Chimeric proteins for modulating cytokine receptor activity

Abstract

The present invention is in the field of immunology and relates to proteins and polypeptides comprising an immunoglobulin single variable domain (ISVD) fused to a cytokine. In particular, the present invention provides a chimeric protein comprising an immunoglobulin single variable domain (ISVD) fused to a cytokine, wherein the internal fusion site of the ISVD is linked to the cytokine, wherein the internal fusion site is located in a loop or turn between two secondary structure elements. The fusion of the cytokine to the ISVD allows for the modulation of cytokine receptor activity and / or downstream signaling upon binding to a cytokine receptor or receptor subunit.

Description

Technical Field

[0001] This invention belongs to the field of immunology and relates to proteins and peptides comprising an immunoglobulin single variable domain (ISVD) fused to a cytokine, wherein the fusion is obtained by inserting the cytokine into a fusion site in the ISVD, the fusion site being located in a loop or turn of the ISVD, the fusion site being not a complementarity-determining region, and / or wherein the cytokine is a loop-arranged variant of a wild-type cytokine. The binding of the cytokine-ISVD chimeric protein to a cytokine receptor or receptor subunit allows for the modulation of cytokine receptor activity and / or (downstream) signaling. Background Technology

[0002] Cytokines are small signaling proteins and are known to play a crucial role in the body's response to inflammation and immune attacks. Pro-inflammatory cytokines alert the immune system to potential infection or danger. However, unregulated cytokine production can lead to an inflammatory state.

[0003] Cytokines have been widely used as therapies for cancer, infections, and other diseases. Interleukin-2 (IL-2) was the first cytokine to be found to have therapeutic benefits, discovered by Robert Gallo and Francis Ruscetti in 1976. The team demonstrated that this cytokine can significantly stimulate the growth of T cells and natural killer (NK) cells, which are essential for the human immune response (Morgan DA, Ruscetti FW, Gallo R., “Selective invitro growth of T lymphocytes from normal human bone marrows”, Science, 1976, 193(4257):1007-8). In 1988, the Rosenberg SA group published a preliminary report on the use of IL-2 to treat patients with metastatic melanoma (Rosenberg SA et al., “Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report [Tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma: A preliminary report]”, N Engl J Med. [New England Journal of Medicine], 1988, 319(25):1676-80). IL-2 became the first cancer immunotherapy approved by the US FDA and is still used in clinical settings to treat metastatic melanoma and renal cell carcinoma (…). https: / / ccr.cancer.gov / news / landmarks / article / cytokines-as-therapy Meanwhile, IFN-α has also been approved by the FDA as an anti-cancer therapy.

[0004] Cytokine receptors are cell surface glycoproteins that specifically bind to cytokines and transduce cytokine signals. Biological responses can vary between cytokine receptors and between cells, but typically involve gene expression, changes in the cell cycle, and the release of mediators such as the cytokines themselves. Cytokine receptors usually function as oligomeric complexes, which are typically composed of two to four potentially identical or different subunits. Once cytokines bind to their surface receptors, they induce receptor aggregation or oligomerization (e.g., heterodimerization or heterotrimerization), followed by receptor activation and intracellular signaling (downstream signal transduction) (Christopher J. et al., “The structural and functional basis of cytokine receptor activation: lessons from the common β subunit of the granulocyte-macrophage colony-stimulating factor, Interleukin-3 (IL-3), and IL-5 Receptors”, Blood, 1997; 89(5): 1471-1482).

[0005] Heterotrimer receptors, such as the IL-2 receptor (IL-2R), exist in three forms: IL-2Rα (or CD25), IL-2Rβ (or CD122), and IL-2Rγ (or CD132). The α-chain receptor binds to IL-2 with low affinity, while the β and γ chains together form a complex that binds to IL-2 with moderate affinity (forming a heterodimer), primarily on memory T cells and NK cells. Furthermore, all three receptor chains form a complex (heterotrimer), which binds to IL-2 with high affinity (approximately 10 Kd) on activated T cells and regulatory T cells. -11M) binds to IL-2. The intermediate and high affinity receptor forms are functional and cause cellular changes when IL-2 binds to them (Liao W, Lin JX and Leonard WJ, “IL-2 family cytokines: new insights into the complex roles of IL-2 as a broad regulator of T helper cell differentiation”, Curr Opin Immunol, 2011, 23(5):598-604).

[0006] An example of a heterodimer receptor is the IFN-α receptor (IFNAR). It is a nearly ubiquitous membrane receptor that binds to IFN-α. IFNAR contains two subunits, IFNAR1 and IFNAR2, which are coupled to the FERM domains of tyrosine protein kinases TYK2 and JAK1, respectively. The biological functions of type I IFNs (such as IFN-α) include different subtypes in different cells (vanBoxel-Dezaire et al., “Complex modulation of cell type-specific signalling in response to type I interferons”, Immunity, 2006, 25(3):361-72).

[0007] The IL-18 receptor (IL-18R) is another example of a heterodimeric receptor. It consists of two distinct but structurally related immunoglobulin-like domains, members of the IL-1 receptor family: IL-18Rα and IL-18Rβ. Secretory mature IL-18 interacts with IL-18Rα. This complex heterodimerizes with the signal transduction accessory protein IL-18Rβ, which promotes a conformational change in the receptor to allow for high-affinity binding of the ligand (Stylianou E., “Interleukins IL-1 and IL-18”, Encyclopedia of Respiratory Medicine, 2006, 350-354). It has been proposed that IL-18Rβ does not directly interact with IL-18, and that IL-18Rα alone is responsible for IL-18 binding. Given the difference in affinity for IL-18 binding between IL-18Rα and the IL-18Rα / β complex, IL-18Rα and the IL-18Rα / β complex may present different contact sites for IL-18. These differences may involve conformational changes, resulting in different orientations and different numbers of contact sites (Chengbin Wu et al., “IL-18 receptor β-induced changes in the presentation of IL-18 binding sites affect ligand binding and signal transduction”, J Immunol, 2003, 170 (11): 5571-5577).

