Muteins derived from human interleukin 2 with superagonist activity

Abstract

The present invention relates to the field of biotechnology and is based on the identification of a set of IL-2 mutations in the vicinity of the interface with the receptor beta chain by selecting variants from a filamentous phage library by their affinity to the extracellular domain of the beta chain. Recombinant proteins derived from these variants show a very favorable developability profile in terms of high expression levels, low aggregation tendency and high thermostability. In addition, compared to the original unmutated IL-2 and other described superagonist mutates, they have a higher ability to stimulate immune effector populations with dimeric IL-2 receptors and a higher antitumor activity in vivo.

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology, and in particular to a family of mutant proteins (muteins) derived from human interleukin-2 (IL-2) that have enhanced binding capacity to the beta chain of the IL-2 receptor, have a favorable developability profile, and confer potent superagonist activity in vitro and in vivo. [Background technology]

[0002] IL-2 was originally described as a T cell growth factor (Smith, KA Immunol. Rev. 51:337-357, 1980) and is now known as a dual-function regulator of immune responses (Malek, TR Annu. Rev. Immunol. 26:453-479, 2008; Hoyer KK et al., Immunol. Rev. 226:19-28, 2008), either promoting or negatively modulating effector functions of the immune system. While effector cells of the immune system constitutively have dimeric intermediate affinity beta / gamma receptors that mediate the potent immunostimulatory functions of IL-2, the high levels of alpha chains on the surface of regulatory T cells result in the presence of high affinity trimeric receptors, allowing this cell population to preferentially use the cytokine over other immune cells (Malek, TR and Castro, I. Immunity. 33:153-165, 2010). As a result, an essential role of IL-2 in physiological conditions is the maintenance of immune tolerance by stimulating regulatory T cells (Malek, TR and Bayer, AL Nat. Rev. Immunol. 4:665-674, 2004).

[0003] The functional duality of IL-2 has led to opposing therapeutic effects on the immune system, greatly expanding its potential for use in modulating immune responses in either direction in a variety of settings. The use of IL-2 at high doses has demonstrated its immune-enhancing potential, useful for stimulating antitumor responses (Klapper, JA et al., Cancer. 113:293-301, 2008). Beyond pharmacological manipulation, protein engineering of the molecule itself has selectively modulated its interactions with different receptor subunits, resulting in changes in the natural balance between immune-stimulating and immune-modulating functions. For example, mutant variants of IL-2 with superagonistic properties have been described, in which increased binding capacity to the beta subunit results in potent stimulation of effector CD8+ and NK T cells, as well as potent antitumor effects (Levin, AM et al., Nature. 484:529-533, 2012). Enhanced interaction with dimeric beta / gamma receptors present at high levels on effector cells reduces the relative contribution of alpha subunits characteristic of regulatory T cells, thus creating molecules with a biased function towards immunopotentiation of immune responses. These muteins, the best studied of which is the so-called H9 superkine, originated from selections from libraries displayed on the surface of yeast cells (Levin, AM et al., Nature. 484:529-533, 2012). Such technology platforms have dominated in vitro evolution procedures for IL-2 (Rao, BM et al., Protein Eng. 16:1081-1087, 2003). Although IL-2 was displayed on filamentous phage more than 20 years ago (Buchli, PJ et al., Arch. Biochem. Biophys. 339:79-84, 1997; Vispo, NS et al., Immunotechnology. 3:185-193, 1997), this technology has not previously been useful in finding new sets of mutations that modulate the interaction of IL-2 with its receptor.

[0004] Modifying the functional properties of cytokines by introducing individual mutations or sets of distinct mutations often adversely affects the stability and solubility of the molecules, negatively impacting their expression levels as recombinant proteins, their developability profile as therapeutic agents, and ultimately their biological activity. These limitations have led to more drastic approaches to redesign stable agonists de novo based on extensive in silico optimization and receptor affinity selection (Silva, D. A. et al., Nature. 565:186-191, 2019). These molecules deviate significantly from the primary sequence of the native cytokine, raising concerns about their potential immunogenicity and undesirable interactions in the organism. Summary of the Invention

[0005] Surprisingly, the inventors of the present invention have defined, from the directed evolution of human interleukin-2 displayed on filamentous phage, a distinct set of mutations with a regularity that has not been described before and cannot be predicted from the structural analysis of human IL-2, the introduction of which increases the ability of the cytokine to bind to the beta subunit of its receptor, thus stimulating immune system cells bearing dimeric IL-2 receptors. The IL-2 variants modified in this way are distinguished from other superagonists described in the prior art by their improved exploitability in terms of expression level, low aggregation tendency and thermostability, and confer greater superagonistic potential and antitumor activity in vivo. This finding lays the foundation for the exploitation of these variants on an industrial scale to obtain therapeutic products useful for the ex vivo stimulation of immune cells for the immunotherapy of cancer, immune deficiencies and adoptive immunotherapy protocols.

[0006] Brief description of the invention In one embodiment, the invention is based on the identification of a set of mutations located in the vicinity of the IL-2 receptor beta chain binding interface from a library of human IL-2 variants displayed on filamentous phage. Phage selection for affinity to the recombinant extracellular domain of the beta chain leads to the isolation of variants with increased binding capacity. The set of mutations identified in this pathway is diverse, but with rules that determine the properties of the selected variants. In particular, they are distinguished by the presence of changes in at least two of positions 81, 83, 84 and 87 of the primary amino acid sequence of IL-2, resulting in a net negative charge of the segment 81-87 of -2 or higher. Another distinguishing feature is the preferential presence of an I92L substitution or the original Ile residue at position 92, with no other substitutions at that position. The order of the sequences is shown in Table 1 (SEQ ID NOs: 1-13).

[0007] If necessary, position 80 can be mutated to the amino acid Phe, Val, Met or Ile, position 82 to Ala, position 85 to Ile, Val or Met, position 86 to Val, position 89 to Leu or Met, and position 93 to Ile or Leu (SEQ ID NOs: 14 to 26).

