Self-deactivating interleukin 12

Abstract

The present invention relates to muteins of the α-subunit of human Interleukin 12 as well as to muteins of the human Interleukin 12 comprising said α-subunit (p35) and a β-subunit. Further, the present invention is directed to a cDNA or DNA nucleic acid molecule or mRNA nucleic acid molecule comprising a nucleotide sequence encoding said muteins according to the present invention, to an engineered cell or virus, a recombinant protein, a fusion-protein, an immune modulator, a pharmaceutical composition comprising any of the muteins according to the present invention. Further, the present invention related to the use of the mutein for the manufacture of a medicament for treating a disease in a mammal, the mutein of the present invention for use as a medicament, a method of treating an Interleukin 12-mediated disease in a mammal, and a method for producing the mutein according to the present invention.

Description

International Patent Application based on European Patent Application No. 24 210 666.4Applicant: Technische Universitat MunchenOur ref.: TUM18079PCTDate: 4 November 2025SELF-DEACTIVATING INTERLEUKIN 12FIELD OF THE INVENTION

[0001] The present invention relates to muteins of the a-subunit of human Interleukin 12 as well as to muteins of human Interleukin 12 comprising said a-subunit (p35) and a p-subunit. Further, the present invention is directed to a cDNA or DNA nucleic acid molecule or mRNA nucleic acid molecule comprising a nucleotide sequence encoding said mutein of human Interleukin 12 or said mutein of the a-subunit of human Interleukin 12 according to the present invention. The present invention further relates to an engineered cell / virus, a recombinant protein, a fusion-protein, an immune modulator, a pharmaceutical composition comprising any of the muteins of the a-subunit of human Interleukin 12 as well as the muteins of human Interleukin 12 according to the present invention. Further, the present invention related to the use of said muteins according to the present invention for the manufacture of a medicament for treating a disease in a mammal, the mutein according to the present invention for use as a medicament, a method of treating an Interleukin 12-related disease in a mammal, a method for producing the muteins according to the present invention or the mRNA nucleic acid molecule according to the present invention as well as a engineered cell / virus comprising any mutein according to the present invention.BACKGROUND OF THE INVENTION

[0002] Cell therapies are rapidly developing as a new tool in the arsenal to fight serious diseases from infection to cancer (Labanieh and Mackall, 2023; Tsiverioti et al., 2024). Among those, chimeric antigen receptor (CAR) T cells are most advanced in the clinics. Liquid tumors, including multiple myeloma and B cell lymphoma, can be very efficiently treated and sometimes cured with such CAR T cells (Dabas and Danda, 2023). In contrast, solid tumors remain a major challenge for cell-based therapies. This is partially due to the very often immunosuppressive microenvironment in solid tumors (Albelda, 2024; Guha et al., 2022).

[0003] Cytokines with their potent immunoregulatory functions could in principle alleviate these challenges. Of particular interest in this context is IL-12. IL-12, which was first described in 1989 as natural killer (NK) cell stimulation factor (Kobayashi, 1989), has strong immune- activating properties. It induces the differentiation of naive T cells into T helper 1 (TH1) cells(Hsieh et al., 1993; Manetti et al., 1993; Trinchieri et al., 2003), increases antigen presentation (Shurin et al., 2009; Suzuki et al., 1998), promotes IFNy secretion from T and NK cells (Trinchieri et al., 2003), and inhibits or reprograms otherwise immunosuppressive cells in the tumor microenvironment.

[0004] Together, this constitutes an ideal signature for a cytokine to improve therapies against solid tumors, which is also reflected in the impressive anti-tumor effects of IL-12 in a very large number of preclinical studies (Lasek et al., 2014). Thus far, IL-12 has fallen short of these promises in the clinics. One main reason for this is the dose-limiting toxicity of IL-12, so that only less efficient lower doses of this potent cytokine can be used (Jia et al., 2022). A maximum IL-12 concentration in the tumor microenvironment while at the same time keeping the systemic level minimal is thus a major conundrum when it comes to using the potential of IL- 12 in the clinics.

[0005] To overcome this dilemma, multiple strategies have been developed. These include immunocytokines, i.e. IL-12 fused to tumor-targeting antibodies, which may alleviate, but will likely not completely overcome potential toxicity and need to be developed specifically for certain tumors, thus restricting their use (Greiner et al., 2021). Alternatively, local release systems can be used, for which different approaches are being developed (Chen et al., 2023). Another possibility is to introduce IL-12-encoding nucleic acids directly into the tumor, be it DNA or mRNA. This approach is obviously limited by the accessibility of the tumor and more localized malignant tissue (unlike e.g. metastases) and suffers from different levels of transfection and expression (Rakhmilevich et al., 1996; Wang et al., 2024). Using tumor- targeted viruses for delivery may overcome some of these obstacles, but in return creates new challenges, including their immunogenicity and off-tumor targeting effects (Nguyen et al., 2020).

[0006] With the present invention, the inventors provide a completely novel approach to make IL-12 amenable for therapy. The inventors of the present invention developed a variant of IL- 12 that is only locally active at the site of production / release and thus represents a platform technology that can e.g. be used in nucleic acid-based, virus-based and cell-based approaches. As such, it may make IL-12 broadly usable in the clinics.SUMMARY OF THE INVENTION

[0007] The present application addresses the provision of self-deactivating IL-12-muteins. Due to the introduction of certain mutations, the a- and p-subunits of IL-12 fall apart and lose its IL- 12 functions. This leads to the provision of IL-12 with less side effects and makes it usable in clinical applications.

[0008] In a first aspect, the present invention provides a mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1), wherein at least one of the amino acid residue(s) of said a- subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207, 210 and 211 is / are mutated, and wherein the disulfide bridge at sequence position 96 of said a-subunit is deleted.

[0009] In a second aspect, the present invention relates to a mutein of human Interleukin 12, comprising an a-subunit (p35) and a p-subunit (p40), wherein the a-subunit is the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, and wherein in said mutein of human Interleukin 12 the amino acid residue at sequence position 96 of the a-subunit and / or the amino acid residue at sequence position 199 of the p-subunit is / are mutated.

[0010] In a third aspect, the present invention provides i) a nucleotide sequence encoding the mutein of human Interleukin 12 according to the present invention, or ii) a nucleotide sequence encoding the mutein of the a-subunit of human Interleukin 12 according to the present invention.

[0011] In a fourth aspect, the present invention relates to a vector comprising the cDNA or DNA nucleic acid molecule according to the present invention.

[0012] In a fifth aspect, the present invention provides a host cell comprising the cDNA or DNA nucleic acid molecule according to the present invention and / or the vector according to the present invention.

[0013] In a sixth aspect, the present invention relates to a mRNA nucleic acid molecule comprising i) a nucleotide sequence encoding the mutein of human Interleukin 12 according to the present invention, or ii) a nucleotide sequence encoding the mutein of the a-subunit of human Interleukin 12 according to the present invention.

[0014] The present invention is further directed in a seventh aspect to an engineered cell or virus comprising the mutein according to the present invention, the cDNA or DNA nucleic acid molecule according to the present invention, and / or the vector according to the present invention, and / or the mRNA nucleic acid molecule according to the present invention, or the use of said engineered cell in cell therapy.

[0015] In an eighth aspect, the present invention relates to a recombinant protein comprising the mutein according to the present invention, the cDNA or DNA nucleic acid molecule according to the present invention, and / or the vector according to the present invention, and / or the mRNA nucleic acid molecule according to the present invention.

[0016] In a ninth aspect, the present invention provides a fusion-protein comprising the mutein according to the present invention, the cDNA or DNA nucleic acid molecule according to the present invention, the vector according to the present invention, and / or the mRNA nucleic acid molecule according to the present invention.

[0017] In a tenth aspect, the present invention relates to an immune modulator comprising the mutein according to the present invention, the mRNA nucleic acid molecule according to the present invention, the engineered cell according to the present invention, the recombinant protein according to the present invention, and / or the fusion-protein according to the present invention.

[0018] In an eleventh aspect, the present invention provides a pharmaceutical composition comprising the mutein according to the present invention, the mRNA nucleic acid molecule according to the present invention, the engineered cell or virus according to the present invention, the recombinant protein according to the present invention, the fusion-protein according to the present invention, and / or the immune modulator according to the present invention, preferably further comprising a pharmaceutically acceptable carrier.

[0019] In a twelfth aspect, the present invention relates to the use of the mutein according to the present invention, of the mRNA nucleic acid molecule according to the present invention, of the recombinant protein according to the present invention, of the fusion-protein according to the present invention, and / or of the engineered cell or virus according to the present invention for the manufacture of a medicament for treating a disease in a mammal, preferably a human.

[0020] The present invention provides, in a thirteenth aspect, a method of treating an Interleukin 12-related disease in a mammal, preferably a human, comprising the step of administering a composition comprising the mutein according to the present invention, the DNA or mRNA nucleic acid molecule according to the present invention, the engineered cell or virus according to the present invention, the recombinant protein according to the present invention, the fusion-protein according to the present invention, the immune modulator according to the present invention, and / or the pharmaceutical composition according to the present invention, to said mammal in need thereof.

[0021] In a fourteenth aspect, the present invention relates to a method for producing the mutein according to the present invention, or the DNA, cDNA or mRNA nucleic acid molecule according to the present invention, comprising the steps of:(a) introducing into a DNA, cDNA or mRNA nucleic acid molecule encoding a polypeptide, said polypeptide being(i) the human Interleukin 12 a-subunit polypeptide (SEQ ID NO: 1), or(ii) a polypeptide comprising at least 90% sequence identity to the human Interleukin 12 a-subunit polypeptide (SEQ ID NO: 1), or(iii) the human Interleukin 12 polypeptide, comprising an a-subunit (p35), preferably of (i) or (ii), and a P-subunit, a respective DNA, cDNA or mRNA nucleotide sequence mutating at least one amino acid residue of said a-subunit of said polypeptide selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207, 210and 211 , and introducing a mutation for deletion of the disulfide bridge between sequence position 96 of said a-subunit of said polypeptide and sequence position 199 of said 0-subunit of said polypeptide, preferably mutating the amino acid residue at sequence position 96 of said a-subunit of said polypeptide and / or mutating the amino acid residue at sequence position 199 of said 0-subunit of said polypeptide, and(b) introducing the obtained DNA, cDNA or mRNA nucleic acid molecule for expression into a host cell, cell extract, cell lysate or engineered cell.

[0022] In a fifteenth aspect, the present invention is directed to a virus, preferably a tumor targeting virus, comprising the mutein according to the present invention.

[0023] These aspects of the invention will be more fully understood in view of the following drawings, detailed description and non-limiting examples.BRIEF DESCRIPTION OF THE FIGURES

[0024] The accompanying drawings are included to further an understanding of the embodiments that are incorporated in and constitute a part of this description. The Figures illustrate embodiments and together with the description serve to explain principles of embodiments. Other embodiments and many of the intended advantages of embodiments will be readily appreciated, as they become better understood by reference to the detailed description. The elements of the drawings are not necessarily to scale relative to each other.

[0025] Figure 1 shows the in silico design and experimental verification of a destabilized human IL-12 heterodimer. Figure 1A shows a model of the heterodimeric cytokine IL-12 and the principle of its destabilized variant sdlL-12. sdlL-12 means in the context of the present invention self-deactivating IL-12 as described and defined herein below. Figure 1B shows the energy contribution of every amino acid of the IL-12a-subunit to IL-12 heterodimerization, as obtained from MM / GBSA energy decomposition calculations. Strongly negative values indicate a stabilizing contribution of the specific amino acid to complex formation between IL- 12a and I L-12p, i.e. such amino acid increases the binding affinity between a- and p-subunit. Mutation of such amino acids is supposed to decrease the stability of the complex; hence they are identified as our top mutation candidates to destabilize the IL-12 complex. Figure 1C shows a structural snapshot of the IL-12 complex. Top mutation candidates as identified from MD simulations are highlighted as sticks and labeled in zoomed view (box). Figure 1 D shows a co-immunoprecipitation (co-IP) experiment of the human IL-12 heterodimer and mutants to examine destabilization and subunit dissociation. IL-12a corresponds to SEQ ID NO: 1 , IL- 12aC96Scorresponds to SEQ ID NO: 11 , IL-12aL52A C96Scorresponds to SEQ ID NO: 3, IL- 12aE60A C96Scorresponds to SEQ ID NO: 4, IL-12aP63A C96Scorresponds to SEQ ID NO: 5, IL-12aE68A C96Scorresponds to SEQ ID NO: 6, IL-12aL90A C96Scorresponds to SEQ ID NO: 7, IL- 12aC96S H200Acorresponds to SEQ ID NO: 8, IL-12aC96S 204Acorresponds to SEQ ID NO: 9, IL- 12aC96S V207Acorresponds to SEQ ID NO: 10, IL-12pFLAGcorresponds to SEQ ID NO: 42, IL- 12pci"scorresponds to SEQ ID NO: 12 and ||_-12pc199S FLAGcorresponds to SEQ ID NO: 43. HEK293T cells were co-transfected with IL-12 subunit constructs containing different single amino acid exchanges. IL-12aC96S(SEQ ID NO: 11) forms a non-covalently linked IL-12 together with its pairing partner IL-12pc199S(SEQ ID NO: 12), which served as reference construct for further amino acid exchanges to destabilize the IL-12 heterodimer. Cell supernatant (medium) was analyzed as input (input) or was subjected to aFLAG- immunoprecipitation (IP) of the C-terminally FLAG-tagged IL-12p (SEQ ID NO: 42) or IL- 12pci"s(SEQ ID NO: 43) and analyzed using immunoblots under reducing conditions to analyze co-immunoprecipitating IL-12a (SEQ ID NO: 1) and its variants. Figure 1 E shows the quantification of input and co-IP signals (alL-12a) with signal normalization to the IL-12aC96S(SEQ ID NO: 11) mutant (n = 3-4 ± SD). IL-12a corresponds to SEQ ID NO: 1 , IL-12aC96Scorresponds to SEQ ID NO: 11 , IL-12aL52A C96Scorresponds to SEQ ID NO: 3, IL-12aE60A C96Scorresponds to SEQ ID NO: 4, IL-12aP63A C96Scorresponds to SEQ ID NO: 5, IL-12aE68A C96Scorresponds to SEQ ID NO: 6, IL-12aL90A C96Scorresponds to SEQ ID NO: 7, IL-12aC96S H200Acorresponds to SEQ ID NO: 8, IL-12aC96S 204Acorresponds to SEQ ID NO: 9, IL-12aC96S V207Acorresponds to SEQ ID NO: 10, I L-12pFLAGcorresponds to SEQ ID NO: 42, and | L-12pc199S FLAGcorresponds to SEQ ID NO: 43. Normalized ratios (input / IP) >1 indicate destabilization of the IL-12 heterodimer, whereas ratios <1 indicate stabilizing effects on subunit association in comparison to the IL-12aC96S / 1 L-12pc199Sas reference. Signals for IL-12a were not detectable in co-IPs for IL-12a with the L52A and C96S (SEQ ID NO: 3) or C96S and I204A (SEQ ID NO: 9) point mutations, suggesting that the heterodimer falls apart entirely in the experiment. In IP quantification and subsequent input to IP ratios, the detected background signal was used for calculations. Figure 1 F shows the same as in Figure 1 D, with IL-12aC96Sconstructs containing two combined mutations based on destabilizing single mutants. IL-12aC96Scorresponds to SEQ ID NO: 11 , IL-12aL52A'P63A'C96Scorresponds to SEQ ID NO: 26, IL-12aL52A'C96S 204Acorresponds to SEQ ID NO: 27, IL-12aL90A’C96S 204Acorresponds to SEQ ID NO: 25, and IL- 12aP63A I204A C96Scorresponds to SEQ ID NO: 28, IL-12pc199Scorresponds to SEQ ID NO: 12, and | L-12pc199S FLAGcorresponds to SEQ ID NO: 43. The overexposed blot verifies lacking IL- 12a signals for co-IPs. iso., isotype control beads; MW, molecular weight.