[0008] In light of the above, modulating the receptor-binding functionality of cytokines, such as regulating the responses triggered by cytokines upon binding to receptors / receptor subunits, could be used to develop new cytokine-based therapies or to improve existing ones. For example, the ability to direct activation to a specific cell population (by, for example, the production of IL-2 cytokines with enhanced affinity for a particular receptor subunit) could be beneficial for the treatment of certain conditions, such as cancer.

[0009] Lopes et al. (“ALKS 4230: a novel engineered IL-2 fusion protein with an improved cellular selectivity profile for cancer immunotherapy”, Journal for ImmunoTherapy of Cancer, 2020, 8:e000673) described an engineered fusion protein containing a ring-shaped arrangement of IL-2 with an extracellular domain of IL-2Rα to selectively activate effector lymphocytes carrying intermediate-affinity IL-2R. According to the authors, the extracellular domain of IL-2Rα in the fusion protein spatially inhibits the interaction between the fusion protein's IL-2 and the endogenous IL-2Rα subunit. With the interaction site between IL-2 and the endogenous IL-2Rα subunit blocked, the IL-2 in the fusion protein will be able to retain its activity through memory CD8+. + The ability of constitutively expressed, moderate-affinity IL-2R (subunits β and γ) on T cells and NK cells to signal. Memory CD8 + T cells and NK cells have been shown to be essential for protective anticancer immune responses.

[0010] However, cytokines and methods for producing these cytokines are required, wherein the receptor / receptor subunit binding functionality of the cytokines can be universally regulated, for example, general regulation, and therefore not limited to specific cytokines and specific receptor subunits. Summary of the Invention

[0011] The present invention addresses the aforementioned problems and provides a chimeric protein comprising an immunoglobulin single variable domain (ISVD) fused to a cytokine, wherein an internal fusion site of the ISVD is linked to the cytokine, wherein the internal fusion site is located in a loop or turn between two secondary structural elements, and wherein the cytokine is preferably a cyclically arranged cytokine. Preferably, in the chimeric protein, the internal fusion site of the ISVD is linked to an internal fusion site of the cytokine, wherein in both the ISVD and the cytokine, the internal fusion site is located in a loop or turn between two secondary structural elements. Compared to N / C-terminal end-to-end fusions, the chimeric protein provided by the present invention is more rigid (i.e., it is a "macroantibody" type fusion, see, for example, WO 2019 / 086548). Therefore, compared to N / C-terminal end-to-end fusion proteins, the chimeric protein of the present invention is less susceptible to proteasome degradation or flexible movement. In addition, the ISVD present in the chimeric protein provided by the present invention can bind to its target, so the chimeric protein may thereby include an additional target-binding portion, which can be selected according to the specific characteristics (longer half-life, labeling, specific localization or any other functional requirements) desired by the chimeric protein / peptide containing the chimeric protein.

[0012] Furthermore, as described below, the chimeric protein of the present invention can be linked to other parts with different functions (see the polypeptide of the present invention).

[0013] Therefore, the chimeric protein provided by the present invention allows for targeted regulation of receptor / receptor subunit binding functionality of cytokines, and may also have the additional advantages described above.

[0014] In addition, the present invention provides a polypeptide comprising the chimeric protein of the present invention, optionally wherein the polypeptide further comprises one or more additional groups, residues, portions or binding units, preferably wherein the polypeptide further comprises one or more ISVDs.

[0015] As illustrated in the following examples, the chimeric proteins and / or polypeptides of the present invention are capable of modulating the activity of cytokines contained in the chimeric proteins and / or proteins of the present invention (or the downstream consequences of the cytokines contained in the chimeric proteins binding to at least one of their receptors or receptor subunits).

[0016] Further, a nucleic acid molecule encoding the chimeric protein or polypeptide of the present invention, a vector containing the nucleic acid molecule of the present invention, a host cell containing the chimeric protein and / or polypeptide of the present invention, or a nucleic acid molecule or vector encoding the chimeric protein of the present invention are provided.

[0017] The present invention further provides a method for generating the chimeric proteins and / or peptides of the present invention.

[0018] A further method is provided for modulating the signal transduction and / or affinity of a cytokine to at least one of its receptors or receptor subunits by fusing a cytokine and an ISVD, preferably wherein the cytokine and the ISVD are fused to produce the chimeric protein of the present invention.

[0019] The present invention further provides a fusion protein comprising a cytokine fused directly or via a linker to an ISVD, for regulating the binding affinity of the cytokine contained in the fusion protein to its receptor.

[0020] The present invention also provides the use of the chimeric proteins and / or polypeptides of the present invention in medicine, particularly in the treatment of cancer and / or inflammatory diseases. Attached Figure Description

[0021] The accompanying drawings are illustrative only and not restrictive. For illustrative purposes, the dimensions of some elements may be exaggerated and not drawn to scale in the drawings.

[0022] Figure 1 A circular variant of the scaffold protein inserted into the first β-turn of the β-chains A and B connecting ISVD. The engineering principle of antigen-binding chimeric proteins.

[0023] This protocol illustrates how an immunoglobulin single variable domain (ISVD) can be grafted onto a large scaffold protein via two peptide bonds or two short linkers that connect the antigen-binding domain to the scaffold. The scissors indicate which exposed turns must be cut in the ISVD and scaffold. The dashed lines indicate how the remaining portions of the ISVD and scaffold must be linked using peptide bonds or short linkers to construct the antigen-binding chimeric protein. The CDR, framework residues, and β-turn region of the ISVD are based on IMGT (Lefranc MP, "Immunoglobulin and T Cell Receptor Genes: IMGT( ® (Immunoglobulin and T-cell receptor genes: IMGT® and the birth and rise of immunoinformatics), Front Immunol, 2014, 5:22) defines this concept.

