Combination of IL-2 / IL-15Rβγ agonists with antibody-drug conjugates to treat cancer

Abstract

The present invention relates to an interleukin-2 / interleukin receptor βγ (IL-2 / IL-15Rβγ) agonist for use in treating cancer in a patient, the IL-2 / IL-15Rβγ agonist being administered in combination with a cytotoxic compound capable of inducing immunogenic cell death (ICD) or in combination with the application of a modality capable of inducing ICD.

Description

[Technical field]

[0001] The present invention relates to the combination of an IL-2 / IL-15Rβγ agonist with an antibody-drug conjugate for the treatment of cancer. [Background technology]

[0002] The past decade has seen significant progress in new cancer treatments through the development of highly selective small molecules that target specific genetic abnormalities that cause the disease (Weinstein 2005, McDermott and Settleman 2009). This approach has seen great success in its application to malignant tumors with a single, well-defined oncolytic driver, but resistance is commonly observed in more complex cancer settings (Rosenzweig 2012, Giroux 2013). Traditional cytotoxic agents (cytotoxins) are another approach to treat cancer. However, unlike target-specific approaches, they suffer from adverse effects resulting from non-specific killing of both healthy and cancer cells. Strategies that combine the potent cell-killing capabilities of potent cytotoxic agents with target specificity would represent a potentially new paradigm in cancer treatment. Antibody-drug-conjugates (ADCs), in which the antibody component provides the specificity for the tumor target antigen and the drug confers cytotoxicity, are such an approach. Further development of modalities for antibody-mediated targeting, such as immunotoxins, immunoliposomes and radionuclide conjugates, coupled with recent advances in ADC technology, represent the next wave of cancer therapeutics.

[0003] In contrast, a lack of therapeutic potential or safety considerations has resulted in only four ADCs currently being approved by the US Food and Drug Administration (FDA) for use in the treatment of cancer, with only one approved for the treatment of solid tumors. A HER2-targeted ADC, ado-trastuzumab emtansine (T-DM1, Kadcyla®), combining the humanized antibody trastuzumab with the potent anti-microtubule cytotoxic agent emtansine (a derivative of maytansine) (DM1), has been approved for the treatment of patients with HER2-positive breast cancer (LoRusso, Weiss et al. 2011; Verma, Miles et al. 2012). However, it has been shown that the therapeutic effect of T-DM1 is entirely dependent on HER2 expression, and only patients with tumors positive at the level of 3+ immunohistochemistry (IHC) by Dako Herceptest™ or with a FISH amplification ratio of ≥ 2.0 by Dako HER2 FISH PharmDx™ test kit are suitable for treatment. There are nearly 30 ADCs in advanced stages of clinical development, some of which have already shown higher therapeutic potential than T-DM1. On the one hand, due to safety considerations, many of these ADCs in development are based on toxins with relatively low potency, which may lead to significantly reduced antitumor efficacy, especially in tumors with low to intermediate target expression. On the other hand, several ADCs loaded with highly potent toxins are in development that are expected to be highly effective but have high off-target toxicity despite targeting, resulting in a limited therapeutic window. To overcome this dilemma and broaden the therapeutic potential of this promising class of drugs, novel concepts are needed to increase its therapeutic window and / or reduce the number and severity of serious adverse events. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Weinstein, J.N. (2005). Oncogenomics:Molecular Approaches to Cancer - Linking Drugs and Genes:Pharmacogenomics, Pharmacoproteomics, Bioinformatics, and the NCI-60, John Wiley & Sons,Inc. [Non-Patent Document 2] McDermott, U. and J. Settleman (2009) J Clin Oncol 27(33):5650-5659 [Non-Patent Document 3] Rosenzweig, SA (2012) Biochemical pharmacology 83(8):1041-1048 [Non-Patent Document 4] Giroux, S. (2013) Bioorg Med Chem Lett 23(2):394-401 [Non-Patent Document 5] LoRusso, PM et al. (2011) Clin Cancer Res 17(20):6437-6447 [Non-Patent Document 6] Verma, S. et al. (2012) N Engl J Med 367(19):1783-1791 Summary of the Invention [Means for solving the problem]

[0005] The inventors have surprisingly found that the combination of a particular class of ADC capable of inducing immunogenic cell death in combination with an emerging class of interleukin-2 / interleukin-15 receptor βγ (IL-2 / IL-15Rβγ) agonists results in improved anti-tumor efficacy. Thus, the present invention provides an IL-2 / IL-15Rβγ agonist for use in treating cancer in a patient, the IL-2 / IL-15Rβγ agonist being administered (a) simultaneously with or sequentially to a cytotoxic compound capable of inducing immunogenic cell death (ICD); (b) simultaneously with or sequentially to the application of a modality capable of inducing ICD; (c) simultaneously with a cytotoxic compound capable of inducing ICD and simultaneously with a modality capable of inducing ICD; (d) simultaneously with a cytotoxic compound capable of inducing ICD and sequentially to a modality capable of inducing ICD; (e) sequentially to a cytotoxic compound capable of inducing ICD and simultaneously with a modality capable of inducing ICD; or (f) sequentially to a cytotoxic compound capable of inducing ICD and sequentially to a modality capable of inducing ICD.

[0006] The concept of immunogenic cell death (ICD), its induction and associated therapeutic benefits provide the rationale for the development of various therapeutic agents and treatment modalities. ICD is a specific cell death modality that occurs in a defined temporal sequence that stimulates an immune response to dying cell antigens characterized by an early surface exposure of chaperones including calreticulin (CRT) and heat shock proteins (HSPs, e.g. HSP70 and HSP90) (Kroemer, Galluzzi et al., 2013). This affects dendritic cell maturation, tumor antigen uptake and presentation, as well as the later release of soluble mediators such as HMGB1 that enhance the presentation of antigens from dying tumor cells to dendritic cells via TLR4 (Fucikova, Kralikova et al., 2011). Such signals act on a series of receptors expressed by dendritic cells. ICD is thought to be a prominent pathway for activation of the immune system against cancer, and understanding of its underlying mechanisms may facilitate the design of highly efficient anti-cancer therapies; however, suboptimal regimens (failing to induce ICD), selective alterations in cancer cells (preventing the release of immunogenic signals during ICD), or defects in immune effectors (causing the immune system to lose perception of ICD) may all contribute to treatment failure (Kroemer, Galluzzi et al. 2013).

[0007] On the other hand, immunotherapy, i.e., treatments that utilize the body's own immune system to help fight disease, aims to exploit the immune system's ability to kill malignant or infected cells while leaving healthy tissues intact. Although the immune system has an inherent ability to detect and eliminate malignancies, tumors and persistent infections have developed mechanisms to evade immune surveillance (Robinson and Schluns, 2017). Potential reasons for immune tolerance include failure of innate immune activation, involvement of the dense stroma as a physical barrier, and possible contribution of immunosuppressive oncogenic pathways (Gajewski, Woo et al., 2013). One group of immunotherapies with some clinical success is cytokine therapy, more specifically interleukin 2 (IL-2), commercially available as aldesleukin / PROLEUKIN® (Prometheus Laboratories Inc.), and the innate immune response via NK cells and CD8 + Interleukin 15 (IL-15) therapy is known to activate both T cell-mediated adaptive immune responses (Steel, Waldmann et al., 2012; Conlon, Miljkovic et al., 2019). Impressive tumor regressions have been observed with IL-2 therapy, but responses are limited to a small percentage of patients and even have high levels of life-threatening toxicity. Furthermore, IL-2 inhibits T cell activation-induced cell death and suppresses immune suppressive regulatory T cells (T reg ) proliferation, as well as immunosuppressant activity (Robinson and Schluns, 2017).

[0008] Both IL-2 and IL-15 act through heterotrimeric receptors with α, β, and γ subunits, but they share a common γ chain receptor (γ cor gamma, CD132) and IL-2 / IL-15Rβ (also known as IL-2Rβ, CD122), and the gamma chain receptor is also shared with IL-4, IL-7, IL-9, and IL-21. As a third subunit, the heterotrimeric receptor contains specific subunits for IL-2 or IL-15, namely IL-2Rα (CD25) or IL-15Rα (CD215). Downstream IL-2 and IL-15 heterotrimeric receptors share JAK1 (Janus kinase 1), JAK3, and STAT3 / 5 (signal transducer and activator of transcription 3 and 5) molecules for intracellular signaling leading to similar functions, although both cytokines also have distinct roles, as reviewed in Waldmann (2015, see e.g. Table 1) and Conlon (2019). Thus, activation of different heterotrimeric receptors by binding of IL-2, IL-15 or their derivatives potentially results in differential regulation of the immune system and potential side effects. Recently, novel compounds have been reported to activate NK cells and CD8 + It was designed to specifically target T cell activation.

[0009] These include intermediate affinity IL-2 / IL-15Rβγ, i.e., NK cells, CD8 + IL-2 / IL-15Rβ and γ expressed on T cells, NKT cells, and γδT cells cThese compounds target the IL-15Rβγ receptor, a receptor composed of IL-15Rα subunits. This is important for safe and potent immune stimulation mediated by IL-15 transpresentation, but the designed compounds RLI-15, ALT-803 and hetIL-15 already contain (part of) the IL-15Rα subunit and therefore mimic the transpresentation of the α subunit by antigen-presenting cells. RLI-15 contains a covalently linked sushi+ domain of IL-15Rα and therefore binds only to the intermediate affinity IL-15Rβγ receptor. On the other hand, RLI-15 does not bind to either IL-15Rα or IL-2Rα. Similarly, ALT-803 and hetIL-15 (NIZ985) possess the IL-15Rα sushi domain or soluble IL-15Rα, respectively, and therefore bind to the intermediate affinity IL-15Rβγ receptor. However, due to their non-covalent binding, there is a chance that the complexes will dissociate in vivo, whereby the dissociated fraction of the applied complexes will still exert other binding (see below). Since ALT-803 contains only the sushi domain of IL-15Rα, which is known to mediate only partial binding to IL-15, whereas the sushi+ domain is required for full binding (Wei, Orchardson et al., 2001), the probability of dissociation seems to be higher for ALT-803 versus hetIL-15. Other examples of complexes of IL-15 and IL-15Rα in various formats are XmAb24306 (WO 2014 / 145806 A2), P-22339 (US Pat. No. 10,206,980), and CUG105 (WO 2019 / 246379 A1).

[0010] Another example of targeting the intermediate affinity IL-2 / IL-15Rβγ receptor is PEGylated IL-2, the hydrolysis of which, for example, NKTR-214 to its most active 1-PEG-IL-2 state, generates a species in which the position of the PEG chain at the IL-2 / IL-2Rα interface prevents binding to the high affinity IL-2Rα receptor, but leaves binding to the intermediate affinity IL-2 / IL-15Rβ unhindered (Charych, Hoch et al., 2016). Additionally, THOR-707 is a site-specific mono-PEGylated form of IL-2 with reduced / deleted IL2Rα chain engagement while retaining binding to the intermediate affinity IL-2Rβγ signaling complex (Joseph, Ma et al., 2019) (WO 2019 / 028419A1). Also, the IL-2 / IL-2Rα fusion protein ALKS4230, which contains the extracellular domain of IL-2Rα and IL-2 circularly permuted (to avoid interactions between the linker and the β and γ receptor chains), selectively targets the βγ receptor, since the α-binding side is already occupied by the IL-2Rα fusion moiety (Lopes, Fisher et al., 2020). Further PEGylated IL-2-based therapeutics specific for IL-2 / IL-25Rβγ are TransCon IL-2 (Rosen, Kvarnhammar et al., 2022) (WO 2019 / 7185705 and WO 2021 / 7245130) and ARX102 (WO 2020 / 056066, WO 2021183832).

[0011] Moreover, the IL-2 mutant IL2v, which has abolished binding to the IL-2Rα subunit, is an example of this class of compound (Klein, Inja et al. 2013; Bacac, Fauti et al. 2016). In addition, NL-201 inhibits the IL-2 receptor βγ c Heterodimer (IL-2Rβγ c), but does not have binding sites for IL-2Rα or IL-15Rα (Silva, Yu et al. 2019). Other IL-2 / IL-25Rβγ-selective IL-2 muteins are STK-012 (Sockolosky, Trotta et al. 2018; Mendoza, Escalante et al. 2019) (WO 2019 / 113221) and MDNA11 (Merchant, Galligan et al. 2022) (WO 2018 / 234862).

[0012] In addition, conditionally activated IL-2 derivatives have been developed, such as WTX-124 (Silva 2022) (WO 2020 / 232305) and XTX202 (O'Neil, Guzman et al., 2021, Abstract and Poster) (WO 2020 / 069398).

[0013] Another strategy to target the IL-2 / IL-15Rβ receptor is the use of IL-15 muteins with reduced or no binding to IL-15Rα (WO 2019 / 166946 A1), thereby reducing or completely avoiding activation of the high affinity IL-15Rαβγ receptor. Similarly, IL-15 is PEGylated to reduce binding to IL-15Rα while retaining binding to the IL-2 / IL-15βγ receptor, examples of which are NKRT-255 (WO 2018 / 213341 A1) and THOR-924, -908, -918 (WO 2019 / 165453 A1). In WO 2016 / 060996 A2, PEGylation for half-life extension is combined with mutated IL-15.

[0014] This class of compounds inhibits IL-2-induced T cell proliferation by targeting the intermediate affinity IL-2 / IL-15Rβγ receptor. regThis avoids the liability associated with targeting the high affinity IL-2 and IL-15 receptors, such as activation or the vascular leak syndrome that can be induced by high concentrations of soluble IL-2 or IL-15. This is because the IL-2Rαβγ high affinity receptor is expressed by CD4 + T reg and the fact that it is additionally expressed on the vascular endothelium and activated by IL-2 cis presentation. Therefore, compounds targeting the high affinity IL-2Rαβγ may be able to inhibit T cell proliferation, as observed for native IL-2 or soluble IL-15 (Conlon, Miljkovic et al. 2019). reg It has the potential to lead to proliferation and vascular leak syndrome (VLS). Potentially, VLS could also be caused by dePEGylated NKTR-214. However, dePEGylated NKT2-214 has a short half-life, and it remains to be determined whether and to what extent this side effect plays a role in clinical development.

[0015] The high-affinity IL-15Rαβγ receptor, activated by IL-15 cis-presentation, is constitutively expressed in T-cell leukemia and mediates the expression of inflammatory NK cells, inflammatory CD8 +It is upregulated in T cells and fibroblast-like synoviocytes (Kurowska, Rudnicka et al., 2002; Perdreau, Mortier et al., 2010), i.e., these cells also express the IL-15Rα subunit. Such activation should be avoided, since IL-15 cis presentation on these cells is involved in the development of T cell leukemia and in exacerbating immune responses potentially inducing autoimmune diseases. Similarly, the high affinity IL-15Rαβγ receptor is expressed on vascular endothelium, and soluble IL-15 can also induce VLS. Other compounds targeting the IL-15 / IL-15Rα complex, and similarly the IL-2 / IL-15Rβγ receptor, either do not bind to the high affinity receptors, since they already possess at least the sushi domain of IL-15Rα, which sterically precludes binding to the heterotrimeric IL-15Rαβγ receptor, or their binding to IL-15Rα is reduced / abolished by mutation or sterically hindered by fusion to other moieties such as PEG, albumin, etc. These side effects induced via high affinity IL-15Rαβγ receptor engagement are induced by native IL-15, but also by non-covalent IL-15 / IL-15Rα complexes such as ALT-803 and hetIL-15, if disruption of the complex occurs in vivo.

[0016] Finally, the high-affinity IL-15Rα is constitutively expressed on myeloid cells, macrophages, B cells and neutrophils (Chenoweth, Mian et al. 2012) and can be activated by native IL-15 as well as by non-covalent IL-15 / IL-15Rα complexes such as ALT-803 and hetIL-15, provided disruption of the complex occurs in vivo.

[0017] Similarly, the above IL-2 based compounds also target linked IL-2 / IL-15Rβγ function via reducing / eliminating binding by mutation (STK-012, MDNA11), sterically hindering binding to IL-2Rα by fusion to soluble IL-2Rα (ALKS4230) or to other moieties such as PEG (NKTR-214, SAR245) to avoid the life-threatening side effects of IL-2.

[0018] In summary, IL-15 has similar immune enhancing properties as wild-type IL-2, but reg Although it is not thought to share immunosuppressive activities such as cell activation and does not cause VLS in the clinic (Robinson and Schluns, 2017), drawbacks of IL-15 therapy include its short in vivo half-life and its dependence on transpresentation by other cell types (Robinson and Schluns, 2017). Both IL-15 therapy and improved IL-2 therapy target the same intermediate affinity IL-2 / IL-15Rβγ while simultaneously detargeting from their respective α chains, thus forming a group of similarly acting compounds, IL-2 / IL-15Rβγ agonists.

[0019] In recent years, these findings have led to a growing number of engineered IL-2 / IL-15Rβγ agonists, some of which are mentioned above, some of which have recently entered clinical development. This list of IL-2 / IL-15Rβγ agonists includes RLI-15 (SOT101, SO-C101), ALT-803 (N803, Anktiva), hetIL-15 (NIZ985), XmAb24306, P-22339, CUG105, NKTR-214, SAR245 (THOR-707), Nemvaleukin alpha (ALKS4230), NL-201, NKRT-255, THOR-924, TransCon IL-2, ARX102, STK-012, MDNA11, WTX-124, XTX202, NKRT-255 and THOR-924, -908, -918.

[0020] As shown by the examples below, stimulation of the immune system with the IL-2 / IL-15Rβγ agonist RLI-15 in combination with the ADC T-DM1 results in synergistic tumor cell killing in vivo, and in combination with SOT102 (containing PNU as a toxin) results in synergistic tumor cell killing in vitro. Without being bound by the mechanism below, it is believed that T-DM1 and PNU induce ICD, thereby priming dendritic cells against dying tumor cells and / or upregulating NK cell receptors on tumor cells. However, this does not support the use of NK cell and CD8 receptor agonists by RLI-15 (or another IL-2 / IL-15Rβγ agonist) to result in superior / synergistic tumor cell killing. + Additional stimulation of immune cells such as cells was required.

[0021] Definitions, Abbreviations and Acronyms An "antibody", also called an "immunoglobulin" (Ig), generally comprises four polypeptide chains, two heavy (H) chains and two light (L) chains, and is therefore a multimeric protein, or its equivalent Ig homologues (e.g., camelid-derived antibodies that contain only heavy chains, single domain antibodies (sdAbs), or nanobodies that can be derived from either the heavy or light chains). The term "antibody" includes antibody-based binding proteins, modified antibody formats that retain their target binding ability. The term "antibody" also includes full-length functional mutants, variants, or derivatives thereof (including, but not limited to, murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies) that retain the essential epitope binding characteristics of the Ig molecule, including Igs that recognize two antigens, bispecific Igs, multispecific Igs, and dual variable domain Igs. The Ig molecules may be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) and allotype. The Ig molecules may be mutated, for example, to increase or decrease affinity for Fcγ receptors or neonatal Fc receptors (FcRn), or for other known reasons.