[0026] Figure 2 shows that an IL-12a mutant with reduced IL-12 affinity modulates IL-12 formation and stability. Figure 2A shows the purification strategy for I -12aL90A C96S I204A(SEQ ID NO: 25) expressed in CHO cells. The C-terminally His-tagged (SEQ ID NO: 60) IL- 12aL90A C96S I204A(SEQ ID NO: 25) was co-expressed with IL-12pc199S(SEQ ID NO: 12) to ensure correct folding and secretion into the medium. The p-subunit was separated by washing with up to 2.5 M Guanidine-HCI on the immobilized metal affinity chromatography column. The Hise-tag (SEQ ID NO: 60) was cleaved by addition of TEV protease and two chromatography columns (affinity, size exclusion) ensured IL-12aL90A’C96S 204A(SEQ ID NO: 25) purity. Figure 2B shows the SDS-PAGE of purified IL-12aL90A’C96S 204A(SEQ ID NO: 25) under reducing (abbreviated with “red”; p-Me) and non-reducing (abbreviated with “non-red.”; 20 mM NEM as final concentration) conditions. The mobility shift (non-red.) verifies formation of intramolecular disulfide bonds. Heterogeneity is caused by IL-12a glycosylation. Figure 2C shows immunoblots (alL-12a and alL-12p) under reducing and non-reducing conditions to confirm purity of the IL-12a mutant, i.e. absence of IL-12p. Heterodimeric IL-12 was used for comparison. Figure 2D shows hydrogen-deuterium exchange (HDX) data for IL-12aC96S(SEQ ID NO: 11) and IL-12aL90A’C96S 204A(SEQ ID NO: 25) subunits. A differential Wood’s plot shows the comparison of both a-subunits, with respect to differential deuterium uptake (ADU). No significant protection or deprotection for t = 120 min (p<0.01 , Hybrid method) could be detected. Figure 2E and Figure 2F show isothermal titration calorimetry (ITC) measurement of purified IL-12aC96S(SEQ ID NO: 11) (E) or IL-12aL90A'C96S 204A(SEQ ID NO: 25) (F) subunit with IL-12pc199S(SEQ ID NO: 12) titration revealing thermodynamic parameters of IL-12 heterodimer assembly. A nearly ten-fold higher KD value of 2 pM for IL-12 composed of the mutant IL-12aL90A'C96S 204A(SEQ ID NO: 25) and IL-12pc199S(SEQ ID NO: 12) points towards its destabilization compared to the wildtype-like IL-12 with a KD value around 200 nM. Figure 2G shows kinetically destabilized IL-12 heterodimer with the engineered IL-12a mutant. Binding kinetics of purified IL-12aC96S(SEQ ID NO: 11) or IL-12aL90A 204A'C96S(SEQ ID NO: 25) to I L-12pc199S(SEQ ID NO: 12) were determined via surface plasmon resonance (SPR). Either IL-12aC96S-His6(SEQ ID NO: 67) or IL-12aL90A 204A’C96S-His6(SEQ ID NO: 44) were immobilized as ligand on a CM5 sensor chip pre-coupled to anti-His antibody and association / dissociation kinetics of the analyte I L-12pc199S(SEQ ID NO: 12) were measured by SPR. A roughly 10-fold higher dissociation rate constant (kOff) for the complex of IL-12aL90A 204A’C96S(SEQ ID NO: 25) with IL-12pc199S(SEQ ID NO: 12) confirms kinetic destabilization of the heterodimer when compared to IL-12aC96Swithout any interface mutations. Figure 2H shows a schematic (left) presenting the setup of a NanoBRET™ assay to assess the binding of IL-12 subunits or IL-12 to IL-12 receptor chains. I L-12Rp 1 (SEQ ID NO: 68) is fused to NanoLuc® Luciferase and IL- 12Rp2 (SEQ ID NO: 69) is fused to HaloTag® 618 Ligand, which induce a bioluminescence resonance energy transfer upon dimerization and substrate addition. The NanoBRET™ assayshowed receptor chain dimerization via reconstituted IL-12 protein, significantly different to the negative control PBS, with signals normalized to PBS (n = 3, ± SD, * p<0.01 , One-way ANOVA). Single purified subunits do not induce receptor dimerization. For the mutant IL-12 composed of IL-12aL90A 204A'C96S(SEQ ID NO: 25) and I L-12pc199S(SEQ ID NO: 12) a ten-fold higher concentration (100 nM) for stimulation was necessary for significant increase in NanoBRET signal, with 10 nM IL-12 being sufficient in the case of stimulation with IL-12C96S(SEQ ID NO: 12) or covalently linked IL-12 wildtype. MW, molecular weight. r00271 Figure 3 shows that self-inactivating IL-12 is functional on immune cells and shows concentration-dependent loss of activity. Figure 3A presents a schematic (left) that shows natural killer (NK)-92 cell activation via cell stimulation with IL-12 heterodimer, inducing intracellular STAT4 phosphorylation. Dissociation of IL-12 into its subunits results in loss of NK-92 cell activation and no STAT4 phosphorylation (P-STAT4). HEK293T cells were transiently transfected with IL-12 P2A constructs (SEQ ID NOs: 45-47) or pcDNA3.4 TOPO empty vector (mock) and a dilution series of HEK293T supernatants (1-1 :100) was used for NK-92 cell stimulation. STAT4 phosphorylation, thus immune cell signaling, was analyzed via immunoblots. aSTAT4 signals served as loading control. Figure 3B shows that murine CD4+T-cell activation by self-inactivating IL-12 induces intracellular STAT4 phosphorylation. HEK293T cells were transiently transfected with mlL-12 P2A constructs (SEQ ID NOs: 39-41) or pcDNA3.4 TOPO empty vector (mock) following which medium was quantified for IL-12 expression via immunoblotting. CD4+T cells isolated from mice were stimulated with 100 ng / ml IL-12 using harvested, quantified HEK supernatants. STAT4 phosphorylation was analyzed via immunoblots. Representative blot is shown accompanied by quantification from three independent experiments. MW, molecular weight.

[0028] Figure 4 shows that self-deactivating IL-12 should render a local tumor therapy possible by reducing systemic side effects. Figure 4 shows that in contrast to current tumor therapy approaches, the present invention allows secretion of self-deactivating IL-12 by engineered chimeric antigen receptor (CAR) T cells in the tumor tissue inducing tumor cell killing by activated immune cells. Once IL-12 diffuses away from the site of action, the heterodimer disassembles into its subunits, thus self-deactivates it. This prevents undesired immune cell activation at distal sites and reduces possible side effects on healthy tissue.

[0029] Figure 5 shows secretion and assembly behavior of IL-12a mutants engineered to destabilize the IL-12 heterodimer complex. Figure 5A shows free energy changes upon mutation obtained from alchemical free energy calculations. Positive values indicate a destabilizing effect. Black bars show effects for an unbound a-subunit, gray bars for the IL-12 complex, and white bars (total contribution) the difference between complex and unbound a- subunit. The desired effect is a positive (destabilizing) total contribution. Figure 5B showsexpression and secretion behavior of single point mutants of IL-12aC96S(SEQ ID NO: 11), compared to IL-12a wildtype (SEQ ID NO: 1) and IL-12aC96S(SEQ ID NO: 11) in isolation, showing similar behavior. Lysate (L) and medium (M) of transiently transfected HEK293T cells were analyzed via immunoblotting. The a-subunits alone are retained within the cell. Hsc70 served as loading control, mock means transfection with empty pSVL vector. IL-12a corresponds to SEQ ID NO: 1 , IL-12aC96Scorresponds to SEQ ID NO: 11 , IL-12aL52A C96Scorresponds to SEQ ID NO: 3, IL-12aE60A C96Scorresponds to SEQ ID NO: 4, IL-12aP63A C96Scorresponds to SEQ ID NO: 5, IL-12aL90A C96Scorresponds to SEQ ID NO: 7, IL-12aC96S H200Acorresponds to SEQ ID NO: 8, IL-12aC96S 204Acorresponds to SEQ ID NO: 9, and IL- 12aC96S V207Acorresponds to SEQ ID NO: 10. Figure 5C shows the secretion behavior of IL- 12aC96S-single point mutants when co-transfected with its p-subunit IL-12pc199S(SEQ ID NO: 12) in HEK293T cells. IL-12a corresponds to SEQ ID NO: 1 , IL-12aC96Scorresponds to SEQ ID NO: 11 , IL-12aL52A C96Scorresponds to SEQ ID NO: 3, IL-12aE60A C96Scorresponds to SEQ ID NO: 4, IL-12aP63A C96Scorresponds to SEQ ID NO: 5, IL-12aE68A C96Scorresponds to SEQ ID NO: 6, IL-12aL90A C96Scorresponds to SEQ ID NO: 7, IL-12aC96S H200Acorresponds to SEQ ID NO: 8, IL-12aC96S 204Acorresponds to SEQ ID NO: 9, and IL-12aC96S V207Acorresponds to SEQ ID NO: 10. The p-subunit is efficiently secreted, whereas IL-12a expression levels and I L-12p- induced secretion efficiency varies. Hsc70 served as loading control. Figure 5D shows the same as in Figure 5B, with transfection of IL-12aC96S-constructs with combined destabilizing mutations. IL-12a corresponds to SEQ ID NO: 1 , IL-12aC96Scorresponds to SEQ ID NO: 11 , IL-12aL52A P63A C96Scorresponds to SEQ ID NO: 26, IL-12aL52A 204A’C96Scorresponds to SEQ ID NO: 27, |L-l2aL90A I204A C96Scorresponds to SEQ ID NO: 25, and IL-12aP63A 204A'C96Scorresponds to SEQ ID NO: 28. Figure 5E shows the same as in Figure 5C, with IL-12aC96S- constructs comprising combined amino acid exchanges. IL-12aC96Scorresponds to SEQ ID NO: 11 , |L-12aL52A P63A C96Scorresponds to SEQ ID NO: 26, IL-12aL52A’C96S 204Acorresponds to SEQ ID NO: 27, IL-12aL90A'C96S 204Acorresponds to SEQ ID NO: 25, and IL-12aP63A 204A'C96Scorresponds to SEQ ID NO: 28, and I L-12pc199Scorresponds to SEQ ID NO: 12. MW, molecular weight.[00301 Figure 6 shows that the purified |L-l2aL90A C9SS I204Aprotein (SEQ ID NO: 25) is structurally similar to IL-12aC96S(SEQ ID NO: 11), but shows essential characteristics for a self-deactivating IL-12. Figure 6A shows an analytical ultracentrifugation (AUG) experiment that proves the monomeric state of purified a-subunits. Figure 6B shows that far- UV spectra of circular dichroism (CD) protein measurements show mostly a-helical structure of four-helix bundled IL-12a-subunits and illustrate structural similarity of IL-12aC96Sbearing two extra amino acid exchanges (SEQ ID NO: 25), compared to IL-12aC96S(SEQ ID NO: 11).Figure 6C shows that thermal unfolding of protein structure was measured by CD- spectroscopy at A = 222 nm and fitted sigmoidal (Boltzmann). Both a-subunits reveal a similar melting temperature (Tm). Figure 6D shows that IL-12-subunit interaction of purified subunits was assessed by a co-IP experiment of C-terminally His-tagged I L-12pc199S(SEQ ID NO: 37) protein and HIS-Select® Nickel affinity gel. Purified IL-12aL90A’C96S 204A(SEQ ID NO: 25) still interacts with purified IL-12pc199S’His(SEQ ID NO: 37) but to a much lesser extent compared to IL-12aC96S(SEQ ID NO: 11), illustrating its self-deactivating tendency. MW, molecular weight. Figure 6E shows coverage plots of IL-12a-subunits based on the peptides that were detected in hydrogen deuterium exchange (HDX) experiments. r00311 Figure 7 shows that constructs of human ll_-12 and IL-12a linked with a P2A peptide behave similar to co-transfected IL-12-subunit constructs. Figure 7A shows that a secretion test verifies secretion competency of IL-12a wildtype (SEQ ID NO: 1), C96A (IL- 12aC96A, SEQ ID NO: 29), or L90A C96A I204A (IL-12aL90A'C96A 204A, SEQ ID NO: 31) when equimolarly co-expressed with I L-12p wildtype (SEQ ID NO: 2) or C199A (I L-12pc199A, SEQ ID NO: 30), utilized by P2A peptide (SEQ ID NO: 65). For comparison to the P2A constructs (SEQ ID NOs: 45-47), co-transfection of IL-12a (SEQ ID NO: 1) and IL-12p (SEQ ID NO: 2) was performed. Hsc70 served as loading control. L, lysate; M, medium. Figure 7B shows that a co-immunoprecipitation (co-IP) experiment of medium samples (input) of transiently transfected HEK293T cells with Ustekinumab antibody reveals decreased IL-12a IP signals for iL-l2aL90A C96A I204A(SEQ ID NO: 31) mutant equimolarly expressed (via P2A peptide) with IL-12pc199A(SEQ ID NO: 30), validating reduced subunit interaction, iso., isotype control; MW, molecular weight; mock, empty vector.

[0032] Figure 8 shows that the homologous murine mutations of self-deactivating, human IL-12 destabilizes heterodimeric murine IL-12. Figure 8A shows the heterodimeric interface between murine IL-12a and IL-12p-subunits containing residues homologous to selfdeactivating hl L-12. Figure 8B shows secretion competency of equimolarly co-expressed FLAG-tagged (SEQ ID NO: 59) mlL-12a wildtype (SEQ ID NO: 35), C92A (mlL-12aC92A, SEQ ID NO: 34), or L86A C92A T200A (mlL-12aL86A C92A T200A, SEQ ID NO: 32) with HA-tagged (SEQ ID NO: 66) mlL-12p wildtype (SEQ ID NO: 36) , mlL-12pc197A(SEQ ID NO: 33) or empty vector (pSVL). Hsc70 served as loading control. L, lysate; M, medium. mlL-12aFLAGcorresponds to SEQ ID NO: 54, mlL-12pHAcorresponds to SEQ ID NO: 55, mlL-12aC92A’FLAGcorresponds to SEQ ID NO: 57, mlL-12pc197A HAcorresponds to SEQ ID NO: 56, and mlL-12aL86A'C92A'T200A'FLAGcorresponds to SEQ ID NO: 58. Figure 8C shows HA-IP from input medium samples of transiently transfected HEK293T cells equimolarly co-expressing FLAG-tagged (SEQ ID NO: 59) mlL-12a (SEQ ID NO: 35), mlL-12aC92A(SEQ ID NO: 34) or mlL-12aL86A'C92A'T200A(SEQ IDNO: 32) with HA-tagged (SEQ ID NO: 66) mlL-12p (SEQ ID NO: 36) or mlL-12pc197A(SEQ ID NO: 33) or empty vector. mlL-12aFLAGcorresponds to SEQ ID NO: 54, mlL-12pHAcorresponds to SEQ ID NO: 55, mlL-12aC92A'FLAGcorresponds to SEQ ID NO: 57, mlL-12pc197A'HAcorresponds to SEQ ID NO: 56, and mlL-12aL86A’C92A’T200A’FLAGcorresponds to SEQ ID NO: 58.DETAILED DESCRIPTION OF THE INVENTION

[0033] The following language and descriptions of certain preferred embodiments of the present invention are provided in order to further an understanding of the principles of the present invention. However, it will be understood that no limitations of the present invention are intended, and that further alterations, modifications, and applications of the principles of the present invention are also included.