[0024] Figure 2 Constructed from a cyclically arranged variant of IL-2 inserted into the first β-turn of β-chains A and B connecting ISVD. The engineering principles of antigen-binding chimeric proteins.

[0025] This scheme illustrates how an ISVD can be grafted onto IL-2 via two peptide bonds with two short linkers, which connect the antigen-binding domain to the cyclically arranged IL-2. The scissors indicate which exposed turns are cut in the ISVD and scaffold. Dashed lines indicate how the remaining portions of the ISVD and scaffold are linked using peptide bonds or short peptide linkers to construct an antigen-binding chimeric protein. The CDR, framework residues, and β-turn region of the ISVD are defined according to IMGT.

[0026] Figure 3 Insertion site on IL-2.

[0027] This diagram illustrates the different sites where ISVD can be transplanted onto IL-2 via two peptide bonds. The start and end positions of the cyclically arranged IL-2 (see also...) Figure 4 The reference number for each construct of the corresponding IL-2 "macro antibody protein" is indicated in the figure.

[0028] Figure 4 Schematic diagram of different IL-2 macroantibody proteins.

[0029] This figure illustrates the design of different IL-2 (K35E,C125S) "macro antibody protein". The circularly arranged amino acid (AA) sequence of IL-2 (K35E,C125S) is given in "collier-de-perle" form, starting from amino acid at position 1 and ending at amino acid at position 133. A small GG linker (gray with white lettering) connects the C-terminus of IL-2 (K35E,C125S) to the N-terminal portion of IL-2. For this purpose, the first 3 amino acids of IL-2 (K35E,C125S) are deleted, depicted as strikethrough lines in the figure. Point mutations in K35E and C125S are circled with a light gray background in the sequence. On this IL-2 (K35E,C125S) sequence, the start (NH3) of each IL-2 (K35E,C125S)_ISVD207 "macro antibody protein" construct is indicated. + ) and end (COO) - In some cases, an additional glycine (G) is added between the GSG linker and IL-2 (K35E,C125S), resulting in a linker length of 4 amino acids between IL-2 (K35E,C125S) and ISVD207 instead of 3.

[0030] To fully demonstrate how ISVD can be grafted onto the cyclically arranged IL-2 (K35E,C125S), as an example, the insertion site of IL-2 (K35E,C125S) in construct SA17667 is magnified at the bottom of the figure. This figure shows the location where the cyclically arranged IL-2 (K35E,C125S) is interrupted (the amino acid between the scissors is missing in this construct) and how it fuses with ISVD207. Construct SA17667 begins with residues 1-12 of ISVD207, followed by a 4-amino acid linker (GSGG), which fuses with amino acid 62 of IL-2 (K35E,C125S) up to amino acid 133, which is linked to amino acid 4 of IL-2 (K35E,C125S) via a GG linker, ending at amino acid L59 and connecting to residues 16-126 of ISVD207 via a 4-amino acid linker (GGSG). The GSG linker between the cyclically arranged IL-2 (K35E,C125S) and ISVD207 is indicated by a dashed circle, as is the additional glycine (G). In construct SA17667, there is a 4-amino acid linker between the cyclically arranged IL-2 (K35E,C125S) and ISVD207 at any site.

[0031] Figure 5 A circular variant of IL-2 inserted into the first β-turn of the β-chains A and B, which are anti-GFP ISVD. A model of the α-sheet of a 29 kDa GFP-binding chimeric protein was constructed.

[0032] (A) A model of an antigen-binding chimeric protein prepared by fusing an anti-GFP ISVD (top) and a circular variant of human IL-2 (bottom) via two peptide bonds or linkers connecting the ISVD to a scaffold. (B) The circular gene encoding the circularly arranged IL-2 (K35E,C125S) (bottom) is inserted into the first β-turn of the anti-GFP ISVD (top, SEQ ID NO: 1), which links β-chain A to β-chain B (β-turn AB). (C) The amino acid sequence of the resulting IL-2(K35E,C125S)[GS75-N71G]_ISVD207 antigen-binding chimeric macroantibody protein (SEQ ID NO: 11). Sequences derived from IL-2 (K35E,C125S) are depicted in bold. Sequences derived from ISVD are underlined. The peptide linker connecting the ISVD to IL-2 is underlined in dashed lines. Peptides that link the N-terminus and C-terminus of IL-2 to produce cyclic variants are depicted in italics.

[0033] Figure 6 A circular variant of IL-2 inserted into the first β-turn of the β-chains A and B, which are anti-GFP ISVD. A model of the α-sheet of a 29 kDa GFP-binding chimeric protein was constructed.

[0034] (A) A model of an antigen-binding chimeric protein created by fusing two peptide bonds or linkers that link ISVD to a scaffold, consisting of an anti-GFP ISVD (top right) and a circular variant of human IL-2 (bottom left). (B) The circular gene encoding the circularly arranged IL-2 (K35E,C125S) (bottom) is inserted into the first β-turn of the anti-GFP ISVD (top, SEQ ID NO: 1), which links β-chain A to β-chain B (β-turn AB). (C) The amino acid sequence of the resulting IL-2(K35E,C125S)[GF42-M39G]_ISVD207 antigen-binding chimeric macroantibody protein (SEQ ID NO: 9). Sequences derived from IL-2 (K35E,C125S) are depicted in bold. Sequences derived from ISVD are underlined. The peptide linker connecting ISVD to IL-2 is underlined in dashed lines. Peptides that link the N-terminus and C-terminus of IL-2 to produce cyclic variants are depicted in italics.

[0035] Figure 7 A circular variant of IL-2 inserted into the first β-turn of the β-chains A and B, which are anti-GFP ISVD. A model of the α-sheet of a 29 kDa GFP-binding chimeric protein was constructed.

