliper three times a week. Tumor volume was calculated according to the formula v=0.5x(lx 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 (authorized APAFIS number: 28780; laboratory animal care is certified by CECCAP, ENS Lyon-PBES, France).

[0254] For in vivo Mcl-1 degradation, 5*10 5 When the tumor reached 100 mm 3 At 14:30 pm, mice were intravenously injected with 20 mg / kg NP137 or isotype control antibody. 8 hours after injection, mice were sacrificed and tumors were collected and immediately frozen at -80°C.

[0255] In vitro breast cancer organotypic culture

[0256] A total of eight fresh human breast samples were obtained after surgical resection from chemotherapy-naive patients with invasive carcinoma at the Centre Léon Bérard (CLB, Lyon, France). All patients gave informed consent for the use of their samples for research in accordance with the requirements of the French bioethical law, and the study was approved by the CLB Ethics Committee. Immediately after surgical resection and macroscopic anatomical pathological examination, tumor tissue was stored in PBS at 4°C and sent to the laboratory. Afterwards, tumors were routinely examined and classified by a board-certified pathologist. After removal of excess necrotic tissue, 200 mm 3Fresh tumor fragments are contained in an agarose matrix. MICROM HM650V vibrating slicer (ThermoFisher Scientific, Walldorf, Germany) is used for automatic sectioning, and the slice thickness is set to 250 μm, the vibration amplitude is 8 mm, and the slice speed is 0.6 mm / second. The slices are cultured within 2 hours after the tumor is removed from the patient. Cultivation is carried out at 37 ° C under 5% CO2 and under atmospheric oxygen levels. The slices are incubated in a 24-well plate in a DMEM culture medium supplemented with 5% fetal bovine serum, 1% GlutaMAX, 1% penicillin / streptomycin, 1% MEM, 1% HEPES (all from Life Technologies) for 24 hours. Several continuous sections of each fresh tumor are then treated with 10 μg / ml anti-axon guidance factor 1 (NP137) or isotype control (NP001) antibodies and 1 μM DMSO. Apoptosis in the treated slices is assessed by immunohistochemical staining of active caspase-3.

[0257] Immunohistochemical staining

[0258] For tumors transplanted in nude mice, ex vivo breast cancer sections, and histological examinations from human clinical trials, tumors were fixed in 10% buffered formalin and embedded in paraffin. Formalin-fixed paraffin-embedded tissue sections of 4 μm thickness were prepared according to routine procedures. The sections were then stained with hematoxylin and eosin and examined with light microscopy. Immunohistochemical analysis was performed on an automated immunostainer (Ventana Discovery XT, Roche, Meylan, France) using the Omnimap DAB kit according to the manufacturer's instructions. The sections were incubated with rabbit anti-cleaved caspase-3 (1:500 dilution, #9661S, Cell Signaling) or rabbit anti-Mcl-1 (1:500 dilution, #HPA031125, Sigma-Aldrich) antibodies. HRP-conjugated anti-rabbit antibodies were applied to the sections. The staining was visualized using a DAB solution with 3,3'-diaminobenzidine as a chromogenic substrate. The sections were then counterstained with Gill's hematoxylin. Finally, the sections were scanned at 20 times with a panoramic scan II (3D Histech, Budapest, Hungary). HALO software and improved algorithm cytonuclear v2.0.8 6 (Indica Labs, Albuquerque, NM, USA) were used to quantify caspase-3 and Mcl-1 positive cells. MCL1 intensity was calculated as a percentage on the surface of tumor cells. All immunohistochemical staining and analysis were performed by Research Pathology Platform East (Plateforme Anatomopathologie Recherche, Université de Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon (CRCL), Lyon, 69373, France).

[0259] Statistical analysis

[0260] Statistical tests are shown 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 on GraphPad software.

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Claims

1. A combination of an anti-AGF1 antibody or an antigen-binding fragment thereof and an inhibitor of an anti-apoptotic protein of the Bcl-2 family for use in treating Mcl1+ and AGF1 receptor+, especially UNC5B+ cancers.

2. The combination for use according to claim 1, wherein the combination is used for simultaneously, separately or sequentially administering an anti-axon guidance factor 1 antibody or an antigen-binding fragment thereof and an inhibitor of an anti-apoptotic protein of the Bcl-2 family to a patient.

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

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

9.

5. The combination for use according to claim 4, wherein the anti-axon guidance factor 1 antibody or its antigen-binding fragment is selected from an anti-axon guidance factor 1 antibody and its antigen-binding fragment comprising a VH and VL amino acid sequence pair, wherein the VH and VL amino acid sequence pair is: 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 or SEQ ID NO: 27 and 19.

6. The combination for use according to claim 4 or 5, wherein the anti-AGF1 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. The combination for use according to any one of claims 1 to 6, comprising the VH of sequence SEQ ID NO: 22 and the VL of sequence SEQ ID NO:

16.

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

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

1.

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 nervitoclax.