[0034] Mutein(s) of the a-subunit human Interleukin 12

[0035] In a first aspect, the present invention relates to a mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1), wherein at least one of the amino acid residue(s) of said a- subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207, 210 and 211 is / are mutated, and wherein the disulfide bridge at sequence position 96 of said a-subunit is missing / deleted. Said disulfide bridge, which is missing / deleted in the mutein of the present invention, is the disulfide bridge that forms between sequence position 96 of the a-subunit of IL-12 and the sequence position 199 of the P-subunit of IL-12. Said deletion may be achieved by mutating the cysteine at sequence position 96 of the a-subunit of IL-12 and / or the sequence position 199 of the P-subunit of IL-12 to another amino acid than cysteine, preferably to serine or alanine.

[0036] Said mutein may comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). It is preferred that said mutein comprises at least 90% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). It is more preferred that said mutein comprises at least 95% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). It is even more preferred that said mutein comprises at least 99% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). In the context of the present invention, when referring to the mature protein of human IL-12 a-subunit according to SEQ ID NO: 1 , wherein the signal sequence is cleaved, the numbering of sequence positions changes by 22 residues, i.e. position 52 then becomes position 30, respectively.

[0037] By "identity" or “sequence identity” is meant a property of sequences that measures their similarity or relationship. The term "sequence identity" or "identity" as used in the present invention means the percentage of pair-wise identical residues - following (homology)alignment of a sequence of a polypeptide of the invention with a sequence in question - with respect to the number of residues in the longer of these two sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100.

[0038] The percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version blastp 2.2.5 (November 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res. 25, 3389-3402). It may be that the percentage of homology is based on the alignment of the entire polypeptide sequences (matrix: BLOSLIM 62; gap costs: 11.1 ; cut-off value set to 10'3) including the respective sequences. It is calculated as the percentage of numbers of "positives" (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

[0039] Said mutein may be a mutein, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207 and 210 is / are mutated. Further, said mutein may be a mutein, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204 and 207 is / are mutated. Said mutein may also be a mutein, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200 and 204 is / are mutated. For example, said mutein may be a mutein, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 68, 90, 200 and 204 is / are mutated. Further, said mutein may be a mutein, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 68, 90, 200, 204, 207 and 211 is / are mutated. Further, said mutein may be a mutein, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 90 and 204 is / are mutated.

[0040] In the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 90, 200 and 204 may be replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0041] For the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention it may be that at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 60 and 68 is / are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0042] In some embodiments of the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, the amino acid residue of the a-subunit at sequence position 52 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 52 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0043] In some embodiments of the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, the amino acid residue of the a-subunit at sequence position 60 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 60 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0044] In some embodiments of the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, the amino acid residue of the a-subunit at sequence position 63 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 63 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0045] In some embodiments of the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, the amino acid residue of the a-subunit at sequence position 68 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 68 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0046] In some embodiments of the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, the amino acid residue of the a-subunit at sequence position 90 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 90 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0047] In some embodiments of the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, the amino acid residue of the a-subunit at sequence position 200 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 200 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0048] In some embodiments of the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, the amino acid residue of the a-subunit at sequence position 204 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 204 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0049] In some embodiments of the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, the amino acid residue of the a-subunit at sequenceposition 207 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 207 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0050] In some embodiments of the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, the amino acid residue of the a-subunit at sequence position 210 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 210 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0051] In some embodiments of the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, the amino acid residue of the a-subunit at sequence position 211 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 211 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0052] In some embodiments of the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, the amino acid residues of the a-subunit at sequence positions 90 and 204 are mutated. In this regard, it may be that the amino acid residues of the a-subunit at sequence positions 90 and 204 are replaced by an amino acid other than the amino acid at the respective sequence positions in SEQ ID NO: 1 , preferably by alanine (A).

[0053] In some embodiments of the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, additionally the amino acid residue of the a-subunit at sequence position 96 is mutated. As described herein, for being self-deactivating, the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention is such a mutein that the disulfide bridge at sequence position 96 of said a-subunit is deleted. One possibility for achieving this it that the amino acid residue at sequence position 96 of said a-subunit is mutated, respectively replaced by another amino acid. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 96 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by serine (S) or alanine (A). It is preferred in this regard that the amino acid residue of the a- subunit at sequence position 96 is replaced by serine (S) or alanine (A), preferably by serine (S).

[0054] For example, it may be that in the a-subunit of human Interleukin 12 (SEQ ID NO: 1) according to the present invention, the amino acid residue of the a-subunit at sequence position 96 is replaced by serine (S), the amino acid residue of the a-subunit at sequence position 63 or 90 is replaced by alanine (A), and the amino acid residue of the a-subunit at sequence position 204 is replaced by alanine (A).

[0055] Mutein(s) of human Interleukin 12

[0056] In a further aspect, the present invention is directed to a mutein of human Interleukin 12, comprising an a-subunit (p35) and a p-subunit (p40), wherein the a-subunit is any mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) as described herein, and wherein in said mutein of human Interleukin 12 the amino acid residue at sequence position 96 of the a- subunit and / or the amino acid residue at sequence position 199 of the p-subunit is / are mutated. This means that for the deletion of the disulfide bridge between the amino acid of sequence position 96 of said a-subunit of human Interleukin 12 and the amino acid of sequence position 199 of said P-subunit of human Interleukin 12 the cysteine at sequence position 96 of said a- subunit or the cysteine at sequence position 199 of said P-subunit of human Interleukin 12 is mutated, or both are mutated.

[0057] It may be for said mutein of human Interleukin 12 of the present invention that at least one of the amino acid residues of said a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207, 210 and 211 is / are mutated.

[0058] Said mutein of human Interleukin 12 according to the present invention may be selfinactivating. IL-12 is only active, when both subunits are assembled. Thus, when the subunits of IL-12 fall apart into its a- and p-subunit, IL-12 activity is eliminated. The inventors of the present invention engineered the self-deactivating muteins of IL-12 according to the present invention, for example, by the creation of a destabilized heterodimerization interface such that IL-12 falls apart into its a- and p-subunits, abolishing IL-12 activity. This self-deactivation of IL- 12 is achieved by the mutations of IL-12 and its subunits as described herein.

[0059] Said mutein of human Interleukin 12 according to the present invention may be selfdissociating. The term “self-dissociating” means, in the context of the present invention, that IL-12 falls apart into its respective a- and its p-subunit spontaneously and self-induced. This self-dissociating property of IL-12 may be achieved by the mutations of IL-12 and its subunits as described herein.

[0060] Said mutein of human Interleukin 12 according to the present invention may be kinetically destabilized. Said destabilization may be due to the mutein-formation. Kinetic stability is usually defined as the rate of protein disassembly or unfolding. In the context of the present invention, it means that IL-12 is destabilized such that IL-12 falls apart into its a- and p-subunits. This kinetically destabilizing property of IL-12 may be achieved by the mutations of IL-12 and its subunits as described herein.

[0061] For said mutein of human Interleukin 12 according to the present invention the a-subunit may comprise at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the a-subunit of human Interleukin 12 according to SEQ ID NO: 1. For said mutein of human Interleukin 12 according to the present invention the a-subunit may comprise at least90% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). It may also be for said mutein of human Interleukin 12 according to the present invention that the a-subunit comprises at least 95% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). It may also be for said mutein of human Interleukin 12 according to the present invention that the a-subunit comprises at least 99% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1).

[0062] For example, in said mutein of human Interleukin 12 according to the present invention, at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207 and 210 is / are mutated. Further, it may be for said mutein of human Interleukin 12 according to the present invention that at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204 and 207 is / are mutated. It may be for said mutein of human Interleukin 12 according to the present invention that at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 68, 90, 200 and 204 is / are mutated. For example, in said mutein of human Interleukin 12 according to the present invention, at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 68, 90, 200, 204, 207 and 211 is / are mutated. For example, in said mutein of human Interleukin 12 according to the present invention, at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 90 and 204 is / are mutated.

[0063] In some embodiments of the mutein of human Interleukin 12 according to the present invention, at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 68, 90, 200 and 204 may be replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0064] Further, in some embodiments of the mutein of human Interleukin 12 according to the present invention, the amino acid residue of the a-subunit at sequence position 52 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 52 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0065] In some embodiments of the mutein of human Interleukin 12 according to the present invention, the amino acid residue of the a-subunit at sequence position 60 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 60 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0066] In some embodiments of the mutein of human Interleukin 12 according to the present invention, the amino acid residue of the a-subunit at sequence position 63 is mutated. In thisregard, it may be that the amino acid residue of the a-subunit at sequence position 63 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A).

[0067] In some embodiments of the mutein of human Interleukin 12 according to the present invention, the amino acid residue of the a-subunit at sequence position 68 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 68 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A).

[0068] In some embodiments of the mutein of human Interleukin 12 according to the present invention, the amino acid residue of the a-subunit at sequence position 90 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 90 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A).

[0069] In some embodiments of the mutein of human Interleukin 12 according to the present invention, the amino acid residue of the a-subunit at sequence position 200 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 200 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A).

[0070] In some embodiments of the mutein of human Interleukin 12 according to the present invention, the amino acid residue of the a-subunit at sequence position 204 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 204 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A).

[0071] In some embodiments of the mutein of human Interleukin 12 according to the present invention, the amino acid residue of the a-subunit at sequence position 207 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 207 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0072] In some embodiments of the mutein of human Interleukin 12 according to the present invention, the amino acid residue of the a-subunit at sequence position 210 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 210 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A).

[0073] In some embodiments of the mutein of human Interleukin 12 according to the present invention, the amino acid residue of the a-subunit at sequence position 211 is mutated. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 211 isreplaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0074] For example, in the mutein of human Interleukin 12 according to the present invention, the amino acid residues of the a-subunit at sequence positions 90 and 204 may be mutated.

[0075] In the mutein of human Interleukin 12 according to the present invention, it may also be that the amino acid residues of the a-subunit at sequence positions 90 and 204 are replaced by an amino acid other than the amino acid at the respective sequence positions in SEQ ID NO: 1 , preferably by alanine (A).

[0076] For the mutein of human Interleukin 12 according to the present invention, it may also be that additionally the amino acid residue of the a-subunit at sequence position 96 is mutated. As described herein, this is one possibility for deletion of the disulfide bridge between the amino acid of sequence position 96 of said a-subunit of human Interleukin 12 and the amino acid of sequence position 199 of said P-subunit of human Interleukin 12. In this regard, it may be that the amino acid residue of the a-subunit at sequence position 96 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by serine (S) or alanine (A). It may also be that the amino acid residue of the a- subunit at sequence position 96 is replaced by serine (S) or alanine (A), preferably by serine (S).

[0077] For example, in the mutein of human Interleukin 12 according to the present invention, the amino acid residue of the a-subunit at sequence position 96 may be replaced by serine (S), the amino acid residue of the a-subunit at sequence position 63 or 90 may be replaced by alanine (A) and the amino acid residue of the a-subunit at sequence position 204 may be replaced by alanine (A).

[0078] For the mutein of human Interleukin 12 according to the present invention, the p-subunit may be the p-subunit of human Interleukin 12 (SEQ ID NO: 2). Said p-subunit may comprise at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the p- subunit of human Interleukin 12 (SEQ ID NO: 2). For example, said p-subunit may comprise at least 90% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2). It may also be that said p-subunit comprises at least 95% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2). It may even be that said p-subunit comprises at least 99% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2).

[0079] For example, it may be for the mutein of human Interleukin 12 according to the present invention that the p-subunit is the p-subunit of human Interleukin 12 (SEQ ID NO: 2). Additionally, the amino acid residue at position 199 may be replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by serine (S) or alanine (A). As described herein, this is also one possibility for the deletion of the disulfide bridge between the amino acid of sequence position 96 of said a-subunit of humanInterleukin 12 and the amino acid of sequence position 199 of said P-subunit of human Interleukin 12. It may be in this regard that the amino acid residue at position 199 is replaced by serine (S) or alanine (A), preferably serine (S). It may be preferred in this regard that the amino acid residue at position 199 is replaced by serine (S).

[0080] cDNA or DNA nucleic acid molecule

[0081] ln one further aspect, the present invention comprises a cDNA or DNA nucleic acid molecule comprising1) a nucleotide sequence encoding any mutein of human Interleukin 12 as described herein, or ii) a nucleotide sequence encoding any mutein of the a-subunit of human Interleukin 12 as described herein.

[0082] For said cDNA or DNA nucleic acid molecule according to the present invention, it may be that the nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 as described herein comprises at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the a-subunit of human Interleukin 12 according to SEQ ID NO: 1. For said cDNA or DNA nucleic acid molecule according to the present invention, it may be that the nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 as described herein comprises at least 90% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). In this regard, it may also be that the nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 as described herein comprises at least 95% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). Further, it may also be that the nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 as described herein comprises at least 99% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1).

[0083] For said cDNA or DNA nucleic acid molecule according to the present invention, it may be that said nucleic acid molecule comprises a nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 of any one of SEQ ID NOs: 3 to 11 and 13 to 29, 31 , 32, 38, 44, and 47 to 49. That nucleotide sequence encoding a mutein of the p-subunit of human Interleukin 12 may comprise at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2). That nucleotide sequence encoding a mutein of the p-subunit of human Interleukin 12 may comprise at least 90% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2). Said nucleotide sequence encoding a mutein of the p-subunit of human Interleukin 12 may also comprise at least 95% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO:2). Further, it may be for said nucleotide sequence encoding a mutein of the p-subunit of human Interleukin 12 that it comprises at least 99% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2).

[0084] Said nucleotide sequence may also comprise a nucleotide sequence encoding a mutein of the p-subunit of human Interleukin 12 of SEQ ID NO: 12 or 30.

[0085] For said cDNA or DNA nucleic acid molecule according to the present invention, the nucleic acid molecule may be operably linked to a regulatory sequence for expression of the nucleic acid molecule. Said regulatory sequence may comprise a promoter sequence.

[0086] Vector

[0087] In a further aspect, the present invention is directed to a vector comprising said cDNA or DNA nucleic acid molecule according to the present invention.

