Interleukin-2 Chimera Construct

Abstract

The present invention relates to a chimeric construct comprising i) an interleukin 2 (IL2) moiety and ii) the beta chain of C4b-binding protein (C4BPβ) or at least one fragment or functional variant thereof capable of forming a dimeric protein.

Description

Technical Field

[0001] The present invention relates to an IL2 construct with improved pharmacokinetics and / or pharmacodynamics.

Background Art

[0002] Background of the Invention Interleukin-2 (IL2 or IL-2) is a cytokine that controls important aspects of the immune system. IL2 has been used in attempts to boost the immune response in patients with cancer as well as autoimmune and / or inflammatory diseases. IL2 promotes immune responses including the clonal expansion of antigen-activated T cells, drives the development of CD4+ T helper (Th) 1 and Th2 cells, terminally differentiates CD8+ cytotoxic T lymphocytes (CTLs), and counteracts the development of CD4+ Th17 and T follicular helper (Tfh) cells. IL2 also shapes the T cell memory recall response.

[0003] Low-dose IL2 is used to selectively boost tolerance to suppress unwanted immune responses associated with autoimmune-like attacks on self-tissues. Experience to date is that this treatment is safe and there are no signs of reactivation of self-aggressive T cells, but regulatory T cells (Tregs) associated with clinical improvement increase in almost all patients.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Nevertheless, IL2 as a treatment has significant room for improvement with respect to its short half-life in vivo. For these reasons, new IL2 biopharmaceuticals need to have improved pharmacokinetics and / or pharmacodynamics.

Means for Solving the Problems

[0005] Summary of the Invention The present invention provides a chimeric construct comprising i) an interleukin-2 (IL2) moiety and ii) a beta chain of a C4b-binding protein (C4BPβ) capable of forming a dimeric protein, or at least one fragment or functional variant thereof.

[0006] Such constructs therefore exhibit an improved half-life of the IL2 portion. Furthermore, the inventors have surprisingly shown that such chimeric constructs improve the selectivity of Treg expansion.

[0007] In certain embodiments, the chimeric construct is in dimeric form, where these monomers are linked by a covalent bond between two cysteine ​​molecules of C4BPβ. Homodimers and heterodimers are described in further detail below.

[0008] In a preferred embodiment, the C4BPβ fragment includes or consists of a longer fragment of C4BPβ that is extended at the N-terminus to amino acid residues 194-252 or up to amino acid 135 of C4BPβ.

[0009] In a preferred embodiment, the Il-2 portion is human IL-2 or a homologous variant thereof, where the variant has at least 85% amino acid identity with human wild-type IL-2, and preferably, where the variant is an active analog of human IL-2 having at least 90% amino acid identity with human wild-type IL-2, where the IL-2 portion is preferably an IL2 mutein containing a substitution at the N88 position of SEQ ID NO: 2, and more preferably a substitution N88R. [Brief explanation of the drawing]

[0010] [Figure 1] The images show the dose-response of pSTAT5 induction in Treg (A), Tconv (B), and CD8 T cells (C). Hi2 is human IL-2 (SEQ ID NO: 1), Hi2cb is human IL-2 fused to the C-terminal region of C4BPβ (SEQ ID NO: 6), and Hi2mcb is mutant IL-2 fused to the C-terminal region of C4BPβ (SEQ ID NO: 7).

[0011] [Figure 2] This shows the rate curves of increase in four different T cell compartments and NK cells in mice injected with AAV expressing the IL-2 construct of the 1011 viral genome.

[0012] [Figure 3] This shows the time-course dynamics of human IL-2 in plasma (A) and urine (B) of mice after injection with AAV expressing the IL-2 construct of the 1011 viral genome.

[0013] [Figure 4] This shows the rate curves of increase in four different T cell compartments and NK cells in mice after AAV injection expressing the IL-2 construct of the 1012 viral genome.

[0014] [Figure 5] The survival Kaplan-Meier curves of mice after injection with AAV expressing the IL-2 construct of the 1012 viral genome are shown.

[0015] [Figure 6] This study demonstrates the therapeutic efficacy of the fusion protein Hi2cb or Hi2mcb compared to Hi2 in an experimental autoimmune encephalomyelitis (EAE) model.

[0016] [Figure 7] This document describes an experimental dosing schedule based on a single injection of 25,000 international units of Hi2, Hi2cb, or Hi2mcb daily for five consecutive days. Immunophenotyping was performed daily before injection to evaluate Treg kinetics.

[0017] [Figure 8] The pharmacokinetic profiles of each construct, Hi2, Hi2cb, or Hi2mcb, after a single subcutaneous injection are shown. [Modes for carrying out the invention]

[0018] Detailed Description of the Invention Definitions The "subject" or "patient" to be treated can be any mammal, preferably a human. A human subject can be a pediatric, adult or elderly individual.

[0019] The term "treat" or "treatment" means any amelioration of a disease. It includes the reduction of at least one symptom or the severity or progression of a disease. When the disease is an inflammatory and / or autoimmune disorder, the term more specifically includes the reduction of the risk, occurrence or severity of an acute episode (flare). The term "treat" or "treatment" encompasses the reduction of disease progression. In particular, the present invention encompasses the prevention or deceleration of disease progression. The term "treat" or "treatment" further includes prophylactic treatment by reducing the risk or delaying the onset of disease, particularly in subjects who are asymptomatic but diagnosed as "at risk".

[0020] "Regulatory T cells" or "Tregs" are T lymphocytes having immunosuppressive activity. Natural Tregs are characterized as CD4+CD25+Foxp3+ cells. Tregs play a major role in the control of inflammatory diseases, but the mechanism of action in such diseases is not fully understood. In fact, in most inflammatory diseases, Treg depletion exacerbates the disease and Treg addition reduces it. Most Tregs are CD4+ cells, but there is also a rare population of CD8+Foxp3+ T lymphocytes having suppressive activity.

[0021] Within the scope of the present invention, an "effector T cell" (or "Teff") means a normal T lymphocyte other than a Treg (sometimes referred to as Tconv in the literature) that expresses one or more T cell receptors (TCRs) and exhibits effector functions (e.g., cytotoxic activity, cytokine secretion, etc.). The major populations of human Teffs of the present invention include CD4+ T helper lymphocytes (e.g., Th0, Th1, Th2, Th9, Th17, Tfh) and CD4+ or CD8+ cytotoxic T lymphocytes and can be specific for self or non-self antigens. Teffs do not include Foxp3+ regulatory CD8+ T cells.

[0022] Within the scope of the present invention, “T follicular helper cells” (or “Tfh”) means T CD4+ lymphocytes that express BcL6, CXCR5, and PD1, are Foxp3-, and assist B cells.

[0023] In the context of this invention, "T follicular regulatory cells" (or "Tfr") means CD4+CXCR5+PD-1+Bcl6+Foxp3+CD25-T lymphocytes.

[0024] The sequence list shows the following sequences: Sequence ID 1 is wild-type human IL2 (253 amino acids, including the signal peptide). Sequence ID 2 is mature wild-type human IL2 (233 amino acids, including the signal peptide). Sequence ID 3 is the C4BP beta chain (1-252). Sequence ID 4 is fragment 194-252 of the C4BP beta chain. Sequence ID 5 is fragment 137-252 of the C4BP beta chain. Sequence ID 6 is the amino acid sequence of Hi2cb (including the signal peptide). Sequence ID 7 is the amino acid sequence of Hi2mcb(N88R) containing the signal peptide. Sequence ID 8 is the GGGGS pattern (linker). Sequence ID 9 is the amino acid sequence signal peptide of Hi2cb, which does not have a signal peptide. Sequence ID 10 is the amino acid sequence of Hi2mcb(N88R) which does not have a signal peptide.

[0025] IL-2 part The interleukin-2 (IL-2) used herein includes mammalian wild-type interleukin-2 and its variants. Preferably, IL-2 is human IL-2 or its variant.

[0026] Active variants of IL-2 are disclosed in the literature. Variants of natural IL-2 may be its fragments, analogs, and derivatives. "Fragment" refers to a polypeptide containing only a portion of the polypeptide sequence. "Analog" means a polypeptide containing the natural polypeptide sequence having one or more amino acid substitutions, insertions, or deletions. Muteins and pseudopeptides are specific examples of analogs. "Derivatives" include any modified natural IL-2 polypeptide or its fragments or analogs, such as glycosylation, phosphorylation, fusion to other polypeptides or molecules, polymerization, or chemical or enzymatic modification or addition, to improve the properties of IL-2 (e.g., stability, specificity, etc.). The IL-2 portion of an active variant generally has at least 75%, preferably at least 80%, 85%, more preferably at least 90%, or at least 95% amino acid sequence identity with the amino acid sequence of the target IL-2 polypeptide, e.g., mature wild-type human IL-2.