[0022] As used herein, an "antibody fragment" or "antibody-binding fragment" refers to a molecule comprising at least one polypeptide chain derived from an antibody that is not full-length and exhibits target binding, examples of which include: (i) a Fab fragment, which is a monovalent fragment consisting of a variable light (VL) domain, a variable heavy (VH) domain, a constant light (CL) domain, and a constant heavy 1 (CH1) domain; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region (reduction of the F(ab')2 fragment results in two Fab' fragments with free sulfhydryl groups); (iii) the heavy chain portion of a Fab (Fa) fragment consisting of the VH and CH1 domains; (iv) a variable fragment (Fv) fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a domain antibody (dAb) fragment, which comprises a single variable domain. (vi) isolated complementarity determining regions (CDRs); (vii) single chain Fv fragments (scFv); (viii) diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain and creating two antigen-binding sites; (ix) linear antibodies that contain a pair of tandem Fv segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions with a complementary light chain polypeptide; (x) dual variable domain immunoglobulins; (xi) other non-full-length portions of immunoglobulin heavy and / or light chains, alone or in any combination, or mutants, variants, or derivatives thereof. Engineered antibody variants are reviewed in Holliger and Hudson and Friedman (Holliger and Hudson 2005, Friedman and Stahl 2009). An antibody fragment retains at least some of the binding specificity of the parent antibody, typically at least 10% of the parent's binding activity when activity is expressed on a molar basis. Given the high affinity / avidity of an antibody, even 10% of the parent's binding activity will typically be sufficient to exert its effect and / or such a loss of binding activity could easily be compensated for by a higher dosage.Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more, especially at least 90%, of the binding affinity of the parent antibody for the target.

[0023] The term "modified antibody formats" as used herein encompasses polyalkylene oxide modified scFvs, monobodies, diabodies, camelid-derived antibodies, domain antibodies, bispecific or trispecific antibodies, IgA or two IgG structures linked by a J chain and a secretory component, shark antibodies, New World primate framework and non-New World primate CDRs, IgG4 antibodies with the hinge region removed, IgGs with two additional binding sites engineered into the CH3 domain, antibodies with altered Fc regions to enhance or reduce affinity for Fcγ receptors, dimerization constructs comprising CH3, VL, and VH, etc. Bispecific antibody formats are reviewed, for example, in Godar et al. (2018).

[0024] The Kabat numbering scheme Martin and Allemn (2014) has been applied to the disclosed antibodies.

[0025] As used herein, "antibody-drug conjugate" or "ADC" refers to an antibody (or antibody fragment) to which a pharmacoactive ingredient (API) or payload is covalently linked, such that the API is targeted by the antibody to the antibody's target and exerts its pharmaceutical function primarily in cells expressing the antibody's target. Typically, the API is a cytotoxic drug or toxin capable of effectively killing cells expressing the target. Covalent attachment of the API can be performed non-site-specifically using standard chemical linkers that attach the API to lysine or cysteine ​​residues of the antibody, or preferably site-specifically, for example by mechanisms outlined in Panowski, Bhakta et al. (2014), but preferably using sortase-mediated transpeptidation (as described in WO 2014 / 140317 A2). The linkers used are required to have distinct properties, such as being stable in plasma but releasing the API upon internalization by the (target) cells, as outlined by Jain, Smith et al. (2015), and at the same time being non- or low-immunogenic, increasing solubility to avoid aggregation of the typically hydrophobic APIs used in ADCs. The linker may be cleavable upon binding to the target or in the microtumor environment to increase the bystander effect, or a non-cleavable (non-cleavable) linker to ensure as much as possible release of the API inside the (target) cell. One important feature of ADCs is the average ratio of covalently linked API (drug) to antibody, the so-called drug-antibody ratio (DAR), where typically low variability is preferred for pharmaceuticals, with a DAR of 2-4 (i.e., 2-4 APIs attached to one antibody) being targeted.

[0026] "Immunogenic cell death" or "ICD" as used herein refers to cell death modalities that stimulate an immune response against dead cell antigens (e.g., cancer cells) that exhibit distinct biochemical properties ("ICD markers"), including exposure of so-called DAMPs (Danger-Associated Molecular Patterns), primarily represented by cell surface exposure of calreticulin (CRT), heat shock proteins 70 and 90, secretion of ATP, and release of the non-histone chromatin protein high-mobility group box 1 (HMGB1) (Kroemer, Galluzzi et al., 2013). These markers of ICD can be readily determined as described in the Examples, particularly Example 1.

[0027] A "cytotoxic compound capable of inducing immunogenic cell death (ICD)" or "ICD-inducing compound" is generally a compound or agent that upon incubation induces measurable ICD in vitro, preferably to the same extent as doxorubicin or idarubicin described by Fucikova et al. (2011, 2014), by induction of expression of ICD markers on cell lines, particularly tumor cell lines, by apoptotic, Annexin V positive / DAPI negative cells; a cytotoxic compound is preferably a small molecule, preferably less than about 1000 daltons in size, that can easily enter cells and is cytotoxic, i.e., toxic to cells, due to its low molecular weight.

[0028] A "modality capable of inducing ICD" is generally a therapeutic modality which, when subjected to a tumor cell line, induces measurable ICD in vitro, preferably to the same extent as doxorubicin or idarubicin as described by Fucikova et al., (Fucikova, Kralikova et al., 2011, 2014), by induction of expression of ICD markers in cell lines, particularly tumor cell lines, by apoptotic Annexin V positive / DAPI negative cells.

[0029] "SOT102" is an antibody-drug-conjugate based on the anti-CLDN18.2 antibody hCl1a (SEQ ID NO:20 (heavy chain), SEQ ID NO:21 (light chain)) with the ADCC inactivating heavy chain substitution LALA (L234A / L235A), in which the anthracycline PNU-159682 (PNU) is linked to the C-terminus of the light chain by the non-cleavable linker GGGGSLPQTGG (SEQ ID NO:24)-ethylenediamine (hCl1a-LC-G2-PNU). The preparation of SOT102 is described in Example 7 of WO 2022 / 136642. SEQ ID NO:22 and SEQ ID NO:23 contain the LALA mutations and the non-cleavable linker.

[0030] "Treating," in reference to disease, means providing medical care to a patient, including curative, palliative, or preventative treatment.

[0031] "Low to intermediate HER2 expression" refers to HER2 expression as measured by HercepTest™ having a HER2 protein expression score of 0 to 2+, preferably 0 to 1+ in a surgical or biopsy specimen, i.e., expression levels comparable to those of HercepTest™ MDA-231 to MDA-175 control slides. HercepTest™ is a semi-quantitative immunohistochemical assay to determine HER2 protein overexpression in breast cancer tissues routinely processed for histological evaluation, compared to included control slides representing different levels of HER2 protein expression: MDA-231 (0), MDA-175 (1+) and SK-BR-3 (3+). HER2 3+ refers to high HER2 expression.

[0032] "Interleukin-2", "IL-2" or "IL2" refers to a human cytokine described by NCBI reference sequence AAB46883.1 or UniProt ID P60568 (SEQ ID NO: 1). Its precursor protein has 153 amino acids, with a 20-aa peptide leader, resulting in a 133-aa mature protein. Its mRNA is described in NCBI GenBank reference number S82692.1.

[0033] "IL-2 derivative" refers to a protein having at least 92%, preferably at least 96%, more preferably at least 98%, and most preferably at least 99% identity with the amino acid sequence of mature human IL-2 (SEQ ID NO:2). Preferably, the IL-2 derivative has at least about 0.1%, preferably at least 1%, more preferably at least 10%, more preferably at least 25%, even more preferably at least 50%, and most preferably at least 80% of the activity of human IL-2 as measured by lymphocyte proliferation bioassay. Since interleukins are extremely potent molecules, even low activity such as 0.1% of human IL-2 may still be sufficiently potent, especially when administered at higher doses or when the extended half-life compensates for the loss of activity. The activity is measured by the World Health Organization 1 st Established by the World Health Organization (WHO) International Standard for Interleukin-2 (human), 2 ndIt is expressed in International Units (IUs), which have been replaced by the Second International Standard (Gearing and Thorpe 1988, Wadhwa, Bird et al. 2013). The relationship between potency and protein mass is as follows: 18 million IU PROLEUKIN = 1.1 mg protein. As mentioned above, mutations (substitutions) can be made to extend the half-life, as was done for THOR-707 (Joseph, Ma et al. 2019) (WO 2019 / 028419 A1), or to modify the binding properties of the molecule, for example mutations at L72, F42 and / or Y45, especially F42A, F42B and / or F43, as was done for IL2v (Klein, Inja et al. 2013, Bacac, Fauti et al. 2016) (WO 2012 / 107417 A1). In order to reduce binding to the IL-2α receptor, mutations such as 2G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K, preferably the mutations F42A, Y45A and L72G, may be introduced to specifically link PEG to IL-2. Various other mutations of IL-2 have been described: R38W to reduce toxicity through reduced vascular permeability activity (Hu, Mizokami et al. 2003) (US Patent Application Publication No. 2003 / 0124678); N88R to increase selectivity for T cells over NK cells (Shanafelt, Lin et al. 2000); R38A and F42K to reduce secretion of proinflammatory cytokines from NK cells (Heaton, Ju et al. 1993) (US Patent No. 5,229,109); D20T, N88R and Q126D to reduce VLS (US Patent Application Publication No. 2007 / 0036752); interaction with CD25 and T to enhance efficacy. regR38W and F42K to reduce cell activation (WO 2008 / 003473). Additional mutations may also be introduced, such as T3A to avoid aggregation and C125A to eliminate O-glycosylation (Klein, Waldhauer et al., 2017). Other mutations or combinations of the above may be generated by genetic engineering methods and are well known in the art. Amino acid numbers refer to the 133 amino acid mature IL-2 sequence (SEQ ID NO:2).

[0034] "Interleukin-15", "IL-15" or "IL15" refers to the human cytokine described by NCBI reference sequence NP_000576.1 or UniProt ID P40933 (SEQ ID NO: 3). Its precursor protein has 162 amino acids and has a long 48-aa peptide leader, resulting in a 114-aa mature protein (SEQ ID NO: 4). The complete coding sequence of its mRNA is described in NCBI GenBank reference number U14407.1.

[0035] "IL-15 derivative" or "derivative of IL-15" refers to a protein having at least 92%, preferably at least 96%, more preferably at least 98%, and most preferably at least 99% identity with the amino acid sequence of mature human IL-15 (114aa) (SEQ ID NO: 4). Preferably, the IL-15 derivative has at least 0.1%, preferably 1%, more preferably at least 10%, more preferably at least 25%, even more preferably at least 50%, and most preferably at least 80% of the activity of human IL-15. As for IL-2 above, interleukins are extremely potent molecules and even low activity such as 0.1% of human IL-15 may still be sufficiently potent, especially when administered at higher doses or when an extended half-life compensates for the loss of activity. For IL-15, a number of mutations have also been described to achieve various distinct changes to the molecule: IL-15Rβγβγ cD8N, D8A, D61A, N65D, N65A, Q108R to reduce binding to the receptor (WO 2008 / 143794A1); N72D (in ALT-803) as an activating mutation; N1D, N4D, D8N, D30N, D61N, E64Q, N65D, and Q108E to reduce proliferative activity (U.S. Patent Application Publication No. 2018 / 0118805); L44D, E46K, L47D, V49D, I50D, L66D ... 66E, I67D, and I67E (WO 2016 / 142314A1); N65K and L69R to abrogate IL-15Rb binding (WO 2014 / 207173A1); Q101D and Q108D to inhibit IL-15 function (WO 2006 / 020849A2); S7Y, S7A, K10A, K11A to decrease IL-15Rβ binding (Ring, Lin et al. 2012); Substitution of L45, S51, L52 with D, E, K or R, and substitution of E64, I68, L69 and N65 with D, E, R or K (WO 2005 / 085282 A1); substitution of N71 with S, A or N, substitution of N72 with S, A or N, substitution of N77 with Q, S, K, A or E, and substitution of N78 with S, A or G to reduce deamidation (WO 2009 / 135031 A1); WO 2016 / 060996 A2 discloses IL defines certain regions of -15 as suitable for substitution (see paragraphs 0020, 0035, 00120 and 00130), specifically providing guidance on how to identify potential substitutions to provide anchors for PEG or other modifications (see paragraph 0021); Q108D, which increases affinity for CD122 and impairs recruitment of CD132 to inhibit IL-2 and IL-15 effector function, and N65K, to abrogate CD122 affinity (WO 2017 / 046200 A1);N1D, N4D, D8N, D30N, D61N, E64Q, N65D, and Q108E to gradually reduce the activity of the IL-15 / IL-15Rα complex with respect to the activation of NK cells and CD8 T cells, respectively (see FIG. 51, WO 2018 / 071918A1, WO 2018 / 071919A1). Additionally or alternatively, the skilled artisan can easily make conservative amino acid substitutions. IL-15 derivatives may further be generated by chemical modifications known in the art, for example by PEGylation or other post-translational modifications (see WO 2016 / 060996A2, WO 2017 / 112528A2, WO 2009 / 135031A1);

[0036] Both IL-2 and IL-15 activity can be measured by induction of proliferation of kit225 cells as described by Hori et al. (1987). Preferably, methods such as colorimetry or fluorescence are used to measure proliferation activation by IL-2 or IL-15 stimulation, for example as described by Soman et al. (Soman, Yang et al. 2009) using CTLL-2 cells. As an alternative to cell lines such as kit225 cells, human peripheral blood mononuclear cells (PBMCs) or buffy coats can be used. A preferred bioassay for measuring IL-2 or IL-15 activity is the IL-2 / IL-15 Bioassay Kit (Promega Catalog No. CS2018B03 / B07 / B05) using STAT5-RE CTLL-2 cells.

[0037] "IL-2Rα" refers to the human IL-2 receptor α or CD25.

[0038] "IL-15Rα" refers to the human IL-15 receptor alpha or CD215, described by NCBI reference sequence AAI21142.1 or UniProt ID Q13261 (SEQ ID NO:5). Its precursor protein has 267 amino acids, with a 30-aa peptide leader, resulting in a 231-aa mature protein. Its mRNA is described in NCBI GenBank reference number HQ401283.1. IL-15Rα sushi domain (or IL-15Rα sushi The sushi+ fragment (SEQ ID NO: 6) is a domain of IL-15Rα that is essential for binding to IL-15 (Wei, Orchardson et al. 2001). The sushi+ fragment (SEQ ID NO: 7) contains the sushi domain and a portion of the hinge region C-terminal to the sushi domain, defined as the 14 amino acids located after the IL-15Rα sushi domain, i.e., the IL-15Rα hinge region starts with the first amino acid after the (C4) cysteine ​​residue and ends with the 14th amino acid (counting in the standard "N-terminal to C-terminal" direction). The sushi+ fragment reconstitutes full binding activity to IL-15 (WO 2007 / 046006).

[0039] An "IL-15Rα derivative" refers to a polypeptide comprising an amino acid sequence that is at least 92%, preferably at least 96%, more preferably at least 98%, even more preferably at least 99% identical, and most preferably 100% identical to the amino acid sequence of the sushi domain of human IL-15Rα (SEQ ID NO: 6), preferably the amino acid sequence of the sushi+ domain of human IL-15Rα (SEQ ID NO: 7). Preferably, an IL-15Rα derivative is an N- and C-terminally truncated polypeptide, but lacking the signal peptide (amino acids 1-30 of SEQ ID NO: 5) and the transmembrane domain and intracytoplasmic portion of IL-15Rα (amino acids 210-267 of SEQ ID NO: 5). Thus, a preferred IL-15Rα derivative comprises at least the sushi domain (aa 33-93), but does not extend beyond the extracellular portion of mature IL-15Rα, which is amino acids 31-209 of SEQ ID NO: 5. Particularly preferred IL-15Rα derivatives are the sushi domain of IL-15Rα (SEQ ID NO: 6), the sushi+ domain of IL-15Rα (SEQ ID NO: 7) and soluble forms of IL-15Rα (amino acid 31 to any of amino acids 172, 197, 198, 199, 200, 201, 202, 203, 204 or 205 of SEQ ID NO: 5; see WO 2014 / 066527 (Giron-Michel, Giuliani et al. 2005)). Within the limits provided by this definition, IL-15Rα derivatives may include naturally occurring or introduced mutations. Natural variants and alternative sequences are described, for example, in UniProtKB entry Q13261 (www.uniprot.org / uniprot / Q13261). Furthermore, one skilled in the art can easily identify amino acids that are less conserved among mammalian IL-15Rα homologs or even primate IL-15Rα homologs in order to generate derivatives that are still functional. The respective sequences of mammalian IL-15Rα homologs are set out in WO 2007 / 046006, pages 18 and 19. Additionally or alternatively, one skilled in the art can easily make conservative amino acid substitutions.

[0040] Preferably, the IL-15Rα derivative has at least 10%, more preferably at least 25%, even more preferably at least 50%, and most preferably at least 80% of the binding activity of the human sushi domain for human IL-15 as measured, for example, in (Wei, Orchardson et al. 2001).

[0041] "IL-2Rβ" refers to human IL-Rβ or CD122.

[0042] "IL-2Rγ" refers to the common human cytokine receptor gamma or gamma shared by IL-4, IL-7, IL-9, IL-15, and IL-21. c Or CD132.

[0043] IL-15 / IL-15Rα complex refers to a covalent or non-covalent complex comprising human IL-15 or an IL-15 derivative and human IL-15Rα or an IL-15Rα derivative. Preferably, the complex comprises human IL-15 and the sushi domain of IL-15Rα (SEQ ID NO: 6), the sushi+ domain of IL-15Rα (SEQ ID NO: 7) or a soluble form of IL-15Rα (amino acid 31 to any of amino acids 172, 197, 198, 199, 200, 201, 202, 203, 204 or 205 of SEQ ID NO: 5; see WO 2014 / 066527 (Giron-Michel, Giuliani et al., 2005)).

[0044] "RLI-15" refers to an IL-15 / IL-15Rα complex that is a receptor-linker-interleukin (N-terminus to C-terminus; "RLI") fusion protein of human IL-15Rα sushi+ fragment and human IL-15. Suitable linkers are flexible with low immunogenicity. Examples are described in WO 2007 / 046006 and WO 2012 / 175222. The sushi domain or fragment of human IL-15Rα has the sequence as described by SEQ ID NO:6 from the first to the fourth conserved cysteine, and is optionally extended at the N-terminus by T or IT and at the C-terminus by I. The sushi+ fragment of human IL-15Rα has the sequence as described by SEQ ID NO:7, and further includes and exhibits a portion of the hinge region.

[0045] "RLI2" or "SO-C101" or "SOT101" refers to the IL-15 / IL-15Rα complex, which is a receptor-linker-interleukin fusion protein of human IL-15Rα sushi+ fragment and human IL-15. "RLI2" or "SO-C101" or "SOT101" is represented by SEQ ID NO: 9. The linker used in "RLI2" or "SO-C101" or "SOT101" has the sequence of SEQ ID NO: 8.

