Fusion protein consisting of an antibody and a mutein

Abstract

The present invention relates to the fields of biotechnology and immuno-oncology. Fusion proteins comprising an interleukin-2 agonist mutein linked to an immunoglobulin via a linker are disclosed. These fusion proteins are useful for the treatment of cancer, given their superior properties compared to other similar ones based on non-alpha IL-2 muteins, by simultaneously maintaining the ability of the antibody to perform ADCC and CDC, and further activating NK cells and CD8+ without expanding regulatory T cells. The convergence of these properties results in fusion proteins with superior antitumor properties than parent antibodies and even in combination with their non-alpha muteins.

Description

[Technical field]

[0001] The present invention relates to the fields of biotechnology and immuno-oncology. In particular, the present invention describes a fusion protein that is composed of a non-alpha mutein agonist of interleukin-2 (IL-2) fused to an antibody specific for an antigen expressed on a tumor, and that is capable of recruiting effector functions. [Background technology]

[0002] Immunocytokines (ICs), biopharmaceuticals composed of a cytokine fused to an antibody or antibody fragment, are attractive immunotherapeutic tools for the treatment of cancer, but their use is also expanding to other diseases.

[0003] IL-2 is a potent activator of T lymphocytes and a stimulator of NK cells and their ADCC (antibody-dependent cellular cytotoxicity) (Hank et al. (1990) J Biol Response Mod. 9:5-14). For this reason, it is one of the most used cytokines in the generation of fusion proteins of this kind. Indeed, most of the clinical advances have been made with IL-2 fused to antibodies specific for tumor antigens (Sondel, PM and SD Gillies. (2012) Antibodies. 1(2):149-171).Despite promising results in phase II clinical trials (Albertini,MR et al.(2012)Cancer Immunol Immunother.61(12):2261-2271; Danielli,R.,et al.(2015)Cancer ImmunolImmunother.64(8):999-1009; Kaufman,L.(2014)Journal of Clinical Oncology.32(15); Lansigan,F.et al.(2016)Blood.128(22):620), these ICs are associated with a high toxicity profile (Panelli,MC et al.(2004)J Transl Med.2(1):17;Skrombolas,D.and JG Frelinger.(2014)Expert Rev Clin Immunol.10(2):207-217;Klein,C.,et al.(2017)Oncoimmunology.6(3):e1277306) and the expansion of regulatory T cells, which can potentially inhibit antitumor activity (Ahmadzadeh,M.and SARosenberg.(2006)Blood.107(6):2409-2414;Jensen,HK,et al.(2009)Clin Cancer Res.15(3):1052-1058;Fournier,P.,et al.(2011)Int J Oncol.38(6):1719-1729;Gubbels,JA,et al.(2011)Cancer Immunol Immunother.60(12):1789-1800;Pretto,F.,et al.(2014)Cancer Immunol Immunother.63(9):901-910;Sim,GC,et al.(2014) J Clin Invest.124(1):99-110; Lansigan,F.et al.(2016) Blood.128(22):620), retains similar adverse effects as recombinant IL-2.Further limitations relate to suboptimal biodistribution and tumor targeting due to interaction with the high affinity IL-2 receptor (Klein, C., et al. (2017) Oncoimmunology. 6(3): e1277306; Waldhauer, I., et al. (2021) Mabs. 13(1): 1913791). This situation has prompted the development of ICs based on mutants of IL-2 (non-alpha muteins) that have reduced or no ability to bind to the alpha chain of the IL-2 receptor and are then rationally designed to stimulate specific subsets of immune cells and reduce toxicity (Runbeck, E., et al. (2021) Antibodies. (Basel) 10(1)).

[0004] Among these ICs there is a group that includes those based on IL-2v, characterized by the presence of mutations F42A, Y45A, L72G that abolish binding to the α chain of the IL-2 receptor, promoting the expansion of effector CD8+ T cells and NK cells, but not the selective proliferation of regulatory T cells. These fusion proteins have the format of full-length IgG (IgG1) and do not have classical antibody effector functions such as CDC (complement-dependent cytotoxicity) and ADCC, but are monovalent or monofunctional with respect to the cytokine. Within this group is the IC sergituzumab amnaleukin (CEA-IL2v), which consists of a specific antibody against carcinoembryonic antigen (CEA) fused to one molecule of an IL-2v variant via the C-terminus of one of its heavy chains (Klein, C., y cols. (2017) Oncoimmunology. 6(3): e1277306).

[0005] The set also includes the IC Simlukafusp alfa (FAP-IL2v), specific for fibroblast activation protein alpha (Waldhauer, I., et al. (2021) Mabs. 13(1):1913791) and the IC Anti-PD1-IL2v, which recognizes the programmed death 1 (PD-1) molecule (Klein, C., et al. (2019) Cancer Res. 79(13 Suppl):Abstract nr 1552).

[0006] On the other hand, an IC Erb-Sum-IL2 specific for EGFR (epidermal growth factor receptor) has been reported, which contains a mutant of IL-2 (sum-IL-2) characterized by the mutations L80F, R81D, L85V, I86Y, I92F. This series of changes with respect to the wild-type cytokine determines a decreased binding and an increased interaction of the mutein with the α- and β-chains of the receptor, respectively. It mediates good binding to activated CD8+ T cells and not to regulatory T cells. This IC is presented as a heterodimeric Fc fusion protein containing sum-IL2 monomer in one arm and anti-EGFR Fab in the other arm. For this monovalent IC with respect to antigen binding sites and cytokines, selective expansion of CD8+ T cells compared to regulatory T cells in the tumor microenvironment has been described, but the ability to recruit classical effector functions of antibodies such as ADCC and CDC has not been reported (Sun, Z., et al. (2019) Nat Commun. 10(1):3874).