[0027] The term "wild-type IL-2" used here refers to IL-2, whether natural or recombinant, containing the 133 amino acid sequence typically found in natural human IL-2. This amino acid sequence is described in Fujita, et. al., PNAS USA, 80, 7437-7441 (1983). Sequence ID No. 2 (133 amino acids) is a human IL-2 sequence without the signal peptide consisting of an additional 20 N-terminal amino acids. Sequence ID No. 1 (153 amino acids) is a human IL-2 sequence containing the signal peptide.

[0028] The term "IL-2 mutein" used here refers to a polypeptide in which specific amino acid substitutions have been made to the human mature interleukin-2 protein. Unless otherwise specified, the total numbering of amino acids corresponds to the human mature interleukin-2 protein of Sequence ID No. 2.

[0029] In one embodiment, the cysteine ​​at position 125 is replaced with a neutral amino acid such as serine (C125S), alanine (C125A), threonine (C125T), or valine (C125V).

[0030] For example, the removal of the O-glucosylation site results in a more homogeneous product when the active variant is expressed in mammalian cells such as CHO or HEK cells.

[0031] In one embodiment, the active variant includes a further amino acid mutation that removes the O-glucosylation site of IL-2 at the position corresponding to residue 3 of human IL-2. In one embodiment, the further amino acid mutation that removes the O-glucosylation site of IL-2 at the position corresponding to residue 3 of human IL-2 is an amino acid substitution. Examples of amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. In a particular embodiment, the further amino acid mutation is the amino acid substitution T3A.

[0032] Active variants that selectively promote T-reg cell proliferation, survival, activation, and / or function are particularly useful in treating inflammatory and / or autoimmune disorders.

[0033] "Selectively promoting" means that the active variant promotes activity in T-reg cells, but has limited or no ability to promote activity in unregulated T cells. Further described here are assays for screening active variants that selectively promote T-reg cell proliferation, survival, activation, and / or function.

[0034] Methods for determining whether a variant IL-2 polypeptide is active are available in the art. See, for example, WO2016 / 014428. Active variants are defined as variants that exhibit the ability to stimulate Tregs, and include variants that have improved, similar, or reduced ability to stimulate Tregs compared to wild-type IL-2 or aldesleukin (defined below), provided that they do not stimulate Teffs beyond Treg stimulation. Methods for testing whether a candidate molecule stimulates T cells, particularly Tregs or NK cells, are well known. Variants may be tested for their ability to stimulate effector T cells (e.g., CD8+ T cells), CD4+Foxp3+Tregs, or NK cells. In a preferred embodiment, an active variant exhibits reduced ability to stimulate NK cells compared to wild-type IL2 or aldesleukin. Monitoring STAT5 phosphorylation is a simple method for evaluating a variant's ability to preferentially stimulate Treg cells over Teff cells, as described in Yu et al, Diabetes 2015;64:2172-2183. In certain embodiments, the variant is particularly useful when Treg cells achieve STAT5 phosphorylation levels at doses at least 10 times lower than other immune cells, including Teff cells.

[0035] The active variant induces signaling events that preferentially induce Treg cell survival, proliferation, activation, and / or function. In one embodiment, the IL-2 variant retains the ability to stimulate phosphorylation of STAT5 phosphorylation and / or one or more downstream signaling molecules of IL-2R in Treg cells, such as p38, ERK, SYK, and LCK. In another embodiment, the IL-2 variant retains the ability to stimulate transcription or protein expression of genes or proteins important for Treg cell survival, proliferation, activation, and / or function, such as FOXP3, Bcl-2, CD25, or IL-10 in Treg cells. In yet another embodiment, the IL-2 variant exhibits reduced ability to stimulate endocytosis of the IL-2 / IL-2R complex on the surface of CD25+ T cells. In yet another embodiment, the IL-2 variant exhibits inefficient, reduced, or absent stimulation of PI3-kinase signaling, such as inefficient, reduced, or absent phosphorylation of AKT and / or mTOR (mammalian target of rapamycin). In yet another embodiment, the IL-2 variant retains the wild-type IL-2's ability to stimulate STAT5 phosphorylation and / or phosphorylation of one or more signaling molecules downstream of IL-2R in Treg cells, while phosphorylation of STAT5, AKT, and / or mTOR or other signaling molecules downstream of IL-2R is inefficient, reduced, or absent in FOXP3-CD4+ or CD8+ T cells or NK cells. In yet another embodiment, the IL-2 variant is inefficient or impossible to stimulate the survival, proliferation, activation, and / or function of FOXP3-CD4+ or CD8+ T cells or NK cells.

[0036] In all cases, these variants have the ability to stimulate cell lines such as CTLL-2 or HT-2, which can be universally used to determine biological activity.

[0037] For example, the biological activity of IL-2 is dependent on IL-2 for proliferation in HT-2 cell lines (clone A5E, ATCC® CRL-1841).TM This can be determined by a cell-based assay performed at ). Cell proliferation in the presence of a certain range of test interleukin-2 products is compared to proliferation recorded with the IL-2 international standard (WHO 2nd International Standard for INTERLEUKIN 2 (Human, rDNA derived) NIBSC code: 86 / 500). Cell proliferation is measured after the addition of [3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (intramolecular salt, MTS) and conversion to formazan by active viable cells. The formazan concentration is then measured by spectrophotometric measurement at 490 nm.

[0038] Examples of IL-2 variants are disclosed, for example, in EP109748, EP136489, US4,752,585;EP200280, EP118617, WO99 / 60128, EP2288372, US9,616,105, US9,580,486, WO2010 / 085495, and WO2016 / 164937.

[0039] For example, certain mutations may reduce the affinity of IL-2 receptors (IL-2Rβ / CD122 and / or IL-2Rγ / CD132) to the signaling chain and / or reduce the ability to induce signaling events from one or both subunits of the IL-2 receptor. Other mutations may confer high affinity to CD25 (IL-2Rα). In either case, such mutations define active variants that preferentially induce Treg survival, proliferation, activation, and / or function. This property can be monitored by surface plasmon resonance.

[0040] A specific example of a useful variant is an IL-2 mutaine exhibiting at least one amino acid substitution at positions D20, N30, Y31, K35, V69, Q74, N88, V91, or Q126, in numbering following wild-type IL-2, meaning that the selected amino acid is identified by referring to the position where that amino acid normally appears in the mature sequence of wild-type IL-2 of SEQ ID NO: 2.

[0041] A preferred IL-2 mutain includes at least one substitution at positions D20H, D20I, D20Y, N30S, Y31H, K35R, V69AP, Q74, N88R, N88D, N88G, N88I, V91K, or Q126L.

[0042] In one embodiment, the IL-2 mutein molecule includes a V91K substitution. In one embodiment, the IL-2 mutein molecule includes an N88D substitution. In one embodiment, the IL-2 mutein molecule includes an N88R substitution. In one embodiment, the IL-2 mutein molecule includes substitutions of H16E, D84K, V91N, N88D, V91K, or V91R, or any combination thereof. In one embodiment, these IL-2 mutein molecules also include the 125-position substitutions described herein. In one instance, the IL-2 mutein molecule is T3N, T3A, L12G, L12K, L12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20H, D20I, D20Y, D20F, D20 Includes one or more substitutions selected from the group consisting of G, D20T, D20W, M23R, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q, D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88I, N88F, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91G, V91S, I92K, I92R, E95G, and Q126.In one embodiment, the amino acid sequence of the IL-2 mutein molecule is the amino acid sequence shown in the mature IL-2 sequence, with C125A or C125S substitutions and T3N, T3A, L12G, L12K, L12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D2 It differs by one substitution selected from 0E, D20F, D20G, D20T, D20W, M23R, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q, D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88F, N88I, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91G, V91S, I92K, I92R, E95G, Q126I, Q126L, and Q126F. In one embodiment, the IL-2 mutein molecule differs from the amino acid sequence shown in the mature IL-2 sequence by a C125A or C125S substitution and one substitution selected from D20H, D20I, D20Y, D20E, D20G, D20W, D84A, D84S, H16D, H16G, H16K, H16R, H16T, H16V, I92K, I92R, L12K, L19D, L19N, L19T, N88D, N88R, N88S, V91D, V91G, V91K, and V91S. In one embodiment, the IL-2 mutein contains an N88R and / or D20H mutation.

[0043] These substitutions can be used individually or in combination with each other. In one embodiment, mutain contains each of these substitutions. In another embodiment, mutain contains one, two, three, four, five, six, seven, or eight of these mutations.