[0008] In another embodiment, the present invention includes genetic constructs of novel recombinant molecules by introduction of the above identified mutations alone or in combination with K35E, K35D and K35Q mutations (SEQ ID NOs: 27-52) and / or other substitutions known to affect the properties of IL-2 (SEQ ID NOs: 127-178) and fusion to genes encoding proteins of interest such as antibodies, domains and fragments thereof, albumin, filamentous phage capsid proteins, cytokines, etc.

[0009] The subject of the present invention is also recombinant proteins produced from the above genetic constructs in expression systems in prokaryotic host cells such as E. coli and in eukaryotic host cells such as HEK293, CHO, NS0, insect cells and yeast cells. These proteins have an increased binding capacity to the beta chain of the receptor, a feature for which they are selected and which confers a strong superagonist activity on cells bearing the dimeric beta / gamma IL-2 receptor. Unlike superkine H9 and other similar molecules described in the prior art, these muteins are further distinguished by a favorable developability profile (high expression levels, low aggregation tendency and high thermostability). The overall properties of the muteins obtained as part of the present invention confer a better developability and a greater capacity for the stimulation of immune system effector cells and antitumor activity in vivo compared to the original IL-2 and superkine H9.

[0010] The subject of the present invention is also a pharmaceutical composition comprising a mutein as described herein in a concentration range of 1 mg / ml to 20 mg / ml, and a pharma- ceutically suitable excipient.

[0011] In another embodiment, the subject of the present invention is the use of the muteins and recombinant molecules derived therefrom as drugs in cancer immunotherapy, drugs for immune deficiencies, and in the ex vivo stimulation of immune cells for adoptive immunotherapy protocols. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description of the Invention The present invention is based on the identification of a set of mutations in the IL-2 sequence shown in FIG. 1, in which the C125S change was introduced in contrast to the wild-type IL-2 sequence (shaded in grey). These set of mutations are located in the vicinity of the binding interface between human IL-2 and the beta chain of its receptor and were selected from a filamentous phage library. These set of mutations increase the ability of the cytokine to bind to the beta subunit of the receptor, thereby stimulating immune system cells that have a dimeric IL-2 receptor. A variety of recombinant proteins derived from the mutated IL-2 variants are constructed, including additional substitutions and fusions to other protein domains. The developability profile of the modified molecules has been shown to be advantageous due to their high expression levels, low aggregation tendency and high thermostability, which confer enhanced superagonistic potential and antitumor activity in vivo. The properties of the novel muteins have been proven to be superior to comparable recombinant molecules obtained in the same format and expression system as the unmutated IL-2 and H9 superkine already known as part of the prior art.

[0013] Selection of a set of mutations that enhances interaction with the beta chain of the receptor from a library of IL-2 variants displayed on filamentous phage Selection of human IL-2 derived polypeptides carrying mutations that increase their ability to bind to the beta subunit of the receptor can be performed from a library of IL-2 variants displayed on filamentous phage. The genes corresponding to these variants are inserted into a phagemid-type expression vector (fused to one of the genes encoding the capsid protein of the filamentous phage) and used to produce viral particles that expose the protein variants on their surface. The library can contain various degrees of diversification (soft or hard randomization) along the entire sequence or along a set of predefined positions. Each of the original residues at these positions can be replaced with a mixture of 20 amino acids or with a subset of selected residues. Diversification can be achieved by random or site-directed mutagenesis.

[0014] The selection of phages with the highest beta chain binding capacity is based on incubating a mixture of phages from the library on a support immobilized with a recombinant protein containing the extracellular domain of the IL-2 receptor beta subunit, removing unbound phages by washing, and eluting bound phages. Several similar screening cycles can be performed. Analysis of the DNA sequences inserted into the selected phages reveals regularities that allow the identification of the most likely substitutions (and their combinations) associated with increased binding capacity. Screening of the initial library can be used to identify hotspots that modulate the interaction, and these hotspots and their vicinity can then be further explored via secondary libraries.

[0015] The ability of the variants to interact with the beta subunit can be assessed in a binding assay such as a solid-phase enzyme-linked immunosorbent assay (ELISA) or a similar phage assay. To separate direct effects on beta chain binding ability (which is the intended effect) from possible changes in the display level of the IL-2 variants on the phage, the signal obtained in the binding assay can be normalized to the signal detected using a molecule that reacts equally with all variants, such as an antibody directed against a common tag peptide fused to them.

[0016] As a result of the selection process, different variants with different sequences are obtained, but they follow certain regularities. The first is the presence of non-silent mutations (with changes in the encoded amino acids) in at least two of the selected positions in the group including residues 81, 83, 84 and 87 of the primary sequence of IL-2, so that the net negative charge of the 81-87 segment is -2 or more. Another regularity is the high frequency of the I92L mutation (predominant) and the absence of other substitutions at that position. Various combinations of residues that satisfy these regularities are illustrated in Table 1, which includes 13 classes of muteins (SEQ ID NOs: 1-13). These changes can be accompanied, if necessary, by other modifications at positions 80, 82, 85, 86, 89 and 93 (SEQ ID NOs: 14-26). Such combinations of mutations increase the reactivity of the IL-2 variants on the phage with the beta subunit of the receptor compared to the original IL-2.

[0017] [Table 1-1] [Table 1-2]

[0018] The 13 classes of mutational variants of IL-2 that meet the rules identified as part of the present invention are named using the letters of the Latin alphabet (A through M). Group I (basic) amino acids (aa) include Arg, Lys and His residues. Group II (acidic) aa include Asp and Glu residues. Group III (neutral) aa include Ala, Asn, Gly, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr, Val, Trp, and Tyr. Cys is excluded as a possible substitution.

[0019] Introduction of defined mutations in recombinant IL-2 derived proteins and characterization of their binding properties Once a set of mutations that increase interaction with the beta subunit of the receptor is identified, they are introduced by site-directed mutagenesis into the IL-2-encoding gene contained in different expression vectors (both plasmids and viral vectors) designed for the modification of bacterial host cells, yeast cells, insect-derived or mammalian-derived cell systems. These sets of mutations may correspond to the combinations of substitutions isolated directly from the phage array, but new combinations may also be designed between them.