[0007] Considering the background information described above, the inventors of the present application have created a type of IC for cancer therapy, whose design is based on the fusion of an IL-2 agonist mutein (IL2 non-alpha) to the carboxyl terminus of the heavy chain of an IgG antibody with the ability to recruit antibody effector functions, in order to make it a bifunctional molecule with respect to antigen binding site and cytokine. Surprisingly, this type of molecule has properties superior to other similar molecules based on non-alpha muteins, by maintaining or simultaneously improving the ability of the antibody to perform direct lysis, ADCC and / or CDC, and, moreover, activating NK and CD8+ T cells without expanding regulatory T cells. The convergence of these properties results in fusion proteins with antitumor properties superior to the parent antibodies and even to their combination with IL2 non-alpha. Finally, the formats and compositions of the fusion proteins listed in this invention do not interfere with the biological activity associated with the variable region of the antibody portion. This also applies to the ability to induce apoptosis or to induce cytotoxicity independently of effector functions. Summary of the Invention

[0008] In one embodiment, the subject of the present invention is a fusion protein, characterized in that it comprises an IL-2 agonist mutein (non-alpha mutein) bound to an immunoglobulin of IgG1 or IgG3 isotype, said immunoglobulin being recognized by Fcγ receptors and by molecules of the complement cascade. This immunocytokine is characterized in that it is a bivalent molecule with respect to the antigen binding site and the cytokine. Such a mutein and said immunoglobulin are linked via a linker. In particular, the ability of the mutein to bind to the alpha chain of the IL-2 receptor is affected by at least two orders of magnitude, the mutein being bound to the immunoglobulin via the carboxy terminus of each heavy chain of said immunoglobulin. In particular, the non-alpha mutein has a sequence selected from the group comprising SEQ ID NOs: 16 to 43.

[0009] In certain embodiments, the linker is 4 Ser) n ThrGly (SEQ ID NO: 44) and (Gly 4 Ser)n (SEQ ID NO:45), wherein n is Gly 4 This is the number of repeats of the Ser fragment and has a value of 1 to 5.

[0010] In a particular embodiment, the immunoglobulin heavy chain belongs to the human IgG1 or IgG3 subclass and comprises the sequence of SEQ ID NO: 11 or 12 or has greater than 97% identity to SEQ ID NO: 11 or 12.

[0011] In a particular embodiment, the light chain of the immunoglobulin is characterized as belonging to the human K isotype and has a sequence corresponding to SEQ ID NO:9.

[0012] In a particular embodiment, the light chain of the immunoglobulin is characterized as belonging to the human λ isotype and has a sequence corresponding to SEQ ID NO:10.

[0013] The immunocytokines of the present invention are further characterized by recognizing tumor antigens, including CD20, CD19, NGcGM3, PDL1, Her1, Her2 and Epcam.

[0014] In a further embodiment, the present invention relates to a pharmaceutical composition comprising a fusion protein as described herein in a concentration range of 0.5 mg / ml to 20 mg / ml and a pharma- ceutically acceptable vehicle.

[0015] Furthermore, the present invention relates to the use of the fusion protein disclosed in the present invention in the treatment of cancer, in particular the use of a nucleic acid molecule encoding the fusion protein of the present invention for subcutaneous, intramuscular or intratumoral injection.

[0016] In a further embodiment, the present invention relates to a method of treatment for a subject in need thereof, comprising subcutaneous, intravenous, intradermal, intramuscular or intraperitoneal administration of the pharmaceutical composition of the present invention in a dose range of 0.01 mg / Kg to 0.6 mg / Kg weight, particularly wherein the administration of said pharmaceutical composition is performed 1 to 3 times per week for 1 to 30 cycles, with the time between cycles being 7 days to 8 weeks.

[0017] IC Description In one embodiment, the object of the invention is a fusion protein or IC based on an IL-2 agonist non-alpha mutein (non-alpha mutein) with at least two orders of magnitude reduced binding affinity to the alpha chain of the receptor (Leon et al. (2018) Semin Oncol. 45(1-2):95-104), linked to an immunoglobulin heavy chain with the ability to recruit classical effector functions of antibodies, making it a bifunctional molecule with respect to antigen binding site and cytokine.

[0018] The term "fusion protein" as used herein refers to a polypeptide comprising the amino acid sequence of an antibody and the amino acid sequence of a heterologous polypeptide or protein, i.e., a polypeptide or protein that is not normally part of an antibody, linked via a peptide linker.

[0019] The term immunocytokine (IC) as used herein refers to an antibody format that binds a cytokine, such cytokine being fused to the antibody by a linker at the carboxy terminus of the heavy chain.

[0020] The terms "fusion protein" and "IC" are used interchangeably in the present invention.

[0021] The muteins forming part of the ICs described in the present invention are affected by their binding ability to high affinity receptors, specifically the alpha chain, constitutively expressed in regulatory T cells, by at least two orders of magnitude. The fusion proteins of the present invention exhibit the ability to activate NK and CD8+ T cells without expanding regulatory T cells, while recruiting effector functions such as direct lysis, ADCC and / or CDC, a property not possessed by other non-alpha mutein based ICs. These muteins are referred to herein as non-alpha muteins.

[0022] In particular, the IC antibody moieties are human IgG1 or IgG3 subclass immunoglobulins whose amino acid sequences correspond to natural or modified allelic variants of IgG1 or IgG3 with greater than 97% identity to those identified as SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and bind to Fcγ receptors and complement molecules. They are therefore compatible with ADCC and CDC activity. The constant regions of the light chains of the antibodies forming part of the fusion proteins of the invention are of the human kappa or lambda isotype, whose sequences are set out in SEQ ID NO: 9 and SEQ ID NO: 10. On the other hand, the variable regions of the antibody moieties may be murine, humanized or fully human and may be derived from immunoglobulins capable of exerting ADCC and CDC. The variable regions of the antibody moieties of the ICs of the invention recognize antigens expressed in human tumors, such as CD20, CD19, ganglioside NGcGM3, PDL1, Her1, Her2 and Epcam.

[0023] The non-alpha muteins according to the present invention are 4 Ser) n It is linked to the carboxyl terminus of each antibody heavy chain via ThrGly (SEQ ID NO: 44) or (Gly4Ser)n (SEQ ID NO: 45), where n is the number of repeats of the Gly4Ser fragment, and has the ability to impair the expansion of regulatory T cells and selectively expand CD8+ and NK effector T cells.