[0044] In one embodiment, IL-2 mutaine contains an N88R or N88D mutation, preferably N88R. In another embodiment, IL-2 mutaine contains a C125A or C125S mutation. These substitutions may be used individually or in combination with each other. In another embodiment, mutaine contains one, two, three, four, five, six, seven, or eight of these mutations. In yet another embodiment, mutaine contains each of these substitutions.

[0045] In certain embodiments, the IL-2 moiety is aldesleukin. Aldesleukin is the active ingredient of Proleukin®. Aldesleukin is a variant of mature human IL-2, comprising two amino acid modifications compared to the mature human IL-2 sequence (SEQ ID NO: 2): a deletion of the first amino acid (alanine) and a serine substitution of cysteine ​​at position 125.

[0046] This includes conservative modifications of IL-2 and substitutions at other positions (i.e., those that have minimal impact on the secondary or tertiary structure of mutein). Such conservative substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978) and by Argos in EMBO J., 8: 779-785 (1989). For example, amino acids belonging to one of the following groups represent conserved changes: -ala, pro, gly, gln, asn, ser, thr; -cys, ser, tyr, thr; -val, ile, leu, met, ala, phe; -lys, arg, his; -phe, tyr, trp, his; and -asp, glu.

[0047] Variants with mutations that interfere with the binding of IL-2R to the α subunit are undesirable because they may reduce the ability of such mutants to stimulate Tregs.

[0048] • Active variants that promote Teff cell proliferation, survival, activation, and / or function may be useful in the treatment of cancer.

[0049] Each of these active IL-2 variants contains at least one amino acid mutation that, compared to the wild-type IL-2 polypeptide, eliminates or reduces the affinity of the heterologous IL-2 polypeptide to the α-subunit (CD25) of the IL-2 receptor, while retaining the affinity of the mutant IL-2 polypeptide to the intermediate affinity IL-2 receptor. This property can be monitored using surface plasmon resonance.

[0050] Preferred active variants include IL-2 muteins containing F42A, K43N, Y45A and / or E62A substitutions.

[0051] Active variants, such as human IL-2 (hIL-2) mutants with reduced affinity for CD25, can be produced by amino acid substitutions at amino acid positions 35, 38, 42, 43, 45, 62, or 72 or combinations thereof (numbering corresponds to the human IL-2 sequence of SEQ ID NO: 2). Examples of amino acid substitutions include K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, K43E, Y45A, Y45G, Y45S, This includes Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, E62G, E62A, E62S, E62T, E62Q, E62E, E62N, E62D, E62R, E62K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. Specific active variants useful for the chimeric constructs of the present invention involve amino acid mutations at amino acid positions or combinations thereof corresponding to residues 42, 45, or 72 of human IL-2. In one embodiment, the amino acid mutation is an amino acid substitution selected from the group F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K, more specifically an amino acid substitution selected from the group F42A, Y45A, and L72G. These active variants exhibit substantially similar binding affinity to the intermediate affinity IL-2 receptor compared to the wild-type IL-2 mutant, and substantially reduced affinity to the α-subunit of the IL-2 receptor and the high affinity IL-2 receptor (IL2Rαβγ).

[0052] Other characteristics of useful active variants may include the ability to induce proliferation of IL-2 receptor-borne T and / or NK cells, the ability to induce IL-2 signaling in IL-2 receptor-borne T and / or NK cells, the ability to produce interferon (IFN)-γ as a secondary cytokine by NK cells, reduced ability to induce anabolism of secondary cytokines—particularly IL-10 and TNF-α—by peripheral blood mononuclear cells (PBMCs), reduced ability to activate regulatory T cells, reduced ability to induce apoptosis in T cells, and reduced in vivo toxicity profile.

[0053] A particular active variant contains three amino acid mutations that cause the active variant to lose or reduce its affinity for the α-subunit of the IL-2 receptor, but retain its affinity for the intermediate-affinity IL-2 receptor. In one embodiment, the three amino acid mutations are at positions corresponding to residues 42, 45, and 72 of human IL-2. In another embodiment, the three amino acid mutations are amino acid substitutions. In one embodiment, the three amino acid mutations are amino acid substitutions selected from the group F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a particular embodiment, the three amino acid mutations are amino acid substitutions F42A, Y45A, and L72G (numbering corresponds to the human IL-2 sequence of SEQ ID NO: 2).

[0054] In one embodiment, the amino acid mutation reduces the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor by at least 5 times, more specifically at least 10 times, and more specifically at least 25 times. In embodiments where there is more than one amino acid mutation that reduces the affinity of the active variant to the α-subunit of the IL-2 receptor, the combination of amino acid mutations may reduce the affinity of the active variant to the α-subunit of the IL-2 receptor by at least 30 times, at least 50 times, or at least 100 times. In one embodiment, the amino acid mutation or combination of amino acid mutations causes the active variant to lose its affinity to the α-subunit of the IL-2 receptor, resulting in binding being undetectable by surface plasmon resonance.

[0055] Substantially similar binding to the intermediate affinity receptor, i.e., conservation of the affinity of the mutant IL-2 polypeptide to the receptor, is achieved when the active variant exhibits an affinity greater than approximately 70 percent of the affinity of the wild-type IL-2 mutant to the intermediate affinity IL-2 receptor. Active variants useful in the present invention may exhibit such affinities exceeding approximately 80 percent and even exceeding approximately 90 percent.

[0056] Removing the O-glucosylation of IL-2, combined with a reduction in IL-2's affinity for the α-subunit of the IL-2 receptor, results in an improved IL-2 protein.

[0057] In certain embodiments, the active variant can induce one or more cellular responses selected from the group consisting of: proliferation of activated T lymphocytes, differentiation of activated T lymphocytes, cytotoxic T cell (CTL) activity, proliferation of activated B cells, differentiation of activated B cells, proliferation of natural killer (NK) cells, cytotoxic activity of NK cells, differentiation of NK cells, cytokine secretion by activated T cells or NK cells, and NK / lymphocyte-activated killer (LAK) antitumor cytotoxicity.

[0058] In one embodiment, these active variants also include the 125-position substitutions described herein.

[0059] C4BPβ or C4BPβ fragment The C4BP protein is involved in coagulation and the complement system. The main form of C4BP consists of seven identical 75kD alpha chains and one 45kD beta chain. The alpha and beta chains contain eight and three SCR (short consensus repeat) domains, respectively. These motifs are found in many complement regulatory proteins and consist of 50-70 amino acids organized into a beta sheet. The amino acid sequence of the human C4BP beta chain is shown as Sequence ID No. 3.

[0060] The nucleic acid sequence corresponding to this polypeptide sequence was also described by Hillarp and Dahlback (1990, PNAS, vol 87, pp 1183-1187).

[0061] The role of the alpha chain in C4BP protein polymerization has been investigated by Kask et al (Biochemistry 2002, 41, 9349-9357). These authors show that the C-terminal portion of the alpha chain, particularly its helical structure and the presence of two cysteine ​​groups, is required for C4BP protein polymerization when the alpha chain is expressed in a heterologous system.

[0062] European Patent Application 2227030 describes the production of heteromultimeric recombinant proteins by using the C-terminal fragments of the alpha and beta chains of a C4BP protein fused with a polypeptide of interest.

[0063] US Patent 7,884,190 describes the use of the beta chain of the C4BP protein, conjugated to the alpha chain of the C4BP protein, for the production of a dimeric protein, regardless of its intended use.

[0064] The C4BP protein used in carrying out the present invention is preferably human C4BP protein.

[0065] In a preferred embodiment, the IL2 moiety is fused to a C4BPβ chain fragment containing or consisting of at least amino acids 194-252 (SEQ ID NO: 4).

[0066] Sequences encoding longer fragments of the beta chain or the entire beta chain may also be used. For a given application, it is preferable to avoid using sequences encoding a beta chain that can bind to protein S, which participates in coagulation. If the selected sequence encodes a fragment containing two initial SCR motifs of the beta chain, these are preferably mutated versions by amino acid addition, deletion, or substitution to eliminate the possibility of interaction with protein S. SCR motifs and / or [GS] domains may be added to modify, for example, increase the flexibility of the resulting fusion polypeptide or to allow the chimeric protein to adopt a suitable conformation for multimerization, particularly dimerization.

[0067] A long fragment of C4BPβ that extends to a maximum of 135 amino acids at its N-terminus can be used.

[0068] In a particular embodiment, the C4BPβ chain fragment may contain or consist of at least amino acids 185-252, 180-252, 175-252, 170-252, 165-252, 160-252, 155-252, 150-252, 145-252, 140-252, or 135-252 (corresponding to SEQ ID NO: 3).