[0036] (A) A model of an antigen-binding chimeric protein created by fusing two peptide bonds or linkers connecting ISVD to a scaffold, consisting of an anti-GFP ISVD (top left) and a circular variant of human interleukin-2 (IL-2, bottom right). (B) The circular gene encoding the circularly arranged IL-2 (K35E,C125S) (bottom) is inserted into the first β-turn of the anti-GFP ISVD (top, SEQ ID NO: 1), which links β-chain A to β-chain B (β-turn AB). (C) The amino acid sequence of the resulting IL-2(K35E,C125S)[GL85-P82G]_ISVD207 antigen-binding chimeric macroantibody protein (SEQ ID NO: 14). Sequences derived from IL-2(K35E,C125S) are depicted in bold. Sequences derived from ISVD are underlined. The peptide linker connecting ISVD to IL-2 is underlined in dashed lines. Peptides that link the N-terminus and C-terminus of IL-2 to produce cyclic variants are depicted in italics.

[0037] Figure 8 A circular variant of IL-2 inserted into the first β-turn of the β-chains A and B, which are anti-GFP ISVD. A model of the α-sheet of a 29 kDa GFP-binding chimeric protein was constructed.

[0038] (A) A model of an antigen-binding chimeric protein created by fusing an anti-GFP ISVD (bottom) and a circular variant of human IL-2 (top) via two peptide bonds or linkers connecting the ISVD to a scaffold. (B) The circular gene encoding the circularly arranged IL-2 (K35E,C125S) (bottom) is inserted into the first β-turn of the anti-GFP ISVD (top, SEQ ID NO: 1), which links β-chain A to β-chain B (β-turn AB). (C) The amino acid sequence of the resulting IL-2(K35E,C125S)[L132-I129]_ISVD207 antigen-binding chimeric macroantibody protein (SEQ ID NO: 18). Sequences derived from IL-2(K35E,C125S) are depicted in bold. Sequences derived from ISVD are underlined. The peptide linker connecting the ISVD to IL-2 is underlined in dashed lines. Peptides that link the N-terminus and C-terminus of IL-2 to produce cyclic variants are depicted in italics.

[0039] Figure 9 Different IL-2 (K35E, C125S)_ISVD207 chimeric macroantibody proteins were analyzed by yeast display and FACS. An overview of the characteristics of white.

[0040] The display levels of macroantibody proteins on the surface of yeast cells were analyzed according to Uchanski et al. (2021). To demonstrate the functionality of ISVD207 in chimeric proteins, cells were screened by GFP staining. Clones with a signal higher than 0.5% were retained for further analysis. The presence of IL-2 in these macroantibody proteins was confirmed by staining yeast cells with either fluorescent mAbNARA1 or fluorescent mAb5111, respectively. To analyze whether the displayed macroantibody proteins still bind to different IL-2 receptor moieties, cells were pre-incubated with either the fluorescent soluble domain CD25 or CD122 / CD132 heterodimer, respectively. For each binding assay, a dot plot of relative fluorescence intensity was created for each EBY100 yeast cell transformed with a pCTCON2 derivative encoding a different IL-2 (K35E, C125S)_ISVD207 macroantibody protein. Gating was performed using negative and positive controls. The percentage of each EBY100 clone falling into the gate for each clone in different binding assays is given. Constructs with higher relative fluorescence intensity than the negative control were marked with an asterisk.

[0041] Figure 10 The phosphorylation of STAT5 in different immune cell subtypes was shown when treated with compounds containing IL-2.

[0042] CD8+CD25- cells were evaluated in A and B, and CD4+CD25+ cells were evaluated in C and D. Treatments were performed in the absence of (A and C) and in the presence of human serum albumin (HSA) (B and D).

[0043] Figure 11 The study showed the proliferation of different immune cell subtypes when treated with compounds containing IL-2.

[0044] CD8+CD25- cells were evaluated in A and B, and CD4+CD25+ cells were evaluated in C and D. Treatments were performed in the absence of HSA (A and C) and in the presence of HSA (B and D).

[0045] Figure 12 Structure of the quaternary complex of IL-2 receptor α, β and γ with IL-2(K35E,C125S)[GS75-N71G]_ISVD207 macroantibody protein in an α-sheet model.

[0046] Figure 13 Structure of the quaternary complex of IL-2 receptor α, β and γ with IL-2(K35E,C125S)[GF42-M39G]_ISVD207 macroantibody protein in an α-sheet model.

[0047] Figure 14 Structure of the quaternary complex of IL-2 receptor α, β and γ with IL-2(K35E,C125S)[L132-I129]_ISVD207 macroantibody protein (SA17678) in an α-sheet model.

[0048] Figure 15 Structure of the quaternary complex of IL-2 receptor α, β and γ with IL-2(K35E,C125S)[GL85-P82G]_ISVD207 macroantibody protein (SA17659) in an α-sheet model.

[0049] Figure 16 The production of IFNγ in a tetanus toxoid recall assay is shown, which inquires the functionality of anti-PD-L1-IL-2 compounds in donors D1688 (A and B) and donors ABL-0341-02 (C and D).

[0050] Figure 17 The structure of IFNA2a. This figure shows the sites on the IFNA2a (PDB 1ITF) structure where IFNA2a will be opened (between positions 76 and 77) to create new N-terminals and C-terminals, and the N-terminals and C-terminals will be linked together by peptide linkers to form a cyclic variant of IFNA2a.

[0051] Figure 18 Flow cytometry analysis of expression levels of different circularly arranged variants of IFNA2a compared to wild-type IFNA2a. Technical analysis showed that each construct was displayed on the surface of EBY100 yeast cells; anti-human IFNA2a monoclonal antibody and the same Flow cytometry analysis of the binding of the constructs, each of which is displayed on the surface of EBY100 yeast cells.