[0088] Host cell

[0089] In one aspect, the present invention is directed to a host cell comprising the cDNA or DNA or RNA nucleic acid molecule according to the present invention and as described herein and / or the vector according to the present invention and as described herein.

[0090] mRNA nucleic acid molecule

[0091] In a further aspect, the present invention is directed to a mRNA nucleic acid molecule comprising i) a nucleotide sequence encoding the mutein of human Interleukin 12 according to the present invention and as described herein, or ii) a nucleotide sequence encoding the mutein of the a-subunit of human Interleukin 12 according to the present invention and as described herein.

[0092] In said mRNA nucleic acid molecule according to the present invention, the nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 according to the present invention and as described herein may comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the a-subunit of human Interleukin 12 according to SEQ ID NO: 1. In said mRNA nucleic acid molecule according to the present invention, the nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 according to the present invention and as described herein may comprise at least 90% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). It may also be that said nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 according to the present invention and as described herein comprises at least 95% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). It may even be that said nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 according to the present invention and as described herein comprises at least 99% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1).

[0093] The mRNA nucleic acid molecule according to the present invention and as described herein may comprise a nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 of any one of SEQ ID NOs: 3 to 11 , 13 to 29, 31 , 32, 38, 44, 47, 48 or 49.

[0094] It may be for said mRNA nucleic acid molecule according to the present invention that the nucleotide sequence encoding a mutein of the p-subunit of human Interleukin 12 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the - subunit of human Interleukin 12 according to SEQ ID NO: 2. It may be for said mRNA nucleic acid molecule according to the present invention that the nucleotide sequence encoding a mutein of the p-subunit of human Interleukin 12 comprises at least 90% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2). It may even be that said nucleotide sequence encoding a mutein of the p-subunit of human Interleukin 12 comprises at least 95% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2). It may even be that said nucleotide sequence encoding a mutein of the p-subunit of human Interleukin 12 comprises at least 99% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2).

[0095] Further, it may be for said mRNA nucleic acid molecule according to the present invention that it comprises a nucleotide sequence encoding a mutein of the p-subunit of human Interleukin 12 of SEQ ID NO: 12 or 30.

[0096] Additionally or alternatively, it may be for said mRNA nucleic acid molecule according to the present invention that the nucleic acid molecule is operably linked to a 5’-cap and / or a poly(A)-tail for transcription of said nucleic acid molecule.

[0097] Engineered cell or virusIn a further aspect, the present invention is directed to an engineered cell or virus comprising any mutein according to the present invention, the cDNA or DNA nucleic acid molecule according to the present invention, and / or the vector according to the present invention, and / or the mRNA nucleic acid molecule according to the present invention. The present invention is further directed to the use of said engineered cell or virus in therapy. An engineered cell is a living cell that has had its genetic material or other components intentionally modified to perform specific tasks, such as secreting therapeutic molecules or killing cancer cells. An engineered virus is a virus that has been genetically altered to e.g. deliver genetic material, be used in vaccines, or be programmed to specifically infect and kill certain cells like cancer cells.

[0098] Recombinant protein

[0099] The present invention further comprises a recombinant protein comprising any mutein according to the present invention and as described herein, the cDNA or DNA nucleic acid molecule according to the present invention, and / or the vector according to the present invention, and / or the mRNA nucleic acid molecule according to the present invention. In the context of the present invention and as used herein, a recombinant protein is an artificially produced protein, created by genetically engineering a host organism to express a specific gene.

[0100] Fusion-protein

[0101] The present invention is further directed to a fusion-protein comprising any mutein according to the present invention and as described herein, the cDNA or DNA nucleic acid molecule according to the present invention, the vector according to the present invention, and / or the mRNA nucleic acid molecule according to the present invention. A fusion-protein may be in the context of the present invention a single polypeptide chain created by combining two or more different protein-coding genes. Fusion-proteins may be often engineered to have new, combined functionalities.

[0102] Immune modulator

[0103] In one further aspect, the present invention is directed to an immune modulator comprising any mutein according to the present invention and as described herein, the mRNA nucleic acid molecule according to the present invention, the engineered cell or virus according to the present invention, the recombinant protein according to the present invention, and / or the fusion-protein according to the present invention. An immune modulator, in the context of the present invention, may be a substance that stimulates or suppresses the body's immune system to treat diseases. By adjusting the immune response, such modulator may either boost a weak immune system to fight e.g. cancer or an infection, or it may suppress an overactive immune system to treat e.g. autoimmune diseases or prevent organ transplant rejection.

[0104] Pharmaceutical composition

[0105] The present invention is further directed to a pharmaceutical composition comprising any mutein according to the present invention and as described herein, the mRNA nucleic acid molecule according to the present invention, the engineered cell or virus according to the present invention, the recombinant protein according to the present invention, the fusionprotein according to the present invention, and / or the immune modulator according to the present invention, preferably wherein said pharmaceutical composition further comprises a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutical composition” relates to a composition which is suitable for administration to a patient, preferably a human patient. The pharmaceutical composition of the present invention may further comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives and / or adjuvants. Acceptable constituents of the pharmaceutical composition are preferably non-toxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions of the invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0106] Uses of the present invention

[0107] The present invention is further directed to the use of the mutein according to the present invention and as described herein, of the mRNA nucleic acid molecule according to the present invention, of the recombinant protein according to the present invention, of the fusion-protein according to the present invention, and / or of the engineered cell or virusaccording to the present invention for the manufacture of a medicament for treating a disease in a mammal, preferably a human.

[0108] For said use it may be that the disease is a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease, such as Graft-versus-Host-disease, a chronic inflammatory disease, such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma. It is preferred that the disease is cancer or an infectious disease.

[0109] Medical uses

[0110] The present invention is further directed to any mutein according to the present invention and as described herein, the mRNA nucleic acid molecule according to the present invention, the recombinant protein according to the present invention, the fusion-protein according to the present invention, and / or the engineered cell or virus according to the present invention, for use as a medicament.

[0111] Further, the present invention comprises the mutein according to the present invention and as described herein, the mRNA nucleic acid molecule according to the present invention, the recombinant protein according to the present invention, the fusion-protein according to the present invention, and / or the engineered cell or virus according to the present invention, for use in the treatment of a disease.

[0112] For that mutein for use, the mRNA nucleic acid molecule for use, the recombinant protein for use, the fusion-protein for use, or the engineered cell or virus for use according to the present invention, the disease is a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease, such as Graft-versus-Host-disease, a chronic inflammatory disease, such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma.

[0113] Method of treating

[0114] In a further aspect, the present invention is directed to a method of treating an Interleukin 12-related disease in a mammal, preferably a human, wherein said method of treating comprises the step of administering a composition comprising any mutein according to the present invention, the mRNA nucleic acid molecule according to the present invention, the engineered cell or virus according to the present invention, the recombinant protein according to the present invention, the fusion-protein according to the present invention, the immune modulator according to the present invention, and / or the pharmaceutical composition according to the present invention, to said mammal in need thereof.

[0115] For said method of treating it may be that the disease is a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation- related disease, such as Graft-versus-Host-disease, a chronic inflammatory disease, such aschronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma.

[0116] Method for producing the muteins

[0117] In one further aspect, the present invention is directed to a method for producing the mutein according to the present invention, or the cDNA, DNA or mRNA nucleic acid molecule according to the present invention, comprising the steps of:(a) introducing into a DNA, cDNA or mRNA nucleic acid molecule encoding a polypeptide, said polypeptide being(i) the human Interleukin 12 a-subunit polypeptide (SEQ ID NO: 1), or(ii) a polypeptide comprising at least 90% sequence identity to the human Interleukin 12 a-subunit polypeptide (SEQ ID NO: 1), or(iii) the human Interleukin 12 polypeptide comprising an a-subunit (p35), preferably of (i) or (ii), and a P-subunit, a respective DNA, cDNA or mRNA nucleotide sequence mutating at least one amino acid residue of said a-subunit of said polypeptide selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207, 210 and 211 , and introducing a mutation for deletion of the disulfide bridge between sequence position 96 of said a-subunit of said polypeptide and sequence position 199 of said P-subunit of said polypeptide, preferably mutating the amino acid residue at sequence position 96 of said a-subunit of said polypeptide and / or mutating the amino acid residue at sequence position 199 of said P-subunit of said polypeptide, and(b) introducing the obtained DNA, cDNA or mRNA nucleic acid molecule for expression into a host cell, cell extract, cell lysate or engineered cell.

[0118] For said method for producing the mutein according to the present invention, or the mRNA nucleic acid molecule according to the present invention, it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207 and 210.

[0119] For said method for producing the mutein according to the present invention, or the mRNA nucleic acid molecule according to the present invention, it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204 and 207.

[0120] It may also be for said method of producing that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNAor mRNA nucleotide sequence is introduced mutating at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200 and 204.

[0121] Further, it may be for said method of producing that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing at least one of the amino acid residues of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200 and 204 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0122] In some embodiments of said method of producing according to the present invention it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 52. In this regard, it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 52 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0123] In some embodiments of said method of producing according to the present invention it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 60. In this regard, it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 60 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0124] In some embodiments of said method of producing according to the present invention it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 63. In this regard, it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 63 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0125] In some embodiments of said method of producing according to the present invention it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 68. In this regard, it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 68 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0126] In some embodiments of said method of producing according to the present invention it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 90. In this regard, it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 90 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0127] In some embodiments of said method of producing according to the present invention it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 200. In this regard, it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 200 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0128] In some embodiments of said method of producing according to the present invention it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 204. In this regard, it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 204 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0129] In some embodiments of said method of producing according to the present invention it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 207. In this regard, it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 207 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0130] In some embodiments of said method of producing according to the present invention it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 210. In this regard, it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 210 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0131] In some embodiments of said method of producing according to the present invention it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 211. In this regard, it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 211 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).

[0132] In some embodiments of said method of producing according to the present invention it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is additionally introduced replacing the amino acid residue of the a-subunit at sequence position 96 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A) or serine (S).

[0133] For example, it may be for said method of producing according to the present invention that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 96 by serine (S), the amino acidresidue of the a-subunit at sequence position 63 or 90 by alanine (A), and the amino acid residue of the a-subunit at sequence position 204 by alanine (A).

[0134] For said method of producing according to the present invention it may be that in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 96 by alanine (A), the amino acid residue of the a-subunit at sequence position 63 or 90 by alanine (A), and the amino acid residue of the a-subunit at sequence position 204 by alanine (A).

[0135] In one further aspect, the present invention is directed to a virus, preferably a tumor targeting virus, comprising the mutein according to the present invention and as described herein.

[0136] Sequences, as used herein, are depicted in Table 1 below (signal sequence underlined).