[0069] In a particular embodiment, the C4BPβ chain fragment contains or consists of at least amino acids 137-252 (SEQ ID NO: 5).

[0070] Functional variants of C4BPβ may be used. These functional variants retain the ability to form at least one dimer, e.g., a homodimer or heterodimer, trimer, tetramer, or any multimer containing a variety of chimeric proteins.

[0071] Within the scope of the present invention, the term “functional variant of the C4BPβ chain fragment” means a polypeptide sequence modified with respect to the sequence of the β chain fragment by the deletion, substitution, or addition of one or more amino acids, in any case the modified sequence retains the ability to form at least a dimeric protein using the method of the present invention. More precisely, the production of a dimeric protein using the sequence encoding the functional variant of the fragment may be at least 80%, preferably at least 90%, and more preferably 95% equal to that obtained with the native sequence encoding the fragment (SEQ ID NO: 3 or its fragment) in the same expression system. Preferably, the variant is produced in dimeric form in a eukaryotic expression system by the present invention, with more than 80% of the fusion polypeptide it contains being produced.

[0072] In certain embodiments, the variant of the beta-chain fragment is encoded by a nucleic acid that can hybridize with the wild-type sequence encoding the fragment under stringent conditions, as described by Hillarp and Dahlback (1990, PNAS, Vol. 87, pp 1183-1187).

[0073] The term "stringent conditions" refers to conditions that allow for specific hybridization of two single-stranded DNA sequences after washing at approximately 65°C with, for example, a solution of 6*SSC, 0.5% SDS, 5*Denhardt's solution and 100 μg of unspecified DNA or any other solution with equivalent ionic strength, and at 65°C with, for example, a solution of up to 0.2*SSC and 0.1% SDS or any other solution with equivalent ionic strength.

[0074] Preferably, the nucleotide sequence encoding a functional variant of the wild-type fragment and hybridizing under stringent conditions with the sequence encoding the fragment has a length of at least 50%, preferably at least 80%, of the sequence encoding the fragment in the hybridization portion. In a particular execution, the nucleotide sequence encoding a functional variant of the fragment and hybridizing under stringent conditions with the sequence encoding the fragment has a length of substantially the same as the sequence encoding the fragment in the hybridization portion.

[0075] In further implementation, functional variants have a modified sequence in which one or more amino acids of the wild-type fragment not essential for dimerization are removed or substituted and / or one or more amino acids essential for dimerization are replaced with amino acids having equivalent functional groups (conservative substitution). It is particularly desirable that the two cysteines located at positions 201 and 215 and the peptide structures surrounding these cysteines are conserved, for example, by conserving at least three amino acids upstream and downstream of each cysteine, so as to be able to form the disulfide bridges necessary for dimerization. In particular, functional variants can also be obtained by inserting heterologous sequences of the beta chain and especially the alpha chain domain of C4BP between the cysteines responsible for dimerization, or, conversely, by removing certain amino acids present between the cysteines. Alternatively, functional variants can be produced by point modification of certain amino acids, particularly cysteine ​​responsible for dimerization, which is replaced with a neutral amino acid (e.g., amino acids A, V, F, P, M, I, L, and W), taking the dimerization process into consideration, while simultaneously replacing other amino acids with cysteine ​​to preserve the ability to form intracatenary and / or intercatenary disulfide bridges between cysteines. These modifications, therefore, result in variations in the distances between the various cysteines involved in the polymerization process, especially dimerization.

[0076] Preferably, less than 50%, preferably less than 25%, less than 10% (e.g., 5 amino acids or less), or less than 5% (e.g., 1 or 2 amino acids) of the 194-252 fragments are removed or substituted.

[0077] ¥ In a particular embodiment, the functional variant is a) A modified sequence of a fragment of C4BPβ (preferably fragments 194-252), wherein less than 25 percent, preferably less than 10 percent, of the amino acids of the fragment (preferably fragments 194-252) are removed or substituted, thereby preserving the cysteines located at positions 202 and 216 (numbered in accordance with SEQ ID NO: 3) and at least three amino acids upstream and downstream of each cysteine; or b) A modified sequence of a C4BPβ fragment (preferably fragment 194-252), wherein the cysteine ​​responsible for dimerization is preferably substituted with an amino acid selected from alanine, valine, phenylalanine, proline, methionine, isoleucine, leucine, and tryptophan, and the other amino acids of the fragment are substituted with cysteine; or c) A sequence of C4BPβ fragments (preferably fragments 194-252) modified by the insertion of a heterologous sequence into the beta chain between cysteine ​​molecules responsible for dimerization; or d) Sequence of a C4BPβ fragment (preferably fragment 194-252) modified by the removal of an amino acid between cysteine ​​groups responsible for dimerization. It includes or consists of.

[0078] Chimera structure Preferably, the IL2 portion is fused with the N-terminus of C4BPβ or a fragment thereof.

[0079] In a preferred embodiment, the chimeric construct comprises a fusion protein in which one IL2 moiety is fused at the N-terminus of C4BPβ or its fragment, and the other IL2 moiety is fused at the C-terminus of C4BPβ or its fragment. According to such an embodiment, the fusion protein comprises the following sequence from N-terminus to C-terminus: IL2-C4BPβ-IL2.

[0080] The IL2 portion and C4BPβ or its fragments may be fused within the frame (directly) or via an amino acid linker, preferably a poly-G linker.

[0081] The term "linker" refers to a (poly)peptide containing 5 to 80 amino acids, preferably 5 to 30, and more preferably 10 to 20 amino acids. Suitable linkers are known in the art. In one embodiment, the linker contains the GGGGS (SEQ ID NO: 8) repeat, but those skilled in the art will recognize that other sequences may also be used, following general recommendations (Argos, 1990, J Mol Biol. 20;211(4):943-58; George R, Heringa J. An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng. 2002;15:871-879). Linkers consisting of small, nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids provide flexibility and allow for the mobility of functional domain connections.

[0082] In certain embodiments, the chimeric construct includes or comprises SEQ ID NO: 9 or SEQ ID NO: 10. Such a chimeric construct can preferably form a homodimer or be used to produce a heterodimer, as described below.

[0083] Homodimer and heterodimer constructs a) Transfect host cells with a vector that enables the expression of a nucleotide sequence or at least one fragment or functional variant thereof that encodes a chimeric construct which is a fusion polypeptide comprising i) at least one interleukin-2 (IL2) moiety and ii) a beta chain of a C4b-binding protein (C4BPβ) capable of forming a dimeric protein; b) Transfected cells are cultured under conditions suitable for in vivo covalent bonding of two fusion polypeptides for the expression of the nucleotide sequence encoding the fusion polypeptide and the formation of a dimeric protein; c) The formed dimeric protein is recovered and preferably purified. Methods for producing recombinant dimeric proteins, including the method described herein, are provided.

[0084] The transfected cells preferably do not contain any nucleic acids that enable the expression of a nucleotide sequence encoding the C-terminal fragment of the alpha chain of the C4BP protein, which is involved in the polymerization of the C4BP protein.

[0085] In a particular embodiment, a method for producing a heterodimer: a. i. A first fusion polypeptide comprising i) at least one interleukin-2 (IL2) moiety and ii) a beta chain of a C4b-binding protein (C4BPβ) capable of forming a dimeric protein, or at least one fragment or functional variant thereof; and ii. A second fusion polypeptide comprising i) at least one heterologous polypeptide and ii) a beta chain of a C4b-binding protein (C4BPβ) capable of forming a dimeric protein, wherein the heterologous polypeptide is defined as being different from interleukin 2 (part) of the first fusion polypeptide, or at least one fragment or functional variant thereof. Transfect host cells with one or more vectors that enable the expression of one or more nucleotide sequences encoding; b. Transfected cells are cultured under conditions that allow for the in vivo binding of two fusion polypeptides for the expression of one or more nucleotide sequences encoding the first and second fusion polypeptides and for the formation of a heterodimeric protein; c. The formed heterodimer protein is recovered and preferably purified. Methods including this are described here.

[0086] Preferably, in the second fusion polypeptide, C4BPβ or the fragment is fused to the C-terminus of the heterologous polypeptide.

[0087] When referring to heterologous polypeptides, the term "different" means a polypeptide having a primary amino acid sequence that differs from the primary sequence of interleukin-2 (part) of the first fusion polypeptide by at least one amino acid. Alternatively, the term "different" may also include heterologous polypeptides that have the same primary sequence but differ in post-translational modifications, for example, in terms of acetylation, amidation, biotinylation, carboxylation, hydroxylation, methylation, phosphorylation, or sulfation, or by lipid (isoprenylation, palmitoylation, and myristoylation), carbohydrate (glucosylation), or polypeptide (ubiquitination) addition.