[0046] "ALT-803" refers to Altor BioScience Corp.'s IL-15 / IL-15Rα complex, which contains two molecules of an optimized amino acid substitution (N72D) human IL-15 "supergonist" and two molecules of the human IL-15α receptor "sushi" domain fused to a dimeric human IgG1 Fc, which confers stability and potentiates IL-15. N72D :IL-15Rα sushi -Increase the half-life of the Fc complex (see, e.g., US Patent Application Publication No. 2017 / 0088597).

[0047] "Heterodimeric IL-15:IL-Rα", "hetIL-15" or "NIZ985" refers to Novartis' IL-15 / IL-15Rα complex, which is similar to IL-15, and which circulates as a stable molecular complex with human IL-15 and soluble human IL-15Rα (sIL-15Rα), a recombinantly co-expressed non-covalent complex of 170 amino acids of IL-15Rα without the signal peptide and transmembrane and cytoplasmic domains (Thaysen-Andersen, Chertova et al. 2016; see e.g., Table 1).

[0048] An "IL-2 / IL-15Rβγ agonist" is one that does not bind to the IL-2Rα and / or IL-15Rα receptors or has significantly reduced binding to the IL-2Rα and / or IL-15Rα receptors, thereby inhibiting T reg This refers to a molecule or complex that primarily targets the medium affinity IL-2 / IL-15Rβγ receptor, lacking / avoiding stimulation of the IL-2 / IL-15Rβγ receptor. "Significantly reduced binding" in this context means that the binding is reduced by at least 50%, preferably at least 75%, especially at least 90%. An example is IL-15 bound to at least the sushi domain of IL-15Rα, which has the advantage of not being dependent on transpresentation or cell-cell interactions and of having a longer in vivo half-life due to the increased size of the molecule, which has been shown to be significantly more potent than native IL-15 in vitro and in vivo (Robinson and Schluns, 2017). This is because, as outlined above, in addition to IL-15 / IL-15Rα-based complexes, IL-2 / 15Rβ and γ c This can be achieved by mutated IL-2 or chemically modified IL-2 that significantly reduces or timely delays binding to the IL-2α receptor without affecting binding to the receptor or IL-15 muteins.

[0049] "NKTR-214" refers to an IL-2 / IL-15Rβγ agonist based on IL-2, a biological prodrug consisting of IL-2 attached with six releasable polyethylene glycol (PEG) chains (WO 2012 / 065086A1). The presence of multiple PEG chains produces an inactive prodrug, which prevents rapid systemic immune activation upon administration. The use of a releasable linker allows the PEG chains to slowly hydrolyze to successively form active conjugated IL-2 attached by two PEGs or one PEG. The position of the PEG chains at the IL-2 / IL-2Rα interface impedes binding to the high affinity IL-2Rα but leaves binding to the low affinity IL-2Rβ unimpeded, favoring immune activation over inhibition in tumors (Charych, Hoch et al., 2016; Charych, Khalili et al., 2017).

[0050] THOR-707 refers to an IL-2 / IL-15Rβγ agonist (Joseph, Ma et al., 2019) (WO 2019 / 028419 A1) based on a site-specific, mono-PEGylated form of IL-2 with reduced / deleted IL2Rα chain engagement while retaining binding to the intermediate affinity IL-2Rβγ signaling complex.

[0051] ALKS4230 refers to a circularly permuted IL-2 (to avoid interactions between the linker and the β and γ receptor chains) in which the extracellular domain of IL-2Rα selectively targets the βγ receptor, since the α-binding side is already occupied by the IL-2Rα fusion moiety (Lopes, Fisher et al. 2020).

[0052] NL-201 inhibits the IL-2 receptor βγ c Heterodimer (IL-2Rβγ c ) but does not have binding sites for IL-2Rα or IL-15Rα (Silva, Yu et al., 2019).

[0053] NKRT-255 refers to a PEG-conjugated human IL-15-based IL-2 / IL-15Rβγ agonist that retains binding affinity for IL-15Rα, exhibits reduced clearance, and provides a sustained pharmacodynamic response (WO 2018 / 213341 A1).

[0054] THOR-924, -908, -918 refer to IL-2 / IL-15Rβγ agonists based on PEG-conjugated IL-15 with unnatural amino acids used for site-specific PEGylation and reduced binding to IL-15Rα (WO 2019 / 165453 A1).

[0055] "IL2v" refers to an IL-2-based IL-2 / IL-15Rβγ agonist, an IL-2 variant with abolished binding to the IL-2Rα subunit with SEQ ID NO: 10. IL2v is used, for example, in a fusion protein fused to the C-terminus of an antibody. IL2v was designed by destroying the binding ability to IL-2Rα through amino acid substitutions F42A, Y45A and L72G (conserved between human, mouse and non-human primates), by abolishing O-glycosylation through amino acid substitution T3A, and by avoiding aggregation by a C125A mutation as in aldesleukin (numbering based on UniProt ID P60568 excluding the signal peptide) (Klein, Waldhauer et al., 2017). IL2v is used as a fusion partner with an antibody, for example, as a fusion partner with a non-targeting IgG (IgG-IL2v) to increase its half-life (Bacac, Colombetti et al., 2017). In RG7813 (or cergutuzumab amunaleukin, RO-6895882, CEA-IL2v), IL2v is fused to an antibody targeting carcinoembryonic antigen (CEA) with a heterodimeric Fc lacking FcγR and C1q binding (Klein 2014; Bacac, Fauti et al. 2016; Klein, Waldhauer et al. 2017). And in RG7461 (or RO6874281 or FAP-IL2v), IL2v is fused to a tumor-specific antibody targeting fibroblast activation protein alpha (FAP) (Klein 2014).

[0056] "Immune checkpoint inhibitors" or "checkpoint inhibitors" for short refer to a class of drugs that block specific proteins made by some types of immune system cells, such as T cells, and some cancer cells. These proteins help check the immune response and can stop T cells from killing cancer cells. When these proteins are blocked, the "brakes" on the immune system are released and T cells can better kill cancer cells. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2 (see definition from the National Cancer Institute at the National Institute of Health, www.cancer.gov / publications / dictionaries / cancer-terms / def / immune-checkpoint-inhibitor), as reviewed by Darvin et al. (2018). Examples of such checkpoint inhibitors are anti-PD-L1, anti-PD-1, anti-CTLA-4 antibodies, but also antibodies against LAG-3 or TIM-3, or blockers of BTLA currently being tested in the clinic (De Sousa Linhares, Leitner et al., 2018). Further promising checkpoint inhibitors are anti-TIGIT antibodies (Solomon and Garrido-Laguna, 2018).

[0057] "Anti-PD-L1 antibody" refers to an antibody or antibody fragment thereof that binds to PD-L1. Examples are avelumab, atezolizumab, durvalumab, KN035, MGD013 (bispecific for PD-1 and LAG-3).

[0058] "Anti-PD-1 antibody" refers to an antibody or antibody fragment thereof that binds to PD-1. Examples are pembrolizumab, nivolumab, cemiplimab (REGN2810), BMS-936558, SHR1210, IBI308, PDR001, BGB-A317, BCD-100, and JS001.

[0059] "Anti-PD-L2 antibody" refers to an antibody or antibody fragment thereof that binds to anti-PD-L2. An example is sHIgM12.

[0060] "Anti-CTLA4 antibody" refers to an antibody or antibody fragment thereof that binds to CTLA-4. Examples are ipilimumab and tremelimumab (ticilimumab).

[0061] "Anti-LAG-3" antibody refers to an antibody or antibody fragment thereof that binds to LAG-3. Examples of anti-LAG-3 antibodies are leratolimab (BMS986016), Sym022, REGN3767, TSR-033, GSK2831781, MGD013 (bispecific for PD-1 and LAG-3), and LAG525 (IMP701).

[0062] "Anti-TIM-3 antibody" refers to an antibody or antibody fragment thereof that binds to TIM-3. Examples are TSR-022 and Sym023.

[0063] "Anti-TIGIT antibody" refers to an antibody or antibody fragment thereof that binds to TIGIT. Examples are tiragolumab (MTIG7192A, RG6058) and etigilimab (WO 2018 / 102536).

[0064] "Percentage of identity", "percentage of identity" or "% identity" between two amino acid sequences refers to the percentage of identical amino acids between the two sequences compared, obtained using the best alignment of those sequences, where this percentage is purely statistical, and the differences between these two sequences are spread randomly across the amino acid sequences. As used herein, "best alignment" or "optimal alignment" refers to the alignment with the highest determined percent identity (see below). Sequence comparison between two amino acid sequences is usually achieved by comparing these sequences that have been pre-aligned according to the best alignment. This comparison is performed on a comparison segment to identify and compare local regions of similarity. In addition to manual methods, the best sequence alignment for the comparison can be achieved by using the global homology algorithm developed by Smith and Waterman (1981), by using the local homology algorithm developed by Needleman and Wunsch (1970), by using the similarity method developed by Pearson and Lipman (1988), by using computer software that uses such algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI, USA), by using the MUSCLE multiple alignment algorithm (Edgar 2004), or by using CLUSTAL (Goujon, McWilliam et al. 2010). To obtain the best local alignment, the BLAST software can be used, preferably with the BLOSUM62 matrix.The percent identity between two amino acid sequences is determined by comparing the two optimally aligned sequences, which may include additions or deletions relative to the reference sequence in order to obtain optimal alignment between the two sequences. The percent identity is calculated by determining the number of identical positions between the two sequences, dividing this number by the total number of positions compared, and multiplying the result by 100 to obtain the percentage of identity between the two sequences.

[0065] Conservative amino acid substitutions refer to the substitution of an aliphatic amino acid (i.e., glycine, alanine, valine, leucine, isoleucine) with another aliphatic amino acid, a hydroxyl or sulfur / selenium containing amino acid (i.e., serine, cysteine, selenocysteine, threonine, methionine) with another hydroxyl or sulfur / selenium containing amino acid, an aromatic amino acid (i.e., phenylalanine, tyrosine, tryptophan) with another aromatic amino acid, a basic amino acid (i.e., histidine, lysine, arginine) with another basic amino acid, or an acidic amino acid or its amide (aspartic acid, glutamic acid, asparagine, glutamine) with another acidic amino acid or its amide.

[0066] When "administered in combination" is described, this typically does not mean that the two agents are co-formulated and administered simultaneously, but rather that one agent has a label that identifies its use in combination with the other agent. So, for example, an IL-2 / IL-15Rβγ agonist is for use in treating or managing cancer or an infectious disease, and this use includes administering the IL-2 / IL-15Rβγ agonist and an additional therapeutic agent simultaneously, separately, or sequentially, or vice versa. However, nothing in this application should preclude two combined agents from being provided as a package or kit, or even from being co-formulated and administered together when the dosing schedules match. So, "administered in combination" includes (i) the agents are administered together, such as by intra-articular infusion, intra-articular injection, etc., (ii) the agents are administered separately but in parallel according to the given method of administration of each agent, and (iii) the agents are administered separately and sequentially. Concurrent administration in this context preferably means that both treatments are started together, for example, the first administration of each drug in the treatment regimen is administered on the same day.Considering the potential different treatment schedules, it is clear that administration does not always have to occur on the same day during the following days / weeks / months.Generally, concurrent administration aims to have both drugs present in the body at the same time at the beginning of each treatment cycle. Sequential administration in this context preferably means that both treatments are initiated sequentially, e.g., the first administration of the first drug occurs at least one day, preferably several days or a week, before the first administration of the second drug to allow for the body's pharmacodynamic response to the first drug before the second drug becomes active. Thereafter, the treatment schedules may overlap or be intermittent with each other, or may follow directly after each other.

[0067] "About" when used in conjunction with a value means ±10% of that value, preferably ±5% and especially ±1% of that value.

[0068] Where the term "comprising" is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising of". In the following, when a group is defined as comprising at least a certain number of embodiments, this is to be understood as also disclosing a group that preferably consists only of these embodiments.

[0069] Where an indefinite or definite article such as "a", "an" or "the" is used when referring to a singular noun, this includes a plural of that noun, unless something else is specifically stated.

[0070] Thus, the terms "at least one," "at least one," as in "at least one chemotherapeutic agent," may be meant to mean one or more chemotherapeutic agents. The term "a combination thereof," in the same context, refers to a combination including more than one chemotherapeutic agent.

[0071] "wt" is used for wild type. "qxw", derived from the Latin quaque / each, every, means every x weeks, for example q2w means every two weeks. "sc" stands for subcutaneous. "iv" stands for intravenous. "ip" stands for intraperitoneal. "SoC" stands for standard of care.

[0072] Technical terms are used with their common meaning. Where a specific meaning is conveyed to a particular term, the definition of the term is given below in the context in which the term is used.

[0073] In a first aspect, the present invention provides an interleukin-2 / interleukin-15 receptor βγ (IL-2 / IL-15Rβγ) agonist for use in treating cancer in a patient, the IL-2 / IL-15Rβγ agonist comprising: a. Administered simultaneously with or sequentially to a cytotoxic compound capable of inducing immunogenic cell death (ICD); b. Administered simultaneously or sequentially to the administration of a modality capable of inducing an ICD; c. Administered simultaneously with a cytotoxic compound capable of inducing ICD and simultaneously with a modality capable of inducing ICD; d. Administered simultaneously with a cytotoxic compound capable of inducing ICD and sequentially with a modality capable of inducing ICD; e. Administered sequentially to a cytotoxic compound capable of inducing ICD and simultaneously with a modality capable of inducing ICD; or f. Administered sequentially to a cytotoxic compound capable of inducing ICD and sequentially to a modality capable of inducing ICD Concerning IL-2 / IL-15Rβγ agonists.

[0074] Disclosed herein are combination therapies that enhance the antitumor effects of IL-2 / IL-15Rβγ agonists that primarily target the medium affinity IL-2 / IL-15Rβγ receptor, as well as the antitumor effects of cytotoxic compounds capable of inducing ICD and / or modalities capable of inducing ICD. Such enhancement of antitumor effects may result in improved efficacy of the combination treatment compared to each monotherapy, as measurable, for example, in increased response rate, overall survival or progression-free (progression-free) survival, and / or may result in application of lower doses of ICD-inducing cytotoxic compounds / modalities / weaker treatments with ICD-inducing cytotoxic compounds / modalities, without interfering with the antitumor effect compared to monotherapy, thereby reducing their toxicity / side effects. Reducing the dose of highly toxic compounds / modalities in combination with the claimed IL-2 / IL-15Rβγ agonists may lead to an increase in the patient population eligible for such toxic compounds / modalities. This is because patients in the early stages of a given treatment may be receptive to such combination treatments based on a more tolerable side effect profile, or tumor indications in which physicians were previously hesitant to use toxic compounds / modalities due to side effects may now become treatable for such combinations when combined with IL-2 / IL-15Rβγ agonists. More specifically, the combination of an IL-2 / IL-15Rβγ agonist with a cytotoxic compound capable of inducing ICD or a modality capable of inducing ICD results in a synergistic enhancement or antitumor activity of the combination treatment compared to the individual treatments.

[0075] The inventors observed in vitro that activation of NK cells from human PBMCs by IL-2 / IL-15Rβγ agonists (SOT101 / SO-C101 / RLI-15 in this study) as a measure of having a strong innate antitumor response was significantly stronger when dying tumor cells expressed the ICD markers Hsp70, Hsp90 and CRT, as well as increased expression of the NK cell ligands CD112, CD155, ULBP3 and ULBP2 / 5 / 6 induced in this study by incubation with trastuzumab emtansine / Kadcyla®. Trastuzumab emtansine is an antibody-drug conjugate consisting of the humanized monoclonal antibody trastuzumab / Herceptin® against the tumor target HER2, covalently linked to the cytotoxic compound mertansine / DM1. Due to the fact that Kadcyla was washed out before incubation with activated PBMCs, a direct interaction of Kadcyla with immune cells could be excluded and we conclude that the early apoptotic state / ICD of the cell population contributes significantly to this effect and therefore other cytotoxic compounds capable of inducing ICD or modalities capable of inducing ICD would have a very similar synergistic effect.

[0076] Similarly, the combination of SOT102, a CLDN18.2-targeting ADC with the anthracycline PNU-159682 as toxin, synergizes with SOT101 in NK cell-based cytotoxicity assays in vitro, and such effects are caused or contributed to by the induction of ICD. ADCs with PNU as toxin have previously been described to induce ICD (D'Amico, Menzel et al., 2019). Activation of danger signals by toxins / chemotherapy as well as radiotherapy, for example, was reported to result in increased Hsp70 cell surface expression on tumor cells in vitro and in vivo, promoting NK cell-mediated cytotoxicity (Zingoni, Fionda et al., 2017). Recently, externalized CRT, a hallmark of ICD, was identified as an activating ligand for the NKp46 receptor on NK cells, whose binding induces NKp46 signaling, while inhibition of this interaction inhibits NKp46-mediated killing (Santara, Crespo et al., 2021).

[0077] Thus, the inventors conclude that there is a direct mechanistic link between the induction of ICD in tumor cells by ICD-inducing cytotoxic compounds and / or modalities as described herein, rendering them more susceptible to the cytotoxic activity of NK cells, which in addition can be enhanced by the described IL-2 / IL-15Rβγ agonists, such as SOT101, which are potent activators of NK cells. NK cell activation is thought to have predictive value for in vivo antitumor efficacy, and indeed a similar synergistic effect was observed in vivo in a mouse orthotopic breast cancer model.

[0078] In general, the observed synergistic effect of the combination of ICD-inducing cytotoxic compounds or modalities with IL-2 / IL-15Rβγ agonists may be used (i) to reduce the dose of the cytotoxic compound or the intensity of the modality (e.g. non-ablative / low dose radiotherapy) to reduce the side effects induced by the cytotoxic compound or modality while producing at least the same therapeutic benefit to the patient due to the combined action, (ii) to avoid recurrence of the tumor disease due to strong ICD-induced immune surveillance in the combined treatment, and / or (iii) in the case of antibody-drug-conjugates, to broaden the patient population, since also patients with lower target expression (compared to the target level of the label of the respective ADC) will benefit from the combined treatment.

[0079] In one embodiment, the IL-2 / IL-15Rβγ agonist is administered before and / or after the cytotoxic compound capable of inducing said ICD, or sequentially before and / or after the modality capable of inducing said ICD. Considering the different administration / treatment schedules of such cytotoxic compound and IL-2 / IL-15Rβγ agonist or such therapeutic modality and IL-2 / IL-15Rβγ agonist, it is very typical that the IL-2 / IL-15Rβγ agonist is not administered exactly at the same time as such cytotoxic compound or modality.

[0080] In a preferred embodiment in the case of sequential administration, the IL-2 / IL-15Rβγ agonist is administered after the cytotoxic compound capable of inducing ICD or after the modality capable of inducing ICD. Since the induction of ICD by such cytotoxic compounds or such modalities takes some time, it may be beneficial to administer the IL-2 / IL-15Rβγ agonist later, so that after the changes to the cell surface and the release of soluble mediators of ICD have occurred, sufficient time is provided for NK and CD8 cells to be activated to boost the immune system against such tumor cells undergoing ICD. Preferably, the time difference between the last administration / treatment of the cytotoxic compound or modality inducing ICD and the administration of the IL-2 / IL-15Rβγ agonist is about 6 hours to about 2 weeks, more preferably about 1 day to about 7 days, especially about 1 day to about 4 days. This timing may vary depending on the nature of the cytotoxic compound. Free drugs may induce ICD more quickly than, for example, ADCs due to their relatively long in vivo half-life, surface binding, internalization, transport through endosomal / lysosomal pathways, construct degradation, release of the cytotoxic payload from lysosomes, and the necessary processing including activation of cell death pathways, and the timing may further vary from cell type to cell and depending on the target antigen (Bauzon, Drake et al. 2019).