[0020] Substitutions at position 35 of the primary sequence of IL-2 may introduce amino acids E, D or Q (SEQ ID NOs: 27-52), the beneficial effects of which on the secretion level, aggregation resistance and biological activity of IL-2-derived molecules are known (WO2018 / 0910003; Rojas, G et al., Sci. Reports. 9:800, 2019). From these sequences of IL-2 muteins, other recombinant molecules can be constructed by genetic fusion with proteins such as albumin, Fc regions and variable domains of antibodies, whole antibodies and other cytokines.

[0021] The above molecule can be obtained from: a. Transformation of host bacteria (such as Escherichia coli) or yeast cells with the corresponding plasmid. b. Transfection of insect cells with baculovirus-derived vectors or plasmids. c. Transient or stable transfection of host cells such as HEK-293, CHO, NS0 and other mammalian derived cells with the corresponding plasmids, as well as transduction of these host cells with viral vectors containing the gene of interest.

[0022] The increased binding ability of the muteins to the beta subunit of the receptor was measured by ELISA or surface plasmon resonance (BIAcore) assay. (商標)) The binding ability of the recombinant proteins derived from the mutant variants described in the present invention to the beta chain of the IL-2 receptor is higher than that of their counterparts based on the original IL-2.

[0023] Characterization of the developability profile of muteins and mutein-derived recombinant proteins The set of properties grouped under the term "developability profile" includes biophysical characteristics such as aggregation tendency and thermostability, as well as secretion levels of a protein of interest in different recombinant protein production systems, all of which have a direct impact on the feasibility of developing a production process and obtaining a biological product suitable for the final application.

[0024] The aggregation tendency has been studied by molecular exclusion chromatography, which detects the presence and proportion of monomeric species and aggregates of different sizes. The presence of aggregates resistant to denaturation is demonstrated by electrophoresis on SDS-polyacrylamide gels.

[0025] Thermostability is characterized by comparing molecules treated at elevated temperatures to their untreated counterparts and detecting changes in their properties associated with heat stress.

[0026] Secretion levels can be measured in various expression systems based on genetically modified host cells using methods that allow quantification of secreted molecules in supernatant samples. Quantification of purified protein from a given volume of supernatant allows direct determination of yield.

[0027] The mutant variants of human IL-2 described as part of the present invention and recombinant molecules derived therefrom are distinguished by a favorable developability profile characterized by higher levels of secretion as soluble protein from host cells, reduced tendency to aggregate, and increased thermostability compared to their counterparts containing the original IL-2 or H9 superkine sequences. The presence of the I92L change is associated with enhanced these properties. In contrast, all variants containing the I92F change, present in the H9 superkine and other IL-2-derived muteins with increased receptor beta chain binding capacity (Levin, AM et al., Nature. 484:529-533, 2012; WO2020 / 260270A1), show unfavorable developability properties such as reduced levels of secretion by host cells, high tendency to aggregate, and low thermostability. The elimination of the I92F change from the muteins claimed as part of the present invention and the preferential introduction of a Leu residue in its place is key to obtaining a favorable developability profile that distinguishes this group of muteins and gives them an advantage over molecules with similar receptor beta chain binding properties described as part of the prior art.

[0028] Demonstration of superagonist properties of the muteins on cells bearing the dimeric beta / gamma IL-2 receptor in vitro and in vivo The superagonist activity of the novel molecules is demonstrated by in vitro stimulation of purified cells expressing the dimeric beta / gamma IL-2 receptor, the proliferation of which is detected by flow cytometric methods.

[0029] To demonstrate the in vivo effect of the muteins on expanding immunological effector populations, experimental animals were treated with the muteins and the populations of interest were also monitored by flow cytometry.

[0030] Recombinant proteins derived from the muteins described as part of this invention show an increased ability to stimulate cells bearing dimeric IL-2 receptors in vitro compared to those containing native IL-2 and comparable to that of the H9 superkine, and their ability to stimulate this type of cell population in vivo exceeds that of both the native IL-2 based protein and the reported H9 variant.

[0031] Demonstration of antitumor effect of muteins The antitumor effects of recombinant proteins derived from the muteins can be demonstrated in animal models of malignant tumors: administration of these proteins to animals inoculated with allograftable tumor cell lines can prevent tumor formation, slow tumor growth, or reduce metastasis formation in various organs.

[0032] Recombinant proteins derived from the muteins described as part of this invention have antitumor effects as documented in experimental tumor models that are orders of magnitude superior to their counterparts containing native IL-2, and in some experimental systems even surpassing those containing superkine H9 reported in the prior art.

[0033] Pharmaceutical Compositions The muteins which are the subject of the present invention can be found as active ingredients, either on their own or forming part of various pharmaceutical compositions suitable for use in pharma- ceutically acceptable vehicles, the concentration of active ingredient in such pharmaceutical compositions ranging from 1 mg / ml to 20 mg / ml.

[0034] Pharmaceutically suitable 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 a patient may be included in the composition of the present invention. The composition may be a solution suitable for administration, and is usually sterile and free of undesirable particles.

[0035] Therapeutic Uses and Methods of Treatment The invention further includes potential therapeutic applications of the muteins described herein aimed at enhancing innate or vaccine-induced immune responses in diseases such as cancer or immunodeficiencies in which effector T cells are particularly relevant. Furthermore, use in the in vitro expansion of T and NK cells for adoptive transfer therapies (terapias de transferencia adoptiva) is also envisaged.

[0036] For therapeutic use, the muteins of the invention should be administered to a subject with a disease, either alone or in combination with other muteins, or in combination with other substances that promote or enhance their therapeutic action. The route of administration can be any of those described by the prior art for parenteral administration of drugs. Preferably, they may be administered intravenously, intramuscularly, subcutaneously or intratumorally.

[0037] The muteins of the present invention can be used in combination with vaccines for the treatment of cancer.