[0052] Top: A dot plot representing the relative fluorescence intensity of individual EBY100 yeast cells from untransformed cells compared to cells transformed with pCTCON2 derivatives encoding IFNA2a (SEQ ID NO: 56), IFNA2a[D77-W76]V2 (SEQ ID NO: 58), or IFNA2a[D77-W76]V4 (SEQ ID NO: 59), each of these constructs fused with the flexible linkers Aga2p, ACP (SEQ ID NO: 32), and c-myc (SEQ ID NO: 33). Transformed and untransformed yeast cells were incubated with an anti-c-myc monoclonal antibody and their display levels were analyzed by staining with phycoerythrin-conjugated anti-mouse IgG-Fc.

[0053] The figure below shows a dot plot of relative fluorescence intensity of individual EBY100 yeast cells in untransformed cells compared to cells transformed with pCTCON2 derivatives encoding IFNA2a (SEQ ID NO: 56), IFNA2a[D77-W76]V2 (SEQ ID NO: 58), or IFNA2a[D77-W76]V4 (SEQ ID NO: 59), each of which is fused with the flexible linkers Aga2p, ACP (SEQ ID NO: 32), and c-myc (SEQ ID NO: 33). Transformed and untransformed yeast cells were incubated with an anti-human IFNA2a monoclonal antibody (mAb93452) and the presence of IFNA2a was analyzed by staining with phycoerythrin-conjugated anti-mouse IgG-Fc.

[0054] Figure 19 Flow cytometry analysis of expression levels of different circularly arranged variants of IFNA2a compared to wild-type IFNA2a. Technical analysis showed that each construct was displayed on the surface of EBY100 yeast cells; the binding of IFNAR2 to the same construct... Flow cytometry analysis showed that each construct was displayed on the surface of EBY100 yeast cells.

[0055] Top: A dot plot representing the relative fluorescence intensity of individual EBY100 yeast cells from untransformed cells compared to cells transformed with pCTCON2 derivatives encoding IFNA2a (SEQ ID NO: 56), IFNA2a[D77-W76]V2 (SEQ ID NO: 58), or IFNA2a[D77-W76]V4 (SEQ ID NO: 59), each of these constructs fused with the flexible linkers Aga2p, ACP (SEQ ID NO: 32), and c-myc (SEQ ID NO: 33). Transformed and untransformed yeast cells were incubated with an anti-c-myc monoclonal antibody and their display levels were analyzed by staining with phycoerythrin-conjugated anti-mouse IgG-Fc.

[0056] The figure below is a dot plot representing the relative fluorescence intensity of individual EBY100 yeast cells in untransformed cells compared to cells transformed with pCTCON2 derivatives encoding IFNA2a (SEQ ID NO: 56), IFNA2a[D77-W76]V2 (SEQ ID NO: 58), or IFNA2a[D77-W76]V4 (SEQ ID NO: 59); each of these constructs is fused with Aga2p, ACP (SEQ ID NO: 32), and c-myc (SEQ ID NO: 33). Transformed and untransformed yeast cells were incubated with IFNAR2 (human IFN-α / β R2 protein, His tag) and their binding affinity was analyzed by staining with a phycoerythrin-conjugated anti-His antibody.

[0057] Figure 20 Interferon α-2a (IFNA2a) is inserted into the first β-turn of the β-chains A and B that link anti-HSA ISVD. An α-sheet model of a 31 kDa HSA-binding chimeric protein constructed from a circularly arranged variant.

[0058] (A) A model of an antigen-binding chimeric protein prepared by fusing an anti-human serum albumin (HSA) ISVD (bottom) and a circular variant of human IFNA2a (top) via two peptide bonds or linkers connecting the ISVD to a scaffold. (B) A circular gene encoding circular interferon α-2a (bottom) is inserted into the first β-turn of the anti-HSA ISVD (top, SEQ ID NO: 55), which links β-chain A to β-chain B (β-turn AB). (C) The amino acid sequence of the resulting IFNA2a[L9-T6]_ALB23002 antigen-binding chimeric protein (SEQ ID NO: 60). Sequences derived from IFNA2a are depicted in bold. Sequences derived from ISVD are underlined. Peptide linkers connecting ISVD to IFNA2a are underlined and dashed. Peptides connecting the N-terminus and C-terminus of IFNA2a to produce circular variants are depicted in italics.

[0059] Figure 21 Interferon α-2a (IFNA2a) is inserted into the first β-turn of the β-chains A and B that link anti-HSA ISVD. An α-sheet model of a 31 kDa HSA-binding chimeric protein constructed from a circularly arranged variant. .

[0060] (A) A model of an antigen-binding chimeric protein created by fusing two peptide bonds or linkers that link ISVD to a scaffold, consisting of an anti-HSA ISVD (left) and a circular variant of IFNA2a (right). (B) A circular gene encoding circular interferon α-2a (bottom) is inserted into the first β-turn of the anti-HSA ISVD (top, SEQ ID NO: 55), which links β-chain A to β-chain B (β-turn AB). (C) The amino acid sequence of the resulting IFNA2a[S25-K23]_ALB23002 antigen-binding chimeric protein (SEQ ID NO: 61). Sequences derived from IFNA2a are depicted in bold. Sequences derived from ISVD are underlined. The peptide linker connecting ISVD to IFNA2a is underlined and dashed. The peptide connecting the N-terminus and C-terminus of IFNA2a to produce the circular variant is depicted in italics.

[0061] Figure 22 The cyclic arrangement of interferon α-2a inserted into the first β-turn of the β-chains A and B that connect to anti-HSA ISVD A model of the α-sheet of a 31 kDa HSA-binding chimeric protein constructed from variants.