[0088] In a preferred embodiment, the heterologous polypeptide is not IL2.

[0089] In certain embodiments, the heterologous polypeptide may be selected from the group consisting of autoantigens, antibodies or antibody fragments that target such autoantigens, and receptors, for example, those containing the alpha chain or receptor ligand of IL2R. Such constructs are particularly useful for treating autoimmune and / or inflammatory disorders. In such embodiments, the IL-2 portion of the first fusion polypeptide is preferably an active variant that promotes Treg cell proliferation, survival, activation, and / or function.

[0090] In other specific embodiments, the heterologous polypeptide may be selected from the group consisting of tumor antigens, microbial antigens, antibodies or antibody fragments that target such antigens, or receptors that include the alpha chain or receptor ligand of IL2R. Such constructs are particularly useful for the treatment of cancer. In this case, the IL-2 portion of the first fusion polypeptide is preferably an active variant that promotes Teff cell proliferation, survival, activation, and / or function.

[0091] Such heterodimer proteins are also part of the present invention.

[0092] In a particular embodiment, the host cell enables co-expression of two fusion polypeptides: a first fusion polypeptide A comprising i) at least one interleukin-2 (IL2) moiety and ii) a beta chain of C4b-binding protein (C4BPβ) capable of forming a dimeric protein; and a second fusion polypeptide B comprising i) at least one heterologous polypeptide and ii) a beta chain of C4b-binding protein (C4BPβ) capable of forming a dimeric protein, where the heterologous polypeptide is defined as being different from the interleukin-2 (moiety) of the first fusion polypeptide; or at least one fragment or functional variant thereof. In this particular embodiment, co-expression of the two fusion polypeptides may also enable the production of homodimers AA and BB and heterodimer AB.

[0093] Recombinant eukaryotic cells are also provided that enable the synthesis of the dimeric or heterodimeric proteins defined above and are obtained by carrying out step a) of the production method defined above. More detailed host cell production is described below.

[0094] Production method Chimeric constructs, which are fusion proteins and can be homodimer or heterodimer, can be produced by DNA recombination techniques using a suitable expression vector.

[0095] The expression vector is selected depending on the host cell into which the construct is introduced. Preferably, the expression vector is selected from vectors that enable expression in eukaryotic cells, particularly chromosomal vectors or episomal vectors or viral derivatives, particularly plasmids, yeast chromosomes or viruses, such as baculovirus, parvovirus or SV40, retroviruses or combinations thereof, particularly phagemids and cosmids. In certain embodiments, a vector that enables baculovirus expression can infect insect cells.

[0096] If necessary, the sequence encoding the fusion polypeptide preferably also includes, at its 5' portion, a sequence encoding a signal peptide for the secretion of the fusion polypeptide. Conventionally, the signal peptide sequence is 15-20 amino acids and rich in hydrophobic amino acids (Phe, Leu, Ile, Met, and Val).

[0097] The vector contains all the sequences necessary for the expression of the sequence encoding the fusion polypeptide. In particular, it includes an appropriate promoter, which is selected depending on the host cell into which the construct is introduced.

[0098] Within the scope of the present invention, the term "host cell" means a cell that is heterogeneous to another cell and has been introduced into its genome by a transfection method, and is capable of expressing nucleic acid-transported genes.

[0099] Preferably, the host cell is a eukaryotic cell. Eukaryotic host cells are selected from yeast cells, particularly budding yeast cells, filamentous fungal cells, such as those of the genus Aspergillus, insect cells, such as Drosophila S2 cells or Sphodoptera sf9 cells, mammalian cells, and plant cells. Mammalian cells that may be specifically mentioned are mammalian cell lines such as CHO, COS, HeLa, C127, 3T3, HepG2, or L(TK-) cells. In a preferred execution, the host cell is selected from a eukaryotic cell line, preferably Sf9 insect cells. A method for preparing recombinant dimeric proteins in sf9 insect cells is described in US Patent 7,884,190.

[0100] Any transfection method known to those skilled in the art for producing cells expressing heterologous nucleic acids may be used in carrying out step a) of the method. Transfection methods are described, for example, in Sambrook et al, 2001, "Molecular Cloning: A Laboratory Manual", 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0101] Alternatively, chimeric constructs can be produced by chemical peptide synthesis. For example, proteins can be produced by the parallel synthesis of short peptides, which are then combined to produce the complete sequence of a protein with the correct disulfide crosslinks. The synthesis of IL-2 is described, for example, in Asahina et al., Angewandte Chemie International Edition, 2015, Vol.54, Issue 28, 8226-8230, which is disclosed herein by reference.

[0102] In another embodiment, the chimeric protein may be expressed in vivo after administering the nucleic acid encoding the chimeric protein to a subject. In a preferred embodiment, the nucleic acid is delivered by a viral vector such as adenovirus-associated virus (AAV).

[0103] Formulation and route of administration Pharmaceutical compositions comprising the constructs, nucleic acids, vectors, or proteins described herein, preferably in combination with a pharmaceutically acceptable medium, carrier, or additive (e.g., a solution, suspension, or mixture), are also provided.

[0104] Suitable additives include any isotonic solution, saline solution, buffered solution, sustained-release formulation, etc. Liquid, lyophilized, or spray-dried compositions are known in the art and can be prepared as aqueous or non-aqueous solutions or suspensions. Preferably, the pharmaceutical composition includes a suitable stabilizer, buffer, filler, or combination.

[0105] The pharmaceutical composition may further contain other active ingredients or may be administered in combination with some other active ingredients.

[0106] The pharmaceutical composition may be administered by any conventional route, including non-enteral routes such as intradermal, subcutaneous, or intranasal routes. Subcutaneous routes are preferred. Oral, sublingual, or buccal administration is also included.

[0107] Examples of formulations suitable for subcutaneous injection are described in international patent application WO2017 / 068031.

[0108] Treatment of autoimmune and / or inflammatory disorders The pharmaceutical compositions described herein are useful in methods for treating autoimmune and / or inflammatory disorders such as systemic lupus erythematosus, type 1 diabetes mellitus, HCV-associated vasculitis, uveitis, myositis, systemic vasculitis, psoriasis, allergies, asthma, Crohn's disease, multiple sclerosis, rheumatoid arthritis, atherosclerosis, autoimmune thyroid disease, autoinflammatory diseases, neurodegenerative diseases including Alzheimer's disease and amyotrophic lateral sclerosis, acute and chronic graft-versus-host disease, spontaneous abortion and allograft rejection; solid organ transplant rejection, vasculitis, inflammatory bowel disease (IBD) and allergic asthma; spondyloarthritis or ankylosing spondylitis; Sjögren's syndrome, systemic sclerosis, alopecia areata or ulcerative colitis.

[0109] In preferred embodiments, a method for treating autoimmune and / or inflammatory disorders with the composition once or twice weekly or once or twice monthly, preferably via a subcutaneous route, is described herein. In some embodiments, doses of less than 30 MIU / day, preferably less than 20 MIU / day, favorably less than 10 MIU / day, or 1 MIU / day to 8 MIU / day are preferred. In other specific embodiments, doses of 1 to 5 MIU / day, preferably 0.1 to 3.5 MIU / day, are used.

[0110] Generally speaking, doses that allow for a 1.5, 2, 3, 4, or 5-fold increase in the number of Tregs are preferred. The standard unit of IL-2 is the International Unit (IU), which, as determined by the World Health Organization (WHO), is the amount that produces a certain biological effect in a particular cell proliferation assay without technically fixing the weight. This is because i) the weight varies depending on the exact sequence of the molecule and its glucosylation profile, and ii) what matters is the activity, not the weight of the molecule.

[0111] The principle of International Units (IFR) is intended to provide a strict standard for comparison of all IL-2 molecules (regardless of origin or whether the sequence contains wild-type or active variant sequences).

[0112] In practice, the WHO provides ampoules of IL-2 molecules that serve as a target for determining the dosage of a certain IL-2 preparation (regardless of the origin or sequence of the IL-2) that is calibrated and determined by its potency. For example, to determine the dosage of a certain IL-2 preparation, the biological activity of the candidate IL-2 preparation is measured in a standard cell proliferation assay using an IL-2-dependent cell line such as CTLL-2 and compared to the biological activity of the standard. The cells proliferate in the presence of various doses of the standard. The dose-response effect of IL-2 is established by plotting the dose of IL-2 as IU on the X-axis and the proliferation (pr) index on the Y-axis. When we want to determine the activity of some IL-2 product of unknown activity, we use the product to proliferate IL-2-dependent cells and measure the proliferation. Then we plot the pr value on the Y-axis and draw a line parallel to the X-axis from that value. From the point where this line intersects the dose-response line, we then draw a line parallel to the Y-axis. The intersection with the X-axis provides the activity of the candidate IL-2 product in IU.