[0038] To obtain the desired therapeutic effect, the muteins of the invention should be administered at a dose high enough to ensure that the concentration of the mutein in the lymph nodes or peripheral sites relevant to the disease under study is in the range of concentrations where the mutein exerts an immune stimulatory effect. The dose in question must therefore be tailored to the type of disease under study and the route of administration. For example, in the case of tumor therapy, the dose should be adjusted to achieve a concentration of the mutein in the tumor and / or local lymph nodes that ensures stimulation of an anti-tumor immune response. Dose ranges to be considered may include 20 μg / kg body weight up to 200 μg / kg body weight. In applications where the mutein is replacing conventional native IL-2 therapy, the dose of the mutein used should be lower than or of equivalent activity (determined using a CTLL-2-based assay) than that conventionally used for native IL-2.

[0039] The number of doses applied should also be adjusted to the biodistribution of the mutein in question. In general, the effective concentrations mentioned above should last anywhere between 2 days and 30 consecutive days. It should be noted that if, for example, the mutein is fused to a carrier protein, the frequency of administration may be reduced and the dose of the mutein should be adjusted accordingly, in which case the dose range varies from 100 μg / kg bw to 1 mg / kg bw. In applications where native IL-2 is substituted, the administration schedule of the mutein may be similar to that applied in conventional treatments.

[0040] A therapeutic effect should be understood as a complete or partial amelioration of the symptoms of the disease. In cancer, a decrease in the tumor burden or an increase in the time to recurrence are inter alia criteria for disease remission.

[0041] The polypeptides of the present invention are particularly useful in the treatment of tumors, such as melanoma, kidney tumors, ovarian tumors, breast tumors and lung tumors, among others. They are also useful in immunodeficiencies, such as HIV and severe combined immunodeficiency. [Brief description of the drawings]

[0042] [Figure 1] Primary amino acid sequence of the original IL-2 (T3-T133) that served as the basis for constructing muteins on filamentous phage. [Diagram 2] Sequences of modified segments (12–23 and 81–87) in a soft randomized library of the interface of the IL-2 receptor with the beta subunit. [Diagram 3] Diagram of the global profile of sequences isolated from the S1 library after phage targeting of the recombinant extracellular domain of the beta subunit of the receptor. [Figure 4] Charges of segments 81–87 in selected variants of the S1 library. [Diagram 5] Increased binding capacity of phage mixtures selected from the S1 library. [Figure 6]Sequence of the modified segment (80–93) of IL-2 in clones selected from the S2 library. [Figure 7] Reactivity determined by ELISA of IL-2 variants against filamentous phages selected from the S2 library. [Figure 8] Primary amino acid sequence of IL-2 (A1-T133). [Figure 9] Yield of proteins fused to human Fc in a transient transfection system of HEK-293T cells adapted to growth in suspension. [Figure 10] Productivity of stable clones producing fusion proteins of IL-2 muteins and human IgG1 Fc. [Figure 11] Binding ability of human Fc fusion proteins derived from IL-2 muteins selected from the S1 library to the beta chain of the receptor. [Figure 12] Binding ability of human Fc fusion proteins derived from IL-2 muteins selected from the S2 library to the beta chain of the receptor. [Figure 13] Relative reactivity towards the beta chain after treatment of the fusion protein at 50°C. [Figure 14] In vivo expansion of activated effector T cell populations (CD8+CD44hi CD122hi). Cell numbers are shown for each mouse. [Figure 15] In vivo expansion of regulatory T cell populations (CD4+CD25+Foxp3+). Cell numbers are shown for each mouse. [Figure 16] Anti-metastatic effect of human LALA Fc fusion proteins containing IL-2 muteins. [Figure 17] Anti-metastatic effect of a fusion protein containing a combined mutein with increased binding to the beta chain and decreased binding to the alpha chain of the receptor in the MB16 / F0 model. EXAMPLES

[0043] (Example 1) Construction of a soft randomized library of human IL-2 interfaces displayed on phage carrying the IL-2 receptor beta subunit and selection of mutant variants therefrom To initially identify positions modulating the interaction of IL-2 with the beta subunit of the receptor, we used a soft randomization strategy focused on the interface of interest, in contrast to the random diversification used in previous studies (Levin, A. M. et al., Nature. 484:529-533, 2012). We used a described genetic construct (Rojas, G. et al., Immunobiology 218:105-113, 2013; Rojas, G. and Carmenate, T., Methods Mol. Biol. 1701:535-560, 2018) containing the gene encoding human IL-2 (residues T3-T133, C125S, FIG. 1) fused to the filamentous bacteriophage M13 protein III gene. 7 × 10 7 We constructed a library of members of the IL-2 triplex. The oligonucleotides introduced diversity restricted to 14 positions (seven each in amino acid segments 12–23 and 81–95 of IL-2) (Figure 2). The residues to be mutated were selected because they are located at the interface with the beta strand (<5 Å) in the structure of the IL-2 / receptor complex (PDB code 2B5I). The oligonucleotides contained 90% of the nucleotides complementary to the original at each position of the diversifying triplex, and the remaining 10% were a mixture of the other three nucleotides.

[0044] Library phage were generated according to the described protocol (Marks, JD et al., J. Mol. Biol. 222:581-597, 1991) and phage with the ability to bind to the surface of immunotubes coated with recombinant extracellular domain of human beta subunit were selected. Sequencing of a sample of phage obtained after three cycles of selection revealed the presence of mutated variants as shown in Figure 2. The first line of the figure shows the original IL-2 (residues to be modified are shaded in grey) and below that the variants isolated after three cycles of selection are shown. The dashed lines indicate the conservation of the original IL-2 residue at a given position. The frequency of each variant in the sample of 41 clones analyzed after selection is shown in brackets. Asterisks ( * ) indicates the presence of other mutations outside the region intentionally altered.

[0045] The amino acids that showed the most frequent mutations in this panel were R81 (preferentially substituted with D), I92 (I92L) and S87 (S87D). These three positions were selected as hot spots modulating the interaction with the beta subunit of the receptor.