[0137] The invention is further characterized by the following items:1. A mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1), wherein at least one of the amino acid residue(s) of said a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207, 210 and 211 is / are mutated, and wherein the disulfide bridge at sequence position 96 of said a-subunit is deleted, preferably wherein said disulfide bridge is deleted by mutating the amino acid at sequence position 96 of said a-subunit to another amino acid than cysteine, more preferably to alanine or serine.2. The mutein of item 1 , wherein said mutein comprises at least 90% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1).3. The mutein of item 1 or 2, wherein said mutein comprises at least 95% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1).4. The mutein of any one of the preceding items, wherein said mutein comprises at least 99% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1).5. The mutein of any one of the preceding items, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207 and 210 is / are mutated.6. The mutein of any one of the preceding items, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204 and 207 is / are mutated.7. The mutein of any one of the preceding items, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200 and 204 is / are mutated.8. The mutein of any one of the preceding items, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 68, 90, 200 and 204 is / are mutated.9. The mutein of any one of the preceding items, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 90,200 and 204 is / are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). . The mutein of any one of the preceding items, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 60 and 68 is / are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 52 is mutated. . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 52 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 60 is mutated. . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 60 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 63 is mutated. . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 63 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 68 is mutated. . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 68 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).. The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 90 is mutated. . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 90 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 200 is mutated. . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 200 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 204 is mutated. . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 204 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 207 is mutated. . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 207 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 210 is mutated. . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 210 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). . The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 211 is mutated.. The mutein of any one of the preceding items, wherein the amino acid residue of the a- subunit at sequence position 211 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). . The mutein of any one of the preceding items, wherein the amino acid residues of the a- subunit at sequence positions 90 and 204 are mutated. . The mutein of any one of the preceding items, wherein the amino acid residues of the a- subunit at sequence positions 90 and 204 are replaced by an amino acid other than the amino acid at the respective sequence positions in SEQ ID NO: 1 , preferably by alanine (A). . The mutein of any one of the preceding items, wherein the disulfide bridge at sequence position 96 of said a-subunit is deleted by mutating the amino acid residue of the a-subunit at sequence position 96. . The mutein of item 33, wherein the amino acid residue of the a-subunit at sequence position 96 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by serine (S) or alanine (A). . The mutein of item 33 or 34, wherein the amino acid residue of the a-subunit at sequence position 96 is replaced by serine (S) or alanine (A), preferably by serine (S). . The mutein of any one of items 33 to 35, wherein the amino acid residue of the a-subunit at sequence position 96 is replaced by serine (S), the amino acid residue of the a-subunit at sequence position 63 or 90 is replaced by alanine (A), and the amino acid residue of the a-subunit at sequence position 204 is replaced by alanine (A). . A mutein of human Interleukin 12, comprising an a-subunit (p35) and a p-subunit (p40), wherein the a-subunit is the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) of any one of items 1 to 36, and wherein in said mutein of human Interleukin 12 the amino acid residue at sequence position 96 of the a-subunit and / or the amino acid residue at sequence position 199 of the p-subunit is / are mutated.The mutein of item 37, wherein at least one of the amino acid residues of said a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207, 210 and 211 is / are mutated. The mutein of item 37 or 38, wherein said mutein is self-deactivating and / or wherein in said mutein of human Interleukin 12 the amino acid residue at sequence position 96 of the a- subunit is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by serine (S), and / or wherein in said mutein of human Interleukin 12 the amino acid residue at sequence position 199 of the p-subunit is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by serine (S). The mutein of any one of items 37 to 39, wherein said mutein is self-dissociating. The mutein of any one of items 37 to 40, wherein said mutein is kinetically destabilized. The mutein of any one of items 37 to 41 , wherein the a-subunit comprises at least 90% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). The mutein of any one of items 37 to 42, wherein the a-subunit comprises at least 95% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). The mutein of any one of items 37 to 43, wherein the a-subunit comprises at least 99% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). The mutein of any one of items 37 to 44, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207 and 210 is / are mutated. The mutein of any one of items 37 to 45, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204 and 207 is / are mutated. The mutein of any one of items 37 to 46, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 68, 90, 200 and 204 is / are mutated.The mutein of any one of items 37 to 47, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 68, 90, 200 and 204 is / are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). The mutein of any one of items 37 to 48, wherein the amino acid residue of the a-subunit at sequence position 52 is mutated. The mutein of any one of items 37 to 49, wherein the amino acid residue of the a-subunit at sequence position 52 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). The mutein of any one of items 37 to 50, wherein the amino acid residue of the a-subunit at sequence position 60 is mutated. The mutein of any one of items 37 to 51 , wherein the amino acid residue of the a-subunit at sequence position 60 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). The mutein of any one of items 37 to 52, wherein the amino acid residue of the a-subunit at sequence position 63 is mutated. The mutein of any one of items 37 to 53, wherein the amino acid residue of the a-subunit at sequence position 63 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). The mutein of any one of items 37 to 54, wherein the amino acid residue of the a-subunit at sequence position 68 is mutated. The mutein of any one of items 37 to 55, wherein the amino acid residue of the a-subunit at sequence position 68 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). The mutein of any one of items 37 to 56, wherein the amino acid residue of the a-subunit at sequence position 90 is mutated.The mutein of any one of items 37 to 57, wherein the amino acid residue of the a-subunit at sequence position 90 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). The mutein of any one of items 37 to 58, wherein the amino acid residue of the a-subunit at sequence position 200 is mutated. The mutein of any one of items 37 to 59, wherein the amino acid residue of the a-subunit at sequence position 200 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). The mutein of any one of items 37 to 60, wherein the amino acid residue of the a-subunit at sequence position 204 is mutated. The mutein of any one of items 37 to 61 , wherein the amino acid residue of the a-subunit at sequence position 204 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). The mutein of any one of items 37 to 62, wherein the amino acid residue of the a-subunit at sequence position 207 is mutated. The mutein of any one of items 37 to 63, wherein the amino acid residue of the a-subunit at sequence position 207 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). The mutein of any one of items 37 to 64, wherein the amino acid residue of the a-subunit at sequence position 210 is mutated. The mutein of any one of items 37 to 65, wherein the amino acid residue of the a-subunit at sequence position 210 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). The mutein of any one of items 37 to 66, wherein the amino acid residue of the a-subunit at sequence position 211 is mutated.The mutein of any one of items 37 to 67, wherein the amino acid residue of the a-subunit at sequence position 211 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). The mutein of any one of items 37 to 68, wherein the amino acid residues of the a-subunit at sequence positions 90 and 204 are mutated. The mutein of any one of items 37 to 69, wherein the amino acid residues of the a-subunit at sequence positions 90 and 204 are replaced by an amino acid other than the amino acid at the respective sequence positions in SEQ ID NO: 1 , preferably by alanine (A). The mutein of any one of items 37 to 70, wherein additionally the amino acid residue of the a-subunit at sequence position 96 is mutated. The mutein of item 71 , wherein additionally the amino acid residue of the a-subunit at sequence position 96 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by serine (S) or alanine (A). The mutein of any one of items 37 to 72, wherein additionally the amino acid residue of the a-subunit at sequence position 96 is replaced by serine (S) or alanine (A), preferably by serine (S). The mutein of any one of items 37 to 73, wherein the amino acid residue of the a-subunit at sequence position 96 is replaced by serine (S), the amino acid residue of the a-subunit at sequence position 63 or 90 is replaced by alanine (A) and the amino acid residue of the a-subunit at sequence position 204 is replaced by alanine (A). The mutein of any one of items 37 to 74, wherein the p-subunit is the p-subunit of human Interleukin 12 (SEQ ID NO: 2). The mutein of any one of items 37 to 75, wherein the p-subunit comprises at least 90% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2). The mutein of any one of items 37 to 76, wherein the p-subunit comprises at least 95% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2).The mutein of any one of items 37 to 77, wherein the p-subunit comprises at least 99% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2). The mutein of any one of items 37 to 78, wherein the p-subunit is the p-subunit of human Interleukin 12 (SEQ ID NO: 2) and wherein the amino acid residue at position 199 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by serine (S) or alanine (A). The mutein of item 79, wherein the amino acid residue at position 199 is replaced by serine (S) or alanine (A), preferably serine (S). The mutein of item 80, wherein the amino acid residue at position 199 is replaced by serine (S). A cDNA or DNA nucleic acid molecule comprising i) a nucleotide sequence encoding the mutein of human Interleukin 12 of any one of items 37 to 81 , or ii) a nucleotide sequence encoding the mutein of the a-subunit of human Interleukin 12 of any one of items 1 to 36. The nucleic acid molecule of item 82, wherein the nucleotide sequence encodes a mutein of the a-subunit of human Interleukin 12 comprising at least 90% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). The nucleic acid molecule of item 82 or 83, wherein the nucleotide sequence encodes a mutein of the a-subunit of human Interleukin 12 comprising at least 95% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). The nucleic acid molecule of any one of items 82 to 84, wherein the nucleotide sequence encodes a mutein of the a-subunit of human Interleukin 12 comprising at least 99% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). The nucleic acid molecule of any one of items 82 to 85, comprising a nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 of any one of SEQ ID NOs: 3 to 11 , 13 to 29, 31 , 32, 38, 44, 47, 48 or 49.The nucleic acid molecule of any one of items 82 to 86, wherein the nucleotide sequence encodes a mutein of the p-subunit of human Interleukin 12 comprising at least 90% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2). The nucleic acid molecule of any one of items 82 to 87, wherein the nucleotide sequence encodes a mutein of the p-subunit of human Interleukin 12 comprising at least 95% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2). The nucleic acid molecule of any one of items 82 to 88, wherein the nucleotide sequence encodes a mutein of the p-subunit of human Interleukin 12 comprising at least 99% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2). The nucleic acid molecule of any one of items 82 to 89, comprising a nucleotide sequence encoding a mutein of the p-subunit of human Interleukin 12 of SEQ ID NO: 12. The nucleic acid molecule of any one of items 82 to 90, wherein the nucleic acid molecule is operably linked to a regulatory sequence for expression of the nucleic acid molecule. The nucleic acid molecule of item 91, wherein the regulatory sequence comprises a promoter sequence. A vector comprising the cDNA or DNA nucleic acid molecule of any one of items 82 to 92. A host cell comprising the cDNA or DNA nucleic acid molecule of any one of items 82 to 92 and / or the vector of claim 93. An mRNA nucleic acid molecule comprising i) a nucleotide sequence encoding the mutein of human Interleukin 12 of any one of items 37 to 81, or ii) a nucleotide sequence encoding the mutein of the a-subunit of human Interleukin 12 of any one of items 1 to 36. The mRNA nucleic acid molecule of item 95, wherein the nucleotide sequence encodes a mutein of the a-subunit of human Interleukin 12 comprising at least 90% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1).The mRNA nucleic acid molecule of item 95 or 96, wherein the nucleotide sequence encodes a mutein of the a-subunit of human Interleukin 12 comprising at least 95% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). The mRNA nucleic acid molecule of any one of items 95 to 97, wherein the nucleotide sequence encodes a mutein of the a-subunit of human Interleukin 12 comprising at least 99% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1). The mRNA nucleic acid molecule of any one of items 95 to 98, comprising a nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 of any one of SEQ ID NOs: 3 to 11 , 13 to 29, 31 , 32, 38, 44, 47, 48 or 49. . The mRNA nucleic acid molecule of any one of items 95 to 99, wherein the nucleotide sequence encodes a mutein of the p-subunit of human Interleukin 12 comprising at least 90% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2). . The mRNA nucleic acid molecule of any one of items 95 to 100, wherein the nucleotide sequence encodes a mutein of the p-subunit of human Interleukin 12 comprising at least 95% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2). . The mRNA nucleic acid molecule of any one of items 95 to 101 , wherein the nucleotide sequence encodes a mutein of the p-subunit of human Interleukin 12 comprising at least 99% sequence identity to the p-subunit of human Interleukin 12 (SEQ ID NO: 2). . The mRNA nucleic acid molecule of any one of items 95 to 102, comprising a nucleotide sequence encoding a mutein of the p-subunit of human Interleukin 12 of SEQ ID NO: 12. . The mRNA nucleic acid molecule of any one of items 95 to 103, wherein the nucleic acid molecule is operably linked to a 5’-cap and / or a poly(A)-tail for transcription of the nucleic acid molecule. . An engineered cell or virus comprising the mutein of any one of items 1 to 81 , the cDNA or DNA nucleic acid molecule of any one of items 82 to 92, and / or the vector of item 93, and / or the mRNA nucleic acid molecule of any one of items 95 to 104, or use of said engineered cell in cell therapy.. A recombinant protein comprising the mutein of any one of items 1 to 81 , the cDNA or DNA nucleic acid molecule of any one of items 82 to 92, and / or the vector of item 93, and / or the mRNA nucleic acid molecule of any one of items 95 to 104. . A fusion-protein comprising the mutein of any one of items 1 to 81 , the cDNA or DNA nucleic acid molecule of any one of items 82 to 92, the vector of item 93, and / or the mRNA nucleic acid molecule of any one of items 95 to 104. . An immune modulator comprising the mutein of any one of items 1 to 81 , the mRNA nucleic acid molecule of any one of items 95 to 104, the engineered cell or virus of item 105, the recombinant protein of item 106, and / or the fusion-protein of item 107. . A pharmaceutical composition comprising the mutein of any one of items 1 to 81 , the mRNA nucleic acid molecule of any one of items 95 to 104, the engineered cell or virus of item 105, the recombinant protein of item 106, the fusion-protein of item 107, and / or the immune modulator of item 108, preferably further comprising a pharmaceutically acceptable carrier. . Use of the mutein of any one of items 1 to 81 , of the mRNA nucleic acid molecule of any one of items 95 to 104, of the recombinant protein of item 106, of the fusion-protein of item 107, and / or of the engineered cell or virus of item 105 for the manufacture of a medicament for treating a disease in a mammal, preferably a human. 1. The use of item 110, wherein the disease is a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease, such as Graft-versus-Host-disease, a chronic inflammatory disease, such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma. . The mutein of any one of items 1 to 81 , the mRNA nucleic acid molecule of any one of items 95 to 104, the recombinant protein of item 106, the fusion-protein of item 107, and / or the engineered cell or virus of item 105, for use as a medicament. . The mutein of any one of items 1 to 81 , the mRNA nucleic acid molecule of any one of items 95 to 104, the recombinant protein of item 106, the fusion-protein of item 107, and / or the engineered cell or virus of item 105, for use in the treatment of a disease.. The mutein for use, the mRNA nucleic acid molecule for use, the recombinant protein for use, the fusion-protein for use, or the engineered cell for use of item 113, wherein the disease is a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease, such as Graft-versus-Host- disease, a chronic inflammatory disease, such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma. . A method of treating an Interleukin 12-mediated disease in a mammal, preferably a human, comprising the step of administering a composition comprising the mutein of any one of items 1 to 81 , the mRNA nucleic acid molecule of any one of items 95 to 104, the engineered cell or virus of item 105, the recombinant protein of item 106, the fusion-protein of item 107, the immune modulator of item 108, and / or the pharmaceutical composition of item 109, to said mammal in need thereof. . The method of treating of item 115, wherein the disease is a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease, such as Graft-versus-Host-disease, a chronic inflammatory disease, such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma. . Method for producing the mutein of any one of items 1 to 81 , or the DNA, cDNA or mRNA nucleic acid molecule of any one of items 82 to 104, comprising the steps of:(a) introducing into a DNA, cDNA or mRNA nucleic acid molecule encoding a polypeptide, said polypeptide being(i) the human Interleukin 12 a-subunit polypeptide (SEQ ID NO: 1), or(ii) a polypeptide comprising at least 90% sequence identity to the human Interleukin 12 a-subunit polypeptide (SEQ ID NO: 1), or(iii) the human Interleukin 12 polypeptide, comprising an a-subunit (p35), preferably of (i) or (ii), and a P-subunit, a respective DNA, cDNA or mRNA nucleotide sequence mutating at least one amino acid residue of said a-subunit of said polypeptide selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207, 210 and 211 , and introducing a mutation for deletion of the disulfide bridge between sequence position 96 of said a-subunit of said polypeptide and sequence position 199 of said P-subunit of said polypeptide, preferably mutating the amino acid residue at sequence position 96 of said a-subunit of saidpolypeptide and / or mutating the amino acid residue at sequence position 199 of said 0- subunit of said polypeptide, and(b) introducing the obtained DNA, cDNA or mRNA nucleic acid molecule for expression into a host cell, cell extract, cell lysate or engineered cell. . The method of item 117, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207 and 210. . The method of item 117 or 118, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204 and 207. . The method of any one of items 117 to 119, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200 and 204. . The method of any one of items 117 to 120, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing at least one of the amino acid residues of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200 and 204 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). . The method of any one of items 117 to 121 , wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 52. . The method of any one of items 117 to 122, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA,cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 52 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). . The method of any one of items 117 to 123, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 60. . The method of any one of items 117 to 124, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 60 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). . The method of any one of items 117 to 125, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 63. . The method of any one of items 117 to 126, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 63 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A). . The method of any one of items 117 to 127, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 68. . The method of any one of items 117 to 128, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 68 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A).. The method of any one of items 117 to 129, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 90. . The method of any one of items 117 to 130, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 90 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). . The method of any one of items 117 to 131, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 200. . The method of any one of items 117 to 132, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 200 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). . The method of any one of items 117 to 133, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 204. . The method of any one of items 117 to 134, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 204 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). . The method of any one of items 117 to 135, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 207.. The method of any one of items 117 to 136, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 207 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). . The method of any one of items 117 to 137, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 210. . The method of any one of items 117 to 138, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 210 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). . The method of any one of items 117 to 139, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced mutating the amino acid residue of the a-subunit at sequence position 211. . The method of any one of items 117 to 140, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 211 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A). . The method of any one of items 117 to 141 , wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 96 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1, preferably by alanine (A) or serine (S). . The method of any one of items 117 to 142, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA,cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 96 by serine (S), the amino acid residue of the a-subunit at sequence position 63 or 90 by alanine (A), and the amino acid residue of the a-subunit at sequence position 204 by alanine (A). . The method of any one of items 117 to 143, wherein in step (a) into the DNA, cDNA or mRNA nucleic acid molecule encoding the polypeptide of (i), (ii) or (iii) a respective DNA, cDNA or mRNA nucleotide sequence is introduced replacing the amino acid residue of the a-subunit at sequence position 96 by alanine (A), the amino acid residue of the a-subunit at sequence position 63 or 90 by alanine (A), and the amino acid residue of the a-subunit at sequence position 204 by alanine (A). . A virus, preferably a tumor targeting virus, comprising the mutein of any one of items 1 to 81.EXAMPLES

[0138] MATERIALS AND METHODS

[0139] Constructs

[0140] Human interleukin cDNAs (IL-12a and IL-12p, UniProt accession numbers: P29459 and P29460, respectively) were obtained from OriGene and were cloned into the pSVL vector (Amersham) for cell culture experiments. For mammalian protein expression or control experiments, C. cricetus optimized (GeneArt, ThermoFisher) sequences in the pcDNA3.4 TOPO were used. Murine IL-12 subunit sequences, mlL-12a (UniProt accession number: P43431) C-terminally FLAG-tagged and mlL-12p (UniProt accession number: P43432) C- terminally HA-tagged, were ordered at GeneArt (ThermoFisher) as human optimized sequences. The human and murine IL-12 constructs with I L-12p connected by a P2A peptide to IL-12a were obtained from GeneArt (ThermoFisher) with the sequence optimized for human expression and pcDNA3.4 TOPO as vector backbone. Where indicated, I L-12p was equipped with C-terminal (GS)2-linker and FLAG-tag. For protein expression and purification, the IL-12a subunit was C-terminally His-tagged, connected with a TEV cleavage site. For NanoBRET™ assay (Promega), the IL-12 receptor chains were cloned into the pHTC HaloTag® (HT) CMV- neo for IL-12Rp2 (SEQ ID NO: 69) or pNLF1-C NanoLuc® (NL) [CMV / Hygro] vector for IL- 12Rpi (SEQ ID NO: 68). Site-directed mutagenesis PCR using Pfu DNA Polymerase (Promega) was used to generate mutants. All constructs were sequenced.