[0081] In another embodiment, the IL-2 / IL-15Rβγ agonist and the cytotoxic compound capable of inducing the ICD are provided as components of the same pharmaceutical composition or as components of separate pharmaceutical compositions and administered simultaneously. Simultaneous treatment is preferred in order to minimize the effort of the patient to go to a hospital or doctor for administration of the drug. Furthermore, in certain combinations, it may be feasible that the IL-2 / IL-15Rβγ agonist and such a cytotoxic compound can be co-formulated into a single pharmaceutical composition to simplify administration.

[0082] In one embodiment, the cytotoxic compounds capable of inducing ICD are anthracyclines; microtubule destabilizing agents including vinca alkaloids, taxanes, epothilones, eribulin, auristatins (e.g., MMAE or MMAF), maytansine or maytansinoids, and tubulysins; bleomycin; proteasome inhibitors including bortezomib; topotecan, exatecan, and exatecan derivatives such as DS-8201a, DX-8951 / DXd (Kitai, Kawasaki et al. 2017; Iwata, Ishii et al. 2018; Haratani, Yonesaka et al. 2019). 2020); alkylating agents including cyclophosphamide, platinum complexes including oxaliplatin, and pyrrolo-benzodiazepines (PBDs) (Rios-Doria, Harper et al., 2017); and nucleoside analogs including gemcitabine (preferably in combination with inhibitory damage-associated molecular pattern (DAMP) blockade) (Hayashi, Nikolos et al., 2021). Cytotoxic compounds capable of inducing ICD have been repeatedly reviewed (Pol, Vacchelli et al., 2015; Diederich 2019; Zhou, Wang et al., 2019). SN38 is preferably excluded, since other topoisomerase I inhibitors such as DS-8201a have higher potency and induce more immunogenic cell death (Iwata, Ishii et al., 2018).

[0083] Anthracyclines (and derivatives) are a class of cytotoxic compounds of bacterial origin applied in many oncological indications including leukemia, lymphoma, breast, gastric, ovarian, bladder and lung cancer, which act primarily by intercalating into DNA and thus interfering with DNA replication and transcription, e.g., by inhibiting topoisomerase II. Members of this class are daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, nemorubicin and PNU-159682 ((3'-deamino-3",4'-anhydro-[2"(S)-methoxy-3"(R)-oxy-4"-morpholinyl]]) (briefly "PNU") - a metabolite of nemorubicin (Quintieri, Geroni et al., 2005) - which has been shown to induce ICD (Fucikova, Kralikova et al., 2011).

[0084] Microtubule destabilizing agents ("MDAs") are another class of compounds that induce ICD (Diederich 2019), a diverse class of compounds grouped together by their mechanism of action with microtubules as targets, affecting cell proliferation, trafficking, signaling, and migration (Dumontet and Jordan 2010). This class includes vinca alkaloids (vinblastine, vincristine, vinflunine, sevipabulin), taxanes (paclitaxel, docetaxel, etc.), (Dumontet and Jordan), as reviewed by Diederich (2019), Dudek et al. (2013), Dumontet and Jordan (2010), and Gerber et al. (2016), (Dumontet and Jordan). These include eribulin, epothilones including epothilones A-F, 7A7, and patupilone, auristatins including monomethylauristatin E (MMAE) and monomethylauristatin F (MMAF), maytansines and maytansinoids such as mertansine / emtansine (DM1), ansamitocins and ravtansine / sorabutansine (DM4), tubulysins, colchicine, etc. (see, e.g., Figure 1 in Dumontet and Jordan (2010)).

[0085] Further cytotoxic compounds that induce ICD are bleomycin; proteasome inhibitors such as bortezomib and shikonin; alkylating agents such as cyclophosphamide, mitoxantrone, platinum complexes including oxaliplatin, cardiac glycosides (Dudek, Garg et al. 2013; Pol, Vacchelli et al. 2015; Gerber, Sapra et al. 2016), and pyrrolo-benzodiazepines (PBDs) (Zhou, Wang et al. 2019), preferably their prodrugs pro-PBDs (Vlahov, Qi et al. 2017). Shikonin, a bioactive phytochemical that inhibits the 20S subunit of the proteasome (it is a proteasome inhibitor like bortezomib), has been shown to induce ICD in cancer cells, characterized by the induction of HSP70, calreticulin and GRP78 expression, as well as the induction of functional maturation of DCs. In addition, calicheamicins, a class of enediyne antitumor antibiotics derived from Micromonospora echinospora, have been reported to induce immunogenic cell death (Tan, Lam et al., 2018). I (LL-E33288) is the best known member, and further calicheamicin derivatives are described in WO 2019 / 110725.

[0086] Topotecan and DX-8951 / DXd have also been described to induce immunogenic cell death (Kitai, Kawasaki et al. 2017; Iwata, Ishii et al. 2018; Haratani, Yonesaka et al. 2020) since they upregulated the expression of DC maturation and activation markers both in vitro and in vivo, increased intratumoral DC populations in vivo (Iwata, Ishii et al. 2018), and observed the release of HMGB-1 from DXd-treated cancer cells (Haratani, Yonesaka et al. 2020).

[0087] Several ICD-inducing cytotoxic compounds, including anthracyclines (Minotti, Menna et al. 2004), (WO 2016 / 102679A1), MMAE, DM1 (Diederich 2019), PBDs (Rios-Doria, Harper et al. 2017; Zhou, Wang et al. 2019) and tubulysins (Rios-Doria, Harper et al. 2017), are of great interest as payloads for ADCs. It is therefore a preferred embodiment of the present invention that such cytotoxic compounds capable of inducing ICD are covalently linked to antibodies to form antibody-drug conjugates (ADCs). ADCs are a rapidly growing class of anti-cancer drugs that target cytotoxic compounds to molecular targets, typically expressed on the surface of target cells, by chemical conjugation to anti-cancer antibodies, thereby reducing systemic exposure and toxicity. Currently, various design strategies are used, including target selection, design of the antibody moiety, covalent linker between the antibody and the cytotoxic compound, and selection of the cytotoxic compound, or payload as it is often referred to in this context. Currently, four ADC products are commercially available (gemtuzumab ozogamicin / Mylotarg®, brentuximab vedotin / Adcetris®, trastuzumab emtansine / Kadcyla®, and inotuzumab ozogamicin / Besponsa®), and more than 60 ADCs are currently in clinical development (Khongorzul, Ling et al., 2020), with three additional approvals in 2019 (trastuzumab deruxtecan / Enhertu®). (R), enfortumab vedotin / Padcev®, and polatuzumab vedotin (Polivy®). In 2020, sacituzumab govitecan (Trodelvy®) and belantamab mafodotin-blmf (Blenrep®) were approved by the FDA, followed by loncastuximab tesillin-lpyl (Zynlonta®) and tisotumab vedotin-tftv (Tivdak®) in 2021.

[0088] In a preferred embodiment, the cytotoxic compound capable of inducing ICD is an anthracycline, maytansine or maytansinoid, a topoisomerase I inhibitor or a calicheamicin derivative. Specifically, ADCs with anthracyclines and ADCs with maytansine or maytansinoids as payloads have been described to induce ICD. D'Amico et al. (2019) reported that an ADC composed of trastuzumab linked to PNU (anthracycline) (T-PNU) induces ICD in CD8 + (2019) show that maytansine and maytansine-based ADCs induce the three main features of ICD in vitro and conclude that maytansine, MMAE, tubulysin and PBD appear to have similar immune stimulatory activity in vivo. Thus, brentuximab vedotin, an MMAE-conjugated anti-CD30 antibody, inhibits tumor-infiltrating CD8+ / -CD30+ cells in a human HER2-expressing syngeneic breast cancer model that is resistant to trastuzumab and trastuzumab emtansine in a T cell-dependent manner, thus confirming a PNU-mediated antitumor immune response also in the context of ADCs. Moreover, T-PNU promoted the generation of immune memory and protected treated animals from tumor rechallenge (reimplantation). Bauzon et al. (2019) show that maytansine and maytansine-based ADCs induce the three main features of ICD in vitro and conclude that maytansine, MMAE, tubulysin and PBD appear to have similar immune stimulatory activity in vivo. Thus, brentuximab vedotin, an MMAE-conjugated anti-CD30 antibody, inhibits tumor-infiltrating CD8+ / -CD30+ cells in a human HER2-expressing syngeneic breast cancer model that is resistant to trastuzumab and trastuzumab emtansine in a T cell-dependent manner, thus confirming a PNU-mediated antitumor immune response also in the context of ADCs. Moreover, T-PNU promoted the generation of immune memory and protected treated animals from tumor rechallenge (reimplantation). + It increased the numbers of T cells and proved effective in patients who expressed little or no target antigen, suggesting a potential indirect immune-mediated mechanism (summarized in Bauzon, Drake et al., 2019).

[0089] Preferably, the anthracycline is selected from the group consisting of daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone and PNU-159682 (PNU), and the maytansine or maytansinoid is selected from maytansine, mertansine / emtansine (DM1), ansamitocin and ravtansine / soravtansine (DM4).

[0090] In another preferred embodiment, the cytotoxic compound capable of inducing ICD is a topoisomerase I inhibitor, preferably topotecan, exatecan and exatecan derivatives such as DX-8951 / DXd. Both trastuzumab deruxtecan (DS-8201a), an anti-HER2 antibody trastuzumab linked to the exatecan derivative DX-8951 / DXd, and patritumab deruxtecan (U3-1402), an anti-HER3 antibody patritumab linked to DX-8951 / DXd, are approved / clinical stage ADCs whose topoisomerase I payloads have been shown to induce immunogenic cell death.

[0091] In another preferred embodiment, the cytotoxic compound capable of inducing ICD is a calicheamicin derivative, preferably calicheamicin γ1. I (LL-E33288) or a calicheamicin derivative described in WO 2019 / 110725.

[0092] In further embodiments, the antibody is an antibody that specifically binds to HER2, preferably trastuzumab, SYD985 or MEDI4276, more preferably trastuzumab; an antibody that binds to Nectin-4, preferably enfortumab; an antibody that binds to CD33, preferably gemtuzumab or IMGN779, more preferably gemtuzumab; an antibody that binds to CD30, preferably brentuximab; an antibody that binds to CD22, preferably inotuzumab, or CD79B, preferably polatuzumab. Further preferred targets / antibodies are TROP2 / sacituzumab, FOLR1 / mirvetuximab, BCMA / GSK2857916, GPNMB / glembatumumab, mesothelin / anetuzumab, CEACAM5 / labetuzumab or SAR408701, PSMA / antibodies of NCT01695044 and NCT02020135 or MEDI3726, CD19 / coltuximab, EGFR / depatuximab, ENPP3 / AGS-16C3F, EFNA4 / PF-06647263, HER3 / patritumab, CD352A / SGN-CD352A, CD37 / AGS67E, FLT3 / AGS-62P1, ROR-1 / NBE-002 and Claudin 18.2 / zolbetuximab or humanized variants thereof (e.g. as disclosed in WO 2021 / 111003 A1) or humanized antibodies, in particular hCl1a as disclosed in Table 3 of WO 2021 / 130291 A1.

[0093] In another preferred embodiment, the ADC is trastuzumab emtansine / Kadcyla®, trastuzumab deruxtecan / Enhertu®, gemtuzumab ozogamicin / Mylotarg®, inotuzumab ozogamicin / Besponsa®, brentuximab vedotin / Adcetris®, enfortumab vedotin / Padcev® and polatuzumab vedotin / Polivy®. Particularly preferred is trastuzumab emtansine (also called ado-trastuzumab emtansine). Further particularly preferred is enfortumab vedotin. A further preferred ADC is sacituzumab govitecan. A further preferred ADC is belantamab mafodotin-blmf. A further preferred ADC is loncastuximab tesillin-lpyl.A further preferred ADC is tisotumab vedotin-tftv.

[0094] In another preferred embodiment, the IL-2 / IL-15Rβγ agonist is for use in patients suffering from tumors expressing HER2, preferably the patients have been diagnosed with tumors with low to moderate HER2 expression. The inventors have demonstrated synergy with trastuzumab emtansine (Kadcyla®), which is approved for the treatment of patients with HER2-positive tumors, particularly HER2-positive metastatic breast cancer who have previously been treated with trastuzumab and a taxane, either separately or in combination. HER2-positive according to the Kadcyla® label refers to patients with breast cancer that has HER2 overexpression, thus high expression of HER2, defined as 3+ IHC by Dako HercepTest™ or FISH amplification ratio ≧2.0 by Dako HER2 FISH PharmDx™ test kit. In one embodiment, the selection of HER2 patients is done according to the label of trastuzumab emtansine, i.e., patients are selected to have high HER2 expression, e.g., HercepTest™ 3+. For combination treatment with IL-2 / IL-15Rβγ agonists in patients with high HER2 expression, we expect that the high recurrence rate typically observed with Kadcyla treatment will be significantly reduced, as observed in the orthotopic huHER2 / EMT-6 breast cancer model (see examples). Alternatively or additionally, for such combination treatment, the dose of Kadcyla® (or ADC in general) may be reduced to reach at least the same efficacy in combination with IL-2 / IL-15Rβγ agonists, but with reduced side effects.

[0095] In another embodiment, patients with low to intermediate HER2 expression are also selected for combination therapy with Kadcyla® and an IL-2 / IL-15Rβγ agonist, preferably SOT101. Given the synergistic enhancement of treatment compared to monotherapy, the inventors speculate that lower expression may be sufficient to obtain therapeutic benefit for the patient.

[0096] Other HER2-overexpressing tumors are ovarian cancer, gastric cancer, adenocarcinoma of the lung, uterine cancer (e.g., uterine serous endometrial carcinoma), salivary gland ductal carcinoma, renal cancer, endometrial cancer, colorectal cancer, head and neck cancer, urothelial carcinoma, breast cancer and cervical cancer, which preferably have a confirmed status of HER2 overexpression, making them, along with breast cancer, preferred tumor indications for treatment with an IL-2 / IL-15Rβγ agonist in combination with trastuzumab emtansine.

[0097] In another preferred embodiment, the IL-2 / IL-15Rβγ agonist is for use in patients suffering from tumors expressing Nectin-4, preferably diagnosed with locally advanced or metastatic urothelial carcinoma who have previously received a programmed death receptor-1 (PD-1) or programmed death-ligand 1 (PD-L1) inhibitor and a platinum-containing chemotherapy in the neoadjuvant / adjuvant, locally advanced or metastatic setting. Enfortumab vedotin (also called enfortumab vedotin-ejfv) has been approved for this indication, and given the known induction of ICD by its MMAE payload, synergy with the IL-2 / IL-15βγ receptor is expected by the inventors based on the findings of the present invention. Nectin-4 is an adhesion protein located on the surface of cells and was detected in all patients tested in the clinical trials leading to approval. Thus, no testing for patient stratification is required. Administration of enfortumab vedotin is preferably pursued according to its label.Other nectin-4 positive tumors are bladder cancer in general, ovarian cancer, lung cancer, prostate cancer, esophageal cancer, breast cancer, pancreatic cancer, head and neck cancer, cervical cancer, which, together with urothelial carcinoma, are preferred indications for treatment with IL-2 / IL-15Rβγ agonists in combination with enfortumab vedotin.Similarly, the recently approved tisotumab vedotin-tftv uses MMAE as a payload, where it targets tissue factor for the indication cervical cancer.Thus, the combination of tisotumab vedotin-tftv with IL-2 / IL-15Rβγ agonists, preferably SOT101, is a further embodiment of the present invention.

[0098] In another preferred embodiment, the IL-2 / IL-15Rβγ agonist is for use in patients suffering from a tumor expressing CLDN18.2 (or claudin 18.2), preferably the patient has been diagnosed with gastric or pancreatic cancer, for example by using the antibody zolbetuximab (IMAB362) disclosed in WO 2007 / 059997 and WO 2016 / 165762. WO 2016 / 166122 discloses anti-CLDN18.2 monoclonal antibodies that can be efficiently internalized upon CLDN18.2 binding and are therefore suitable for ADC development. Other antibodies suitable for ADC development are, for example, the human variant of zolbetuximab disclosed in WO 2021 / 111003 A1, or the humanized antibodies disclosed in Table 3 of WO 2021 / 130291 A1, in particular hCl1a. Suitable ADCs targeting CLDN18.2 are described in WO 2022 / 136642 A1 and include SOT102, described in Example 7 therein, in particular the combination of SOT102 with an IL-2 / IL-15Rβγ agonist, preferably SOT101, making it another embodiment of the invention.

[0099] In another embodiment, the modality capable of inducing ICD is selected from high hydrostatic pressure (HHP), photodynamic therapy, UV irradiation, radiotherapy, gamma irradiation and hyperthermia. HHP refers to the treatment of tumor cells with high hydrostatic pressure, as described, for example, in WO 2013 / 004708, WO 2015 / 097037, WO 2019 / 145469, WO 2019 / 145471, Fucikova et al. (2014), Obeid et al. (2007) and Adkins et al. (2018). In one embodiment, such a HHP modality is a dendritic cell vaccine, in which whole tumor cells are driven (guided) into ICD by high hydrostatic pressure (HHP) as described in WO 2013 / 004708 and WO 2015 / 097037 (see, e.g., Examples 1-4 of WO 2013 / 004708 and Examples 2 and 3 of WO 2015 / 097037). Briefly, whole tumor cells from cell lines or patients are treated with HHP at 200-300 MPa for 10 min-2 h. Such treatment induces ICD in the treated tumor cells, which may be characterized by the expression of immunogenic molecules such as HSP70, HSP90 and calreticulin on the cell surface and the release of late apoptotic markers HMGB1 and ATP, thus increasing the uptake of these cells by dendritic cells (DCs) and resulting in loaded DCs presenting multiple tumor antigens. Apoptotic tumor cells may be cryopreserved before loading DCs. The whole tumor cells loaded with DC vaccines are preferably allogeneic to the patient, e.g. tumor cell lines with expressed tumor antigens overlapping with typical tumor antigens of the tumor disease being treated. Autologous tumor cells would be better matched with the tumor antigens of the patient, but in practice, it is very complicated to produce DC vaccines from autologous tumor biopsies. Therefore, DCs may be derived from monocytes autologous to the patient being treated. As used herein, the term "monocyte" refers to white blood cells circulating in the blood, characterized by bean-shaped nuclei and the absence of granules. Monocytes can give rise to dendritic cells.Monocytes can be isolated from patient's blood by any technique known to those skilled in the art, and the preferred method is leukopheresis.Leukopheresis allows to collect the autologous monocytes of the patient to be treated, which are used for preparing DC vaccine.Leukopheresis can be carried out by any technique known to those skilled in the art.