[0046] (Example 2) Combinatorial exploration of the vicinity of identified hotspots using secondary phage libraries Two secondary libraries (S1 and S2) were constructed using techniques similar to those described in the previous example. Library S1 included total randomization of hot spots R81 and S87, as well as the neighboring (also solvent exposed) residues R83 and D84, and replacement of the neighboring residue P82 with a Pro / Ala mixture. Library S2 included the above modifications plus replacement of residue I92 (a hot spot) and other hydrophobic core amino acids close to the interface with the beta strand (L80, L85, I86, I89 and V93) with a mixture of hydrophobic residues (Phe / Ile / Met / Leu / Val).

[0047] While previous studies have addressed diversity in the hydrophobic core of IL-2 (Levin, AM et al., Nature. 484:529-533, 2012), the randomization of protruding and solvent-exposed residues to positively modulate the interaction of IL-2 with the beta chain is novel.

[0048] Sequencing a sample of clones obtained from the S1 library after three cycles of selection on immunotubes coated with recombinant extracellular domain of the beta subunit of the receptor revealed a wide diversity. 48 unique sequences (SEQ ID NOs: 53-100) were found, none of which were repeated more than four times, and which fulfilled certain regularities shown in Figure 3, where the letters represent the amino acids encoded at each position in the 81-87 segment. The size of each letter is proportional to the frequency of occurrence of the corresponding residue at a given position.

[0049] The basic Arg residues at positions 81 and 83 were replaced by several amino acids, including Asp and Glu (acidic). The acidic character of residue D84 was mostly retained or preferentially replaced by Glu. Amino acid S87 was frequently replaced by both Glu and Asp. This selection pattern revealed the possible importance of the accumulation of negative charges in the 81-87 segment of IL-2 for increased interactions with the beta chain. While IL-2 has a net positive charge (+1) in this region and the charge of the H9 variant described in the prior art is -1, variants with net negative charges -2 and -3 in segment 81-87 were predominant among the novel variants selected from the S1 library, even though the segment in the unselected library was mostly neutral due to randomization (Figure 4).

[0050] The reactivity of the phage mixtures obtained after two and three cycles of selection was evaluated by ELISA on plates coated with the recombinant extracellular domain of the beta subunit of the receptor and the unrelated protein bovine serum albumin. Bound phages were detected with an anti-phage antibody conjugated with horseradish peroxidase. Phages carrying the original unmutated IL-2 and its H9 variant (superkine) were used as controls. The functional outcome of the selection was an increase in the reactivity of the phage mixture with the beta chain (Figure 5).

[0051] Figure 6 shows the sequences obtained after three cycles of selection of phages from the S2 library on the recombinant extracellular domain of the receptor beta subunit (SEQ ID NOs: 100-112). The first row shows the original starting IL-2 (with diversified residues shown in grey shading) and below it the variants isolated after three cycles of selection on the immobilized recombinant extracellular domain of the receptor beta subunit. The dashed lines indicate the conservation of the original IL-2 residues at the given positions. The number of repetitions of each variant within the sample of 54 clones analyzed is shown in brackets. The numbers at the end of each row represent the net charge of the 81-87 segment in each selected variant.

[0052] As shown in Figure 6, 12 different sequences were obtained (SEQ ID NOs: 100-112), differing in relative abundance. The selected variants were distinguished by the preferential presence of the I92L substitution; the disappearance of the original Arg residues at positions 81 and 83; the frequent finding of acidic residues at positions 81, 83 and 87; as well as the preservation of the acidic character of residue D84 (only replaced by Glu). This sequence pattern resulted in segment 8187 acquiring a significant net negative charge (-2 or higher). The above data reinforced the importance of electrostatic interactions in optimizing binding to the beta subunit. Although there was an overlap between the sequences selected from the S2 library and the H9 superkine described in the prior art (final appearance of the L80F, R81D, L85V and I86V changes), there were notable differences between them. The sequences isolated from the S2 library mostly contained the I92L change, in contrast to the I92F mutation present in H9. The latter retains R83, whereas in the novel variant this residue is substituted. S87 is also conserved in H9, but is frequently replaced by acidic amino acids in the novel variants. The net charge of segment 81-87 is more negative in library variants (-2, -3) than in H9 (-1). This result demonstrated the possibility of obtaining human IL-2 variants with increased beta-chain binding capacity and distinct from that of H9 through various structural solutions.

[0053] The binding capacity of the new variants to the extracellular domain of the IL-2 receptor beta subunit was evaluated by ELISA. For this purpose, plates were coated with the recombinant extracellular domain of the IL-2 receptor beta subunit. Bound phages were detected with an anti-M13 antibody conjugated to horseradish peroxidase. At the same time, their binding to plates coated with the monoclonal antibody 9E10 against the c-myc peptide fused to the protein displayed on the phage was determined to establish the respective display levels. The ratio between the signals obtained with both coating molecules (relative reactivity) is plotted as a measure of the intrinsic binding capacity of each variant to the beta subunit. The original IL-2 and H9 superkine displayed on the phage were also used as controls.

[0054] FIG. 7 shows the increased binding capacity of these variants in contrast to human IL-2 and possibly also H9 displayed on phage.

[0055] Example 3: Design of novel IL-2 variant-derived fusion proteins and production by transient transfection Characterization of the phage-selected variants as soluble proteins was carried out in the form of fusion proteins with the N-terminus of the Fc region of human IgG1 (SEQ ID NO: 113). The expression system chosen was the transient transfection of HEK-293T cells adapted to grow in suspension (Jager et al., BMC Biotechnol. 13:52, 2013). For production in eukaryotic hosts, the first two amino acids of IL-2 (Ala-Pro), not present in the molecule displayed on the phage, were added and the K35E mutation, which has been demonstrated to have a beneficial effect on the exploitability of IL-2 (Rojas, G. et al., Sci. Reports. 9:800, 2019), was introduced. Figure 8 shows the primary sequence of IL-2 obtained as a basis for production in eukaryotic cells, to which relevant changes were introduced at the interface with the beta chain. These changes were designed based on the results of screening the initial soft randomized library at the interface with the secondary libraries S1 and S2.