[0024] The non-alpha muteins can be any of those described in SEQ ID NOs: 16-43 previously disclosed in US Pat. No. 9,206,243 B2 and US Patent Application No. 2019 / 0315826 A1, or IL-2v (SEQ ID NO: 40) and variants thereof described in SEQ ID NOs: 41-43 previously disclosed in WO 2012107417. The muteins described in SEQ ID NOs: 16-21, created at the Center for Molecular Immunology, are IL-2 agonists, and their binding capacity to high affinity receptors constitutively expressed in regulatory T cells is affected by at least two orders of magnitude. As a result of their modifications with respect to wild-type IL-2, they can selectively expand the effector population of NK and memory CD8+ T cells, have the agonistic action of native IL-2, and have a greater antitumor effect in animal models than native IL-2 (US Pat. No. 9,206,24309). The IL2 non-alpha K35E, IL2 non-alpha K35Q and IL2 non-alpha K35D muteins (SEQ ID NOs: 22-39) are variants of IL2 non-alpha obtained by the method described in US Pat. No. 9,206,243, which contain point mutations that facilitate expression in different host systems and are compatible with the interaction profile of the non-alpha muteins with different chains of the IL-2 receptor (Rojas, G., et al. (2015) J Mol Recognit. 28(4):261-268). The IL-2v mutant and its variants (SEQ ID NOs: 40-43) contain mutations with respect to wild-type IL-2 that prevent binding to the alpha chain of the receptor, preventing the selective expansion of regulatory T cells and promoting the expansion of effector TCD8+ and NK cells (Klein, C., et al. (2017) Oncoimmunology. 6(3):e1277306).

[0025] In summary, the fusion protein format of the invention ensures the maintenance or improvement of the classical effector functions of antibodies (direct lysis, ADCC and CDC), which is consistent with the specific immunomodulatory properties of the non-alpha muteins, consisting of the induction of selective proliferation of effector cells versus regulatory T cells.

[0026] Pharmaceutical Compositions The IC subject of the present invention may be found as an active ingredient forming part of a variety of suitable pharmaceutical compositions and pharma- ceutically acceptable vehicles therefor, the concentration of the active ingredient in said pharmaceutical compositions being in the range of 0.5 mg / ml to 20 mg / ml, preferably 1 mg / ml to 10 mg / ml, or lyophilized.

[0027] Pharmaceutically acceptable vehicles include, but are not limited to, saline, pH neutral phosphate buffered saline, etc. Other buffers, dispersants, and non-toxic inert substances suitable for delivery to patients may be included in the compositions of the present invention. The compositions may be in a solution suitable for administration, and are usually sterile and free of undesirable particles.

[0028] Therapeutic Uses and Treatment The novelty of the ICs described in this invention consists of the convergence of the ability, in bifunctional fusion proteins based on IgG1 or IgG3 immunoglobulins and non-α muteins, to selectively expand and activate effector cells such as memory TCD8+ and NK cells over regulatory T cells, as well as to maintain or enhance typical effector functions of antibodies such as direct lysis, ADCC and CDC. This simultaneity has not been described for any of the non-α mutein based ICs described so far. Moreover, the presence of two non-α IL-2 mutein molecules per IC molecule makes it possible to increase the intrinsic immunomodulatory activity of the fusion protein. This further contributes to a potential reduction in toxicity due to non-interaction with high affinity receptors on endothelial cells. The Fc region offers, among other things, the possibility to obtain highly purified fusion proteins by affinity chromatography with protein A, which allows them to be administered as soluble proteins by various routes (subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal), while increasing the half-life of these agents in the circulation and thus their therapeutic efficacy.

[0029] The IC of the present invention is applied to subjects (these subjects are vertebrates such as humans) preferably via subcutaneous or intravenous routes in a dose range of 0.01 mg / Kg weight to 0.6 mg / Kg weight, corresponding to a total of 0.7 and 42 mg, respectively. The IC is administered to the patient 1 to 3 times per week for 1 to 30 cycles. The time between administration cycles is 7 days to 8 weeks.

[0030] Furthermore, the delivery strategies used in the present invention for these ICs may also include injection of the products by gene therapy approaches, such as injection of mRNA and transducing particles encoding them.

[0031] Considering the immunomodulatory properties of these molecules, in addition to enhancing antigen-specific antitumor responses, the systemic immune stimulatory capacity supports the possibility of combination with treatments against other targets in patients with various types of cancer. Similarly, combination with other immunomodulators and classical tumor treatments such as chemotherapy and radiotherapy are also possible. In particular, this type of biopharmaceutical can be used in the treatment of B-cell lymphoproliferative disorders, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), mantle cell lymphoma (MCL), follicular lymphoma, low-grade lymphoma, marginal body lymphoma (MZL), severe lymphoplasmacytic lymphoma (PL), B-cell lymphoproliferative syndrome, advanced solid tumors, head and neck tumors, brain tumors, adult and pediatric gliomas, pancreatic cancer, esophageal cancer, non-small cell lung cancer, and nasopharyngeal tumors.

[0032] Thus, the above-mentioned ICs are intended to constitute a therapeutic frontline that enhances the protective effect of antibodies and IL-2 currently used in the treatment of cancer. This effect is associated with the generation of fusion proteins that simultaneously possess important antitumor mechanisms ADCC and CDC activity and are able to selectively expand cytotoxic T cells and NK cells, but not regulatory T cells, resulting in a more efficient antitumor immune response and thus a delay in tumor growth and an increased survival of the treated individuals. Furthermore, the lower level of toxicity guaranteed by the IL-2 muteins used increases the probability of success compared to treatment with wild-type cytokines. All this translates into an improvement in life expectancy and quality of life in treated patients. [Brief description of the drawings]