[0100] Other therapeutic modalities to induce ICD have been described in the art, including photodynamic therapy, preferably with hypericin; UV radiation, preferably UVC radiation; radiotherapy, including brachytherapy; oncolytic virotherapy; and hyperthermia, all of which have also been described to induce ICD (Dudek, Garg et al. 2013; Adkins, Sadilkova et al. 2017; Zhou, Wang et al. 2019) and are therefore preferred modalities to induce ICD. Briefly summarized, ultraviolet light (UVC) has been described to induce an inflammatory response in the skin and can induce ICD determinants such as calreticulin, HMGB1 and HSP70.

[0101] Similarly, in addition to direct cell killing, radiotherapy can induce the so-called "abscopal effect", i.e. T cell-mediated growth retardation of tumors located far from the irradiated area, which is explained by the ability of radiotherapy to reproducibly induce ICD, again characterized by exposure of calreticulin, HSP70 and release of HMGB1. Exposure / release of DAMPs from irradiated cells is thought to stimulate DCs in vivo (similar to the above-mentioned DC vaccination with tumor cells undergoing ICD ex vivo). Preferably, local high-dose radiotherapy is applied to induce ICD, since it has been shown to increase the number of tumor-infiltrating active DCs. Also, lower doses of non-ablative or sub-ablative radiotherapy have been described to reprogram macrophages towards a beneficial M1 phenotype, so lower doses of non-ablative or sub-ablative radiotherapy may have advantages for the claimed combination. Appropriate doses and fractionation of radiation therapy are summarized in Golden and Apetoh (2015).

[0102] Photodynamic therapy based on the photosensitizer hypericin has also been shown to induce ICD in cancer cells, again establishing highly productive contacts with DCs at the level of phagocytosis and maturation by inducing immunological signatures of ICD in cancer cells, such as calreticulin, HSP70, etc. Nanopulse stimulation with ultrashort electrical pulses in the nanosecond range has also been described to induce ICD, as has treatment with oncolytic viruses, which during oncolytic virus-mediated oncolysis of cancer cells induce calreticulin surface exposure, ATP release and ER stress, also hallmarks of ICD. Further specific treatments described as being able to induce ICD are near-infrared photoimmunotherapy, oxygen-enhanced photodynamic therapy, nanosized drug carriers or hyperthermia (Dudek, Garg et al. 2013; Adkins, Sadilkova et al. 2017; Zhou, Wang et al. 2019). Thus, there is a growing field of therapeutic modalities unified by the specific feature of inducing ICD in tumor cells and thus eliciting a specific anti-immune response likely mediated by DCs, and therefore all of these therapeutic modalities are preferred for combination with IL-2 / IL-15Rβγ agonist treatment.

[0103] In one embodiment, the IL-2 / IL-15Rβγ agonist is an IL-15 / IL-15Rα complex. IL-2 and IL-15 share β and γ receptors and therefore have overlapping downstream intracellular signaling, but the wtIL-2 complex has been shown to mediate the signaling pathways that they mediate. reg and IL-2Rαβγ expressed on the pulmonary endothelium, which activation should be avoided. Different strategies have been used to modify IL-2 to avoid binding to the IL-2α receptor using IL-2 muteins and / or chemical modifications, all of which have certain drawbacks, such as reducing activity (e.g., IL2v, NKTR-255), complex expression systems (THOR-707) or expensive chemical modifications (e.g., PEGylated conjugates). On the other hand, IL-15 itself also activates T regTherefore, complexes containing IL-15 or IL-15Rα derivatives that mimic the transpresentation of IL-15Rα and thus limit binding to the IL-2 / IL-15βγ receptor are preferred.

[0104] Preferably, the IL-15 is mature wtIL-15 having the sequence of SEQ ID NO: 4. Furthermore, many activating or inactivating mutations have been described in the art to achieve various defined changes to the molecule: IL-15Rβγβγ cD8N, D8A, D61A, N65D, N65A, Q108R to reduce binding to the receptor (WO 2008 / 143794A1); N72D as an activating mutation (in ALT-803); N1D, N4D, D8N, D30N, D61N, E64Q, N65D and Q108E to reduce proliferative activity (US 2018 / 0118805); L44D, E46K, L47D, V49D, I50D, L66 to reduce binding to IL-15Rα. D, L66E, I67D, and I67E (WO 2016 / 142314A1); N65K and L69R to abrogate IL-15Rβ binding (WO 2014 / 207173A1); Q101D and Q108D to inhibit IL-15 function (WO 2006 / 020849A2); S7Y, S7A, K10A, K11A to decrease IL-15Rβ binding (Ring, Lin et al., 2012); substitution of L45, S51, L52 with D, E, K or R, and substitution of E64, I68, L69 and N65 with D, E, R or K to reduce deamidation (WO 2005 / 085282 A1); substitution of N71 with S, A or N, substitution of N72 with S, A or N, substitution of N77 with Q, S, K, A or E, substitution of N78 with S, A or G to reduce deamidation (WO 2009 / 135031 A1); defines certain regions of -15 as suitable for substitution (see paragraphs 0020, 0035, 00120 and 00130), specifically providing guidance on how to identify potential substitutions to provide anchors for PEG or other modifications (see paragraph 0021); Q108D, which increases affinity for CD122 and impairs recruitment of CD132 to inhibit IL-2 and IL-15 effector function, and N65K, to abrogate CD122 affinity (WO 2017 / 046200 A1);N1D, N4D, D8N, D30N, D61N, E64Q, N65D, and Q108E to gradually decrease the activity of the IL-15 / IL-15Rα complex on NK cell and CD8 T cell activation, respectively (see Figure 51, WO 2018 / 071918A1, WO 2018 / 071919A1). Mutation of K86 (e.g., K86R) has been described to increase stability, since K86 is a putative site for ubiquitin-dependent degradation, and N112 (e.g., N112) has been described to enhance IL-15 activity (see WO 2018 / 151868). The L52C substitution has been made to introduce an additional cysteine ​​for disulfide bonding with the mutated IL-15Rα sushi domain (Hu, Ye et al., 2018). Additionally or alternatively, one skilled in the art can easily make conservative amino acid substitutions. Considering the high potency of the IL-15 molecule, a reduction in activity of a factor of several thousand times may still be compensated for by a higher dosage or by the pharmacodynamic effect of the molecule having a longer half-life due to a significantly increased molecular weight. An increased molecular weight can be achieved, for example, by fusion or covalent attachment of the IL-15 / IL-15Rα complex to the Fc portion of an antibody, to an antibody, to serum albumin, or by PEGylation;

[0105] Therefore, multiple mutations can be easily combined in a protein without compromising the biological / commercial value of the protein.Thus, preferably, the IL-15 derivative has at least 0.1%, preferably 1%, more preferably at least 10%, more preferably at least 25%, even more preferably at least 50%, and most preferably at least 80% of the activity of human IL-15.In one embodiment, the activity is measured as the effect of IL-15 on the induction of proliferation of kit225 cell line (HORI et al., Blood, vol. 70(4), pp. 1069-72, 1987).

[0106] Furthermore, it is preferable to limit the number of mutations / substitutions, since each additional mutation at least theoretically increases the risk of inducing immunogenicity and thus the possibility of generating anti-drug antibodies in patients, which may limit the activity of the conjugate with increasing administration frequency.Thus, preferably, the IL-15 derivative has at least 92%, preferably at least 96%, more preferably at least 98%, most preferably at least 99% identity with the amino acid sequence of mature human IL-15 (114aa) (SEQ ID NO: 4).

[0107] Furthermore, as in the case of IL-15, chemical modifications known in the art, such as PEGylation or other post-translational modifications (see WO 2016 / 060996 A2, WO 2017 / 112528 A2, WO 2009 / 135031 A1), may be preferably used for the IL-15 / IL-15Rα complex of the present invention.

[0108] The IL-15Rα in the IL-15 / IL-15Rα complex preferably refers to an IL-15Rα derivative that contains at least the sushi domain of wt IL-15Rα, but does not contain the transmembrane and intracellular domains of wt IL-15Rα. Furthermore, it is preferred that the IL-15Rα does not contain the 30 aa peptide leader sequence that is typically cleaved during expression. The IL-15Rα sushi domain (or IL-15Rα sushiThe sushi+ fragment (SEQ ID NO: 6) is the domain of IL-15Rα that is essential for binding to IL-15 (Wei, Orchardson et al. 2001) and is therefore the smallest fragment of IL-15Rα in the IL-15 / IL-15Rα complex. The sushi+ fragment (SEQ ID NO: 7) contains the sushi domain and a portion of the hinge region C-terminal to the sushi domain, defined as the 14 amino acids located after the IL-15Rα sushi domain, i.e., the IL-15Rα hinge region starts with the first amino acid after the (C4) cysteine ​​residue and ends with the 14th amino acid (counting in the standard "N-terminal to C-terminal" direction). The sushi+ fragment reconstitutes full binding activity to IL-15 (WO 2007 / 046006) and is therefore preferred. Thus, preferred IL-15Rα derivatives contain at least the sushi domain (aa 33-93) but do not extend beyond the extracellular portion of mature IL-15Rα, amino acids 31-209 of SEQ ID NO: 5. Specifically, preferred IL-15Rα derivatives are the IL-15Rα sushi domain (SEQ ID NO: 6) and the IL-15Rα sushi+ domain (SEQ ID NO: 7). As has been done for hetIL-15, IL-15Rα sushi+ can be extended further C-terminally in order to enlarge the molecule and thereby increase its serum half-life. Thus, another preferred IL-15Rα derivative is a soluble form of IL-15Rα (amino acid 31 to any of amino acids 172, 197, 198, 199, 200, 201, 202, 203, 204 or 205 of SEQ ID NO:5; see WO 2014 / 066527 (Giron-Michel, Giuliani et al. 2005)).

[0109] In another embodiment, IL-15Rα derivatives may contain naturally occurring or introduced mutations. Natural variants and alternative sequences are described, for example, in UniProtKB entry Q13261 (https: / / www.uniprot.org / uniprot / Q13261). Furthermore, one skilled in the art can easily identify amino acids that are less conserved among mammalian IL-15Rα homologs or even primate IL-15Rα homologs to generate derivatives that are still functional. IL-15Rα derivatives bind to IL-15 and thereby promote the activation of immune effector cells (e.g., NK cells or CD8 + It functions by forming a complex that mimics the transpresentation of IL-15 at the immunological synapse by antigen-presenting (e.g., dendritic) cells to IL-15 (T cells). Furthermore, the IL-15Rα derivative, due to its presence, blocks binding to the IL-15αβγ receptor. It is clear to those skilled in the art that, in particular, in a covalent fusion protein containing both IL-15 (or its derivative) and an IL-15Rα derivative, the binding of the IL-15Rα derivative to IL-15 (or its derivative) can be significantly reduced without losing its activity, since the covalent bond compensates for the reduced binding and the molecules still form a stable complex. Furthermore, the substitution S40C in IL-15Rα has been made to introduce an additional cysteine ​​to form a disulfide bond with the mutated IL-15 (Hu, Ye et al., 2018).

[0110] The respective sequences of mammalian IL-15Rα homologues are described in WO 2007 / 046006, pages 18 and 19. Again, the number of mutations compared to the wt sequence should be limited in order to avoid increased immunogenicity, and thus the IL-15Rα derivative preferably comprises an amino acid sequence having at least 92%, preferably at least 96%, more preferably at least 98%, even more preferably at least 99% identity with the respective wt sequence of the same length, more preferably the amino acid sequence of the sushi domain of human IL-15Rα in the overlapping sequence (SEQ ID NO: 6), in particular the amino acid sequence of the sushi+ domain of human IL-15Rα in the overlapping sequence (SEQ ID NO: 7), and most preferably 100% identity with the respective wt sequence of the same length, more preferably the amino acid sequence of the sushi domain of human IL-15Rα in the overlapping sequence (SEQ ID NO: 6), in particular the amino acid sequence of the sushi+ domain of human IL-15Rα in the overlapping sequence (SEQ ID NO: 7).

[0111] Preferably, the IL-15Rα derivative has at least 10%, more preferably at least 25%, even more preferably at least 50%, and most preferably at least 80% of the binding activity of the human sushi domain for human IL-15, as determined, for example, in (Wei, Orchardson et al. 2001).

[0112] In one embodiment, the IL-2 / IL-15Rβγ agonist is an interleukin-15 (IL-15) / interleukin-15 receptor alpha (IL-15Rα) complex, which is a fusion protein comprising the sushi domain of human IL-15Rα or a derivative thereof, a flexible linker, and human IL-15 or a derivative thereof, preferably the human IL-15Rα sushi domain comprises the amino acid sequence of SEQ ID NO: 6 and the human IL-15 comprises the amino acid sequence of SEQ ID NO: 4. Such a fusion protein is preferably ordered (from N-terminus to C-terminus) IL-15Rα-linker-IL-15 (RLI-15). Other examples of fusion proteins are described in WO 2018 / 071919A1, in which the sushi domain of IL-15Rα is fused to IL-15 via a disulfide bond (e.g., XENP22004), to a heterodimeric Fc via a covalent bond (e.g., XENP22013, XENP22357, XENP22639, or to two IL-15Rα(sushi) / IL-15 fusions (e.g., XENP22634)). WO 2015 / 103928 also discloses alternative formats for constructing IL-15 / IL-15Rα complexes, for example by forming a stable complex through the interaction of a first and a second Fc variant, one linked to IL-15 (or a derivative thereof) and the other to an IL-15Rα derivative. Hu et al. (Hu, Ye et al. 2018) described the IL-15 / IL-15Rα complex P22339, in which IL-15 is covalently linked to the sushi domain of IL-15Rα by introducing a novel disulfide bond between L52C of IL-15 and S40C of IL-15Rα.

[0113] A particularly preferred IL-2 / IL-15Rβγ agonist is a fusion protein designated RLI2 having the sequence of SEQ ID NO: 9. RLI2 (also known as SO-C101 or CYT101) is the subject of clinical trial NCT04234113 and is therefore particularly suitable for development in combination with cytotoxic compounds and / or modalities that induce ICD.

[0114] In a preferred embodiment, the IL-15 / IL-15Rα complex is a fusion protein comprising, and in particular consisting of, the amino acid sequence of SEQ ID NO:9, and the ADC comprises an antibody that specifically binds to HER2, preferably the antibody is trastuzumab. As the inventors have shown, SOT101 / RLI2 has been shown to act synergistically with trastuzumab emtansine both in vitro and in vivo, thus making this combination particularly preferred. Another preferred combination is SOT101 / RLI2 with SOT102 as described herein. In one embodiment, SOT101 is particularly preferred as it offers several advantages over other IL-2 / IL-15Rβγ agonists. SOT101 binds with high affinity to the intermediate affinity receptor composed of β and γ chains, whereas IL-2 and IL-15 based molecules with steric or mutational hindrance of α chain binding bind to the intermediate affinity receptor with lower affinity. However, transpresentation of IL-15 by membrane-bound IL-15Rα or soluble IL-15 / IL-15Rα complexes is believed to result in metabolically more active, larger sized and more proliferative T cells compared to cells stimulated by soluble IL-15 itself through stronger and more sustained signaling, i.e., intermediate affinity binding to intermediate affinity receptors results in stronger phenotypic responses (Arneja, Johnson et al., 2014). Furthermore, SOT101 is a fusion protein, avoiding dissociation of non-covalent IL-15 / IL-15Rα complexes such as hetIL-15, ALT803 or other IL-15Rα / Fc fusions that non-covalently bind IL-15 (which may dissociate at the respective dilution in blood and thus lose both their specificity and high affinity). Furthermore, SOT101, which has a relatively short in vivo half-life, is a very potent stimulator of NK cells even at low doses (Antosova, Podzimkova et al., 2020), and the inventors have shown that SOT101 synergizes with ICD induction in a model that is completely dependent on NK cells (in the absence of T cells, see Examples).Thus, in one embodiment, the present invention provides SOT101 in combination with a cytotoxic compound capable of inducing ICD or a modality capable of inducing ICD.

[0115] Preferably, IL-2 / IL-15Rβγ agonists are administered subcutaneously (sc) or intraperitoneally (ip), with sc being even more preferred. ICD-inducing cytotoxic compounds are preferably administered according to their approved label, e.g., as approved by the FDA, typically intravenously (iv).

[0116] In a preferred embodiment, the IL-2 / IL-15Rβγ agonist is further combined with an immune checkpoint inhibitor (or checkpoint inhibitor for short). Checkpoint inhibitors, or more precisely immune checkpoint inhibitors, refer to a type of drug that blocks certain proteins made by some types of immune system cells, such as T cells, and some cancer cells. These proteins help check the immune response and can stop T cells from killing cancer cells. When these proteins are blocked, the "brakes" on the immune system are released and T cells can better kill cancer cells. Thus, checkpoint inhibitors are antagonists of immune inhibitory checkpoint molecules or antagonists of agonist ligands of inhibitory checkpoint molecules. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2 (see National Cancer Institute definition at the National Institute of Health, https: / / www.cancer.gov / publications / dictionaries / cancer-terms / def / immune-checkpoint-inhibitor), as reviewed for example by Darvin et al. (2018). Examples of such checkpoint inhibitors are anti-PD-L1 antibodies, anti-PD-1 antibodies, anti-CTLA-4 antibodies, but also antibodies against LAG-3 or TIM-3, or blockers of BTLA, are currently being tested in the clinic (De Sousa Linhares, Leitner et al. 2018). A further promising checkpoint inhibitor is the anti-TIGIT antibody (Solomon and Garrido-Laguna 2018). Examples of anti-PD-L1 antibodies are avelumab, atezolizumab, durvalumab, KN035, MGD013 (bispecific for PD-1 and LAG-3), examples of anti-PD-1 antibodies are pembrolizumab, nivolumab, cemiplimab (REGN2810), BMS-936558, SHR1210, IBI308, PDR001, BGB-A317, BCD-100, JS001, and an example of an anti-PD-L2 antibody is sHIgM12.Examples of anti-CTLA-4 antibodies are ipilimumab and tremelimumab (ticilimumab), examples of "anti-LAG-3" antibodies are leratolimab (BMS986016), Sym022, REGN3767, TSR-033, GSK2831781, MGD013 (bispecific for PD-1 and LAG-3), LAG525 (IMP701), examples of anti-TIM-3 antibodies are TSR-022 and Sym023, and examples of anti-TIGIT antibodies are tiragolumab (MTIG7192A, RG6058) and etigilimab (WO 2018 / 102536). Preferably, the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody or an anti-CTLA4 antibody, more preferably an anti-PD-L1 antibody or an anti-PD-1 antibody.