[0056] Two fusion proteins were designed from the S1 library products that have frequently selected mutations (R81D / R81V, R83Q / R83E, and S87E) and a net negative charge of -3 in segment 81-87. These variants are hereafter referred to as the variants of their primary sequences in this segment. 81 DPQDDLIE 87 (SEQ ID NO: 114) and 81 VPEDLIE 87 (SEQ ID NO: 115). Introduction of an additional I92L change (iteratively selected from an initial screen of a soft randomized library) yielded two additional fusion proteins: 81 DPQDLIE 87+I92L (SEQ ID NO: 116) and 81 VPEDLIE 87 +I92L (sequence number 117).

[0057] Among the molecules selected from the S2 library, the variants displayed on phages 1A and 2B were selected due to their high reactivity towards the beta chain, their repeated presence in the selected clones, and the net negative charge of the 81-87 segment being -2. Two fusion proteins were constructed from these and are hereafter referred to as: 80 FDPHDVVE 87 +I92L+V93L (SEQ ID NO: 118) and 80 VPPEDLIA 87 This is designated +I92L (SEQ ID NO: 119).

[0058] variant 80 VPPEDLIA 87 +I92L (SEQ ID NO: 119) does not share any mutations with the H9 superkine and therefore represents a new structural solution to increase the interaction with the beta subunit. In order to optimize this, another variant was designed ( 80 VPPEDLIE 87 +I92L) (SEQ ID NO: 120), which also incorporates the S87E mutation, was frequently selected from the library and increased the net negative charge at the interface (-3).

[0059] In contrast, the sequences of other clones selected from the S2 library overlap with that of the H9 superkine, so combining the changes present in H9 with novel identifiable features of variants selected from our library was considered as a possible optimization. The L80F, R81D, L85V and I86V changes in H9 (also selected from a phage library) were retained, but R83 was replaced with Ala or Glu (both frequently selected from the phage library) and an S87E change (a recurrent mutation among novel variants) was introduced. Position 92 in H9 is occupied by a Phe residue, and since the I92L change was found in most of the selected variants, both residues (Phe and Leu) were evaluated at this position. The variants designed and constructed were as follows: 80 FDPADVVE 87 +I92L (SEQ ID NO: 121) 80 FDPADVVE 87 +I92F (SEQ ID NO: 125) 80 FDPEDVVE 87 +I92L (SEQ ID NO: 122) 80 FDPEDVVE 87 +I92F (SEQ ID NO: 126)

[0060] Figure 9 illustrates the mass yields of Protein A affinity purification from 1 L of culture supernatant of different fusion proteins in a transient transfection experiment of HEK-293T cells in suspension. There was a significant decrease in the secretion levels of the fusion protein containing the H9 superkine (30 mg per 1 L of supernatant) and the other two engineered muteins containing the I92F change in their sequences (<33 mg / L). In contrast, the remaining muteins designed from the phage library screening results were produced at higher levels (>67 mg / L). This result was the first evidence of a good developability profile associated with the novel muteins and suggested a negative impact of the I92F mutation present in the H9 superkine on the production of the novel muteins.

[0061] Example 4: Stable production of IL-2 mutein-based fusion proteins from lentiviral transduction of suspension culture-adapted HEK-293 cells Cloning of the expression cassettes of the gene constructs used for transient transfection into the lentiviral vector pL6WBlast allowed the generation of stable clones producing a subset of the fusion proteins described in the previous examples: 80 VPPEDLIA 87 +I92L (SEQ ID NO: 119) 80 VPPEDLIE 87 +I92L (SEQ ID NO: 120) 80 FDPADVVE 87 +I92L (SEQ ID NO: 121) 80 FDPEDVVE 87 +I92L (SEQ ID NO: 122)

[0062] Clones were obtained by lentiviral transduction of the gene constructs of interest derived from the pL6WBlast vector into HEK-293 cells adapted to grow in suspension. The top 5 clones producing each variant were included in the study. The concentration of fusion protein in the supernatant was determined by ELISA on microtiter plates coated with anti-IL-2 antibody. Fusion protein was detected with an anti-human IgG antibody conjugated to horseradish peroxidase. An IL-2 / Fc(K35E) fusion protein preparation was used as a standard. Statistical analysis used one-way ANOVA followed by Tukey's multiple comparison test (p<0.05).

[0063] Figure 10 illustrates the productivity of the obtained clones. Despite the heterogeneity between clones, possibly due to differences in the location and multiplicity of integration of the foreign DNA into the host cell genome, it was observed that the clones producing fusion proteins containing the novel muteins were clearly superior to those producing those containing the H9 superkine. One of the variants (80FDPEDVVE87+I92L) (SEQ ID NO: 122) is even better produced than the others. These data reaffirmed the idea of ​​a favorable developability profile associated with the novel variants.

[0064] Example 5: Demonstration of increased beta-chain binding ability and reduced aggregation tendency of fusion proteins containing novel muteins Fusion proteins (at 60 ng / mL) purified from the supernatants of HEK-293-T cells adapted to suspension growth and transfected with the corresponding gene constructs were incubated on ELISA plates coated with recombinant extracellular domains of the beta subunits of the receptors and detected with an anti-human Fc antibody conjugated to horseradish peroxidase. The fusion proteins derived from the novel muteins were named according to the sequences contained in their interface with the beta chain (segments 81-87 and positions 92). The original IL-2 and superkine H9 derived fusion proteins were used as controls.

[0065] First generation fusion proteins (derived from screening of the S1 library) showed increased ability to bind to the extracellular domain of the receptor beta subunit compared to their original IL-2-containing counterparts (Figure 11), and were in the same range as fusion proteins based on the H9 superkine. Introduction of the I92L change into these molecules had no further effect on receptor beta subunit binding ability.

[0066] Molecular exclusion chromatography profiling of the 81DPQDLIE87 (SEQ ID NO: 114) and 81VPEDLIE87 (SEQ ID NO: 115) variants revealed the presence of a major peak corresponding to a homodimer, as expected according to the specific dimerization capacity of the Fc fragment (92-95%), and at least one minor peak of aggregates of high molecular size (Table 2). For the versions of these fusion proteins containing the I92L change (81DPQDLIE87+I92L (SEQ ID NO: 116) and 81VPEDLIE87+I92L (SEQ ID NO: 117)), no aggregates were observed and virtually all of the protein (>98%) was found in its homodimeric form. Despite these differences and the positive impact of the I92L mutation, all fusion proteins based on the novel muteins showed a lower tendency to aggregate compared to their counterparts based on the H9 superkine, which were mostly found in the form of large aggregates (Table 2).