[0141] Cell culture and transient transfections

[0142] Human embryonic kidney (HEK) 293T and COS7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing high glucose (4500 mg / l) and L-Ala-L-GIn (Sigma-Aldrich) supplemented with 10% (v / v) fetal bovine serum (FBS; Gibco) and 1 % (v / v) antibiotic-antimycotic solution (25 pg / ml amphotericin B, 10 mg / ml streptomycin and 10,000 units of penicillin; Sigma-Aldrich) at 37 °C and 5% CO2. Transient transfections of HEK 293T cells were conducted in poly D-lysine coated p35 dishes (BioCoat, Corning or VWR) using GeneCellin (Eurobio) or Lipofectamine 3000 (Thermo Fisher Scientific) or Metafectene PRO (Biontex) according to the manufacturer’s instructions. 2 pg DNA was used for transfection of cells in p35 dishes. When a-subunits were co-transfected with the p-subunit or empty vector, a 1 :1 ratio was used.

[0143] Secretion and co-immunoprecipitation (co-IP) experiments

[0144] For secretion experiments, HEK 293T cells were transfected for 8 h and then supplemented with 0.5 ml fresh medium for another 16 h. To analyze protein secretion, the medium was centrifuged (300 g, 5 min, 4 °C) and the supernatant was transferred into a newreaction tube, supplemented with 0.1 volumes of 500 mM Tris / HCI, pH 7.5, 1.5 M NaCI, complemented with Roche complete Protease Inhibitor w / o EDTA (Roche Diagnostics). To analyze cell lysate, cells were washed twice with ice-cold phosphate-buffered saline (PBS, Sigma-Aldrich) before lysis with RIPA buffer (50 mM Tris / HCI, pH 7.5, 150 mM NaCI, 1.0% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% SDS, 1x Roche complete Protease Inhibitor w / o EDTA; Roche Diagnostics) for 20 min on ice. Medium and lysate samples were centrifuged (20,000 g, 15 min, 4 °C) and samples were supplemented with 0.2 volumes of 5x Laemmli buffer (0.3125 M Tris / HCI, pH 6.8, 10% SDS, 50% glycerol, bromphenol blue) containing 10% (v / v) p-mercaptoethanol (p-Me) for SDS-PAGE.

[0145] Co-IPs were performed with anti-FLAG agarose beads (Sigma-Aldrich, A2220), Pierce™ anti-HA magnetic beads (Thermo Fisher, 88837) or mouse IgG-beads (Sigma- Aldrich, A0919) for isotype control as indicated in the figures. Harvested medium was supplemented with 25 pl beads, rotated for 1 hour at 4 °C, and beads were washed three times with NP40 wash buffer (50 mM Tris / HCI, pH 7.5, 400 mM NaCI, 0.5% NP40, 0.5% DOC). Elution from beads was performed with 2x Laemmli buffer containing 10% p-Me (95°C, 5 min).

[0146] For co-IP experiments of 2 pg purified protein, IL-12a protein variants were coincubated with I L-12pc199S’His(SEQ ID NO: 37) (25 °C, 1 h) or used alone, as control. Samples were supplemented with 30 pl HIS-Select® Nickel affinity gel (Sigma-Aldrich) and rotated for 1 hour (4°C). Bead washing and elution was performed as described above.

[0147] For antibody based-IPs, harvested medium was supplemented with 1 pg Ustekinumab antibody (ProteoGenix, TA1011) and rotated for 1 hour at 4°C, before adding 25 pl Protein A / G PLUS agarose beads (Thermo Fisher, 20421) for another 1 hour at 4°C with rotation. Rabbit IgG (Thermo Fisher, 10500C) was used as isotype control. Beads were washed three times with NP40 wash buffer (50 mM Tris / HCI, pH 7.5, 400 mM NaCI, 0.5% NP40, 0.5% DOC). Elution from beads was performed with 2x Laemmli buffer containing 10% p-Me (95°C, 5 min).

[0148] Immunoblotting

[0149] For immunoblots, samples were run on 12% SDS-PAGE gels, transferred to polyvinylidene difluoride (PVDF) membranes by blotting overnight at 30 V. After blocking the membrane with 5% (w / v) milk powder in Tris-buffered saline (25 mM Tris / HCI, pH 7.5, 150 mM NaCI; TBS) and 0.05% (v / v) Tween-20 (M-TBST), respective primary antibody was added: anti-Hsc70 (Santa Cruz Biotechnology, sc-7298, 1 :1 ,000), anti-IL-12p (abeam, ab133752, 1 :500), anti-IL-12a (abeam, ab1337511 , 1 :1 ,000), anti-FLAG-tag (Sigma-Aldrich, F7425, 1 :1 ,000), anti-HA-tag (Biolegend, 902302 1 :500) in M-TBST containing 0.002% NaNs. Species-specific HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, 1 :10,000 in M-TBST) were used to detect the proteins with ECL prime reagent (Cytiva) and a Fusion FX7 Edge V0.7 imager (Vilber Lourmat).

[0150] Molecular Dynamics Simulations

[0151] The crystal structure determined by Yoon et al. (2000) (pdb 1f45) served as starting structure for the simulations carried out by the inventors of the present invention. Protonation states were captured with the pdb2pqr server (Dolinsky et al., 2004). Afterwards, the simulation system was prepared with the xleap-module of the Amber18 package (Case et al., 2018). Thereby, disulfide-bonds between cysteine residues were defined, and the IL-12 complex was solvated into an octahedral simulation system with a minimum distance of 1 nm between protein and box boundary. Water molecules were described by the OPC model and sodium counter-ions were added to electrostatically minimize the system and a final salt-concentration of 250 mM NaCI was adjusted. The proteins were described with the ff14SB force field (Maier et al., 2015). The generated simulation system was energy-minimized in 2,000 steps of steepest descent. The energy-minimized system was equilibrated in 8 consecutive MD simulations. In the first three simulations, the system was equilibrated from 100K to 300K (Berendsen-thermostat) in steps of 100K including positional restraints with a force constant of 25.0 kcal / mol / AA2 on the protein atoms. In the first of the three simulations (at 100K) a timestep of 1 fs was used, all consecutive simulations use a time-step of 2 fs. In the next equilibration stage, the restraints were gradually removed in the following five simulations and the Berendsen-barostat was activated with a reference pressure of 1 bar. The whole equilibration phase covers a simulation-time of 1.4 ns. The output structure of the equilibration phase was used as input structure for the production simulation. The production simulation covers 200 ns, and data was written out every 5000 steps.

[0152] Alchemical Free Energy Calculations

[0153] For each of our top mutation candidates, the inventors of the present invention have performed Alchemical Free Energy simulations, in which a specific candidate is mutated into an alanine, to obtain more precise free energy contributions of the mutation candidate to IL-12 complex formation. To this purpose, the inventors of the present invention have generated a merged-topology (concatenating topologies of the WT simulation system and the mutation simulation system) with the parmed module of the AMBER18 package. In the Alchemical Free Energy simulations, the state of the candidate amino acid is controlled by a A-variable (A = 0 means WT, A=1 means mutation). Simulations were conducted at 9 different A-values (A = 0.01592, 0.0820, 0.1933, 0.3379, 0.5000, 0.6621 , 0.8067, 0.9180, 0.9841). Note that the potential energy for these simulations is given by:

[0155] The free energy difference between WT and mutated system is then computed according to:

[0157] The inventors of the present invention calculated the integral in equation 2 based on the Gaussian quadrature rule. Note that simulations for each A were energy minimized and equilibrated following an analogous procedure as described in the previous section. In addition, all alchemical free energy simulations were not only conducted for the IL-12 complex, but also for the alpha subunit alone, allowing the inventors of the present invention to compute t GWT MUTvalues that quantify changes in the binding affinity due to the mutation.

[0158] Recombinant protein expression

[0159] IL- 12aC96S(SEQ ID NO: 11), IL-12aL90A'C96S 204A(SEQ ID NO: 25), and IL-12pc199S(SEQ ID NO: 12) were produced using the pcDNA3.4 vector and the ExpiCHO Expression System (Gibco) according to the manufacturer’s protocol. For IL-12a purification, IL-12a was C-terminally His-tagged (SEQ ID NOs: 60, 69), connected with a TEV cleavage site. Transient transfections were conducted with a subunit ratio of 1 :1 (a:p) for 6 days at 32°C (high titer). After expression, the medium was supplemented with SigmaFAST protease inhibitor (Sigma- Aldrich), centrifuged, and applied to a HisTrap HP column (Cytiva) in PBS buffer, pH 7.4. For IL-12aC96S(SEQ ID NO: 11) and IL-12aL90A'C96S 204A(SEQ ID NO: 25), IL-12pc199S(SEQ ID NO: 12) is washed away with a guanidinium hydrochloride gradient up to 2.5 M Gua in PBS buffer, pH 7.4, and IL-12a is refolded with the same but inverse gradient to PBS buffer (see International Application No. PCT / EP2024 / 059944). Elution was performed with an imidazole concentration step gradient up to 500 mM imidazole in PBS. The His-tag (SEQ ID NO: 60) was cleaved by His-tagged TEV protease, overnight at 4 °C. A subsequent HisTrap HP column in PBS buffer was performed to purify the cleaved IL-12a protein. The final purification was performed by size exclusion chromatography (HiLoad 26 / 600 Superdex 200pg, GE Healthcare) in PBS buffer, pH 7.4. IL-12 heterodimer was also expressed in ExpiCHO cells (standard protocol) and purified using a HisTrap HP column (Cytiva) in PBS buffer, pH 7.4 for harvested supernatant and elution with an imidazole concentration step gradient. TEV protease cleavage (cleavage site, SEQ ID NO: 64) as well as further purification steps were performed same as for IL-12a purification. Expression and purification of I L-12pc199S(SEQ ID NO: 12) was performed as described previously (Meier S., et al., 2019).

[0160] To assess purity and folding status, a 12% SDS-PAGE, supplementing protein samples with 0.2 volumes 5x Laemmli buffer with 10% p-Me (reducing) or 100 mM N- ethylmaleimide (NEM, non-reducing) instead, and Coomassie staining of the gel or immunoblotting with indicated antibodies were performed.

[0161] Hydrogen-deuterium exchange (HDX) mass spectrometry (MS)

[0162] HDX MS experiments were performed using an ACQUITY UPLC M-class system equipped with automated HDX technology (Waters, Milford, MA, USA). HDX kinetics were determined in technical triplicates, tacking data points at 0, 10, 60, 600, 1800 and 7200 s at20 °C. At each data point, 3 pl of a solution of 25 pM IL-12a protein was diluted automatically 1 :20 into 99.9% D2O-containing PBS, pH 7.4. The reaction mixture was quenched by the 1 :1 addition of 200 mM KH2 O4, 200 mM Na2HPO4, pH 2.3 (titrated with HCI), containing 4 M guanidine hydrochloride and 200 mM TCEP at 1 °C and 50 pl of the resulting sample were subjected to on-column peptic digest on a Waters Enzymate BEH pepsin column 2.1 x 30 mm at 20 °C. Peptides were separated by reversed phase chromatography at 0 °C using a Waters Acquity LIPLC C18, 1.7 pm, 2.1 x 5.0 mm, 130 A trapping column and a Waters Acquity LIPLC BEH C18, 1.7 pm, 1 x 100 mm, 130 A separation column. For separation, a gradient increasing the acetonitrile concentration stepwise from 5-35% in 6 min, from 35-40% in 1 min and from 40-95% in 1 min was applied and the eluted peptides were analyzed using an in-line Synapt G2-S QTOF HDMS mass spectrometer (Waters, Milford, MA, USA). UPLC was performed in protonated solvents (0.1 % formic acid), allowing deuterium to be replaced with hydrogen from side chains and amino / carboxyl termini that exchange much faster than backbone amide linkages. All experiments were performed in duplicates. Deuterium levels were not corrected for back exchange and are therefore reported as relative deuterium levels. The use of an automated system handling all samples at identical conditions avoids the need for back exchange correction. MS data were collected over an m / z range of 100-2000. Mass accuracy was ensured by calibration with Glu-fibrino peptide B (Waters, Milford, MA, USA) and peptides were identified by MSEramping the collision energy automatically from 20-50 V. Data were analyzed in PLGS 3.0.3 and DynamX 3.0 software packages (Waters, Milford, MA, USA). For illustration Deuteros 2.0 was used (Lau et al., 2021).

[0163] Isothermal titration calorimetry (ITC)

[0164] All proteins were buffer matched by dialysis in PBS, pH 7.4, before measurement. Experiments were performed with a MicroCai PEAQ-ITC instrument (Malvern Panalytical). Titration of IL-12pc199S(SEQ ID NO: 12) (300-350 pM) in the syringe to IL-12aC96S(SEQ ID NO: 11) (26-23 pM) or IL-12aL90A'C96S 204A(SEQ ID NO: 25) (17-27 pM), respectively, in the cell was conducted at 25 °C. Injection spacing was chosen to allow for signal to return to a stable baseline. Data were analyzed using the PEAQ-ITC analysis software v1.41 (Malvern Panalytical).

[0165] Analytical ultracentrifugation (AUC)

[0166] Sedimentation velocity analytical ultracentrifugation was conducted on a Beckman Coulter Optima™ analytical ultracentrifuge equipped with absorbance optics (Beckman Coulter, Brea, CA, USA) with an An-50 Ti rotor at 20 °C, 42,000 rpm (with an initial test run at 3,000 rpm). 350 pl of 10 pM IL-12a in PBS buffer, pH 7.4, were loaded into a standard 12 mm double-sector epon-filled centerpiece, covered with quartz windows, alongside the reference buffer solution. Scans were acquired at 235 nm with a total of 280 scans and a radial step sizeof 0.001 cm. Resulting sedimentation velocity profiles were analyzed using SedFit software (Dam et al., 2005) with a non-model based continuous Svedberg distribution method (c(s)). The density (p) and viscosity (q) of the PBS buffer used for data analysis was experimentally determined.

[0167] Far-UV circular dichroism (CD) spectroscopy

[0168] Far-UV CD spectra were measured with Jasco J-1500 spectropolarimeter at 25 °C in a 0.2 mm quartz cuvette with a protein concentration of 50 pM in PBS, pH 7.4. Spectra were recorded 10 times, averaged and buffer corrected. Temperature transition measurements of IL-12a-subunits were performed with 10 pM protein in PBS, pH 7.4, in a 1 mm quartz cuvette at a heating rate of 60 °C / h from 20 °C to 90 °C, recorded at 222 nm.