[0117] The IL-2 / IL-15Rβγ agonists are for use in treating cancer, where the cancer is a hematological or solid cancer. The mechanism of action of these agonists is activation of the innate immune response through activation of NK cells and CD8 +Being the activation of adaptive immune responses by T cell activation, it is generally assumed that these agonists have great potential to treat both (advanced) solid tumors and hematological malignancies, as they have already been tested in a number of mouse cancer models and in several clinical trials in various tumor indications (Robinson and Schluns 2017). Thus, IL-2 / IL-15Rβγ agonists have been tested in colorectal cancer, melanoma, renal cell carcinoma, adenocarcinoma, carcinoid tumors, leiomyosarcoma, breast cancer, ocular melanoma, osteosarcoma, thyroid cancer, bile duct cancer (cholangiocarcinoma), salivary gland cancer, adenoid cystic carcinoma, gastric cancer, head and neck squamous cell carcinoma, ovarian cancer, urothelial cancer (Conlon, Leidner et al. 2019). ALT-803 has been tested in AML and MDS as examples of hematological malignancies (Rome, Cooley et al. 2018). Patients with particularly advanced tumor diseases, such as metastatic tumors, may preferably benefit from such treatment. In this regard, accordingly, ALT-803 has been tested in metastatic non-small cell lung cancer (Wrangle, Velcheti et al., 2018). In a Phase 1 / 1b clinical trial with SO-C101, patients with renal cell carcinoma, non-small cell lung cancer, small cell lung cancer, bladder cancer, melanoma, Merkel cell carcinoma, cutaneous squamous cell carcinoma, high-frequency microsatellite instability solid tumors, triple-negative breast cancer, mesothelioma, thyroid cancer, thymic cancer, cervical cancer, biliary tract cancer, hepatocellular carcinoma, ovarian cancer, gastric cancer, head and neck squamous cell carcinoma, and anal cancer have been recruited. Examples of hematological cancers are leukemias such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) and acute monocytic leukemia (AMoL), lymphomas such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, and myeloma. For combination with enfortumab vedotin, bladder cancer, urothelial cancer, kidney cancer, cervical cancer, endometrial cancer, ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, head and neck cancer, esophageal cancer and breast cancer are preferred, in particular urothelial cancer, lung cancer, head and neck cancer, pancreatic cancer, kidney cancer, breast cancer, cervical cancer and endometrial cancer.

[0118] Thus, renal cell carcinoma, lung cancer (especially non-small cell lung cancer, small cell lung cancer), bladder cancer (especially urothelial carcinoma), melanoma, Merkel cell carcinoma, cutaneous squamous cell carcinoma, microsatellite instability high solid tumors, breast cancer (especially triple negative breast cancer), mesothelioma, prostate cancer, thyroid cancer, thymic cancer, cervical cancer, biliary tract cancer, hepatocellular carcinoma, ovarian cancer, gastric cancer, esophageal cancer, squamous cell carcinoma of the head and neck, and anal cancer, as well as ALL, AML, CLL, CML, AMoL, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and myeloma, are preferred cancer indications.

[0119] In one embodiment, the IL-2 / IL-15Rβγ receptor agonist is (a) a first period of up to 3 weeks during which a cytotoxic compound capable of inducing ICD is administered; (b) optionally, a second period during which the cytotoxic compound is not administered and the IL-2 / IL-15Rβγ agonist is not administered; (c) a third period of up to 2 weeks during which an IL-2 / IL-15Rβγ agonist is administered. and the first to third periods are repeated at least twice, more preferably at least three times, and more preferably until disease progression.

[0120] In one embodiment, the first period is two weeks. Administration of a cytotoxic compound capable of inducing ICD may be carried out according to its label.

[0121] In one embodiment, the second period of time is at least the in vivo half-life or at least twice the in vivo half-life of the cytotoxic compound capable of inducing ICD.

[0122] In one embodiment, the third period of time is one week.

[0123] In one embodiment, the IL-2 / IL-15Rβγ receptor agonist is (a) a first period of up to three weeks, preferably two weeks, more preferably one week, during which a cytotoxic compound capable of inducing ICD is administered according to its label during such first period; (b) optionally, a second period of time that is at least 1 or 2 in vivo half-life of the cytotoxic compound, preferably 1 in vivo half-life, during which the cytotoxic compound is not administered; (c) a third period of up to 2 weeks, preferably up to 1 week, during which an IL-2 / IL-15Rβγ agonist is administered. and is administered in a cyclic administration regimen comprising repeating the first to third periods at least twice, more preferably at least three times, and more preferably until disease progression.

[0124] Optionally, the cyclic dosing regimen further comprises a fourth period (d) in which the ICD-inducing cytotoxic compound is not administered and the IL-2 / IL-15Rβγ agonist is not administered, the fourth period (d) being at least 1 week to 1 in vivo half-life of the IL-2 / IL-15Rβγ agonist, which fourth period is added after each third period before resuming the cycle.

[0125] Regarding the combined administration schedule of the IL-2 / IL-15Rβγ agonist and the cytotoxic compound that induces ICD, the treatment schedules of both compounds should be aligned for best treatment results at convenient, preferably weekly, intervals and best coordinated according to the approved drug labeling instructions.

[0126] Many chemotherapeutics, such as anthracyclines, vinca alkaloids, taxanes, epothilones, eribulin, auristatins, maytansine or maytansinoids, microtubule destabilizing agents including tubulysin, bleomycin, proteasome inhibitors including bortezomib, alkylating agents including cyclophosphamide, platinum complexes including oxaliplatin, pyrrolo-benzodiazepines, calicheamicin derivatives, topoisomerase I inhibitors, and nucleoside analogs, are typically administered daily for longer periods of time. To obtain the combined effect of inducing ICD and the immune stimulating effects of IL-2 / IL-15Rβγ agonists, the inventors envision that such chemotherapy treatments will be applied according to their label, but only for a maximum of two weeks, preferably only one week, to allow for intermittent treatment with IL-2 / IL-15Rβγ agonists.

[0127] Indeed, the inventors have demonstrated enhanced antitumor activity in an in vivo MC38 mouse colon cancer model for the combined administration of a platinum complex in combination with an IL-2 / IL-15Rβγ agonist, in this case oxaliplatin in combination with SOT101.

[0128] Regarding ADCs, many of them are administered every 3 weeks, taking into account their typical half-lives between about 2 and about 12 days. The in vivo half-lives of ADCs are shown in Table 2 of Mahmood et al. (2021). Kadcyla, Adcetris, Enhertu and Trodelvy are administered in a 3-week / 21-day cycle, while Padcev is administered in a 4-week cycle (see Table 1).

[0129] [Table 1]

[0130] Thus, the three-week cycle of ADCs is administered according to its label on day 1 (e.g., Kadcyla, Adcetris in a three-week scheme, Enhertu) or days 1 and 8 (Trodelvy) of the first period. The four-week cycle of Padcev is preferably administered according to its label on days 1, 8 and 15.

[0131] Prior to immune activation by administration of IL-2 / IL-15Rβγ agonists, cytotoxic compounds capable of inducing ICD should be absent or present only in residual amounts in the patient's plasma in order not to interfere with the induced proliferation of immune cells. Therefore, a treatment interruption of 1 or 2 in vivo half-life is introduced to remove the compound from circulation. In the case of short-lived chemotherapy, such a treatment interruption may be as short as 1 day, but may also be 1 week for the convenience of the patient. In a continuous treatment regimen, an optional treatment period (b) without administration of cytotoxic compounds and without administration of IL-2 / IL-15Rβγ agonists, at least 1 or 2 in vivo half-life of the cytotoxic compound, preferably 1 in vivo half-life, is preferred.

[0132] For example, Kadcyla is given every 3 weeks with an in vivo half-life of 4 days, so the ADC by label is typically cleared from plasma before re-administration anyway. Thus, an additional period (b) for clearance is not required. Thus, for an ADC with a 3-week schedule administered on day 1, the IL-2 / IL-15Rβγ agonist is preferably administered after a 1- or 2-week treatment break starting on day 8 or day 15, after which the ADC is administered again on day 22 (new day 1). For ADCs administered on days 1 and 8 of a 3-week cycle, the IL-2 / IL-15Rβγ agonist is preferably administered from day 15 onwards, as such more frequently administered ADCs typically have a fairly short half-life (e.g., Todelvy, only 16 hours) and are therefore cleared from plasma within a few days. For ADC on a 4 week schedule administered on days 1, 8 and 15, IL-2 / IL-15Rβγ agonist treatment is preferably initiated on day 22.

[0133] The IL-2 / IL-15Rβγ agonist is administered for up to 2 weeks, preferably 1 week, according to its label / prior use in the most advanced clinical trials. The frequency of administration also depends on its half-life. IL-2 / IL-15Rβγ agonists with short half-lives of a few hours to a day are preferably administered on days 1, 2, 3 and 4 within a treatment week, preferably on days 1 and 2, and in the case of a 2-week treatment period, on days 1, 2, 3, 4, 8, 9, 10 and 11 of such treatment period, preferably on days 1, 2, 8 and 9. For example, the administration schedule of SO-C101 is disclosed in WO2020 / 234387. Optionally, SO-C101 may be administered potently by split administration on days 1, 2, 8 and 9. IL-2 / IL-15Rβγ agonists with longer half-lives are preferably administered only once per treatment week, on day 1, or on days 1 and 8 for a two week treatment period.

[0134] Optionally, an additional treatment break of at least one week, preferably one week, is introduced after each cycle (a)-(c) to allow sufficient time for the activated immune cells to kill tumor cells.

[0135] If necessary, the initiation of a new treatment period (a) of the ADC with the label is delayed in one week increments to conform to the time requirements of periods (b), (c) and optionally (d).

[0136] Example of a dosing schedule for Kadcyla and SO-C101 (a) Administration of Kadcyla given iv at a dose of 3.6 mg / kg on day 1 (b) None (c) administration of SO-C101 at a dose of 12 μg / kg per day given sc (as a single dose or divided into two doses separated by 4-14 hours) on days 8, 9, 15, and 16 (days 1, 2, 8, and 9 of the SO-C101 treatment period); (d) None or (a) Administration of Kadcyla given i.v. at a dose of 3.6 mg / kg on day 1 (b) None (c) administration of SO-C101 at a dose of 12 μg / kg per day given sc (as a single dose or divided into two doses separated by 4-14 hours) on days 15 and 16 (days 1 and 2 of the SO-C101 treatment period); or (a) Administration of Kadcyla given i.v. at a dose of 3.6 mg / kg on day 1 (b) None (c) administration of SO-C101 at a dose of 12 μg / kg per day given sc (as a single dose or divided into two doses separated by 4-14 hours) on days 15, 16, 22, and 23 (days 1, 2, 8, and 9 of the SO-C101 treatment period); (d) 1 week, This will extend the Kadcyla schedule to 4 weeks, starting again on day 29.

[0137] Another embodiment is the use of an IL-2 / IL-15Rβγ agonist in the manufacture of a kit of parts for the treatment of cancer, the kit of parts comprising several doses of an IL-2 / IL-15Rβγ agonist of the invention, instructions for the administration of such an IL-2 / IL-15Rβγ agonist in combination with a cytotoxic compound capable of inducing ICD and / or a modality capable of inducing ICD, and optionally an administration device for the IL-2 / IL-15Rβγ agonist. In a preferred embodiment, the kit further comprises a checkpoint inhibitor and instructions for the use of the checkpoint inhibitor.

[0138] The present invention relates to a method for treating cancer with the above-mentioned combination treatment, and to a method for treating NK cells and / or CD8 + Methods for stimulating T cells are also disclosed.

[0139] In one embodiment, the invention relates to an IL-2 / IL-15Rβγ agonist for use in treating cancer in a patient, the IL-2 / IL-15Rβγ agonist being administered in combination with a cytotoxic compound capable of inducing ICD.

[0140] In one embodiment, the present invention relates to an IL-2 / IL-15Rβγ agonist for use in treating cancer in a patient, the IL-2 / IL-15Rβγ agonist being administered in combination with the application of a modality capable of inducing ICD.

[0141] The present invention also provides a pharmaceutical combination (combination medicament) comprising an IL-2 / IL-15Rβγ agonist and a cytotoxic compound capable of inducing ICD.

[0142] The present invention further provides a pharmaceutical combination comprising an IL-2 / IL-15Rβγ agonist and a modality capable of inducing ICD.

[0143] Administration of the IL-2 / IL-15Rβγ agonist may be simultaneous with administration of a cytotoxic compound capable of inducing ICD and / or application of a modality capable of inducing ICD, or sequential to administration of a cytotoxic compound capable of inducing ICD and / or application of a modality capable of inducing ICD.

[0144] In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is Kadcyla. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is an ADC comprising an anti-CLDN18.2 antibody and an anthracycline. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is SOT102. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the modality capable of inducing ICD is radiation therapy. In a particularly preferred embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the modality capable of inducing ICD is non-ablative or sub-ablative radiation therapy. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is gemtuzumab ozogamicin. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is brentuximab vedotin. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is trastuzumab emtansine. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is inotuzumab ozogamicin. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is trastuzumab deruxtecan. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is enfortumab vedotin. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is polatuzumab vedotin. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is sacituzumab govitecan. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is belantamab mafodotin-blmf. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is loncastuximab tesillin-lpyl. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is tisotumab vedotin-tftv. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is an anthracycline, preferably doxorubicin. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is a taxane, preferably paclitaxel. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is bortezomib. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is a platinum complex, preferably oxaliplatin or cisplatin, more preferably oxaliplatin. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is topotecan or exatecan. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is gemcitabine. In one embodiment, the IL-2 / IL-15Rβγ agonist is SOT101 and the cytotoxic compound capable of inducing ICD is cyclophosphamide.

[0145] In a preferred embodiment, the cytotoxic compounds are administered at lower dosages and / or less frequently compared to their labels for use in cancer therapy. [Brief description of the drawings]