[0067] [Table 2]

[0068] The second generation fusion proteins (derived from screening of the S2 library) exhibited heterogeneous behavior in terms of aggregation levels: two molecules containing the I92F change by design showed a complex pattern of unwanted aggregation and a small percentage of nonaggregated homodimers (Table 3), whereas the fusion protein containing the mutein selected directly from the library and one designed to contain a Leu residue at position 92 had a very low tendency to aggregate (>95% homodimer purified) (Table 3).

[0069] [Table 3]

[0070] The two fusion proteins with a high tendency to aggregate were already distinguished from the rest by their lower secretion levels (Example 3) and were excluded from further analysis. The remaining fusion proteins at a concentration of 30 ng / mL, purified from the supernatants of suspension-grown HEK-293-T cells transfected with the corresponding gene constructs, were incubated on ELISA plates coated with recombinant extracellular domains of the receptor beta subunit and detected with an anti-human Fc antibody conjugated to horseradish peroxidase. The original IL-2 and H9 superkine-derived fusion proteins were used as controls.

[0071] In addition to their reduced aggregation tendency, the second generation muteins tested showed increased beta-chain binding capacity, which in ELISA was at least 50% higher than that of the H9 superkine-based protein (Figure 12).

[0072] This result was obtained by BIAcore (商標) This was confirmed by measuring the affinity and kinetic parameters of the interaction with the beta subunit by the muteins described in the present invention. The fusion proteins derived from the muteins according to the present invention were significantly shorter than the unmutated IL-2 (dissociation equilibrium constant K D was reduced by more than 90-fold compared to that of the control) and superkine H9 (K D This increase was due to the increased dissociation rate constant (k off ), coupled with a decrease in the association kinetics (k on ) is now faster.

[0073] [Table 4]

[0074] The purified proteins were incubated at 50°C for 48 and 72 hours and then assessed by ELISA on microtiter plates coated with recombinant extracellular domain of the beta subunit of the receptor. Bound proteins were detected with an anti-human Fc antibody conjugated to horseradish peroxidase. The relative reactivity of each treated protein was calculated by taking the reactivity of the same untreated protein as reference (100%). A fusion protein based on H9 superkine was included as a control. Figure 13 shows that the muteins of the invention have a higher thermal stability, as evidenced by the preservation of the beta chain binding capacity of the fusion protein by more than 82%. This result contrasts with the dramatic loss of reactivity (more than 76%) when a fusion protein containing H9 superkine was subjected to similar conditions.

[0075] Taken together, these results demonstrate that the incorporation of mutations selected from our phage library confers on IL-2 and recombinant proteins derived therefrom an increased beta subunit binding capacity coupled with a favorable developability profile in terms of expression levels, low aggregation tendency and high thermostability, this last set of properties representing a substantial advantage over similar molecules described in the prior art (Levin, AM et al., Nature. 484:529-533, 2012).

[0076] Example 6: Demonstration of in vitro and in vivo superagonist potential of novel muteins in cells presenting the dimeric IL-2 receptor beta / gamma To investigate the in vitro effect of fusion proteins based on IL-2 muteins, CD8+ T cells were purified from unimmunized mice and labeled with CTV. They were stimulated with each fusion protein under study, and the decrease in intracellular fluorescence was evaluated by flow cytometry as an indication of the ability of the protein to induce proliferation of this cell population. Unstimulated cells and cells treated with fusion proteins containing the original IL-2 and the superkine H9 were used as controls. The fusion proteins containing the four novel IL-2 muteins studied induced proliferation in 93-96% of CD8+ T cells, behaving similarly to the H9 superkine-based proteins (Table 5). In contrast, the human IL-2 / Fc fusion protein had a moderate effect, inducing proliferation in only 55.3% of the cells. These results demonstrated the in vitro superagonistic potential of the novel variants for cells bearing the dimeric beta / gamma IL-2 receptor.

[0077] [Table 5]

[0078] In in vivo experiments, the LALA Fc-containing version of the fusion protein (with L234A and L235A mutations) (SEQ ID NO: 123) was used to prevent their binding to Fc gamma receptors and the generation of Fc gamma receptor-mediated immunological effects beyond the action of the muteins. These novel fusion proteins were named as follows: 80 VPPEDLIA 87 +I92L / LALA (SEQ ID NO: 119) 80 VPPEDLIE 87 +I92L / LALA (SEQ ID NO: 120) 80 FDPADVVE 87 +I92L / LALA (SEQ ID NO: 121) 80 FDPEDVVE 87 +I92L / LALA (SEQ ID NO: 122)

[0079] In contrast to the previous scenario, in vivo stimulation experiments showed a greater effect of the novel mutein-based fusion proteins compared to their counterparts containing superkine H9. Each group of animals was treated intraperitoneally daily with 5.6 μg of fusion protein each for 4 consecutive days, after which the mice were sacrificed on the 5th day, their spleens were removed and macerated, and the cells were analyzed by flow cytometry. Statistical analysis used one-way ANOVA followed by Tukey's multiple comparison test (p<0.05).

[0080] The number of activated CD8+ T cells (CD8+CD44hiCD122hi) was significantly increased in the groups treated with the fusion protein containing the novel mutein compared to animals treated with IL-2 / FcLALA, H9 / FcLALA and the control group that received only phosphate buffered saline (PBS) (Figure 14).

[0081] In contrast, the regulatory T cell population (CD4+CD25+FoxP3+) was expanded in animals treated with all fusion proteins compared to the control group, but no differences were noted between animals receiving the novel fusion proteins (Figure 15) and those inoculated with the IL-2 / Fc and H9 / Fc proteins used as controls. These results demonstrate that the novel muteins with increased affinity for the beta subunit of the receptor have a selective advantage over the IL-2 and H9 superkines described in the prior art for the in vivo activation of immune system effector cells bearing dimeric beta / gamma receptors. Since their increased effector potential is not accompanied by an increase in regulatory function, the net balance should be to enhance immune system effector functions.