[0169] NanoBRET™ receptor assay

[0170] COS7 cells were transiently seeded in uncoated tissue culture 6-well plates (VWR) and transfected with in total 2 pg receptor chain DNA per well with a ratio of 100:1 HT:NL using GeneCellin (Eurobio) according to the manufacturer’s protocol. After 16 h, transfected cells were detached by Accutase® solution (Sigma-Aldrich), resuspended in assay medium (DMEM w / o Phenolred, 4% (v / v) FBS) to a cell number of 2.2 x 105cells / ml and divided into two pools to which 1 pl HaloTag® NanoLuc® 618 Ligand (Promega) or 1 pl DMSO per ml cells were added. 2 x 104cells were seeded into white bottom 96-well plates and incubated for another 20 h. Purified, reconstituted proteins (1 :1), single subunits, or the covalently linked IL-12- heterodimer were incubated at 25 °C for one hour. Cells were stimulated with respective cytokine subunits, heterodimeric cytokines, or PBS for 30 min at a final concentration of 10 nM or 100 nM. Measurement was conducted with a CLARIOstar® platereader (BMG Labtech) after addition of the Nano-Gio® substrate. NanoBRET™ ratios were calculated by dividing the blank- corrected acceptor emission (610 nm) by blank-corrected donor luminescence (450-480 nm) and multiplication by 1 ,000. Mean NanoBRET™ ratios from technical triplicates were determined averaging the experimental ratios and subtracting the DMSO control mean from the experimental mean.

[0171] NK-92 activation assay

[0172] The human NK-92 cell line (ATCC® CRL-2407™) was cultured in a-MEM without ribonucleosides (Gibco) with 1 .5 g / l NaHCOs, 12.5% FBS (Gibco), 12.5% horse serum (Gibco), 0.2 mM myo-inositol, 0.1 mM p-Me, 0.02 mM folic acid and 100 U / ml IL-2 (Peprotech) at 37°C and 5% CO2. To assess NK cell activation by IL-12-constructs, STAT4 phosphorylation was determined by immunoblotting. Before cell stimulation, cells were starved overnight in medium without sera and IL-12. Cells were seeded in a tissue-culture treated 48-well plate with a cell number of 0.4 x 106 / well and were stimulated with a dilution series of supernatants of transiently transfected HEK 293T cells, secreting IL-12 protein. After 30 minutes of stimulation, cells were transferred into reaction tubes, centrifuged (300 g, 5 min, 4 °C) and lysed with RIPAbuffer (50 mM Tris, pH 7.5, 150 mM NaCI, 1% NP40, 0.5% DOC, 0.1 % SDS), supplemented with Roche complete Protease Inhibitor w / o EDTA (Roche Diagnostics) and phosphatase inhibitor (Serva), for 20 min at 4 °C. After centrifugation (20,000 g, 5 min, 4 °C), lysates were supplemented with 0.2 volumes 5x Laemmli buffer with p-Me and boiled at 95°C for 5 min. Samples were run on 12% SDS-PAGE gels, transferred to PVDF membranes, blocked with 5% (w / v) BSA in TBS with 0.1 % Tween-20 and blotted with aSTAT4 (Cell Signaling, #2653S, 1 :1 ,000 in 5% BSA) oraPSTAT4 (Cell Signaling #5267S, 1 :1 ,000 in 5% BSA). Species-specific HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, 1 :10,000 in 5% BSA) were used to detect the proteins using Amersham ECL prime (Cytiva) and Fusion-FX7.Edge V0.70 imager (Vilber).

[0173] Murine CD4+ T cell stimulation

[0174] C57BL / 6J mice were obtained from Charles River Laboratories and maintained under specific pathogen-free conditions at the Helmholtz Center Munich. The animal experiments were approved by the local authorities (Regierung von Oberbayern). Female and male 12- week-old mice were sacrificed and CD4 T cells were isolated from the spleen via magnetic sorting (mouse CD4+ T Cell Isolation Kit, Miltenyi Biotec). Medium from HEK 293T cells transiently transfected with murine IL-12 P2A constructs was quantified via immunoblotting with the help of a dilution series from known concentrations of purified IL-12. Murine CD4+ T cells were stimulated for 30 min in RPMI + 0.5% BSA with 100 ng / ml IL-12 using quantified IL- 12 secreting HEK medium and STAT4 phosphorylation was analyzed through immunoblotting.

[0175] Structural analyses and schematics

[0176] Based on the IL-12 crystal structure from the PDB database (3HMX), missing loops were modelled using Yasara Structure (www.yasara.org) with a subsequent steepest decent energy minimization. Structures were depicted with PyMOL (PyMOL Molecular Graphics System, Version 2.1 Schrodinger, LLC; www.pymol.org) and schematic illustrations were created with BioRender.

[0177] Quantifications and statistics

[0178] Immunoblot signals were quantified using the Bio-1 D software (Vilber Lourmat). Statistical analyses were performed using Prism (GraphPad Software). Applied statistical tests, experimental sample sizes and types of error calculation are stated in the figure legends. Where no statistical data are shown, experiments were repeated at least two times.

[0179] Surface plasmon resonance (SPR) measurement

[0180] SPR measurements were performed on a Biacore X100 instrument (Cytiva). His- tagged IL-12aC96S(SEQ ID NO: 11) or IL-12aL90A'C96S 204A(SEQ ID NO: 25) (IL-12aC96S-His6corresponds to SEQ ID NO: 67 and IL-12aL90A I204A C96S-HiS6 corresponds to SEQ ID NO: 44) was immobilized on a CM5 sensor chip (Cytiva) previously treated with the His Capture Kit(Cytiva) to covalently couple anti-His antibody on the surface. With a low density of captured His-tagged ligand / a-subunit (70 responsive units (RU) or up to 10 pg / ml of injected ligand as per kit recommendation), binding of IL-12pc199S(SEQ ID NO: 12) was measured at room temperature with a flow rate of 30 pl / min in PBS with 0.05% Tween 20, pH 7.5. Contact of the ligand was performed for 180 s followed by a 120 s stabilization phase. Analyte was injected for 120 s and dissociation was performed for 180 s. Following each cycle, the chip was regenerated with 10 mM Glycine-HCI, pH 1.5. Binding curves were fitted to calculate the kinetics of association and dissociation using BIAevaluation software (Cytiva).[001811 RESULTS

[0182] Example 1 : Design and validation of a self-deactivating IL-12 variant

[0183] IL-12 is a heterodimeric cytokine composed of an a (IL-12a, p35) and a p (IL-12p, p40) subunit (see Fig. 1A). IL-12 is only active when both subunits are assembled (Gubler et al., 1991 ; Yoon et al., 2000). The inventors of the present invention considered these features to be ideal starting points for engineering a self-deactivating variant of IL-12 that would only be functional close to its site of production / release from cells. The hypothesis of the inventors of the present invention was that they could design an IL-12 variant with a destabilized heterodimerization interface so that upon extended times and at low enough subunit concentrations it would fall apart into its a- and p-subunit, abolishing IL-12 activity (see Fig. 1A).

[0184] To achieve this, the inventors of the present invention used the available crystal structure of human IL-12 (Yoon et al., 2000) and computationally analyzed the energy contribution of each residue within IL-12a to heterodimerization (see Fig. 1 B). The inventors of the present invention focused on IL-12a since it depends on IL-12p for release from cells (Reitberger et al., 2017). On the one hand, this gave the inventors an easy readout for the non- disruptive character of the respective mutants according to the present invention. On the other hand, this avoids that a slightly altered I L-12p would be set free from cells which could have a distinct behavior from wildtype I L-12p (Hildenbrand et al., 2022).

[0185] The approach of the inventors of the present invention allowed them to identify ten amino acids within IL-12a that were predicted to strongly contribute to IL-12 heterodimer stability (see Fig. 1 B and Fig. 1C). For all other residues, the inventors individually mutated these to alanine to assess the effects on IL-12. The inventors additionally deleted the disulfide bond in IL-12 that normally connects IL-12a and I L-12p, but is dispensable for IL-12 secretion and function (Reitberger et al., 2017; Yoon et al., 2000). Its presence would of course preclude dis-assembly of IL-12. First, the inventors of the present invention monitored if the mutated IL- 12a subunits still showed wildtype-like secretion behavior. For all mutants, IL-12a was retainedin cells in isolation and I L-12p induced its secretion (see Fig. 5B and Fig. 5C), like for wildtype IL-12a (Reitberger et al., 2017). Two important conclusions can be drawn from this experiment. First, that the mutations did not alter IL-12a retention, and second, since all mutants were secreted in the presence of IL-12p, that the mutations were compatible with proper IL-12 assembly in cells. Building on this, the inventors of the present invention next tested if the mutants led to a destabilization of heterodimeric IL-12 assembly as intended. Among the mutants the inventors tested, exchange of either L52, P63, L90, or I204 against Ala significantly destabilized the IL-12 heterodimer as judged from co-immunoprecipitation experiments of IL-12a with FLAG-tagged (SEQ ID NO: 59) IL-12p (see Fig. 1 D and Fig. 1 E).

[0186] Based on these findings, the inventors next tested if combining two mutations would be an amenable approach to even further destabilize IL-12 without compromising its initial assembly in cells. Indeed, for all four combined mutants the inventors have tested they observed IL-12p-induced secretion of IL-12a, similar to the wildtype pair (see Fig. 5D and 5E), but complete disassembly in co-immunoprecipitation experiments (see Fig. 1 F). Together, the data of the inventors of the present invention show that rational engineering of the IL-12- heterodimerization interface allows to obtain IL-12 variants that are properly assembled and secreted as heterodimers - but whose assembly once outside the cell is drastically destabilized, exactly as the inventors of the present invention intended to.

[0187] Example 2: An IL-12 variant with destabilized heterodimerization, but preserved functionality

[0188] Building on the cell-based experiments, the inventors of the present invention next decided to analyze one of the IL-12-variants in biochemical and biophysical detail and to assess its functionality.

[0189] For this, the inventors focused on the IL-12aL90A I204A(SEQ ID NO: 38) variant, always combined with the exchange of the disulfide bond-forming cysteines in IL-12aC96S(SEQ ID NO: 11) and I L-12pc199S(SEQ ID NO: 12). The inventors of the present invention decided to focus on this variant as it showed a very beneficial signature of computed destabilization of the heterodimeric IL-12 interface while hardly affecting IL-12a stability (see Fig. 5A), which was in agreement with cell-based secretion and co-immunoprecipitation experiments (see Fig. 5E).

[0190] To purify IL-12aL90A’C96S 204A(SEQ ID NO: 25), the inventors of the present invention furnished it with a C-terminal, TEV-cleavable Hise-tag (SEQ ID NO: 60) and co-expressed it (SEQ ID NO: 44) with IL-12pc199S(SEQ ID NO: 12) (inducing its secretion) in CHO cells and developed a simple two-column purification procedure (for details see legend to Fig. 2A) that gave rise to pure IL-12aL90A’C96S 204A(SEQ ID NO: 25) (see Fig. 2B). The purified protein was devoid of any detectable I L-12p subunit (see Fig. 2C) so that it allowed us to perform a largearray of biophysical analyses on its biophysical characteristics (Fig. 6A-C). A direct comparison of |L-l2aL90A C96S I204A(SEQ ID NO: 25) with IL-12aC96S(SEQ ID NO: 11) by hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed no significant differences in amide proton protection (see Fig. 2D and Fig. 6E), showing that the structure and dynamics of IL-12a itself was not altered by the interface mutants. In contrast, isothermal titration calorimetry (ITC) revealed a roughly ten-fold lower affinity of IL-12aL90A’C96S 204A(SEQ ID NO: 25) to I L-12p than for IL-12a not containing any interface mutation (approx. 0.2 versus 2.0 iM KD value, see Fig. 2E and 2F).

[0191] To assess the functionality of IL-12 containing IL-12aL90A’C96S 204A(SEQ ID NO: 25), the inventors of the present invention first tested if it was able to induce IL-12 receptor heterodimerization, which is a prerequisite for IL-12 signaling. To this end, the inventors of the present invention used a bioluminescence resonance energy transfer (BRET)-based assay they recently developed. In this assay, IL-12 containing IL-12aL90A’C96S 204A(SEQ ID NO: 25) was functional when used at a 10-fold higher concentration than the wildtype IL-12 (or IL-12 only devoid of its interchain disulfide bond), which is fully consistent with the destabilized heterodimerization in IL-12 containing our interface mutant (see Fig. 2H). Together, these combined analyses confirmed the design by the inventors of the present invention and showed by mutating two interface residues in IL-12a that the inventors of the present invention successfully engineered a kinetically and thermodynamically destabilized IL-12 heterodimer without compromising the stability of IL-12a itself. Importantly, this kinetically and thermodynamically destabilized IL-12 remained functional. Therefore, the inventors of the present invention termed the IL-12-variant composed of IL-12aL90A’C96S 204A(SEQ ID NO: 25) and I L-12pc199S(SEQ ID NO: 12) self-deactivating IL-12 (sdlL-12).

[0192] Example 3: SPR experiments

[0193] In this Example, kinetically destabilized IL-12 heterodimers with the engineered IL-12a mutant were created and binding kinetics of purified IL-12aC96S(SEQ ID NO: 11) or IL- 12aL90A I204A C96S(SEQ ID NO: 25) to I L-12pc199S(SEQ ID NO: 12) were determined via surface plasmon resonance (SPR) (see Figure 2G). Either IL-12aC96S-Hise (SEQ ID NO: 67) or IL- 12aL90A I204A C96S-HiS6 (SEQ ID NO: 44) were immobilized as ligand on a CM5 sensor chip precoupled to anti-His antibody and association / dissociation kinetics of the analyte IL-12pc199S(SEQ ID NO: 12) were measured by SPR. A roughly 10-fold higher dissociation rate constant (koff) for the complex of IL-12aL90A 204A'C96S(SEQ ID NO: 25) with IL-12pc199S(SEQ ID NO: 12) confirms kinetic destabilization of the heterodimer when compared to IL-12a without any interface mutations.REFERENCESAlbelda, S.M., 2024. CAR T cell therapy for patients with solid tumours: key lessons to learn and unlearn. Nat. Rev. Clin. 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Claims

CLAIMS1. A mutein of the a-subunit of human Interleukin 12 according to SEQ ID NO: 1 , wherein at least one of the amino acid residue(s) of said a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207, 210 and 211 is / are mutated and wherein the disulfide bridge at sequence position 96 of said a-subunit is deleted, optionally by mutating the amino acid at sequence position 96 of said a-subunit to another amino acid than cysteine.

2. The mutein of claim 1 , wherein the disulfide bridge at sequence position 96 of said a-subunit is deleted by mutating the amino acid at sequence position 96 of said a-subunit to another amino acid than cysteine, optionally to alanine or serine.

3. The mutein of claim 1 or 2, wherein said mutein comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the a-subunit of human Interleukin 12 according to SEQ ID NO: 1.

4. The mutein of any one of the preceding claims, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207 and 210 is / are mutated.

5. The mutein of any one of the preceding claims, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204 and 207 is / are mutated.

6. The mutein of any one of the preceding claims, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200 and 204 is / are mutated.