[0146] [Figure 1] Antitumor efficacy of chemotherapy. Chemotherapeutic agents activate molecular pathways that induce upregulation and / or release of stress molecules (danger-associated molecular patterns, DAMPs; NK cell ligands) that promote tumor cell recognition and elimination by NK cells. In addition, chemotherapy can also downregulate the expression of ligands such as inhibitory receptors PD-L1 and (MHC)-I. [Figure 2A] Dose-dependent cytotoxic effect of Kadcyla in AGS tumor cell lines. Gastric adenocarcinoma cell lines (AGS) were treated with Kadcyla at the indicated concentrations (5, 7, 8, 10 μg / ml) for 72 h. Data represent the average of two independent experiments. (A) Cell surface expression of Her-2 analyzed by FACS in untreated AGS human gastric adenocarcinoma cell lines (Her-2 IHC1+). [Figure 2B(i)]Dose-dependent cytotoxic effect of Kadcyla in AGS tumor cell line. Gastric adenocarcinoma cell line (AGS) was treated with Kadcyla at the indicated concentrations (5, 7, 8, 10 μg / ml) for 72 h. Data represent the average of two independent experiments. (B) Tumor cell viability at endpoint was analyzed by FACS analysis using Annexin V (x-axis) / DAPI (y-axis) staining. Representative dot plots are shown with Kadcyla concentrations of 5 μg / ml, 7 μg / ml, 8 μg / ml, and 10 μg / ml, with NT representing untreated. [Figure 2B(ii)] This is a continuation of Figure 2B(i). [Figure 2C] Dose-dependent cytotoxic effect of Kadcyla in AGS tumor cell line. Gastric adenocarcinoma cell line (AGS) was treated with Kadcyla at the indicated concentrations (5, 7, 8, 10 μg / ml) for 72 h. Data represent the average of two independent experiments. (C) Individual populations of early apoptotic (Annex+ / DAPI-, hatched), late apoptotic (Annex+ / DAPI+, grey) and necrotic (Annex- / DAPI+, black) cells at a given concentration. For analysis of ICD markers and NK ligands, only the early apoptotic cells (Annex+ / DAPI- population) were selected. [Figure 3A] Dose-dependent induction of cell surface exposure of CRT, HSP70, and HSP90 by Kadcyla in AGS tumor cell line. Early apoptotic (Annex+ / DAPI-) populations from Figure 2 were analyzed for expression of CRT, HSP70, and HSP90 on the cell surface. Data represent the average of two independent experiments. (A) Mean fluorescence intensity (MFI) of individual markers of antigen presenting cells (APCs) CRT, HSP70, and HSP90. [Figure 3B]Dose-dependent induction of cell surface exposure of CRT, HSP70, and HSP90 by Kadcyla in AGS tumor cell lines. The early apoptotic (Annex+ / DAPI-) population from Figure 2 was analyzed for expression of CRT, HSP70, and HSP90 on the cell surface. Data represent the average of two independent experiments. (B) Correlated percentage of binding of individual primary antibodies to the individual markers CRT, HSP70, and HSP90. [Figure 4(i)] Dose-dependent induction of expression of NK cell ligands on the surface of AGS tumor cell lines by Kadcyla. The early apoptotic (Annex+ / DAPI-) population from Figure 2 was analyzed for expression of NK activating ligands CD112, CD155 and ULBP1, 2 / 5 / 6 and 3 and expressed as mean fluorescence intensity (MFU). Data represent the average of two independent experiments. [Figure 4(ii)] This is a continuation of Figure 4(i). [Figure 5A(i)] Dose-driven in vitro synergy of RLI-15 with Kadcyla for stimulation of CD56-positive cells. Human PBMCs isolated from three different donors were incubated for 72 h in the presence of 2.5 ng / ml RLI-15. In parallel, AGS tumor cells were treated with Kadcyla at the indicated concentrations (5, 7, 8, 10 μg / ml) for 72 h and after washing, the two cell cultures were mixed in a ratio of 10 (PBMC):1 (tumor cells) and incubated for the next 4 h. The entire population was then analyzed by flow cytometry for the markers CD3, CD56, CD107a and IFNγ. (A) A representative gating strategy is shown. [Figure 5A(ii)] This is a continuation of Figure 5A(i). [Figure 5B]Dose-driven in vitro synergy of RLI-15 with Kadcyla for stimulation of CD56-positive cells. Human PBMCs isolated from three different donors were incubated for 72 hours in the presence of 2.5ng / ml RLI-15. In parallel, AGS tumor cells were treated with Kadcyla at the indicated concentrations (5, 7, 8, 10μg / ml) for 72 hours and after washing, the two cell cultures were mixed in a ratio of 10 (PBMC):1 (tumor cells) and incubated for the next 4 hours. The entire population was then analyzed by flow cytometry for the markers CD3, CD56, CD107a and IFNγ. (B) Percentage of CD3-CD56+ cells representing NK cells for increasing concentrations of Kadcyla from 0 (KadNT) to 10 μg / ml Kadcyla in combination with RLI-15 (RLI+ADC) or Kadcyla alone (ADC), versus RLI-15 only control and untreated PBMCs (PBMC CTR). RLI-15 treatment results in an increase in the total number of CD3-CD56+ cells. Gating strategy is shown in panel A. Data represent the average of three independent experiments. [Figure 5C] Dose-driven in vitro synergy of RLI-15 versus Kadcyla for stimulation of CD56-positive cells. Human PBMCs isolated from three different donors were incubated for 72 h in the presence of 2.5 ng / ml RLI-15. In parallel, AGS tumor cells were treated with Kadcyla at the indicated concentrations (5, 7, 8, 10 μg / ml) for 72 h and after washing, the two cell cultures were mixed in a ratio of 10 (PBMC):1 (tumor cells) and incubated for the next 4 h. The entire population was then analyzed by flow cytometry for the markers CD3, CD56, CD107a and IFNγ. (C) Their activation measured by the release of CD107a (LAMP1). The gating strategy is shown in panel A. Data represent the average of three independent experiments. [Figure 5D]Dose-driven in vitro synergy of RLI-15 with Kadcyla for stimulation of CD56-positive cells. Human PBMCs isolated from three different donors were incubated for 72 h in the presence of 2.5 ng / ml RLI-15. In parallel, AGS tumor cells were treated with Kadcyla at the indicated concentrations (5, 7, 8, 10 μg / ml) for 72 h and after washing, the two cell cultures were mixed in a ratio of 10 (PBMC):1 (tumor cells) and incubated for the next 4 h. Afterwards, the entire population was analyzed by flow cytometry for the markers CD3, CD56, CD107a and IFNγ. (D) Their activation measured by the release of IFNγ. The gating strategy is shown in panel A. Data represent the average of three independent experiments. [Table 2] [Figure 6A] Direct comparison of the cytotoxic effects caused by selected ICD-induced SoCs (doxorubicin, cisplatin) and Kadcyla in the AGS tumor cell line. Gastric tumor cell line (AGS) was treated in vitro for 48 h with doxorubicin or cisplatin (Table 3) at defined concentrations (titration data not shown), respectively, or for 72 h with Kadcyla at previously defined concentrations most likely to synergize with RLI-15 (Figure 6B-D) (Table 3). Data represent the average of two independent experiments. [Table 3] (A) Early apoptosis at given concentrations of SoC and Kadcyla (Table 3) (Annex + / DAPI - , hatched), late apoptosis (Annex + / DAPI + , grey) and necrosis (Annex - / DAPI + , black) individual populations of cells. [Figure 6B(i)]Direct comparison of the cytotoxic effects caused by selected ICD-inducing SoC (doxorubicin, cisplatin) and Kadcyla in AGS tumor cell line. Gastric tumor cell line (AGS) was treated in vitro for 48 h with doxorubicin or cisplatin (Table 3) at defined concentrations (titration data not shown), respectively, or for 72 h with Kadcyla at previously defined concentrations most likely to synergize with RLI-15 (Table 3). Data represent the average of two independent experiments. (B) Induction of cell surface exposure of CRT, HSP70, HSP90 by SoC and Kadcyla in AGS tumor cell line. The early apoptotic (Annex+ / DAPI-) population from Figure 6A was analyzed for expression of CRT, HSP70, and HSP90 on the cell surface. Mean fluorescence intensity (MFI) of the individual markers CRT, HSP70 and HSP90 of antigen presenting cells (APC) and the correlated percentage of binding of the individual primary antibodies to the individual markers CRT, HSP70 and HSP90. Data represent the average of two independent experiments. [Figure 6B(ii)] This is a continuation of Figure 6B(i). [Figure 6B(iii)] This is a continuation of Figures 6B(i) and 6B(ii). [Figure 7A]Comparison of in vitro synergistic effect of RLI-15 with SoC (doxorubicin, cisplatin) or Kadcyla for stimulation of CD56 positive cells. Human PBMCs isolated from three different donors were incubated for 72 hours in the presence of 2.5 ng / ml RLI-15. In parallel, AGS tumor cells were treated with the indicated concentrations of SoC (doxorubicin or cisplatin, respectively) for 48 hours or with 5, 7, 8 or 10 μg / ml Kadcyla for 72 hours (as above, see Table 2), washed, and then the two cell cultures were mixed in a ratio of 10 (PBMC):1 (tumor cells) and incubated for the next 4 hours. The total cell population was then analyzed by flow cytometry for the markers CD3, CD56, CD107a and IFNγ. Panel A: % of CD3-CD56+ cells representing the total NK cell fraction in samples treated with SoC (chemistry: Doxo stands for doxorubicin, CisPt stands for cisplatin) or Kadcyla (Kad, ADC) alone or in combination with RLI-15 (RLI-15+chemistry; RLI-15+ADC), marked as control. Gating strategy is similar to that shown in Figure 5A. Data represent the average of three independent experiments. [Figure 7B] Comparison of in vitro synergism of RLI-15 with SoC (doxorubicin, cisplatin) or Kadcyla for stimulation of CD56 positive cells. Human PBMCs isolated from three different donors were incubated for 72 h in the presence of 2.5 ng / ml RLI-15. In parallel, AGS tumor cells were treated with a defined concentration of SoC (doxorubicin or cisplatin, respectively) for 48 h or with 5, 7, 8 or 10 μg / ml Kadcyla for 72 h (as described above, see Table 2) and after washing, the two cell cultures were mixed in a ratio of 10 (PBMC):1 (tumor cells) and incubated for the next 4 h. The total cell population was then analyzed by flow cytometry for the markers CD3, CD56, CD107a and IFNγ. Panel B: % of activated NK cells measured by release of CD107a (LAMP1). The gating strategy is similar to that shown in Figure 5A. Data represent the mean of three independent experiments. [Figure 7C] Comparison of in vitro synergism of RLI-15 with SoC (doxorubicin, cisplatin) or Kadcyla for stimulation of CD56 positive cells. Human PBMCs isolated from three different donors were incubated for 72 hours in the presence of 2.5 ng / ml RLI-15. In parallel, AGS tumor cells were treated with a defined concentration of SoC (doxorubicin or cisplatin, respectively) for 48 hours or with 5, 7, 8 or 10 μg / ml Kadcyla for 72 hours (as described above, see Table 2) and after washing, the two cell cultures were mixed in a ratio of 10 (PBMC):1 (tumor cells) and incubated for the next 4 hours. The total cell population was then analyzed by flow cytometry for the markers CD3, CD56, CD107a and IFNγ. Panel C: % of cytotoxic NK cells measured by release of IFNγ. The gating strategy is similar to that shown in Figure 5A. Data represent the mean of three independent experiments. [Figure 8A(i)] Antitumor efficacy of RLI-15 in combination with Kadcyla in a mouse orthotopic huHER2 EMT-6 breast cancer model. Groups of n=8 animals (Balb / c AnN, fully immunocompetent mice) were implanted with the EMT-6 breast cancer cell line carrying an engineered human HER2 receptor. Treatment with Kadcyla (15 mg / kg; days 0, 7) was initiated when tumors reached a mean tumor volume of 140 mm3 in the individual groups. Two weeks later, animals were given RLI-15 (1 mg / kg; days 15-18) and outcomes were evaluated on study day 23. (A) Representative example of ex vivo Her-2 expression FC analysis in tumors from one of the study mice (all n=3 mice were euthanized at the randomization stage and subjected to ex vivo analysis of Her-2 tumor expression). [Figure 8A(ii)] This is a continuation of Figure 8A(i). [Figure 8A(iii)] This is a continuation of Figures 8A(i) and 8A(ii). [Figure 8B]Antitumor efficacy of RLI-15 in combination with Kadcyla in a murine orthotopic huHER2 EMT-6 breast cancer model. Groups of n=8 animals (Balb / c AnN, fully immunocompetent mice) were implanted with the EMT-6 breast cancer cell line carrying an engineered human HER2 receptor. Treatment with Kadcyla (15 mg / kg; days 0, 7) was initiated when tumors reached a mean tumor volume of 140 mm3 in the individual groups. Two weeks later, animals were given RLI-15 (1 mg / kg; days 15-18) and outcomes were evaluated on study day 23. (B) Mean absolute tumor volumes (mm3) with SEM dependent on time shown as study days for treatment groups. G1: vehicle, sc on days 1-4; G2: RLI-15, 1 mg / kg sc on days 15-18; G4: Kadcyla iv on days 0 and 7; and G7: Kadcyla iv on days 0 and 7 and RLI-15, 1 mg / kg sc on days 15-18. Vertical arrows indicate a single iv dose of Kadcyla. Horizontal arrows indicate 4 days of sc administration of RLI-15. [Figure 8C(i)] Antitumor efficacy of RLI-15 in combination with Kadcyla in a mouse orthotopic huHER2 EMT-6 breast cancer model. Groups of n=8 animals (Balb / c AnN, fully immunocompetent mice) were implanted with the EMT-6 breast cancer cell line with engineered human HER2 receptors. When tumors reached a mean tumor volume of 140 mm3 in individual groups, treatment with Kadcyla (15 mg / kg; days 0, 7) was initiated. Two weeks later, animals were given RLI-15 (1 mg / kg; days 15-18) and outcomes were evaluated on study day 23. (C) Individual animal data are shown for the four treatment groups G1, G2, G4, and G7 in panel B. [Figure 8C(ii)] This is a continuation of Figure 8C(i). [Figure 8D]Antitumor efficacy of RLI-15 in combination with Kadcyla in a mouse orthotopic huHER2 EMT-6 breast cancer model. Groups of n=8 animals (Balb / c AnN, fully immunocompetent mice) were implanted with the EMT-6 breast cancer cell line with engineered human HER2 receptors. Treatment with Kadcyla (15 mg / kg; days 0, 7) was initiated when tumors reached a mean tumor volume of 140 mm3 in each group. Two weeks later, animals were given RLI-15 (1 mg / kg; days 15-18) and outcomes were evaluated on study day 23. (D) Intermediate levels of HER2 expression in the huHER2 / EMT-6 model in Balb / c mice. Paraffin-embedded sections were prepared from residual tumors in all study mice and stained with HercepTest™. Individual photographs are representative of the staining pattern that most closely matched the mean H-score in the individual treatment groups G1, G2, G4, and G7. [Figure 9] In vitro cell killing assay of anti-CLDN18.2 ADCs in combination with SOT101. A549-CLDN18.2 cells expressing claudin 18.2 were incubated with the indicated concentrations of anti-CLDN18.2 antibody hCl1a WT (with unmodified IgG1 Fc) or SOT102 (hCl1a-derived ADC with PNU as toxin and LALA Fc IgG1 Fc substitution) with freshly isolated human NK cells at an E:T ratio of 10:1. SOT101 was added to reach a concentration of 0.1 nM where indicated. After 24 hours, cytotoxicity was measured using lactate dehydrogenase assay (LDH). Data are plotted as mean ± SEM, % cytotoxicity compared to cells permeabilized with lysis buffer. n=2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0147] Example 1 - General Method Flow cytometry analysis of HSP70, HSP90 and CRT on the cell surface (Fucikova, Moserova et al. 2014) Total 1×10 6Cells are plated in 12-well plates and then treated with compounds or modalities that induce ICD for 6, 12 or 24 hours. Cells are harvested and washed twice with PBS. The cells are then incubated with primary antibodies diluted in cold blocking buffer (2% fetal bovine serum in PBS) for 30 minutes, followed by washing and incubation with Alexa 648-conjugated monoclonal secondary antibodies in blocking solution. Each sample was then analyzed using a FACScan Aria (BD Bioscience). Cell surface expression of HSP70, HSP90 and CRT is analyzed in non-permeabilized Annexin V-positive / DAPI-negative cells.

[0148] Detection of HMGB1 release (Fucikova, Moserova et al. 2014) After treatment of cells with compounds or modalities that induce ICD, supernatants are collected at different time points (6, 12, 24 and 48 hours). Dying tumor cells are removed by centrifugation, and the supernatants are isolated and immediately frozen. Quantification of HMGB1 in the supernatants can be assessed using an enzyme-linked immunosorbent assay according to the manufacturer's instructions (IBL, Hamburg, Germany).

[0149] ATP detection (Adkins, Sadilkova et al. 2017) For the measurement of extracellular ATP release, cell culture supernatants are used, and for intracellular ATP detection, cells are centrifuged at 2200 rpm for 2 min and the pellet is resuspended in cell lysis buffer (eBioscience). ATP content can be determined according to the manufacturer's instructions (ATP assay kit, Sigma-Aldrich).

[0150] Example 2 – In vitro killing of AGS tumor cells by Kadcyla 5 × 10 of gastric AGS tumor cell line (HER2 FC: 1–2+) 6 cells (75cm 2Culture flasks) were incubated for 72 hours in the presence of increasing concentrations of Kadcyla (5, 7, 8, 10 μg / ml).

[0151] Survival (Annexin V - / DAPI - ), early apoptosis (Annexin V + / DAPI - ), late apoptosis (Annexin V + / DAPI + ) and necrotic cell populations (Annexin V - / DAPI + The viability of tumor cells upon treatment with Kadcyla was analyzed by flow cytometry using Annexin V (Exbio, Czech Republic) and DAPI dilactate (Thermo-Fisher Scientific, USA) staining for the analysis of the amount of apoptotic cell death (see Figure 2A and B). An increase in the concentration of Kadcyla increased the amount of Annexin V, which represents an early apoptotic cell population. + / DAPI - This resulted in an increase in the population of cells.

[0152] Expression of ICD markers HSP70, HSP90 and CRT as well as NK cell ligands was analyzed using anti-calreticulin (Abcam, USA), anti-HSP70 (R&D Systems, USA), anti-HSP90 (Enzo Life Sciences, USA) antibodies against these early apoptotic (Annexin V)-associated proteins. + / DAPI -) cell populations by flow cytometry. APC AffiniPure F(ab')2 Fragment Goat Anti-Mouse (Jackson ImmunoResearch) was used as the secondary antibody. For all tested ICD markers, Kadcyla treatment led to a strong increase in ICD markers compared to non-treated cells. There was no or only a weak trend for the mean fluorescence intensity to increase with increasing concentrations of Kadcyla, but this trend was stronger when looking at the % of marker-positive cells (see Figure 3A and B). Similarly, expression of NK cell ligands CD112, CD155, and ULBP3 and ULBP2 / 5 / 6, as well as ULBP1, was examined using ULBP-2 / 5 / 6 (Biocompare, USA), CD155 (Biolegend, USA), Nectin-2 / CD112 (R&D Systems, USA), ULBP-1 (Biocompare, USA) and ULBP-3 antibodies (Biocompare, USA) to assess the early apoptotic (Annexin V) expression in these cells. + / DAPI - ) cell populations were determined by flow cytometry. The early apoptotic cell population showed increased expression of the NK cell ligands CD112, CD155, ULBP3 and ULBP2 / 5 / 6, which reached a maximum already at 7 μg / kg for CD155 and at 8 μg / kg for ULBP3 and ULBP2 / 5 / 6. The NK cell-independent ligand ULBP1 did not show any significant change upon Kadcyla treatment (see Figure 4).

[0153] In summary, the data indicate that Kadcyla-mediated cell killing results in the majority of tumor cells undergoing ICD, even in tumor cell lines with low to intermediate Her-2 expression.

[0154] Example 3 –PBMC isolation and RLI-15 treatment To mimic the combined administration of Kadcyla and RLI-15 on a sequential schedule, human PBMCs from three donors were isolated from fresh human blood using a Ficoll-Paque gradient and then incubated in the presence of 2.5 ng / ml RLI-15 for 72 hours.

[0155] Example 4 – In vitro activation of NK cells by tumor cells treated with Kadcyla After the incubation period, the dying tumor cells prepared in the examples were washed with increasing concentrations of Kadcyla, transferred to fresh culture medium, and added to the RLI-15-treated PBMCs prepared in the examples and examples at a 1:10 ratio (30,000 tumor cells:300,000 PBMCs). These mixed cell populations were incubated for an additional 4 hours to induce CD3 - CD56 + The percentage of cells (NK cells) was analyzed by flow cytometry using the markers CD3, CD56, CD107a and IFNγ (Figure 5A). As controls, untreated tumor cells were incubated with untreated PBMCs (KadNT in ADC group) and untreated tumor cells were incubated with RLI-15 treated PBMCs (KadNT in RLI+ADC group).

[0156] Treatment of tumor cells with Kadcyla reduced the expression of CD3 - CD56 + ) did not result in a subsequent proliferation of NK cells. On the other hand, RLI-15 alone led to the expected strong proliferation of NK cells, which was somewhat weaker when RLI-15-incubated PBMCs were cocultured with Kadcyla-treated tumor cells (Figure 5B).

[0157] CD107a is an activation marker for NK cells, but not all NK cells (CD107a + and CD107a - (including both populations of cells) +Examining the activated NK cells by plotting the % of NK cells, incubation of PBMCs with both RLI-15 alone (RLI-15) or Kadcyla-treated tumor cells (ADC group) resulted in only a moderate activation of NK cells up to 20% (compared to PBMC CTR), whereas the combination of RLI-15-treated PBMCs and Kadcyla-treated tumor cells resulted in a robust activation of NK cells, reaching a plateau of approximately 70% for 7-8 μg / ml Kadcyla (Figure 5C).

[0158] Treatment of PBMCs with RLI-15 was sufficient for NK activation (see KadNT group), but the effect was considerably higher when combined with Kadcyla-treated tumor cells (RLI+ADC Kad 5–10 μg / ml group), which upregulated CD107a + An increase in Kad expression from <40% (KadNT) to approximately 70% (Kad 7 μg / ml and Kad 8 μg / ml) of cells was observed. + The increase in the number of cells expressing annexin V + / DAPI - This also corresponded to tumor cell viability as measured by cell proliferation (see Fig. 2B, C).

[0159] IFN-γ as another measure of NK cell activation + A very similar picture was seen when looking at the % of NK cells (Figure 5D), again showing only moderate IFNγ-producing NK cells for the Kadcyla only group (up to about 10%) and the RLI-15 only group, but the combination of RLI-15-incubated PBMCs with Kadcyla-treated tumor cells resulted in up to about 40% IFNγ-producing NK cells, peaking at 7-8 μg / ml Kadcyla.

[0160] In summary, we have shown that RLI-15 as a single agent can significantly stimulate NK cell proliferation (up to about 40% compared to control PBMCs), but after co-incubation with Kadcyla-pretreated tumor cells, we observed a dramatic increase in NK cell activation compared to those treated with RLI-15 alone (up to 10% CD3+ cells compared to PBMCs treated with RLI-15 alone). - CD56 + CD107 + Up to 70% of cells and CD3 - CD56 + IFNγ + We observed up to 40% of cells in mice, indicating a strong synergy in vitro between Kadcyla-mediated induction of ICD and the immunostimulatory effects of RLI-15 that may have predictive value for in vivo efficacy.

[0161] Interestingly, we observed that in this in vitro setting, NK cell activation already peaks at approximately 7-8 μg / ml Kadcyla. Such 7.7 μg / ml is equivalent to a dose of approximately 0.5 mg / kg of Kadcyla in mice, which is much lower than the typically applied dose of 15 mg / kg. Although this finding is difficult to translate to an in vivo or even human situation, it is still expected that such an additional therapeutic effect of co-treatment with an IL-2 / IL-15Rα agonist will result in increased efficacy of the ICD inducer at lower doses, thereby increasing the therapeutic window.

[0162] Example 5 – Testing of other ICD Inducing Agents or ICD Inducing Modalities A similar set-up of in vitro experiments described in Examples 2 and 4 was used to screen for synergistic effects against other ICD-inducing agents / modalities (selected standard of care chemotherapy "SoC": doxorubicin or cisplatin).

[0163] When investigating other ADCs, tumor cell lines expressing the respective target to which the antibody is directed are required. For cytotoxic small molecules such as anthracyclines, microtubule destabilizers (Diederich 2019) (vinca alkaloids, taxanes such as paclitaxel, epothilones, eribulin, auristatin E, maytansine derivatives), bleomycin, bortezomib, cyclophosphamide, platinum complexes (oxaliplatin, cisplatin) and nucleoside analogues, customary cell lines showing sensitivity to such drugs should be used. Appropriate conditions for bortezomib are described in Spisek et al. (2007).