[0033] [Figure 1A] Scheme representation of IC IgG-IL2 non-α. [Figure 1B] Showing the format of IC IgG-IL2 non-α. [Figure 2A] Assessment of IC expression in the supernatants of transduced CHO cells. [Figure 2B] Assessment of IC expression in transiently transfected HEK293T cells in suspension. [Figure 2C] Assessment of IC expression in transiently transfected HEK293T cells in suspension. [Figure 2D] Assessment of IC expression in transiently transfected HEK293T cells in suspension. [Figure 2E] Assessment of IC expression in the supernatants of transduced CHO cells. [Figure 2F] Assessment of IC expression in the supernatants of transduced CHO cells. [Figure 3A] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of anti-CD20-IgG1h-IL2non-α (IC1) under non-reducing conditions. [Figure 3B]Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of anti-CD20-IgG1h-IL2 non-α (IC1) under reducing conditions. [Figure 4A] Immunoidentification of IC1 and simple controls by Western blot with a specific antibody against human IgG under non-reducing conditions. [Figure 4B] Immunoidentification of IC1 and simple controls by Western blot with a specific antibody against human IgG under reducing conditions. [Figure 4C] Immunoidentification of IC1 and simple controls by Western blot with a specific antibody against IL-2 under non-reducing conditions. [Figure 5A] Size-exclusion high-performance liquid chromatography (SEC-HPLC) analysis of IC1 on a TSK gel 3000 column. [Figure 5B] Size-exclusion high-performance liquid chromatography (SEC-HPLC) analysis of rituximab on a TSK gel 3000 column. [Figure 6A] SDS-PAGE of IC anti-CD20-IgG2a-IL2 non-α (IC7) under non-reducing conditions. [Figure 6B] SDS-PAGE of IC anti-CD20-IgG2a-IL2 non-α (IC7) under reducing conditions. [Figure 7] Immunoidentification of IC7 and simple controls by Western blot with a specific antibody against IL-2 under non-reducing conditions. [Figure 8] SEC-HPLC analysis of IC7 on a TSKgel 3000 column. [Figure 9A] SDS-PAGE of IC anti-NGcGM3-IL2 non-α (IC8) under non-reducing conditions. [Figure 9B] SDS-PAGE of IC anti-NGcGM3-IL2 non-α (IC8) under reducing conditions. [Figure 10] Immunoidentification of IC8 and simple controls by Western blot with a specific antibody against IL-2 under non-reducing conditions. [Figure 11] SEC-HPLC analysis of IC8 (1) and 14F7m (2) on a TSKgel 3000 column. [Figure 12] Immunoidentification of IC4 and simple controls by Western blot with a specific antibody against human IgG under non-reducing conditions. [Figure 13A] Recognition of the EL4-huCD20+ cell line by RTX antibodies and IC1. [Figure 13B] Recognition of the EL4-huCD20+ cell line by mouse RTX-IgG2a antibody and IC7. [Figure 14A] Recognition of CD20 molecules on tumor cells by IC1. [Figure 14B] Ratio of mean fluorescence intensity (MFI) of RTX and IC1 to MFI of the conjugate. [Figure 15] Induction of CDC by IC1. [Figure 16] Induction of apoptosis by IC1. [Figure 16A] Early apoptosis in Ramos cells treated with IC1. [Figure 16B] Apoptosis in Ramos cells treated with IC1 (sum of all Annexin V+ cells). [Figure 16C] Activation of caspase 3 in Ramos cells treated with IC1. [Figure 17A] Induction of ADCC by IC1. [Figure 17B] In vitro depletion of B cells (CD19+) from healthy donors with IC1. [Figure 17C] In vitro depletion of B cells (CD19+) from patients with diffuse large B-cell lymphoma by IC1. [Figure 18A] Recognition of NGcGM3 ganglioside by IC8 measured by ELISA. [Figure 18B] Recognition of NGcGM3 ganglioside by IC8 measured by flow cytometry. [Figure 19] Induction of complement-independent cytotoxicity by IC8. [Figure 20A] IL2 non-α activity of IC1 measured by proliferation assay of CTLL-2 cell line. [Figure 20B] IL2 non-alpha activity of IC7 and IC8 measured by proliferation assay of CTLL-2 cell line. Concentration values ​​refer to the non-alpha IL-2 fraction contained in each sample. [Figure 21A] Measurement of CD16, a surface marker of IC1-induced NK cell activation. [Figure 21B] Measurement of CD69, a surface marker of IC1-induced NK cell activation. [Figure 21C] Measurement of CD107a, a surface marker of IC1-induced NK cell activation. [Figure 22] Antitumor effect of IC7 in the EL4-huCD20 model. [Figure 22A] Comparison with parent antibody at high doses. [Figure 22B] Comparison with the combination of parent antibody and IL2 non-alpha mutein (equimolar amounts). [Diagram 23] Antitumor effect of IC1 in the EL4-huCD20 model. [Figure 23A] Comparison with high-dose rituximab. [Figure 23B] Compared to equimolar amounts of rituximab. [Figure 23C] Long-term protective effect of IC1 in surviving animals rechallenged with tumor cells. [Figure 23D] Antitumor efficacy of IC8 for equimolar amounts of antibody and IL2 non-alpha mutein in the 3LL-D122 model. [Figure 24] In vivo immunomodulation by IC1 in C57BL / 6 mice bearing EL4-hCD20 tumors. [Figure 24A] Absolute CD8+ T cell counts. [Figure 24B] Absolute number of NK1.1+CD3- cells. [Figure 24C] Percentage of FOXP3+CD4+ / CD8+ cells. [Figure 24D] Percentage of FOXP3+CD4+ / NK1.1+CD3+ cells. [Figure 24E] Absolute number of FOXP3+CD4+ T cells. EXAMPLES

[0034] The present invention is further illustrated by the following examples and figures, which should not, however, be construed as limiting the scope of the invention.

[0035] Example 1. Designing and obtaining an IC. Human ICs were designed that consist of bivalent molecules with respect to the antigen-binding site, are bifunctional with respect to the mutated IL-2 moiety and are based on the full-length IgG format, specifically IC1, IC2, IC3, IC4, IC5 and IC6 correspond to the formats as depicted in Figure 1 (A-B).

[0036] IC1 and IC2 are specific for the human CD20 molecule, and the sequence of their variable domain is that of rituximab, the leading monoclonal antibody in passive therapy for B-cell lymphoproliferative disorders (Pierpont, TM, et al. (2018) Front Oncol 8:163; Sehn, LH & G. Salles. (2021) N Engl J Med. 384(9):842-858). The light chain of each of these ICs is formed by the VL variable domain (SEQ ID NO:1) and a human Cκ constant domain (SEQ ID NO:9). The heavy chain consists of the VH variable domain of SEQ ID NO:2 followed by a linker peptide (Gly) fused to the carboxy terminus. 4 It is composed of a human IgG1 constant region (SEQ ID NO: 11) with Ser)3ThrGly (SEQ ID NO: 44) followed by an IL2 non-alpha molecule (SEQ ID NO: 21 for IC1 and SEQ ID NO: 37 for IC2). ICs of this specificity containing mutants of IL-2 have not been described before.