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

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

13. A pharmaceutical composition comprising an anti-axon guidance factor 1 antibody or an antigen-binding fragment thereof, an anti-apoptotic protein of the Bcl-2 family, and a pharmaceutically acceptable carrier or excipient. The composition according to claim 13 , wherein the anti-apoptotic protein of the Bcl-2 family is a Bcl-2 inhibitor.

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

16. The 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 navitoc.

17. An anti-AGF1 antibody or an antigen-binding fragment thereof for use as a tumor-specific Mcl-1 inhibitor for treating Mcl-1+ and AGF1 receptor+, especially UNC5B+ tumors.

18. The anti-AGF1 antibody or antigen-binding fragment thereof for use according to claim 17, wherein the tumor is a Mcl-1+, AGF1+ and UNC5B+ tumor. 19 . The anti-axon guidance factor 1 antibody or an antigen-binding fragment thereof for use according to claim 17 or 18 , wherein the anti-axon guidance factor 1 antibody or an antigen-binding fragment thereof is as defined in any one of claims 4 to 9 .

20. The anti-axon guidance factor 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 equal to or higher than grade 3, or equal to or higher than grade 4, or equal to grade 5, as measured by antibody staining using an antibody that binds to Mcl-1, counting tumor cells in the cytoplasm, and evaluating the percentage of stained positive tumor cells.

21. An anti-AGF1 antibody or antigen-binding fragment thereof for use according to any one of claims 17 to 20, wherein the antibody or fragment thereof is used for patients with Mcl1+ and AGF1 receptor+, particularly UNC5B+ tumors, and is further treated with an inhibitor of anti-apoptotic proteins of the Bcl-2 family, preferably an inhibitor of Bcl-2 protein.

22. A method for treating Mcl1+ and NGFR1+, particularly UNC5B+ cancers, comprising administering to a patient suffering from such cancers an effective amount of an anti-NGFR1 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-AGF1 antibody and antigen-binding fragments thereof exert tumor-specific Mcl-1 inhibition or degradation in the patient.

24. The method according to claim 22 or 23, wherein the anti-AGF1 antibody and antigen-binding fragment thereof inhibit the binding of AGF1 and UNC5B, and the dissociated UNC5B allows the CRL3-COMMD2 complex to ubiquitinate Mcl-1, targeting it for proteasomal degradation.

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

26. The method according to any one of claims 22 to 25, wherein the anti-axon guidance factor 1 antibody and the antigen-binding fragment thereof comprises A variable domain VH comprising: - H-CDR1 having the sequence shown in SEQ ID NO: 28; - H-CDR2 having the sequence shown in SEQ ID NO: 29; - H-CDR3 having the sequence shown in SEQ ID NO: 30; A variable domain VL comprising: - L-CDR1 having the sequence shown in SEQ ID NO: 31; - L-CDR2 having the sequence shown in SEQ ID NO: 32; - L-CDR3 having the sequence shown in SEQ ID NO:

9.

27. The method of claim 26, wherein the anti-axon guidance factor 1 antibody and antigen-binding fragments thereof are selected from anti-axon guidance factor 1 antibodies and antigen-binding fragments thereof comprising a VH and VL amino acid sequence pair, wherein the VH and VL amino acid sequence pairs are: 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, and SEQ ID NO: 27 and 19.

28. The method according to claim 26 or 27, wherein the anti-axon guidance factor 1 antibody and the antigen-binding fragment thereof comprises a VH sequence of SEQ ID NO: 22 and a VL sequence of SEQ ID NO:

16.

29. The method of claim 26 or 27, wherein the anti-AGF1 antibody further comprises human IgG1 CH and human IgG1 CLκ.

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

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

33. The method of claim 32, wherein the anti-AGF1 antibody and antigen-binding fragment thereof inhibits the binding of AGF1 to UNC5B, and the dissociated UNC5B allows the CRL3-COMMD2 complex to ubiquitinate Mcl-1, targeting it for proteasomal degradation.

34. The method according to claim 32 or 33, wherein the anti-axon guidance factor 1 antibody and the antigen-binding fragment thereof comprise A variable domain VH comprising: - H-CDR1 having the sequence shown in SEQ ID NO: 28; - H-CDR2 having the sequence shown in SEQ ID NO: 29; - H-CDR3 having the sequence shown in SEQ ID NO: 30; A variable domain VL comprising: - L-CDR1 having the sequence shown in SEQ ID NO: 31; - L-CDR2 having the sequence shown in SEQ ID NO: 32; - L-CDR3 having the sequence shown in SEQ ID NO:

9.

35. The method of claim 34, wherein the anti-axon guidance factor 1 antibody and antigen-binding fragments thereof are selected from anti-axon guidance factor 1 antibodies and antigen-binding fragments thereof comprising a VH and VL amino acid sequence pair, wherein the VH and VL amino acid sequence pairs are: 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, and SEQ ID NO: 27 and 19.

36. The method of claim 35, wherein the anti-axon guidance factor 1 antibody and antigen-binding fragment thereof comprises a VH sequence of SEQ ID NO: 22 and a VL sequence of SEQ ID NO:

16.

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 a Bcl-2 protein.

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

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