[0164] This setup may also be used for ICD-inducing therapeutic modalities, such as high hydrostatic pressure (HHP), X-rays, gamma or UV rays, photodynamic therapy or hyperthermia / hyperthermia, where susceptible tumor cells are subjected to such physical stresses under conditions that induce ICD before co-culturing with pre-treated PBMCs. Suitable conditions for inducing ICD physical stresses are described, for example, in WO 2013 / 004708, Adkins et al. (2014), and Adkins et al. (2017).

[0165] Obviously, RLI-15 can be replaced by other IL-2 / IL-15βγ agonists known in the art to pre-treat PBMCs.

[0166] For the selected SoC, we defined the optimal concentration and treatment period (Table 2) for efficient induction of ICD in a similar manner as previously done for Kadcyla analyzing cell death and cell surface exposure of ICD markers (Figure 6, panels A, B). Doxorubicin and cisplatin were equally (cisplatin) or even better suited to induce early apoptosis in AGS tumor cell lines (see Figure 6A). Looking at the % of positive cells for calreticulin, HSP70 and HSP90, Kadcyla was more efficient compared to cisplatin and doxorubicin, but in terms of the mean fluorescence intensity (MFI) indicator, cisplatin had a stronger induction and doxorubicin seemed almost equal (potentially stronger for calreticulin and HSP70, but weaker for HSP90). This may be interpreted as chemotherapy induces relatively few cells to enter ICD, but the induced cells show a stronger ICD marker expression.

[0167] Next, the inventors investigated NK cell proliferation (CD3 - CD56 + The synergistic potential of doxorubicin and cisplatin on RLI-15 was evaluated, as measured by CD3+ cell counts, activation (CD107a release), and cytotoxicity (IFNγ). Figure 7, panels A-C, compares this data with data previously collected for Kadcyla (Figure 5B-D). RLI-15 treatment increased CD3+ - CD56 +Combination with SoC or Kadcyla also resulted in a significant activation of these cells compared to PBMCs treated with RLI-15 alone, as shown by the release of CD107a (panel B) and a higher cytotoxic potential as shown by the analysis of IFNγ secretion (panel C). Furthermore, Kadcyla showed a higher synergistic potential against RLI-15 in vitro than either doxorubicin or cisplatin, mainly as represented by the higher cytotoxic potential of NK cells after treatment with Kadcyla (panel C). Specifically, the activation status and cytotoxic potential of NK cells after treatment with Kadcyla were significantly higher than those of NK cells after treatment with SoC (Figure 7, panel C). This may be due to the antibody-dependent cellular cytotoxicity (ADCC) activity of trastuzumab, which is present in the vicinity of the payload (DM1) in Kadcyla and may contribute to the synergistic effect in combination with RLI-15. This is because ADCC has been described as being synergistic with the activity of IL-2 / IL-15βγ agonists.

[0168] Example 6 – Orthotopic huHER2 / EMT-6 in vivo breast cancer model The combination of Kadcyla and RLI-15 was tested in vivo in an orthotopic huHER2 / EMT-6 breast cancer model in Balb / c AnN immunocompetent mice. 3The study was initiated when the tumor size reached 100%. Kadcyla was administered twice on study days 0 and 7 at a human equivalent dose (15 mg / kg) to potentially induce ICD prior to RLI-15 treatment. RLI-15 was administered in four consecutive doses on study days 15-18 to amplify the number of immune cells and activate them. Antitumor efficacy was assessed at the level of absolute tumor volume change. Safety was monitored by weight loss in individual animals. HER2 expression in individual tumors was analyzed at the end point using HercepTest™ (Dako) to map potential heterogeneity of the model (staining was performed according to the instructions given by the manufacturer).

[0169] Three pre-test animals were sacrificed prior to study initiation to collect tumors for ex vivo analysis of HER2 expression by flow cytometry, which revealed that approximately 90% of huHER2 / EMT-6 tumor cells were positive for HER2 ex vivo (Figure 8A).

[0170] For all remaining tumors collected at the end of the study and analyzed by IHC for HER2 expression, it was shown that tumor expression levels in vivo could be considered rather intermediate with a mean H-score of 91.66 to 121.10 (see Table 4).

[0171] [Table 4]

[0172] During the course of the study, we observed very homogeneous tumor growth among the individual groups, and a synergistic effect of the combination of RLI-15 and Kadcyla was seen compared to the single agent activity of both compounds (Figure 8B). Both RLI-15 single agent treatment at 1 mg / kg sc on days 15-18 and Kadcyla single agent treatment at 15 mg / kg iv on days 0 and 7 resulted in an intermediate increase in tumor volume, whereas the combined treatment of these treatments resulted in a significant decrease in tumor volume at the end of the study.

[0173] Single-animal data (Figure 8C) showed that single-agent activity of RLI-15 resulted in complete tumor eradication in 2 of 8 mice, whereas Kadcyla therapy alone resulted in 3 complete and durable responses and partial responses with tumor recurrence in another 3 animals. Such tumor recurrence is also frequently observed in patients and represents one of the major trends of Kadcyla antitumor therapy. However, we showed that combining Kadcyla with RLI-15 can prevent such recurrence. This combination resulted in durable and complete tumor eradication in 6 of 8 animals, indicating the possible therapeutic prospects of this combination that may be used in common practice.

[0174] In summary, the combination of RLI-15 and Kadcyla demonstrated synergistic antitumor efficacy in immune-resistant huHER2 / EMT-6 tumors in vivo.

[0175] Example 7 – In vitro cell killing assay The combination of anti-CLDN18.2-directed ADC SOT102 in combination with SOT101 (RLI-15) was evaluated in an in vitro cell killing assay. SOT102 is an antibody-drug-conjugate based on the anti-CLDN18.2 antibody hCl1a (SEQ ID NO: 20, SEQ ID NO: 21) with the ADCC-inactivating heavy chain substitution LALA (L234A / L235A), in which the anthracycline PNU-159682 (PNU) is linked to the C-terminus of the light chain by the non-cleavable linker GGGGSLPQTGG (SEQ ID NO: 24)-ethylenediamine (hCl1a-LC-G2-PNU) (SEQ ID NO: 22, SEQ ID NO: 23), which is further described in Example 7 of WO 2022 / 136642.

[0176] Cell lines. Human A549 cells overexpressing CLDN18.2 (A549-CLDN18.2) were grown in DMEM medium (Gibco) supplemented with 10% fetal bovine serum, 2 mM glutamine (GlutaMAX, Gibco), 100 U / ml penicillin, 0.1 mg / ml streptomycin (Invitrogen), and 2 μg / ml puromycin (Gibco). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

[0177] Isolation of human NK cells: First, donor blood (buffy coat, approximately 70 ml of blood) was processed by ficoll density gradient centrifugation. Peripheral blood mononuclear cells (PBMCs) were collected and human NK cells (hNK) were isolated using EasySep Human NK Cell Isolation Kit (STEMCELL) according to the manufacturer's protocol. NK cells were washed and used directly in the assay. The purity of the NK cell fraction was assessed by flow cytometry and reached more than 70%.

[0178] Cell killing assay: A549_CLDN18.2 cells were seeded in 96-well plates (20,000 cells / well) and incubated overnight. Freshly isolated human NK (hNK) cells were resuspended in assay medium-RPMI1640 (without phenol red) supplemented with 2 mM glutamine and 10% heat-inactivated (20 min at 56 °C) pooled complemented human serum (Innovative Research). Media from the 96-well plates containing adherent cells was aspirated and target cells were mixed with hNK cells to reach an E:T ratio of 10. Tested proteins were added at a concentration range of 0-100 μg / ml and SOT101 was added to the appropriate wells to a concentration of 0.1 nM. The mixture was incubated at 37°C for 24 hours, and then cytotoxicity was measured as the activity of lactate dehydrogenase enzyme released from dead cells using the LDH Cytotoxicity Assay (Abcam, ab65393) according to the manufacturer's protocol. 10 μl of the supernatant was transferred to a new 96-well plate, mixed with LDH substrate, and the resulting color change was measured using a spectrophotometer. Cytotoxicity was calculated as a percentage of the signal obtained from wells where all plated cells were permeabilized with lysis buffer (100% cytotoxicity).

[0179] Flow cytometry: CLDN18.2 expression levels were measured by flow cytometry (BD LSRFortessa). Cells were harvested by trypsinization, washed, and labeled with human primary anti-CLDN18.2 antibody (2 μg / ml) for 30 min at 4° C., followed by goat anti-human secondary antibody conjugated with phycoerythrin (PE; eBiosciences, 12-4998-82) and DAPI to detect dead cells. For negative controls, cells were labeled with secondary antibody and DAPI only. The purity of isolated hNK cells was measured by staining the NK fraction with a set of fluorescently labeled antibodies to identify immune cell populations: anti-CD3 (APC-ef780, Thermo-Fisher Scientific), anti-CD16 (PE-Cy7, Biolegend), anti-CD56 (A700, Biolegend), anti-CD11c (APC, Exbio), and Zombie Aqua Viability Dye (BV510, Biolegend). - CD11c - CD16 + CD56 + All flow cytometry data obtained were analyzed with FlowJo software.

[0180] The ADCC-capable anti-CLDN18.2 antibody itself (hCl1a WT) showed slight cytotoxic activity against target cells under test conditions with freshly isolated NK cells, which was only slightly improved by the addition of SOT101. The ADC SOT102 alone, which contains the same CDRs as hCl1a WT but has a LALA substitution that minimizes the ADCC activity of the antibody, showed some cell killing, which was therefore mediated by the linked PNU toxin. The combination of SOT102 and SOT101 then exerted significantly higher cell killing activity (see Figure 9). Since the combination of hCl1a WT and SOT101 showed no significant difference, such an increase is not mediated by ADCC (ADCC is further reduced in SOT102), i.e., the above effect must be due to the combination of NK cell activation by SOT101 and an ICD-inducing toxin linked to a very inactive antibody. Also, given the absence of T cells and dendritic cells in this assay, the observed synergy is based on the direct effect of the delivered anthracycline on tumor cells interacting with SOT101-stimulated NK cells. In other words, the anti-CLDN18.2 ADCs SOT102 and SOT101 synergize in killing CLDN18.2-expressing target tumor cells in the presence of freshly isolated human NK cells, and such synergy is not based on ADCC or tumor antigen presentation to cytotoxic T cells by, for example, dendritic cells, and therefore may be due to ICD induced by the anthracycline PNU.

[0181] Example 8 –Anti-tumor efficacy of SOT101 and oxaliplatin in vivo in the MC38 colon cancer model C57BL / 6 mice were treated with 5 × 10 5MC38 colon cancer cells were injected sc. Starting from day 3 after tumor cell inoculation, mice were treated ip with 7.5 mg / kg oxaliplatin Q2W or sc with 2×2 mg / kg SOT101 on W1 and W2 or a combination of both according to combination schedules 1 (oxaliplatin 7.5 mg / kg ip D3 and D17 + SOT101 sc 2×2 mg / kg D4,5 and D18,19), 2 (oxaliplatin 7.5 mg / kg ip D3 and D17 + SOT101 sc 2×2 mg / kg D4,5 and D11,12) and schedule 3 (oxaliplatin 7.5 mg / kg ip D3 + SOT101 sc 2×2 mg / kg D4,5 and D11,12). Individual mouse weights and tumor growth were monitored. On day 21, mice were euthanized. Combination treatment with SOT101 and oxaliplatin was well tolerated, given that there were no significant differences in relative mouse body weights (data not shown). Results indicate increased efficacy of combination administration (data not shown).

[0182] array [ka] [ka] [ka]

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Claims

Claim 1 An interleukin-2 / interleukin-15 receptor βγ (IL-2 / IL-15Rβγ) agonist for use in treating cancer in a patient, wherein the IL-2 / IL-15Rβγ agonist is a. administered simultaneously with or sequentially relative to a cytotoxic compound capable of inducing immunogenic cell death (ICD), b. administered simultaneously with or sequentially relative to the application of a modality capable of inducing ICD, c. administered simultaneously with a cytotoxic compound capable of inducing ICD and simultaneously with a modality capable of inducing ICD, d. administered simultaneously with a cytotoxic compound capable of inducing ICD and sequentially relative to a modality capable of inducing ICD, e. administered sequentially relative to a cytotoxic compound capable of inducing ICD and simultaneously with a modality capable of inducing ICD, or f. administered sequentially relative to a cytotoxic compound capable of inducing ICD and sequentially relative to a modality capable of inducing ICD The IL-2 / IL-15Rβγ agonist for use. Claim 2 In the case of sequential administration, the IL-2 / IL-15Rβγ agonist is administered before and / or after the cytotoxic compound capable of inducing the ICD, or before and / or after the modality capable of inducing the ICD. The IL-2 / IL-15Rβγ agonist for use according to claim 1. Claim 3 In the case of sequential administration, the IL-2 / IL-15Rβγ agonist is administered after the cytotoxic compound capable of inducing the ICD, or after the modality capable of inducing the ICD. The IL-2 / IL-15Rβγ agonist for use according to claim 1 or claim 2. Claim 4 In the case of simultaneous administration, the IL-2 / IL-15Rβγ agonist and the cytotoxic compound capable of inducing the ICD are provided as components of the same pharmaceutical composition or as components of separate pharmaceutical compositions. The IL-2 / IL-15Rβγ agonist for use according to claim 1. Claim 5 The cytotoxic compound capable of inducing the ICD is selected from the group consisting of anthracyclines; vinca alkaloids, taxanes, epothilones, eribulin, auristatins, and maytansine or maytansinoids, and microtubule destabilizing agents including tubulysin; bleomycin; proteasome inhibitors including bortezomib; platinum complexes including cyclophosphamide, oxaliplatin, and alkylating agents including pyrrolo-benzodiazepines, calicheamicin derivatives and topoisomerase I inhibitors, and nucleoside analogs, for use in the IL-2 / IL-15Rβγ agonist according to claim 1 or claim 2.

6. The cytotoxic compound capable of inducing the ICD is covalently linked to an antibody to form an antibody-drug conjugate (ADC), for use in the IL-2 / IL-15Rβγ agonist according to claim 1 or claim 2.

7. The cytotoxic compound is an anthracycline, maytansine or maytansinoid, topoisomerase I inhibitor, or calicheamicin derivative, preferably the topoisomerase I inhibitor is not SN38 for use in the IL-2 / IL-15Rβγ agonist according to claim 5.

8. The anthracycline is selected from the group consisting of daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone and PNU-159682, or the maytansine or maytansinoid is selected from the group consisting of maytansine, mertansine / emtansine (DM1), ansamitocin and ladostatin / sorladostatin (DM4), or the topoisomerase I inhibitor is topotecan, exatecan or an exatecan derivative, especially DS-8201a or DX-8951, or The calicheamicin is calicheamicin γ １ Ｉ is for use in the IL-2 / IL-15Rβγ agonist according to claim 5.

9. The antibody is an antibody that specifically binds to HER2, preferably trastuzumab; an antibody that binds to HER3, preferably patritumab; an antibody that binds to nectin-4, preferably enfortumab; an antibody that binds to CD33, preferably gemtuzumab or IMGN779, more preferably gemtuzumab; an antibody that binds to CD30, preferably brentuximab; an antibody that binds to CD22, preferably inotuzumab, or CD79B, preferably polatuzumab; an antibody that binds to ROR-1, preferably NBE-002; or an antibody that binds to CLDN18.2, preferably zolbetuximab or a humanized variant thereof, the IL-2 / IL-15Rβγ agonist for use according to claim 6.

10. The ADC is trastuzumab emtansine, trastuzumab deruxtecan, gemtuzumab ozogamicin, inotuzumab ozogamicin, brentuximab vedotin, enfortumab vedotin and polatuzumab vedotin, especially trastuzumab emtansine or enfortumab vedotin, the IL-2 / IL-15Rβγ agonist for use according to claim 6.

11. The patient suffers from a tumor expressing HER2, preferably, the patient is diagnosed with a tumor having low to moderate HER2 expression, the IL-2 / IL-15Rβγ agonist for use according to claim 1 or claim 2.

12. The modality capable of inducing the ICD is selected from high hydrostatic pressure (HHP), photodynamic therapy, UV irradiation, radiotherapy, oncolytic virus therapy and hyperthermia, the IL-2 / IL-15Rβγ agonist for use according to claim 1 or claim 2.

13. The IL-2 / IL-15Rβγ agonist is an interleukin 15 (IL-15) / interleukin-15 receptor α (IL-15Rα) complex, the IL-2 / IL-15Rβγ agonist for use according to claim 1 or claim 2.

14. The IL-2 / IL-15Rβγ agonist is an interleukin 15 (IL-15) / interleukin-15 receptor α (IL-15Rα) complex, and the complex is a fusion protein comprising a sushi domain of human IL-15Rα or a derivative thereof, a flexible linker, and human IL-15 or a derivative thereof. Preferably, the human IL-15Rα sushi domain contains the amino acid sequence of SEQ ID NO: 6, and the human IL-15 contains the amino acid sequence of SEQ ID NO: 4 The IL-2 / IL-15Rβγ agonist for use according to claim 1 or claim 2

15. The IL-2 / IL-15Rβγ agonist is an interleukin 15 (IL-15) / interleukin-15 receptor α (IL-15Rα) complex, and the complex is a fusion protein containing the amino acid sequence of SEQ ID NO:

9. The IL-2 / IL-15Rβγ agonist for use according to claim 1 or claim 2

16. The IL-2 / IL-15Rβγ agonist is an IL-15 / IL-15Rα complex, the complex is a fusion protein containing the amino acid sequence of SEQ ID NO: 9, and the ADC contains an antibody that specifically binds to HER2. Preferably, the antibody is trastuzumab. The IL-2 / IL-15Rβγ agonist for use according to claim 6

17. The IL-2 / IL-15Rβγ agonist is administered subcutaneously (s.c.) or intraperitoneally (i.p.), preferably s.c. The IL-2 / IL-15Rβγ agonist for use according to claim 1 or claim 2

18. The IL-2 / IL-15Rβγ agonist is further combined with an immune checkpoint inhibitor. Preferably, the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody or an anti-CTLA4 antibody, more preferably an anti-PD-L1 antibody or an anti-PD-1 antibody. The IL-2 / IL-15Rβγ agonist for use according to claim 1 or claim 2

19. The cancer is a hematological cancer or a solid cancer, preferably, renal cell carcinoma, lung cancer (especially, non-small cell lung cancer, small cell lung cancer), bladder cancer (especially, urothelial cancer), melanoma, Merkel cell carcinoma, cutaneous squamous cell carcinoma, high-frequency microsatellite instability solid tumor, breast cancer (especially, triple-negative breast cancer), mesothelioma, prostate cancer, thyroid cancer, thymic cancer, cervical cancer, biliary tract cancer, hepatocellular carcinoma, ovarian cancer, gastric cancer, pancreatic cancer, esophageal cancer, head and neck squamous cell carcinoma, and anal cancer, and ALL, AML, CLL, CML, AMoL, Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma, and the IL-2 / IL-15Rβγ agonist for use according to claim 1 or claim 2 selected from the group consisting of.

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