[0062] (A) A model of an antigen-binding chimeric protein created by fusing two peptide bonds or linkers of ISVD to a scaffold, consisting of a circular variant of anti-HSA ISVD (left) and human interferon α-2a (IFNA2a) (right). (B) A circular gene encoding circularly arranged interferon α-2a (bottom) is inserted into the first β-turn of anti-HSA ISVD (top, SEQ ID NO: 55), which links β-chain A to β-chain B (β-turn AB). (C) The amino acid sequence of the resulting Mb_IFNA2a[D32-L30]_ALB23002 antigen-binding chimeric protein (SEQ ID NO: 62). Sequences derived from IFNA2a are depicted in bold. Sequences derived from ISVD are underlined. The peptide linker connecting ISVD to IFNA2a is underlined in dashed lines. Peptides that link the N-terminus and C-terminus of IFNA2a to produce cyclic variants are depicted in italics.

[0063] Figure 23 The cyclic arrangement of interferon α-2a inserted into the first β-turn of the β-chains A and B that connect to anti-HSA ISVD A model of the α-sheet of a 31 kDa HSA-binding chimeric protein constructed from variants.

[0064] (A) A model of an antigen-binding chimeric protein created by fusing two peptide bonds or linkers of ISVD to a scaffold via a circular variant of anti-HSA ISVD (right) and human interferon α-2a (left). (B) A circular gene encoding circular interferon α-2a (bottom) is inserted into the first β-turn of anti-HSA ISVD (top, SEQ ID NO: 55), which links β-chain A to β-chain B (β-turn AB). (C) The amino acid sequence of the resulting Mb_IFNA2a[P109-T106]_ALB23002 antigen-binding chimeric protein (SEQ ID NO: 63). Sequences derived from IFNA2a are depicted in bold. Sequences derived from ISVD are underlined. Peptide linkers connecting ISVD to IFNA2a are underlined. Peptides connecting the N-terminus and C-terminus of IFNA2a to produce circular variants are depicted in italics.

[0065] Figure 24 The structure of the human ternary complex IFNA2a-IFNAR compared with the α-sheet model of the IFNA2a[L9-T6]_ALB23002 protein.

[0066] Figure 25 The structure of the human ternary complex IFNA2a-IFNAR compared with the α-sheet model of the IFNA2a[P109-T6]_ALB23002 protein.

[0067] Figure 26 The structure of the human ternary complex IFNA2a-IFNAR compared with the α-sheet model of the IFNA2a[S25-K23]_ALB23002 protein.

[0068] Figure 27 The structure of the human ternary complex IFNA2a-IFNAR compared with the α-sheet model of the IFNA2a[D32-L30]_ALB23002 protein.

[0069] Figure 28 The phosphorylation of STAT1 in A549 cells was shown when treated with a compound containing IFNA2a.

[0070] Processing is performed in the absence of (A) and in the presence of HSA (B).

[0071] Figure 29 The results of treatment with compounds containing IFNA2a are shown for RPMI 8226 (A and B) and NCI-H929 (C and D). Cell proliferation .

[0072] Processing is performed in cases where (A and C) are absent and (B and D) are present.

[0073] Figure 30 Design of IL-18 ring-shaped variant.This figure shows the location of sites on the IL-18 (PDB 3F62) structure where IL18 is opened (between positions 69 and 70) to create new N-terminals and C-terminals, and when the circumferential arrangement variant of IL18 is designed, the N-terminals and C-terminals are joined together.

[0074] Figure 31 Flow cytometry analysis of expression levels of different circularly arranged variants of IL18 compared to wild-type IL18. Analysis showed that each construct was displayed on the surface of an EBY100 yeast cell.

[0075] Dot plots representing the relative fluorescence intensity of individual EBY100 yeast cells in untransformed cells compared to cells transformed with pCTCON2 derivatives encoding IL-18 (SEQ ID NO: 64), IL-18[K70-E69]V1b (SEQ ID NO: 66), IL-18[K70-E69]V5b (SEQ ID NO: 68), or IL-18[K70-E69]V7 (SEQ ID NO: 70), each of these constructs fused with the flexible linkers Aga2p, ACP (SEQ ID NO: 32), and c-myc (SEQ ID NO: 33). Transformed and untransformed yeast cells were incubated with anti-c-myc mAb and their display levels were analyzed by staining with phycoerythrin-conjugated anti-mouse IgG-Fc.

[0076] Figure 32 A schematic diagram of gene fusion used to obtain the chimeric protein of the present invention.

[0077] Figure 33 : mAb D044-3 is a circular variant that binds to IL18. Similar to the variants shown on the cell surface of yeast cells. Flow cytometry analysis of mAbD044-3 binding to IL18 and its circular variant. As described by Uchanski et al. (2021), the display levels of IL18 and its circular variants on the surface of yeast cells were analyzed. Correct folding of IL18 or its circular variants was confirmed by staining yeast cells with fluorescent mAbD044-3. A dot plot representing the relative fluorescence intensity of each EBY100 yeast cell transformed with a pCTCON2 derivative encoding a different IL18 variant is shown. Gating was performed using negative and positive controls.

[0078] Figure 34 IL18BP binds to some circular variants of IL18. IL18-BP binds to certain variants of IL18 on the cell surface of yeast cells. Flow cytometry analysis of IL18 and its circular variants is shown.

[0079] As described by Uchanski et al. (2021), the display levels of IL18 and its circular variants on the surface of yeast cells were analyzed. To analyze whether the displayed IL18 or IL18 circular variants still bound to IL18-BP, cells were pre-incubated with fluorescent IL18-BP. Dot plots representing the relative fluorescence intensities of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding different IL18 variants are shown. Gating was performed using negative and positive controls.