[0113] Any changes to the WHO standard ampoule will not affect international units or the determination of dosages for any IL-2 preparations.

[0114] The first standard (WHO International Standard Code 86 / 504, dated 1987) contained purified glycosylated IL-2 derived from Jurkat cells, with a potency of 100 IU / ampoule arbitrarily assigned. Due to a shortage of the stock solution of the first international standard (IS), the WHO had to replace it. The WHO provided other calibrated IL-2 ampoules, this time produced by saturating E. coli. The second standard ampoule contained 210 IU of biological activity per ampoule. A change in the standard ampoule does not imply a change in IU. Therefore, the determination of the dosage of the test IL-2 preparation remains unchanged whether the first standard ampoule, the second standard ampoule, or any subsequent standard ampoule is used as the target.

[0115] In one embodiment, for example, a chronic administration regimen is performed, including administration once every three days to once every three months. Such a series of administrations may be repeated as needed.

[0116] In another embodiment, IL-2 is administered every other day for 1 to 2 weeks in cycles that can be repeated after interruption of administration, lasting from 3 days to 3 months, preferably 1 to 4 weeks.

[0117] In other embodiments, the treatment may include a first course, also called an induction course, and a second course, which is a maintenance course.

[0118] In certain embodiments, the treatment comprises at least a first course of administering the pharmaceutical composition once daily for at least about two or three consecutive days, preferably three to seven consecutive days, and more preferably four to five consecutive days, and preferably a maintenance dose continuing for, for example, about six days or about one to four weeks.

[0119] The maintenance dose is generally administered for at least one month, preferably at least about three months, and more preferably at least about six months. In a preferred embodiment, the maintenance dose is administered for about three to twelve months, preferably at about six to twelve months.

[0120] In a preferred embodiment, the maintenance treatment consists of administering the pharmaceutical composition once or twice a week, or every one or two weeks, or once a month.

[0121] In a preferred embodiment, the maintenance treatment consists of administering interleukin-2 once or twice a week, every week or every two weeks, or once a month for a period of at least one month, preferably about three to twelve months.

[0122] Preferably, the maintenance dose may be substantially the same as, or lower than, the dose of the first course.

[0123] Cancer treatment The pharmaceutical compositions described herein are useful in methods for treating cancer. In some embodiments, the subject has locally advanced or metastatic cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is colon cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, breast cancer, kidney cancer, esophageal cancer, or prostate cancer.

[0124] In one embodiment, the dosage is less than 30 MIU / day, preferably less than 20 MIU / day, and more preferably less than 10 MIU / day or 3 to 5 MIU / day.

[0125] In other embodiments, IL2 is administered at a dose of 400,000 to 750,000 IU / kg or 550,000 to 750,000 IU / kg, preferably 600,000 to 700,000 IU / kg. The dose may be similar to, but is expected to be less than, the amount prescribed with PROLEUKIN®.

[0126] The composition may be administered at least once daily to at least once weekly, including once every other day. Those skilled in the art will recognize that certain factors, including disease severity, prior treatment, the subject's general health and / or age and other pre-existing conditions, may influence the dosage and timing required to effectively treat the subject. Furthermore, treatment of the subject may consist of a single treatment or a series of treatments.

[0127] The above protocols relating to autoimmune and / or inflammatory disorders may be applied in an equivalent or similar manner to their use in cancer treatment. Alternatively, in other examples, the composition may be administered every 8 hours for 5 days, followed by a rest period of 2 to 14 days, for example, 9 days, followed by administration every 8 hours for another 5 days. In one embodiment, administration is given three times, every 4 days.

[0128] Examples and figures illustrate the present invention without limiting its scope. [Examples]

[0129] Example 1: Production and characterization of IL-2 / C4BP fusion protein Lentiviral vectors were used to produce IL-2 fusion proteins. Specifically, human IL-2 (Hi2, SEQ ID NO: 1), human IL-2 fused to the C-terminal region of C4BPβ (Hi2cb, SEQ ID NO: 6), or the same molecule with a mutant IL-2 (N88R variant; Hi2mcb, SEQ ID NO: 7) were integrated into lentiviral plasmids under the spleen-focusing virus (SFFV) promoter. HEK 293T cells were transfected with lentiviral plasmids using polyethyleneimine (PEI) at 70% confluence and cultured in serum-free medium for 48 hours. The supernatant was then filtered, concentrated by ultracentrifugation, resuspended in appropriate buffer, and stored at -80°C. To obtain stable transfected cells, HEK 293T cells were infected with lentiviruses at various MOIs, and the transduction efficiency was evaluated by flow cytometry using the produced green fluorescent protein (GFP) as a selection marker. Cells with at least 50% transduction efficiency and 80% viability were cultured in complete medium for one week, and then GFP+ cells were sorted every other day to ensure that nearly 100% of the cells produced the IL-2 fusion protein. To perform in vitro evaluation of the functional design of the constructs (Western blotting, human whole blood STAT5 phosphorylation), stable cell lines were cultured in serum-free medium for 48 hours, the supernatant was collected, filtered, concentrated, purified by chromatography, and then used. Recombinant adeno-associated virus (AAV) was produced by transfection of HEK 293T cells with an AAV2 / 8 vector containing the same intended transgene and auxiliary plasmid (virus containing a serotype 2 genome encapsulated in a capsid from serotype 8 AAV). The AAV vector was extracted from the cell supernatant, purified by centrifugation, and the virus was purified using a cesium chloride gradient and dialyzed. Next, Hi2cb and Hi2mcb were characterized by Western blotting using primary anti-human IL-2 antibody or primary anti-human C4BPβ antibody. Under reducing conditions, Hi2cb and Hi2mcb were detected at a molecular weight of approximately 23 kDa, corresponding to the monomer. Under normal conditions, two bands were detected, with the primary signal at a molecular weight of approximately 46 kDa, corresponding to the dimer. A second, weaker signal was approximately 23 kDa, corresponding to the monomer.

[0130] Example 2: Treg selectivity of fusion protein in human whole blood pSTAT5 response Immunophenotype. Whole blood was collected from healthy adults with informed consent. The effects of Hi2, Hi2cb, or Hi2mcb on STAT5 phosphorylation (pSTAT5) were evaluated using flow cytometry in human CD4+ regulatory T cells (Treg; CD4+Foxp3+CD127lo / -), CD4+ conventional T cells (Tconv; CD4+Foxp3-), and CD8+ T cells. Tenfold diluted Hi2, Hi2cb, and Hi2mcb were mixed with 100 μl of whole blood for 15 minutes at 37°C, and then stained for pSTAT5. Figure 1 shows the dose-response of pSTAT5 induction in the target population. Hi2 can induce STAT5 phosphorylation in Treg, Tconv, and CD8 T cells. Both IL-2-C4BPβ proteins can induce STAT5 phosphorylation in Treg cells but not in Tconv and CD8 T cells, indicating that the fusion protein has the ability to selectively target Treg cells within a large therapeutic window.

[0131] Example 3: In vivo evaluation of IL-2-C4BPβ-encoded AAV on T cells and glomerular filtration Mice. Female C57BL / 6 (Jrj) mice, 6-8 weeks old, were given an AAV encoding Hi2, Hi2cb, or Hi2mcb. 11 The viral genome (vg) was injected via the intraperitoneal route. Immunophenotyping. Blood samples were collected weekly in heparinized tubes to avoid clot formation. After hemolysis, immunotherapeutic cells were stained and analyzed by flow cytometry to determine the percentages of Treg (CD4+CD25+Foxp3+), Teff (CD4+CD25+Foxp3-), CD8+Treg (CD8+CD25+Foxp3+), and CD8+Teff (CD8+CD25+Foxp3-). Dosage. Blood and urine samples were collected at various time points in response to IL-2 administration using a human IL-2 uncoated ELISA kit (ThermoFisher). Figure 2 shows the reaction rate curves of the increase factor in four different T cell compartments compared to the control. Regarding regulatory T cells, the three proteins induced large and comparable increases in Treg (Figure 2A) and CD8+ Treg (Figure 2C), with a slightly better increase with the fusion protein and a high plateau compared to Hi2. Hi2, on the other hand, showed large peak increases in Teff (3x, Figure 2B) and CD8+ Teff (10x, Figure 2D) and NK cell expansion (2x, Figure 2E) after one week, while Hi2cb blocked only a slight increase of about 1.5x for both CD4+ and CD8+ T cells, appearing to provide complete control over NK cell expansion. The mutant form of the fusion protein, Hi2mcb, showed complete control of the effector compartments of both CD4+ and CD8+ T cells, as well as control despite a transient increase in NK cells, demonstrating the ability of this mutation to support Treg selectivity. Figure 3 shows the kinetics of human IL-2 in plasma (A) and urine (B) over time. Significant differences are observed between the kinetics of Hi2 and IL-2-C4BPβ fusion protein in both plasma and urine. Hi2 has a peak with a sustained plateau of approximately 20 pg / mL after one week (Figure 3A). In contrast, the fusion protein has a late peak with a very high and sustained plateau of approximately 300 pg / mL after two weeks. In urine, human IL-2 is only detectable after hIL-2-encoding AAV, while human IL-2 is not detectable in urine after IL-2-C4BPβ-encoding AAV administration. These results highlight the ability of these fusion proteins not to be filtered by the kidney and suggest an extension of the half-life of these IL-2-C4BPβ fusion proteins in plasma.