[0037] IC3 and IC4 are specific for NGcGM3 ganglioside, which is absent or only detectable in small amounts in normal tissues (Varki, A. (2009) Glycoconj J 26(3):231-245). The sequence of their variable regions is that of the 14F7hT antibody, currently in clinical trials for the treatment of advanced solid tumors (EC-IICRDEC168). The light chain of both ICs is formed by the VL variable region (SEQ ID NO: 3) and the human Cκ constant region (SEQ ID NO: 9). The heavy chain consists of the VH variable region of SEQ ID NO: 4, followed by a human IgG1 constant region (SEQ ID NO: 11) with a linker peptide (Gly4Ser)3ThrGly (SEQ ID NO: 44) fused to the carboxyl terminus, followed by an IL2 non-alpha molecule (SEQ ID NO: 21 for IC3, SEQ ID NO: 37 for IC4). So far, no IC based on this antibody, nor any other IC specific for NGcGM3 ganglioside has been obtained.

[0038] IC5 and IC6 recognize the EGFR receptor and the sequence of their variable domain corresponds to that of the antibody nimotuzumab (US Pat. No. 5,891,996 B2), which is widely used in the treatment of tumors of epithelial origin (Crombet T, et al. (2002) Int J Cancer 101(6):567-75 Ramos-Suzarte M et al (2012) Cancer Biol Ther 13(8):600-5 Suarez Martinez G and BencomoYanes A. (2014) Biotecnol APL [Internet] 31(2):159-167, Crombet T. (2014) Handbook of therapeutic antibodies. 1679-94). The light chain is composed of the VL variable domain (SEQ ID NO:5) and the human Cκ constant domain (SEQ ID NO:9). The heavy chain is composed of the VH variable domain of the sequence SEQ ID NO:6 followed by a linker peptide (Gly) fused to the carboxy terminus. 4 Ser) 3It is composed of a human IgG1 constant region (SEQ ID NO: 11) with ThrGly (SEQ ID NO: 44) followed by an IL2 non-alpha molecule (SEQ ID NO: 21 for IC5 and SEQ ID NO: 37 for IC6).

[0039] Two other ICs were designed in parallel, based on the full mouse IgG format, that were bivalent with respect to the antigen-binding site and bifunctional with respect to the mutated IL-2 moiety (Figure 1A-B).

[0040] IC7 is specific for the human CD20 molecule, and the sequence of its variable region is that of rituximab. The light chain is composed of the VL variable region (SEQ ID NO: 1) and the Cκ mouse constant region (SEQ ID NO: 13). The heavy chain is composed of the VH variable region of SEQ ID NO: 2, followed by the constant region of mouse IgG2a (SEQ ID NO: 15) with the linker peptide (Gly4Ser)3ThrGly (SEQ ID NO: 44) fused to the carboxyl terminus, followed by the IL2 non-alpha molecule (SEQ ID NO: 21).

[0041] IC8 is specific for the NGcGM3 ganglioside. The sequence of its variable region is that of the 14F7 antibody (US Pat. No. 6,429,295 B1), the murine ancestor of 14F7hT. The light chain is formed by the VL variable region (SEQ ID NO: 7) and the Cκ murine constant region (SEQ ID NO: 13). The heavy chain is formed by the VH variable region of SEQ ID NO: 8, followed by a linker peptide (Gly) fused to the carboxy terminus. 4 Ser) 3 It is composed of the constant region of mouse IgG1 (SEQ ID NO: 14) with ThrGly (SEQ ID NO: 44) followed by an IL2 non-alpha molecule (SEQ ID NO: 21).

[0042] To obtain human ICs, genes encoding the variable regions of the light chains of the rituximab, nimotuzumab or 14F7hT antibodies were cloned into the vector pFUSE(Ckh) containing the human Cκ constant region gene, thus constructing genes encoding the respective light chains.

[0043] Subsequently, a linker peptide (Gly) was inserted via its N-terminus.4 Ser) 3 The gene segment encoding the IL2 non-alpha mutein (SEQ ID NO:21 or SEQ ID NO:37) fused to ThrGly (SEQ ID NO:44) was amplified by using the polymerase chain reaction (PCR) and then cloned EcoRV / XbaI into the vector pFUSE(IgG1h) previously modified for the insertion of an EcoRV site. In the resulting gene construct, the corresponding VH gene previously amplified by PCR was cloned EcoRI / NheI.

[0044] The IC1 heavy and light chain genes (anti-CD20) were cloned independently into the pLV-CMV-IRES-Neo vector to generate the respective transfer vectors with which we proceeded to obtain lentiviral transduction particles. For the generation of recombinant stable clones, the lentiviral particles obtained above were transduced into CHO-K1 cells. Using a specific ELISA against the human Fc region, the IC1 fusion protein (anti-CD20) was detected in the supernatant of transduced CHOK1 cells (Figure 2A).

[0045] IC2 (anti-CD20) was obtained by transient expression from plasmid DNA corresponding to gene constructs encoding the light and heavy chains in the pFUSE vector (FIG. 2B).

[0046] IC3 and IC4 (anti-NGcGM3) were obtained by transient expression using plasmid DNA corresponding to gene constructs encoding the heavy and light chains in the pFUSE vector. HEK293T cells were transiently transfected with these gene constructs, and IC expression was verified after 7 days using a specific ELISA against the human Fc region (Figure 2C).

[0047] IC5 and IC6 (anti-EGFR) were obtained by transient expression in HEK293T cells with plasmid DNA corresponding to gene constructs encoding the heavy and light chains in the pFUSE vector. Supernatants from HEK293T cells grown in suspension and transfected with PEI were evaluated by a specific ELISA against the human Fc region to confirm the presence of the fusion protein (Figure 2D).

[0048] To obtain mouse IC, a gene encoding the variable region of the light chain of the rituximab antibody or 14F7 was cloned into the vector pFUSE (Ckm) containing the mouse Cκ constant region gene to construct a gene encoding each light chain.

[0049] In the case of IC7 (anti-CD20), the heavy chain gene was composed of the VH region of rituximab (SEQ ID NO: 2), the CH region of mouse IgG2a (SEQ ID NO: 15), a connecting peptide (Gly 4 Ser) 3 It was obtained by PCR assembly of gene segments encoding ThrGly (SEQ ID NO: 44) and IL2 non-alpha molecule (SEQ ID NO: 21). The obtained heavy and light chain genes contained in the pFUSE vector were amplified by PCR and cloned independently into the pLV-CMV-IRES-Neo vector. The transfer vector thus obtained was used to generate lentiviral particles that were further used to transduce CHO-K1 cells. IC7 (anti-CD20) fusion protein was detected in the supernatant of transduced CHOK1 cells using a specific ELISA against the mouse Fc region (Figure 2E).