[0080] Figure 35 : A circularly arranged variant of IL18 inserted into the first β-turn of the β-chains A and B, which are anti-GFP ISVD. An α-sheet model of 33 kD GFP binding chimeric protein was constructed. (A) A model of an antigen-binding chimeric protein created by fusing an anti-GFP ISVD (top) and a circular variant of human IL18 (bottom) via two peptide bonds or linkers connecting the ISVD to a scaffold. (B) The circular gene encoding the circularly arranged IL18 (bottom) is inserted into the first β-turn of the anti-GFP ISVD (top, SEQ ID NO: 1), which links β-chain A to β-chain B (β-turn AB). (C) The amino acid sequence of the resulting IL18_ISVD207 antigen-binding chimeric macroantibody protein (IL18[Y1-D157]_ISVD207_V1 macroantibody protein, SEQ ID NO: 230). Sequences derived from IL18 are depicted in bold. Sequences derived from ISVD are underlined. The peptide linker connecting ISVD to IL18 is underlined in dashed lines. The peptide connecting the N-terminus and C-terminus of IL18 to produce the circular variant is depicted in italics.

[0081] Figure 36 : A circularly arranged variant of IL18 inserted into the first β-turn of the β-chains A and B, which are anti-GFP ISVD. An α-sheet model of 33 kD GFP binding chimeric protein was constructed. (A) A model of an antigen-binding chimeric protein created by fusing an anti-GFP ISVD (top) and a circular variant of human IL18 (bottom) via two peptide bonds or linkers connecting the ISVD to a scaffold. (B) The circular gene encoding the circularly arranged IL18 (bottom) is inserted into the first β-turn of the anti-GFP ISVD (top, SEQ ID NO: 1), which links β-chain A to β-chain B (β-turn AB). (C) The amino acid sequence of the resulting IL18_ISVD207 antigen-binding chimeric macroantibody protein (IL18[K70-E69]_ISVD207_V2 macroantibody protein, SEQ ID NO: 233). Sequences derived from IL18 are depicted in bold. Sequences derived from ISVD are underlined. The peptide linker connecting ISVD to IL18 is underlined in dashed lines. The peptide connecting the N-terminus and C-terminus of IL18 to produce the circular variant is depicted in italics.

[0082] Figure 37 : A circularly arranged variant of IL18 inserted into the first β-turn of the β-chains A and B, which are anti-GFP ISVD. An α-sheet model of 33 kD GFP binding chimeric protein was constructed.(A) A model of an antigen-binding chimeric protein created by fusing an anti-GFP ISVD (top) and a circular variant of human IL18 (bottom) via two peptide bonds or linkers connecting the ISVD to a scaffold. (B) The circular gene encoding the circularly arranged IL18 (bottom) is inserted into the first β-turn of the anti-GFP ISVD (top, SEQ ID NO: 1), which links β-chain A to β-chain B (β-turn AB). (C) The amino acid sequence of the resulting IL18_ISVD207 antigen-binding chimeric macroantibody protein (IL18[P57-Q56]_ISVD207_V1 macroantibody protein, SEQ ID NO: 237). Sequences derived from IL18 are depicted in bold. Sequences derived from ISVD are underlined. The peptide linker connecting ISVD to IL18 is underlined and dashed. The peptide connecting the N-terminus and C-terminus of IL18 to produce the circular variant is depicted in italics.

[0083] Figure 38 mAb D044-3 binds to the IL18-ISVD macroantibody protein. This is similar to the protein displayed on the cell surface of yeast cells. Flow cytometry analysis of mAbD044-3 bound to IL18_ISVD207 macroantibody protein As described by Uchanski et al. (2021), the display levels of different proteins on the surface of yeast cells were analyzed. To demonstrate the binding of mAb D044-3 to the displayed IL18_ISVD207 macroantibody protein, cells were pre-incubated with fluorescent mAb D044-3. For each binding assay, dot plots of relative fluorescence intensity were created for each EBY100 yeast cell transformed with either a pCTCON2 derivative encoding a control protein or a different IL18_ISVD207 macroantibody protein. Gating was set up using negative and positive controls.

[0084] Figure 39 : IL18BP binds to the IL18-ISVD macroantibody protein. IL18-BP spreads on the cell surface of yeast cells. Flow cytometry analysis of IL18_ISVD207 macroantibody protein binding. As described by Uchanski et al. (2021), the display levels of different proteins on the surface of yeast cells were analyzed. To demonstrate the binding of IL18-BP to the displayed IL18_ISVD207 macroantibody protein, cells were pre-incubated with fluorescent IL18-BP. Changes in the binding of IL18-BP to different IL18_ISVD207 macroantibody proteins were observed. For each binding assay, dot plots representing the relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding either the control protein or different IL18_ISVD207 macroantibody proteins were created. Gating was performed using negative and positive controls.

[0085] Figure 40 GFP binds to the IL18-ISVD macroantibody protein. This binds to IL18_ displayed on the cell surface of yeast cells. Flow cytometry analysis of ISVD207 macroantibody protein-bound GFPAs described by Uchanski et al. (2021), the display levels of different proteins on the surface of yeast cells were analyzed. To demonstrate the functionality of ISVD207 in the chimeric protein, cells were stained with GFP. For each binding assay, dot plots of relative fluorescence intensity were created for each EBY100 yeast cell transformed with either a pCTCON2 derivative encoding a control protein or a different IL18_ISVD207 macroantibody protein. Gating was set up using negative and positive controls.

[0086] Figure 41 Experimental setup for harvesting and displaying proteins from yeast cell walls. The IL18[K70-E69]V5b and IL18_ISVD207 macroantibody proteins, fused with acyl carrier proteins and numerous accessory peptides, displayed on the surface of yeast cells can be biotin-labeled with biotin-PEG3-coenzyme A and SFP synthase and released from the cell wall by adding DTT.

[0087] Figure 42 Biological layer interferometry (BLI) analysis of GFP binding to IL18_ISVD207 macroantibody protein To analyze whether the IL18_ISVD207 macroantibody protein released from the cell wall and captured on the SA biosensor binds to GFP, the loaded SA sensor was incubated with GFP. BLI sensing maps of each construct were recorded and compared with IL18[K70-E69]V5b, which served as a negative control.