[0132] Example 4: Toxicity of Hi2, Hi2cb, or Hi2mcb after high-dose administration of AAV Mice. Female C57BL / 6 (Jrj) mice, 6-8 weeks old, were given 10 AAVs encoding Hi2, Hi2cb, or Hi2mcb. 12 It was injected via the intraperitoneal route using VG. Immunophenotyping. Blood samples were collected weekly in heparinized tubes to avoid clot formation. After hemolysis, immunotherapeutic cells were stained and analyzed by flow cytometry to determine the percentages of Treg (CD4+CD25+Foxp3+), Teff (CD4+CD25+Foxp3-), CD8+Treg (CD8+CD25+Foxp3+), and CD8+Teff (CD8+CD25+Foxp3-). Figures 4A–4E show the rate curves of increase in four different T cell compartments compared to the control. The effects of the three constructs on effector and regulatory T cells were remarkably similar to those of the low doses. In short, Hi2mcb increases both CD4+ and CD8+ Tregs while simultaneously completely regulating both effector levels and NK cells. This regulatory T cell selectivity is highly likely to underpin the favorable safety profile. The dynamics after Hi2cb-encoded AAV show only partial control over the increase in CD4+ (3-fold increase at peak, Figure 4B) and CD8+ (9-fold increase at peak, Figure 4D) effector compartments. However, both Treg compartments increased after 2 weeks, reaching nearly a 4-fold increase in Tregs (Figure 4A) and a 7-fold increase in CD8+ Tregs (Figure 4C). Finally, all Hi2-treated mice died within 1 week, but there was a large increase in CD4+ and CD8+ Tregs (Figures 4A, C) and a dramatic expansion of Teff (25-fold increase, Figure 4B), CD8+ Teff (62-fold increase, Figure 4D), and NK cells (3-fold increase, Figure 4E), which likely causes a severe imbalance in immune homeostasis leading to rapid death. Figure 5 shows the Kaplan-Meier curves for mice after high-dose AAV administration. High-dose toxicity assessments of the three proteins show a remarkably favorable safety profile for the IL-2-C4BPβ fusion protein compared to classical Hi2 (Figure 5). In fact, all mice injected with Hi2 died within 7 days. A delay in death was observed with Hi2cb, with two out of three mice dying for the first time on day 17. Finally, all mice treated with Hi2mcb were still alive at 20 weeks after the start of the experiment, highlighting the complete safety of the fusion protein.

[0133] Example 5: Therapeutic efficacy of fusion proteins in an experimental autoimmune encephalomyelitis model Mice. Female C57BL / 6(Jrj) mice aged 6-8 weeks were given an AAV encoding Hi2, Hi2cb, or Hi2mcb 7 days before induction of an experimental autoimmune encephalomyelitis model (EAE). 11 The IL-2-based molecules were administered intraperitoneally via injection to ensure Treg enlargement at disease onset. Prophylactic treatment with all IL-2-based molecules delayed clinical onset (Figure 6A). However, 3–5 days after disease onset, standard IL-2-treated mice (black dots) showed similar clinical symptom dynamics compared to control mice (white dots, Figure 6C). This result was confirmed and observed across all analytical parameters, with significant weight loss (Figure 6B) and 100% of mice developing clinical symptoms (Figure 6A). Conversely, mice treated with both fusion proteins showed the same delayed onset but with control of clinical symptom severity (black squares and triangles, respectively), complete control of weight loss (Figure 6B), and 40% complete disease prevention, meaning no clinical symptoms at all (Figure 6A).

[0134] Example 6: Production and evaluation of fusion protein 6.1. Production of Fusion Proteins The purified fusion protein was obtained from stable cell line production. Specifically, HEK 293T cells were cultured, the supernatant was collected, and purified AKTA was obtained. TM The samples were purified by size exclusion chromatography using a specific system. Positive fractions were collected by ELISA and Western blotting. Finally, the collected samples were subjected to anion exchange chromatography.

[0135] 6.2. Immunophenotypic testing Female C57BL / 6 (Jrj) mice, 6-8 weeks old, were injected subcutaneously with Hi2, Hi2cb, or Hi2mcb (25000UI) daily for 5 days. Immunophenotyping. Blood samples were collected once daily in heparin tubes before injection to avoid clot formation. After hemolysis, immunocells were stained and analyzed by flow cytometry to determine the percentage of Tregs (CD4+CD25+Foxp3+) and the mean CD25 fluorescence intensity of Tregs. Figure 7A shows an experimental dosing schedule based on a single daily injection of 25,000 international units of protein for five consecutive days. Immunophenotyping was performed daily for five days immediately before injection. Figures 7B–7C show the reaction rate curves of T cell compartment and NK cell expansion ratios compared to controls. Regulatory T cell expansion was very similar for Hi2 and fusion proteins, with a 1.6-fold increase in CD25 MFI in Tregs.

[0136] 6.3. Dosage Blood and urine samples were collected at various time points in response to IL-2 administration using a human IL-2 uncoated ELISA kit (ThermoFisher). Six- to eight-week-old C57BL / 6(Jrj) female mice were administered a single dose of Hi2, Hi2cb, or Hi2mcb supernatant protein via subcutaneous route, and plasma concentrations of hIL-2 were determined at various time points. Major pharmacokinetic differences were observed between Hi2 and the fusion proteins (Figure 8). Indeed, the fusion proteins were able to remain in the plasma compartment longer and had reduced excretion compared to Hi2. These results highlight the fact that these fusion proteins have an extended half-life with reduced clearance. This disclosure relates, for example, to the following: [1] i) a chimeric construct comprising at least one interleukin-2 (IL2) moiety and ii) a beta chain of a C4b-binding protein (C4BPβ) or at least one fragment or functional variant thereof capable of forming a dimeric protein. [2] The chimeric construct of [1] comprising or consisting of a longer fragment of C4BPβ, in which the fragment of C4BPβ is extended at the N-terminus to amino acid residues 194–252 or up to 135 of C4BPβ. [3] a) A modified sequence of a C4BPβ fragment, wherein less than 25 percent, preferably less than 10 percent, of the amino acids of the fragment are removed or substituted, so that the cysteines located at positions 202 and 216, as well as at least three amino acids upstream and downstream of each cysteine, are preserved; or b) A modified sequence of the C4BPβ fragment, wherein the cysteine ​​responsible for dimerization is preferably replaced with an amino acid selected from alanine, valine, phenylalanine, proline, methionine, isoleucine, leucine, and tryptophan, and the other amino acids of the fragment are replaced with cysteine; or c) A C4BPβ fragment modified by the insertion of a heterologous sequence into the beta chain between the cysteine ​​molecules responsible for dimerization; or d) Sequence of C4BPβ fragments modified by removal of amino acids between cysteine ​​groups responsible for dimerization. A chimeric construct of [1] or [2] comprising a functional variant of C4BPβ, including the above. [4] A chimeric construct of any of [1] to [3], wherein the IL-2 portion is human IL-2 or a homologous variant thereof, where the variant has at least 85% amino acid identity with human wild-type IL-2, preferably an active analog of human IL-2 having at least 90% amino acid identity with human wild-type IL-2, wherein the IL-2 portion is preferably substituted at the N88 position of SEQ ID NO: 2, more preferably an IL2 mutein containing the substituted N88R. [5] A chimeric construct of any of the [1] to [4], wherein the IL2 portion and C4BPβ or a fragment thereof are fused within a frame or via an amino acid linker, preferably a poly-G linker. [6] A chimeric construct of any of [1] to [5], wherein the IL2 portion is fused with the N-terminus of C4BPβ or a fragment thereof, preferably the chimeric protein is the fusion protein, where one IL2 portion is fused with the N-terminus of C4BPβ or a fragment thereof, and the other IL2 portion is fused with the C-terminus of C4BPβ or a fragment thereof. [7] A homodimer protein comprising two fusion polypeptides, each consisting of a chimeric product of one of the above [1] to [6]. [8] A method for producing a recombinant dimeric protein as defined in [6] above: a) Transfect a host cell with a vector that enables the expression of a nucleotide sequence encoding a fusion polypeptide which is a chimeric construct as defined in any of [1] to [6] above; b) Transfected cells are cultured under conditions suitable for in vivo covalent bonding of two fusion polypeptides for the expression of the nucleotide sequence encoding the fusion polypeptide and the formation of a dimeric protein; c) Collect the formed dimeric protein. A method that includes doing so. [9] A heterodimer protein comprising two fusion polypeptides, wherein the first fusion polypeptide consists of a chimeric product of any of [1] to [6] above, and the second comprises i) at least one heterologous polypeptide and ii) a beta chain of a C4b-binding protein (C4BPβ) or at least one fragment or functional variant thereof capable of forming a dimer protein, wherein the heterologous polypeptide differs from the IL-2 portion of the first fusion polypeptide, preferably, wherein the heterologous polypeptide is an autoantigen or a tumor antigen.