[0050] IC8 (anti-NGcGM3) is linked to a linker peptide (Gly) via its N-terminus. 4 Ser) 3The gene segment coding for the IL2 non-alpha mutein fused to ThrGly (SEQ ID NO: 21) was amplified by PCR and cloned EcoRV / XbaI into a pFUSE vector (mouse IgG1) previously modified for the insertion of an EcoRV site. In the resulting gene construct, the gene VH14F7 previously amplified by PCR was cloned EcoRI / NheI. The heavy and light chain genes contained in the pFUSE vector were amplified by PCR and cloned independently into the pLV-CMV-IRES-Neo vector. The transfer vector thus obtained was used to generate lentiviral particles that were further used for co-transduction of CHO-K1 cells. The IC8 fusion protein (anti-NGcGM3) was detected in the supernatant of transduced CHOK1 cells using a specific ELISA for the mouse Fc region (Figure 2E).

[0051] Example 2. IC is expressed as an intact and functional protein. From the culture supernatants of recombinant CHO-K1 clones, we proceeded to purify the ICs anti-CD20-IL2 non-alpha (IgG1h) (IC1), anti-CD20-IL2 non-alpha (mouse IgG2a) (IC7) and anti-NGcGM3-IL2 non-alpha (mouse IgG1) (IC8). Gel filtration was used followed by a Protein A capture step and subsequent molecular exclusion chromatography on a preparative scale. The size and immunochemical identity of the purified proteins were then assessed. It was confirmed that the IC was expressed as a protein whose electrophoretic mobility corresponds to the approximate theoretical size of 180 kDa for non-reducing conditions (Figures 3A, 6A and 9A), while under reducing conditions the presence of both chains of the recombinant molecule was observed with a mobility corresponding to the theoretical size: approximately 65 kDa for the heavy chain (15 kDa) and 25 kDa for the light chain due to the presence of the IL2 non-alpha mutein (Figures 3B, 6B and 9B). The identity of the antibody moiety was verified by performing a Western blot assay specific for human IgG (Figures 4A-B and 12), and the presence of the cytokine in the structure of the IC was demonstrated by the same technique using a specific antibody against human IL-2 (Figures 4C, 7 and 10). The purity of the fusion proteins was analyzed by SEC-HPLC on an analytical scale using a TSK3000 column (Figures 5A, 8 and 11). As they have a higher molecular weight compared to the IgG antibody (150 kDa), they showed a shorter retention time than the reference mAb (Figures 5A-B and 11). A high percentage of monomeric species (>95%) was also observed, suggesting their high purity (Figures 5A, 8 and 11).

[0052] Example 3: IC preserves and enhances the biological activity of the corresponding antibody moiety in vitro. To determine whether the antibody moieties are active in the resulting anti-CD20 IC structures, in vitro experiments were performed using equimolar amounts with respect to the parent or reference molecules. Recognition of human CD20 molecules by ICs anti-CD20-IL2 non-alpha (IgG1h) (IC1) and anti-CD20-IL2 non-alpha (IgG2a mouse) (IC7) was determined by flow cytometry using the mouse tumor cell line EL4-huCD20, which expresses high levels of human CD20 on its membrane (Di Gaetano, N., et al. (2003) J Immunol. 171(3):1581-1587). The recognition levels of EL4-huCD20 cells by IC1 and rituximab (positive control), as well as those of IC7 and its parent antibody (Rtx-IgG2a), were similar (Figure 13A-B). The above results demonstrate the ability of both ICs to bind and recognize the human CD20 molecule. Recognition of the EL4 parental cell line by IC was negative, supporting the specificity of labeling observed with the EL4-huCD20 mouse lymphoma cell line (Figure 13A-B). In addition, two human cell lines of Burkitt's lymphoma expressing CD20 were evaluated. As Figure 14A and B shows, no differences in the recognition patterns of IC1 compared to Rituximab were observed for each of these lines. The specificity of labeling is supported by the lack of recognition of unrelated ICs and the inability of IC1 to bind to Jurkat cells, a CD20 non-expressing T lymphoma (Figure 14A-B).

[0053] Activation of the complement cascade is one of the proposed mechanisms for the in vivo therapeutic activity of rituximab (Smith, MR (2003) Oncogene. 22 (47): 7359-7368; Seyfizadeh, N., et al. (2016) Crit Rev Oncol Hematol. 97: 275-290). For this reason, the ability of IC1 to perform CDC against Ramos tumor cell lines was assessed by flow cytometry via the incorporation of propidium iodide (PI). Human AB serum at a final dilution of 1:5 was used as a complement source. The mean ± standard deviation of the percentage of positive PI cells obtained for three independent samples is displayed (Figure 15). IC1 at 6 nM retained the cytotoxic effect observed with rituximab.

[0054] The irrelevant IC did not induce cytotoxicity at the concentrations tested (Figure 15). This result indicates that this activity is not affected for this molecule, as it occurs for other ICs of similar format (Gillies, SD, et al. (2005) Blood. 105(10):3972-3978; Gillies, SD, et al. (1999) Cancer Res. 59(9):2159-2166).

[0055] Apoptosis in tumor cells is another of the mechanisms proposed to explain the therapeutic action of rituximab in the clinic (Smith, MR (2003) Oncogene. 22 (47): 7359-7368; Seyfizadeh, N., et al. (2016) Crit Rev Oncol Hematol. 97: 275-290). Phosphatidylserine exposure is an event that characterizes the early stages of apoptosis (Koopman, G., et al. (1994) Blood. 84 (5): 1415-1420). Exposure of this phospholipid in the outer monolayer of the plasma membrane was evaluated in Ramos cells treated with IC1. Phosphatidylserine levels were measured by flow cytometry by labeling with Annexin V. Mean values ​​obtained for triplicate samples ± standard deviation are displayed. Early apoptotic cells were identified as those presenting Annexin V+ / PI-labeling (Figure 16A); late apoptotic cells were Annexin V+ / PI+ labeled cells, and apoptosis was defined as the sum of these two populations (Figure 16B). Surprisingly, IC1 had a better effect than rituximab (Figures 16A-B). Considering that phosphatidylserine exposure can occur in non-apoptotic events, caspase-3 activation as a marker of late apoptosis was further evaluated. The 17 / 19 kDa active fragment of caspase-3 was determined by flow cytometry using specific rabbit serum and Alexa Fluor-488-conjugated rabbit antiserum. The mean ± standard deviation of the percentage of active caspase-3 cells obtained for three independent samples is displayed. Incubation of Ramos cells with 6 nM IC1 for 48 h induced activation of this enzyme (Figure 16C).