[0082] These results demonstrate a selective advantage of the novel muteins with increased affinity for the beta subunit of the receptor for the in vivo activation of immune effector cells bearing dimeric beta / gamma receptors over the IL-2 and H9 superkines described in the prior art.

[0083] (Example 7) Demonstration of antitumor effect of novel muteins The antitumor effect of fusion proteins based on the novel human FcLALA fusion mutein was studied in an experimental melanoma metastasis model MB16 / F0. A quantity of 100,000 cells of this tumor line were inoculated into the tail vein of C57BL / 6 mice, and then each group of animals was treated by intraperitoneal injection of 5.6 μg of each fusion protein containing the IL-2 mutein. Treatment with the fusion proteins was repeated every 24 hours until the fifth day. Lung metastases were counted 21 days after the first inoculation. For statistical analysis, one-way ANOVA was used, followed by Tukey's multiple comparison test (p<0.05).

[0084] Animals treated with fusion proteins containing the novel muteins showed a significant reduction in the number of lung metastases compared to PBS-treated control animals and to animals receiving IL-2 / FcLALA or H9 / FcLALA proteins (Figure 16). Thus, the antitumor effect of the novel muteins was demonstrated in a scenario where neither the original IL-2 nor the H9 superkine in the equivalent fusion protein format had an anti-metastatic effect. These results confirmed the enhanced in vivo biological activity of the novel superagonist muteins compared to the original IL-2 as well as to the superagonists described in the prior art.

[0085] Example 8: Combining a mutation identified at the interface with the receptor beta chain with a substitution known to disrupt interaction with the alpha chain. Aligned at the interface with the beta chain of the receptor 80 VPPEDLIE 87A human Fc fusion protein (LALA version) was designed and constructed containing the mutations R38A, F42A, Y45A and E62A, known to break the interaction with the alpha chain in a variant of IL-2 with +I92L and in a variant called IL-2 alphaless, as well as the mutation K35E, known to have an effect on increasing IL-2 secretion (Rojas, G et al., Sci. Reports. 9:800, 2019). The above set of mutations is described in SEQ ID NO: 4 of patent EP 3 543 252 B1. The protein was produced in a transient transfection system of HEK293-T cells adapted to grow in suspension as described above.

[0086] The antitumor effect of the fusion protein based on the combined muteins (SEQ ID NO: 124) was studied in an experimental melanoma metastasis model MB16 / F0 in comparison with a fusion protein containing only changes at one of the interfaces. A quantity of 100,000 tumor cells was inoculated via the tail vein of C57BL / 6 mice, and then each group of animals was treated by intraperitoneal injection of 1.9 μg of each fusion protein containing the IL-2 mutein. Treatment with the fusion proteins was repeated every 24 hours until the fifth day. Lung metastases were counted 21 days after the first inoculation. For statistical analysis, one-way ANOVA was used, followed by Tukey's multiple comparison test (p<0.05). Figure 17 shows the results of the comparison of the fusion protein with PBS, the control fusion protein containing the original IL-2, and the fusion protein containing only one change at the two interfaces (alpha-less mutein, 80 VPPEDLIE 87 +I92L variant)) These results demonstrate the utility of combining the mutations at the interface with the IL-2 receptor beta chain described as part of the present invention with other substitutions known to affect the properties of IL-2.

Claims

1. A mutein derived from human interleukin-2 (IL-2), comprising amino acid substitutions at at least two of positions 81, 83, 84 and 87 of the primary amino acid sequence of IL-2, which amino acid substitutions result in a net negative charge of segment 81-87 of -2 or greater, and optionally containing the I92L change.

2. 2. The mutein of claim 1, wherein positions 81 and 83 are replaced with amino acids selected from the group including Lys, His, Asp, Glu, Ala, Ala, Asn, Gly, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr, Val, Trp and Tyr.

3. 3. The mutein according to any one of claims 1 and 2, wherein positions 84 and 87 are replaced with amino acids selected from the group comprising Asp, Glu, Ala, Asn, Gly, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr, Val, Trp and Tyr.

4. 4. The mutein according to any one of claims 1 to 3, wherein position 80 is replaced with an amino acid selected from the group comprising Phe, Val, Met and Ile.

5. 5. A mutein according to any one of claims 1 to 4, wherein position 82 is replaced by Ala.

6. 6. The mutein according to any one of claims 1 to 5, wherein position 85 is replaced with an amino acid selected from the group comprising Ile, Val and Met.

7. 7. A mutein according to any one of claims 1 to 6, wherein position 86 is replaced by Val.

8. 8. The mutein according to any one of claims 1 to 7, wherein position 89 is replaced with an amino acid selected from the group comprising Leu and Met.

9. 4. The mutein according to any one of claims 1 to 3, wherein position 93 is replaced with an amino acid selected from the group comprising Ile and Leu.

10. 10. The mutein according to any one of claims 1 to 9, wherein position 35 is replaced with an amino acid selected from the group comprising Glu, Asp and Gln.

11. 11. A mutein according to any one of claims 1 to 10, in which the mutations R38A, F42A, Y45A and E62A are introduced.

12. - capsid proteins of filamentous phages, - albumin, - the Fc region of an antibody, - complete antibody, - an antibody fragment comprising its variable domain, and - Other cytokines 12. A mutein according to any one of claims 1 to 11, characterized in that it is fused to any one of the proteins selected from the group comprising:

13. A pharmaceutical composition comprising a mutein according to any one of claims 1 to 12 in a concentration range of 1 mg / ml to 20 mg / ml and a pharma- ceutically suitable excipient.

14. Use of a mutein according to any one of claims 1 to 13 for therapeutic purposes in the treatment of cancer and immune disorders.

15. Use of a mutein according to any one of claims 1 to 13 for the in vitro expansion of T cells and NK cells for adoptive transfer therapy.

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