7. The mutein of any one of the preceding claims, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 68, 90, 200 and 204 is / are mutated.

8. The mutein of any one of the preceding claims, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 68, 90, 200, 204, 207 and 211 is / are mutated.

9. The mutein of any one of the preceding claims, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 90, 200 and 204 is / are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A), and / or wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 60 and 68 is / are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

10. The mutein of any one of the preceding claims, wherein the amino acid residue of the a- subunit at sequence position 52 is mutated, optionally wherein the amino acid residue of the a-subunit at sequence position 52 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).11 . The mutein of any one of the preceding claims, wherein the amino acid residue of the a- subunit at sequence position 60 is mutated, optionally wherein the amino acid residue of the a-subunit at sequence position 60 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

12. The mutein of any one of the preceding claims, wherein the amino acid residue of the a- subunit at sequence position 63 is mutated, optionally wherein the amino acid residue of the a-subunit at sequence position 63 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

13. The mutein of any one of the preceding claims, wherein the amino acid residue of the a- subunit at sequence position 68 is mutated, optionally wherein the amino acid residue of the a-subunit at sequence position 68 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

14. The mutein of any one of the preceding claims, wherein the amino acid residue of the a- subunit at sequence position 90 is mutated, optionally wherein the amino acid residue of the a-subunit at sequence position 90 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

15. The mutein of any one of the preceding claims, wherein the amino acid residue of the a- subunit at sequence position 200 is mutated, optionally wherein the amino acid residue of the a-subunit at sequence position 200 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

16. The mutein of any one of the preceding claims, wherein the amino acid residue of the a- subunit at sequence position 204 is mutated, optionally wherein the amino acid residue of the a-subunit at sequence position 204 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

17. The mutein of any one of the preceding claims, wherein the amino acid residue of the a- subunit at sequence position 207 is mutated, optionally wherein the amino acid residue of the a-subunit at sequence position 207 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

18. The mutein of any one of the preceding claims, wherein the amino acid residue of the a- subunit at sequence position 210 is mutated, optionally wherein the amino acid residue of the a-subunit at sequence position 210 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

19. The mutein of any one of the preceding claims, wherein the amino acid residue of the a- subunit at sequence position 211 is mutated, optionally wherein the amino acid residue of the a-subunit at sequence position 211 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

20. The mutein of any one of the preceding claims, wherein the amino acid residues of the a- subunit at sequence positions 90 and 204 are mutated, optionally wherein the amino acid residues of the a-subunit at sequence positions 90 and 204 are replaced by an amino acid other than the amino acid at the respective sequence positions in SEQ ID NO: 1 , optionally by alanine (A).

21. The mutein of any one of the preceding claims, wherein the disulfide bridge at sequence position 96 of said a-subunit is deleted by mutating the amino acid residue of the a-subunit at sequence position 96 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by serine (S) or alanine (A).

22. The mutein of claim 21 , wherein the amino acid residue of the a-subunit at sequence position 96 is replaced by serine (S) or alanine (A), optionally by serine (S).

23. The mutein of claim 21 or 22, wherein the amino acid residue of the a-subunit at sequence position 96 is replaced by serine (S), the amino acid residue of the a-subunit at sequenceposition 63 or 90 is replaced by alanine (A), and the amino acid residue of the a-subunit at sequence position 204 is replaced by alanine (A).

24. A mutein of human Interleukin 12, comprising an a-subunit (p35) and a p-subunit (p40), wherein the a-subunit is the mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1) of any one of claims 1 to 23, and wherein in said mutein of human Interleukin 12 the amino acid residue at sequence position 96 of the a-subunit and / or the amino acid residue at sequence position 199 of the p-subunit is / are mutated, optionally wherein said mutein is self-deactivating and / or self-dissociating and / or kinetically destabilized.

25. The mutein of claim 24, wherein at least one of the amino acid residues of said a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207, 210 and 211 is / are mutated, optionally from the group consisting of sequence positions 52, 68, 90, 200, 204, 207 and 211 is / are mutated.

26. The mutein of claim 24 or 25, wherein said mutein is self-deactivating and / or selfdissociating and / or kinetically destabilized.

27. The mutein of any one of claims 24 to 26, wherein in said mutein of human Interleukin 12 the amino acid residue at sequence position 96 of the a-subunit is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by serine (S), and / or wherein in said mutein of human Interleukin 12 the amino acid residue at sequence position 199 of the p-subunit is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by serine (S).

28. The mutein of any one of claims 24 to 27, wherein the a-subunit comprises at least 90%, 91 %, 92%, 93% 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the a-subunit of human Interleukin 12 according to SEQ ID NO: 1.

29. The mutein of any one of claims 24 to 28, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204, 207 and 210 is / are mutated.

30. The mutein of any one of claims 24 to 29, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 63, 68, 90, 200, 204 and 207 is / are mutated.31 . The mutein of any one of claims 24 to 30, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 60, 68, 90, 200 and 204 is / are mutated.

32. The mutein of any one of claims 24 to 28, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 68, 90, 200, 204, 207 and 211 is / are mutated.

33. The mutein of claim 32, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 52, 68, 90, 200, 204, 207 and 211 is / are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

34. The mutein of any one of claims 24 to 33, wherein the amino acid residue of the a-subunit at sequence position 52 is mutated, optionally wherein the amino acid residue of the a- subunit at sequence position 52 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

35. The mutein of any one of claims 24 to 31 , wherein the amino acid residue of the a-subunit at sequence position 60 is mutated, optionally wherein the amino acid residue of the a- subunit at sequence position 60 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

36. The mutein of any one of claims 24 to 30, wherein the amino acid residue of the a-subunit at sequence position 63 is mutated, optionally wherein the amino acid residue of the a- subunit at sequence position 63 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

37. The mutein of any one of claims 24 to 33, wherein the amino acid residue of the a-subunit at sequence position 68 is mutated, optionally wherein the amino acid residue of the a- subunit at sequence position 68 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

38. The mutein of any one of claims 24 to 33, wherein the amino acid residue of the a-subunit at sequence position 90 is mutated, optionally wherein the amino acid residue of the a-subunit at sequence position 90 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

39. The mutein of any one of claims 24 to 33, wherein the amino acid residue of the a-subunit at sequence position 200 is mutated, optionally wherein the amino acid residue of the a- subunit at sequence position 200 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

40. The mutein of any one of claims 24 to 33, wherein the amino acid residue of the a-subunit at sequence position 204 is mutated, optionally wherein the amino acid residue of the a- subunit at sequence position 204 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).41 . The mutein of any one of claims 24 to 33, wherein the amino acid residue of the a-subunit at sequence position 207 is mutated, optionally wherein the amino acid residue of the a- subunit at sequence position 207 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

42. The mutein of any one of claims 24 to 29, wherein the amino acid residue of the a-subunit at sequence position 210 is mutated, optionally wherein the amino acid residue of the a- subunit at sequence position 210 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

43. The mutein of any one of claims 24 to 33, wherein the amino acid residue of the a-subunit at sequence position 211 is mutated, optionally wherein the amino acid residue of the a- subunit at sequence position 211 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , optionally by alanine (A).

44. The mutein of any one of claims 24 to 33, wherein the amino acid residues of the a-subunit at sequence positions 90 and 204 are mutated, optionally wherein the amino acid residues of the a-subunit at sequence positions 90 and 204 are replaced by an amino acid other than the amino acid at the respective sequence positions in SEQ ID NO: 1 , optionally by alanine (A).

45. The mutein of any one of claims 24 to 44, wherein additionally the amino acid residue of the a-subunit at sequence position 96 is mutated, optionally wherein additionally the amino acid residue of the a-subunit at sequence position 96 is replaced by an amino acid otherthan the amino acid at the respective sequence position in SEQ ID NO: 1, optionally by serine (S) or alanine (A).

46. The mutein of any one of claims 24 to 45, wherein additionally the amino acid residue of the a-subunit at sequence position 96 is replaced by serine (S) or alanine (A), optionally by serine (S).

47. The mutein of any one of claims 24 to 46, wherein the amino acid residue of the a-subunit at sequence position 96 is replaced by serine (S), the amino acid residue of the a-subunit at sequence position 63 or 90 is replaced by alanine (A) and the amino acid residue of the a-subunit at sequence position 204 is replaced by alanine (A).

48. The mutein of any one of claims 24 to 47, wherein the p-subunit is the p-subunit of human Interleukin 12 according to SEQ ID NO: 2.

49. The mutein of any one of claims 24 to 48, wherein the p-subunit comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the p-subunit of human Interleukin 12 according to SEQ ID NO: 2.

50. The mutein of any one of claims 24 to 49, wherein the p-subunit is the p-subunit of human Interleukin 12 according to SEQ ID NO: 2 and wherein the amino acid residue at position 199 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, optionally by serine (S) or alanine (A).

51. The mutein of claim 50, wherein the amino acid residue at position 199 is replaced by serine (S) or alanine (A), optionally serine (S).

52. The mutein of claim 51 , wherein the amino acid residue at position 199 is replaced by serine (S).

53. A cDNA or DNA nucleic acid molecule comprising i) a nucleotide sequence encoding the mutein of human Interleukin 12 of any one of claims 24 to 52, or ii) a nucleotide sequence encoding the mutein of the a-subunit of human Interleukin 12 of any one of claims 1 to 23.

54. The nucleic acid molecule of claim 53, wherein the nucleotide sequence encodes a mutein of the a-subunit of human Interleukin 12 comprising at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the a-subunit of human Interleukin 12 according to SEQ ID NO: 1.

55. The nucleic acid molecule of claim 53 or 54, comprising a nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 of any one of SEQ ID NOs: 3 to 11 , 13 to 29, 31 , 32, 38, 44, 47, 48 or 49.

56. The nucleic acid molecule of any one of claims 53 to 55, wherein the nucleotide sequence encodes a mutein of the p-subunit of human Interleukin 12 comprising at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the -subunit of human Interleukin 12 according to SEQ ID NO: 2.

57. The nucleic acid molecule of any one of claims 53 to 56, comprising a nucleotide sequence encoding a mutein of the p-subunit of human Interleukin 12 of SEQ ID NO: 12.

58. The nucleic acid molecule of any one of claims 53 to 57, wherein the nucleic acid molecule is operably linked to a regulatory sequence for expression of the nucleic acid molecule.

59. The nucleic acid molecule of claim 58, wherein the regulatory sequence comprises a promoter sequence.

60. A vector comprising the cDNA or DNA nucleic acid molecule of any one of claims 53 to 59.61 . A host cell comprising the cDNA or DNA nucleic acid molecule of any one of claims 53 to 59 and / or the vector of claim 60.

62. An mRNA nucleic acid molecule comprising i) a nucleotide sequence encoding the mutein of human Interleukin 12 of any one of claims 24 to 52, or ii) a nucleotide sequence encoding the mutein of the a-subunit of human Interleukin 12 of any one of claims 1 to 23.

63. The mRNA nucleic acid molecule of claim 62, wherein the nucleotide sequence encodes a mutein of the a-subunit of human Interleukin 12 comprising at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity to the a-subunit of human Interleukin 12 according to SEQ ID NO: 1.

64. The mRNA nucleic acid molecule of claim 62 or 63, comprising a nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 of any one of SEQ ID NOs: 3 to 11 , 13 to 29, 31 , 32, 38, 44, 47, 48 or 49.

65. The mRNA nucleic acid molecule of any one of claims 62 to 64, wherein the nucleotide sequence encodes a mutein of the p-subunit of human Interleukin 12 comprising at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the p- subunit of human Interleukin 12 according to SEQ ID NO: 2.

66. The mRNA nucleic acid molecule of any one of claims 62 to 65, comprising a nucleotide sequence encoding a mutein of the p-subunit of human Interleukin 12 of SEQ ID NO: 12.

67. The mRNA nucleic acid molecule of any one of claims 62 to 66, wherein the nucleic acid molecule is operably linked to a 5’-cap and / or a poly(A)-tail for transcription of the nucleic acid molecule.

68. An engineered cell or virus comprising the mutein of any one of claims 1 to 52, the cDNA or DNA nucleic acid molecule of any one of claims 53 to 59, and / or the vector of claim 60, and / or the mRNA nucleic acid molecule of any one of claims 62 to 67, or use of said engineered cell in cell therapy.

69. A recombinant protein comprising the mutein of any one of claims 1 to 52, the cDNA or DNA nucleic acid molecule of any one of claims 53 to 59, and / or the vector of claim 60, and / or the mRNA nucleic acid molecule of any one of claims 62 to 67.

70. A fusion-protein comprising the mutein of any one of claims 1 to 52, the cDNA or DNA nucleic acid molecule of any one of claims 53 to 59, the vector of claim 60, and / or the mRNA nucleic acid molecule of any one of claims 62 to 67.

71. An immune modulator comprising the mutein of any one of claims 1 to 52, the mRNA nucleic acid molecule of any one of claims 53 to 59, the engineered cell or virus of claim 68, the recombinant protein of claim 69, and / or the fusion-protein of claim 70.

72. A pharmaceutical composition comprising the mutein of any one of claims 1 to 52, the mRNA nucleic acid molecule of any one of claims 53 to 59, the engineered cell or virus of claim 68, the recombinant protein of claim 69, the fusion-protein of claim 70, and / or the immune modulator of claim 71 , optionally further comprising a pharmaceutically acceptable carrier.

73. Use of the mutein of any one of claims 1 to 52, of the mRNA nucleic acid molecule of any one of claims 53 to 59, of the recombinant protein of claim 69, of the fusion-protein of claim 70, and / or of the engineered cell or virus of claim 70 for the manufacture of a medicament for treating a disease in a mammal, optionally a human.

74. The use of claim 73, wherein the disease is a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease, Graft-versus-Host-disease, a chronic inflammatory disease, chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma.

75. The mutein of any one of claims 1 to 52, the mRNA nucleic acid molecule of any one of claims 53 to 59, the recombinant protein of claim 69, the fusion-protein of claim 70, and / or the engineered cell or virus of claim 68, for use as a medicament.

76. The mutein of any one of claims 1 to 52, the mRNA nucleic acid molecule of any one of claims 53 to 59, the recombinant protein of claim 69, the fusion-protein of claim 70, and / or the engineered cell or virus of claim 68, for use in the treatment of a disease.

77. The mutein for use, the mRNA nucleic acid molecule for use, the recombinant protein for use, the fusion-protein for use, or the engineered cell for use of claim 76, wherein the disease is a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease, Graft-versus-Host- disease, a chronic inflammatory disease, chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma.

78. A method of treating an Interleukin 12-mediated disease in a mammal, optionally a human, comprising the step of administering a composition comprising the mutein of any one of claims 1 to 52, the mRNA nucleic acid molecule of any one of claims 53 to 59, the engineered cell or virus of claim 68, the recombinant protein of claim 69, the fusion-protein of claim 70, the immune modulator of claim 71 , and / or the pharmaceutical composition of claim 72, to said mammal in need thereof.

79. The method of treating of claim 78, wherein the disease is a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease, Graft-versus-Host-disease, a chronic inflammatory disease, chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma.

80. A virus, optionally a tumor targeting virus, comprising the mutein of any one of claims 1 to 52.90