[10] A method for producing a recombinant heterodimeric protein as defined in [9] above: a. i. A first fusion polypeptide which is a chimeric construct as defined in any of [1] to [6] above; and ii. A second fusion polypeptide comprising i) at least one heterologous polypeptide and ii) a beta chain of a C4b-binding protein (C4BPβ) capable of forming a dimeric protein, wherein the heterologous polypeptide differs from the interleukin 2 portion of the first fusion polypeptide, or at least one fragment or functional variant thereof. Transfect host cells with one or more vectors that enable the expression of one or more nucleotide sequences encoding; b. Transfected cells are cultured under conditions that allow for the in vivo binding of two fusion polypeptides for the expression of one or more nucleotide sequences encoding the first and second fusion polypeptides and for the formation of a heterodimeric protein; c. Recover the formed heterodimer protein. A method that includes doing so.

[11] A nucleic acid encoding one of the chimeric constructs described in [1] to [6] above.

[12] A vector comprising the nucleic acid described in

[11] above.

[13] A host cell comprising the nucleic acid of

[11] or the vector of

[12] .

[14] The homodimeric protein of [7] or the heterodimeric protein of [9] for use in treating inflammatory and / or autoimmune disorders in a subject.

[15] The homodimer protein of [7] or the heterodimer protein of [9] for use in treating cancer in a subject.

Claims

1. i) a chimeric construct comprising at least one interleukin-2 (IL-2) moiety and ii) a beta chain of a C4b-binding protein (C4BPβ) capable of forming a dimeric protein, or at least one fragment or functional variant thereof, The IL-2 portion is human IL-2 or a homologous variant thereof, wherein the variant has at least 90% amino acid identity with the human wild-type IL-2 of SEQ ID NO: 1 or SEQ ID NO:

2. The C4BPβ fragment comprises or consists of a longer fragment of C4BPβ that extends at the N-terminus to amino acid residues 194-252 of C4BPβ in SEQ ID NO: 3, or up to amino acid 135 of SEQ ID NO:

3. The functional variant comprises or consists of a modified sequence of the fragment in which less than 10% of the amino acids of the fragment are removed or substituted. Chimera structure.

2. a) A modified sequence of the C4BPβ fragment in which the cysteines located at positions 202 and 216 of SEQ ID NO: 3, and at least three amino acids upstream and downstream of each cysteine ​​are conserved; or b) A modified sequence of the C4BPβ fragment, wherein the cysteine ​​responsible for dimerization is substituted with another amino acid; or c) The sequence of the C4BPβ fragment modified by the insertion of a heterogeneous sequence into the beta chain between the cysteine ​​molecules responsible for dimerization; or d) Sequence of the C4BPβ fragment modified by removal of the amino acid between the cysteine ​​responsible for dimerization. A chimeric construct according to claim 1, comprising a functional variant of C4BPβ, including the above.

3. The chimeric construct of claim 2, comprising a functional variant of C4BPβ containing a modified sequence of the C4BPβ fragment, wherein the cysteine ​​responsible for dimerization is substituted with an amino acid selected from alanine, valine, phenylalanine, proline, methionine, isoleucine, leucine, and tryptophan, and the other amino acids of the fragment are substituted with cysteine.

4. A chimeric construct according to any one of claims 1 to 3, wherein the IL-2 portion is an IL-2 mutaine that includes a substitution at position N88 of sequence number 2.

5. The chimeric construct of claim 4, wherein the IL-2 mutaine contains the substitution N88R.

6. A chimeric construct according to any one of claims 1 to 5, wherein the IL-2 moiety and C4BPβ or a fragment thereof are fused within a frame or via an amino acid linker.

7. The chimeric construct according to claim 6, wherein the amino acid linker is a poly-G linker.

8. A chimeric construct according to any one of claims 1 to 7, wherein the IL-2 portion is fused with C4BPβ or the N-terminus of the fragment thereof.

9. A chimeric construct according to any one of claims 1 to 7, wherein the chimeric protein is a fusion protein, in which one IL-2 portion is fused at the N-terminus of C4BPβ or its fragment, and the other IL-2 portion is fused at the C-terminus of C4BPβ or its fragment.

10. A homodimer protein comprising two fusion polypeptides, each consisting of a chimeric construct according to any one of claims 1 to 9.

11. A method for producing a recombinant dimer protein as defined in claim 10: a) Transfecting a host cell with a vector that enables the expression of a nucleotide sequence encoding a fusion polypeptide which is a chimeric construct as defined in any of claims 1 to 9; b) The transfected cells are cultured under conditions suitable for in vivo covalent bonding of two fusion polypeptides for the expression of the nucleotide sequence encoding the fusion polypeptide and for the formation of a dimeric protein; c) Collect the formed dimeric protein. A method that includes doing so.

12. A heterodimer protein comprising two fusion polypeptides, wherein the first fusion polypeptide consists of a chimeric construct of any of claims 1 to 9, and the second comprises i) at least one heterologous polypeptide and ii) a beta chain of a C4b-binding protein (C4BPβ) or at least one fragment or functional variant thereof capable of forming a dimer protein, wherein the heterologous polypeptide differs from the IL-2 portion of the first fusion polypeptide. The C4BPβ fragment comprises or consists of a longer fragment of C4BPβ that extends at the N-terminus to amino acid residues 194-252 of C4BPβ in SEQ ID NO: 3, or up to amino acid 135 of SEQ ID NO:

3. The functional variant comprises or consists of a modified sequence of the fragment in which less than 10% of the amino acids of the fragment are removed or substituted. Heterodimeric protein.

13. The heterodimer protein of claim 12, wherein the heterologous polypeptide is an autoantigen or a tumor antigen.

14. A method for producing a recombinant heterodimer protein as defined in claim 12: a. i. A first fusion polypeptide which is a chimeric construct as defined in any of claims 1 to 9; and ii. A second fusion polypeptide comprising i) at least one heterologous polypeptide and ii) a beta chain of a C4b-binding protein (C4BPβ) or at least one fragment or functional variant of C4BPβ capable of forming a dimeric protein, wherein the heterologous polypeptide differs from the interleukin 2 portion of the first fusion polypeptide, the C4BPβ fragment comprises or consists of a longer fragment of C4BPβ that extends at the N-terminus to amino acid residues 194-252 of SEQ ID NO: 3 or up to amino acid 135 of SEQ ID NO: 3, and the functional variant comprises or consists of a modified sequence of the fragment in which less than 10% of the amino acids of the fragment are removed or substituted. Transfect a host cell with one or more vectors that enable the expression of one or more nucleotide sequences encoding; b. Transfected cells are cultured under conditions that allow for the in vivo binding of two fusion polypeptides for the expression of one or more nucleotide sequences encoding the first and second fusion polypeptides and for the formation of a heterodimeric protein; c. Collect the formed heterodimer protein. A method that includes doing so.

15. A nucleic acid encoding a chimeric construct according to any one of claims 1 to 9.

16. A vector comprising the nucleic acid of claim 15.

17. A host cell comprising the nucleic acid of claim 15 or the vector of claim 16.

18. A homodimer protein of claim 10 or a heterodimer protein of claim 12 for use in treating inflammatory and / or autoimmune disorders in a subject.

19. A homodimer protein of claim 10 or a heterodimer protein of claim 12 for use in treating cancer in a subject.

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