[0056] ADCC is another of the mechanisms responsible for the antitumor effects of rituximab (Smith, MR (2003) Oncogene. 22 (47): 7359-7368; Seyfizadeh, N., et al. (2016) Crit Rev Oncol Hematol. 97: 275-290). The ability of IC1 to mediate ADCC was determined by evaluation of the activity of LDH enzyme released by target cells (Ramos) as a measure of cell damage. The experiment was performed at a suboptimal setting of rituximab with respect to the effector:target cell ratio (10:1) and the use of non-activated effector cells. Peripheral blood mononuclear cells (PMBC) were used as effector cells and Ramos were used as target cells. As shown in Figure 17A, IC1 induced a higher percentage of specific lysis with respect to rituximab and the combination of rituximab with IL2 non-alpha. The induced cytotoxicity was concentration-dependent.

[0057] Given the superiority of IC1 in inducing ADCC against Ramos cells, we investigated its ability to eliminate malignant cells (CD19+) in vitro from non-Hodgkin's lymphoma patients using PBMCs as a source of target and effector cells. In this experiment, PBMCs were incubated with IC and rituximab (18 nM) for 24 h, and the percentage of B cells in PBMCs was measured by flow cytometry. As shown in Figure 17B, IC1 and rituximab were able to kill normal B cells from healthy donors. Notably, only IC1 significantly reduced the percentage of CD19+ cells in PBMCs of diffuse large B-cell lymphoma patients (Figure 17B). This indicates the potential use of this IC1 in patients where resistance to rituximab is associated with insufficient or suboptimal ADCC.

[0058] Mouse immunocytokine anti-NGcGM3-IL2 non-alpha (IC8) recognized ganglioside NGcGM3 by ELISA, similar to the parent antibody 14F7. The specificity of the binding is supported by the non-reactivity of an irrelevant mAb used as an isotype control (Figure 18A). As a complement to the solid-phase immunoenzymatic assay, flow cytometry experiments were performed. L1210 mouse tumor cells were used, which express high levels of ganglioside NGcGM3 (Roque-Navarro, L., et al. (2008) Mol Cancer Ther. 7(7):2033-2041). IC8 was able to bind to this tumor cell line, similar to the 14F7 antibody (Figure 18B).

[0059] We then proceeded to evaluate the cytotoxic activity in L1210 cells independent of effector functions, previously described for 14F7 (Roque-Navarro, L., et al. (2008) Mol Cancer Ther. 7(7):2033-2041). For this, the increase in membrane permeability to nuclear DNA labeling by PI was analyzed as an indicator of cell death. Surprisingly, the percentage of non-viable cells was higher for IC anti-NeuGcGM3-IL2nonα compared to untreated cells and parental antibody (Figure 19).

[0060] Example 4. The IC retains the functionality of the non-α IL-2 portion. The in vitro biological activity corresponding to the IL2 non-α portion of the IC was evaluated in a proliferation assay of the IL-2-dependent murine cell line CTLL-2, which expresses a high affinity mutant of the IL-2R (Figures 20A-B). The concentration values ​​shown in Figure 20 refer to the non-α IL-2 portion contained in each sample.

[0061] Incubation of CTLL-2 cells with the muteins and IC resulted in stimulation of their proliferation. In all cases, the extent of proliferation showed a concentration-dependent behavior. This result demonstrates that the IL2 non-α part maintains its functionality in the context of the fusion protein (Figure 20A-B).

[0062] Example 5. IC promotes NK cell activation Activation of NK cells is a critical step for ADCC, and furthermore, these cells have been shown to be important mediators of the antitumor function of IL2 non-alpha muteins (Carmenate, T., et al. (2013) J Immunol. 190(12):6230-6238). After 20 hours of assay setup, the ability of IC1 to activate NK cells was determined at 6.6 nM in the presence of target (Ramos) and effector (PMBC) cells at a ratio of 1:10. A greater reduction in the expression of the surface marker CD16 was observed in the NK cell population of IC1-treated samples compared to Rituximab (Figure 21A), indicating a higher activation of this cell type (Bowles, JA and GJ Weiner. (2005) J Immunol Methods. 304(1-2):88-99). Similarly, increased CD69 expression was verified as evidence of NK cell activation, an effect that was also observed in the presence of rituximab (Figure 21B). Expression of CD107a, a surface marker associated with lysosomal degranulation, was increased three-fold in cells incubated with IC1 compared to other treatments (Figure 21C). Taken together, these results demonstrate a greater activation of NK cells associated with ADCC activity in cells treated with this fusion protein compared to the rituximab antibody, which could possibly be due to the enhancing effect provided by IL2-α.

[0063] Example 6. IC has excellent antitumor effect. To prove the concept that fusion of a non-α mutein to an antibody can result in superior antitumor efficacy, we compared the antitumor activity of a murine IgG2a version of rituximab and the corresponding IL2 non-α based IC (IC7). For this, C57BL / 6 mice were inoculated with 2.5 × 10 5EL4-huCD20+ cells were inoculated intravenously, followed by three injections of 20 μg of IC7 (days 1, 4 and 7) or intraperitoneal treatment with five doses (days 1, 4, 7, 10 and 13) of 200 μg of the parental antibody anti-CD20 IgG2a, which has demonstrated therapeutic efficacy in previous studies (Abes, R., et al. (2010) Blood. 116(6): 926-934; Casadesus, AV, et al. (2020) Oncoimmunology. 9(1): 1770565). Interestingly, treatment with a low dose (20ug) of IC7 allowed survival rates similar to those obtained in mice receiving a high dose of the IgG2a version of rituximab (Figure 22A). However, its combination with 20ug of IC7 and an equimolar dose of the antibody and an equimolar amount of the L2 non-alpha mutein had no antitumor effect (Figure 22B).

[0064] Due to the cross-reactivity between human antibodies and mouse effector cells, as well as between human IL-2 and mouse IL-2 receptors (Arenas-Ramirez, N., et al. (2015) Trends Immunol. 36(12):763-777), we evaluated the efficacy of IC1 in immunocompetent mice. C57BL / 6 mice were injected with 5 × 10 5 EL4-huCD20 cells were inoculated intravenously, followed by intraperitoneal treatment on days 1, 4, and 7 with 20 μg of IC1 and 150 μg of rituximab, which has been shown to be therapeutic in previous studies (Di Gaetano, N., et al (2003) J Immunol. 171(3):1581-1587) (Figure 23A). It was observed that a 7.5-fold lower dose of IC1 had the same antitumor effect as the antibody compared to rituximab (Figure 23A). Furthermore, an equimolar amount of rituximab (20 ug) to IC1 had no antitumor effect, unlike IC (Figure 23B). Furthermore, after surviving mice were challenged with EL4-huCD20 tumor cells and prior treatment with IC1 on day 65, it was confirmed that IC at low doses maintained the long-term protective effect described for the high dose of the murine IgG2a variant of rituximab (Abes, R., et al. (2010) Blood. 116(6):926-934) (Figure 23C).

[0065] To compare the antitumor effect of anti-NGcGM3-IL2 non-α (IC8) versus control, C57BL / 6 mice were injected with 2 × 10 of 3LL-D122 in a volume of 200 μL. 5 Cells were inoculated subcutaneously (sc) on day 0. Six days later, after confirming their attachment, equimolar amounts of antibodies, cytokines, and IC (85 μg for antibodies, 16.6 μg for IL2non-α, and 100 μg for IC8) were administered intraperitoneally. Treatment was repeated on days 8 and 10. IC8, unlike the control antibody 14F7, was able to reduce tumor volume in treated animals (Figure 23D).

[0066] Example 7. ICs selectively expand NK cells and CD8+ cells over regulatory T cells. To evaluate the effect of non-alpha mutein-based ICs on specific populations of immune cells in the tumor context, EL4hu-CD20 tumor-bearing C57BL / 6 mice were treated with anti-CD20-IL2 non-alpha (IC1) as described in Example 6. Ten days after tumor challenge, spleens were removed and the different cell populations were analyzed by flow cytometry. A significant increase in TCD8+ (Figure 24A) and NK1.1+CD3- (Figure 24B) cells was observed for IC1, but not for treatment with rituximab or PBS. It is noteworthy that IC1 promoted the expansion of effector cells more than regulatory T cells, as shown by the ratios of FOXP3+CD4+ / TCD8+ and FOXP3+CD4+ / NK1.1+CD3- (Figure 24C-D). It is noteworthy that not only was the effector cells more expanded, but also that no increase in regulatory T cells was observed (Figure 24E). The results demonstrate that IC1 achieves immune modulation in favor of effector cells to the detriment of regulatory T cells, which may contribute to the superior antitumor effects observed.

[0067] Taken together, previous results point to the therapeutic superiority of non-α mutein-based ICs with the previously described formats, demonstrating that the fusion of an anti-CD20 antibody with proven direct lytic effect, ADCC and / or CDC to a non-α IL-2 mutein leads to the activation of qualitatively or quantitatively different molecular and cellular mechanisms, eliciting an enhanced anti-tumor response beyond that obtained with the antibody and non-α mutein controls.

Claims

1. A fusion protein comprising an interleukin 2 (IL-2) agonist tutein linked to an immunoglobulin of the IgG1 or IgG3 isotype, the immunoglobulin being recognized by Fcγ receptors and molecules of the complement cascade.

2. The fusion protein of claim 1, characterized as a bivalent molecule with respect to the antigen binding site and the cytokine.

3. The fusion protein of claim 1, wherein the ability of the mutein to bind to the α-chain of the IL-2 receptor is affected by at least two orders of magnitude.

4. The fusion protein of claim 1 , wherein the mutein is linked to the immunoglobulin by a linker.

5. The fusion protein of claim 1 , wherein the mutein binds to a heavy chain of an immunoglobulin.

6. The fusion protein of claim 5 , wherein the mutein is linked to the carboxy terminus of the immunoglobulin.

7. The fusion protein according to any one of claims 1 to 6, wherein the mutein has a sequence selected from the group comprising SEQ ID NOs: 16 to 43.

8. The linker is ４ Ser) ｎ ThrGly (SEQ ID NO: 44) and (Gly ４ a fragment Gly(Ser)n (SEQ ID NO: 45), wherein n has a value from 1 to 5; ４ The fusion protein according to claim 4, wherein the number of repeats of Ser is 1 or more.

9. The fusion protein of claim 1, wherein the IgG1 or IgG3 subclass comprises the sequence of SEQ ID NO: 11 or 12, respectively, or has greater than 97% identity to SEQ ID NO: 11 or 12.

10. The fusion protein of claim 1, wherein the light chain of the immunoglobulin is a human kappa isotype having a sequence corresponding to SEQ ID NO:

9.

11. The fusion protein of claim 1 , wherein the light chain of the immunoglobulin is of the human lambda isotype having a sequence corresponding to SEQ ID NO:

10.

12. 2. The fusion protein of claim 1, wherein the target tumor antigen is selected from the group including CD20, CD19, NGcGM3, PDL1, Her1, Her2 and Epcam.

13. Concentration range: 0.5mg / ml to 20mg / ml A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 12 and an acceptable pharmaceutical vehicle.

14. 14. Use of the pharmaceutical composition of claim 13 in the treatment of cancer.

15. Use of a nucleic acid molecule encoding a fusion protein according to any one of claims 1 to 12 for subcutaneous, intramuscular or intratumoral injection.

16. 14. A method of treating a subject in need thereof comprising administering the pharmaceutical composition of claim 13 subcutaneously, intravenously, intradermally, intramuscularly or intraperitoneally in a dose range of 0.01 mg / Kg to 0.6 mg / Kg body weight.

17. The method of claim 16, wherein the administration of the pharmaceutical composition is performed for 1 to 30 cycles, 1 to 3 times a week, at intervals of 7 days to 8 weeks.

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