[0088] Figure 43 Biological layer interferometry of GFP bound to IL18_ISVD207 IL18[K70-E69]_ISVD207_V1 (BLI) analysis To demonstrate the functionality and affinity of ISVD207 in IL18_ISVD207 IL18[K70-E69]_ISVD207_V1, biolayer interferometry analysis using GFP was performed. For each concentration, association and dissociation isotherms were recorded and analyzed using Octet. ® Analysis Studio software analyzes data.

[0089] Figure 44 Blood containing IL-2 compounds after intravascular treatment in untreated female C57Bl / 6N mice Pulp PK curve. Results are expressed as mean ± SD. LOQ: Limit of quantitation for plasma pharmacokinetic assay.

[0090] Figure 45 In vivo intravascular treatment of untreated female C57Bl / 6N mice with a compound containing IL-2 Phosphorylation of STAT5 (pSTAT5) in different immune cells at two time points after treatment (24 h and 48 h) CD3+CD4-CD8+ cells are shown in A, CD3+CD4+CD8-CD25-Foxp3- cells are shown in B, CD3+CD4+CD8-CD25+Foxp3+ cells are shown in C, and CD3-NK1.1+ cells are shown in D. Results are presented as median (grey bars) and individual data (symbols).

[0091] Figure 46 In vivo intravascular treatment of untreated female C57Bl / 6N mice with a compound containing IL-2 Ki67 expression in different immune cells at two time points after treatment (48 h and 72 h)CD3+CD4-CD8+ cells are shown in A, CD3+CD4+CD8-CD25-Foxp3- cells are shown in B, CD3+CD4+CD8-CD25+Foxp3+ cells are shown in C, and CD3-NK1.1+ cells are shown in D. Results are presented as median (gray bars) and individual data (symbols):

[0092] When comparing each treatment group with the vector group, there were statistically significant differences ( p < 0.0001);

[0093] #: When comparing the TP208 control group with TP207 and TP206, there were statistically significant differences (#p < 0.05; ##p < 0.01; ###p < 0.001; ####p < 0.0001).

[0094] ns: No significant difference.

[0095] Figure 47 In vivo intravascular treatment of untreated female C57Bl / 6N mice with a compound containing IL-2 Proliferation of different immune cells 72 hours after treatment CD3+CD4-CD8+ cells are shown in A, CD3+CD4+CD8-CD25+Foxp3+ cells are shown in B, and CD3-NK1.1+ cells are shown in C. Results are presented as median (gray bars) and individual data (symbols):

[0096] When comparing each treatment group with the vector group, there were statistically significant differences ( p < 0.0001);

[0097] #: When comparing the TP208 control group with TP207 and TP206, there was a statistically significant difference (####p < 0.0001).

[0098] ns: No significant difference.

[0099] Figure 48 In vivo intravascular treatment of untreated female C57Bl / 6N mice with a compound containing IL-2 The ratio between different immune cell populations 72 hours after treatment. The ratio between CD3+CD4-CD8+ cells and CD3+CD4+CD25+Foxp3+ cells is shown in A, and the ratio between CD3-NK1.1+ cells and CD3+CD4+CD25+Foxp3+ cells is shown in B. Results are presented as median (grey bars) and individual data points (symbols):

[0100] When comparing the TP208 control group with TP207 and TP206, there was a statistically significant difference ( p < 0.0001);

[0101] ns: No significant difference. Detailed Implementation

[0102] The invention will be described with reference to specific embodiments and certain accompanying drawings, but the invention is not limited thereto, but is limited only by the claims. No reference numerals in the claims should be construed as limiting the scope. It should be understood, of course, that not all aspects or advantages can be achieved according to any particular embodiment of the invention. Therefore, for example, those skilled in the art will recognize that the invention may be embodied or practiced in a manner that achieves or optimizes one or more advantages as taught herein, without necessarily achieving other aspects or advantages as may be taught or suggested herein. The invention (both in terms of organization and operation) and its features and advantages can be best understood by referring to the following detailed description when read in conjunction with the accompanying drawings. Aspects and advantages of the invention will become apparent from one or more embodiments described below, and will be set forth with reference to these embodiments. Throughout this specification, reference to “one embodiment” or “embodiment” means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the phrases “in one embodiment” or “in an embodiment” appearing in various places throughout this specification do not necessarily refer to the same embodiment, but may refer to the same embodiment. Similarly, it should be understood that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, drawing, or description thereof for the purpose of simplifying this disclosure and aiding in understanding one or more of the various inventive aspects. However, the approach of this disclosure should not be construed as reflecting an intention to include more features required by the claimed invention than expressly recited in each claim. Rather, as reflected in the following claims, the inventive aspect lies in fewer features than all of the features in a single foregoing disclosed embodiment.

[0103] definition

[0104] Unless otherwise indicated or defined, all terms used have their usual meaning in the art, which will be clear to a person skilled in the art. For example, reference standard manuals include Sambrook et al., 1989 (Molecular Cloning: A Laboratory Manual, 2nd ed., Volumes 1-3, Cold Spring Harbor Laboratory Press); Ausubel et al., 1987 (Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York); Lewin 1985 (Genes II, John Wiley & Sons, New York, NY); Old et al., 1981 (Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2nd ed., University of California Press, Berkeley, CA); and Roitt et al., 2001 (Immunology, 6th ed., Mosby / Elsevier). Edinburgh (Mosby / Elsevier), Roitt et al., 2001 (Roitt's Essential Immunology, 10th edition, Blackwell Publishing, UK), and Janeway et al., 2005 (Immunobiology, 6th edition, Garland Science Publishing / Churchill Livingstone, New York), as well as the general background techniques cited herein.

[0105] Unless otherwise instructed, as will be clear to those skilled in the art, all methods, procedures, techniques and operations not specifically described in detail herein can be performed and have been performed in a manner known per se. For example, refer again to the standard manuals and general background techniques mentioned in this article and other references cited therein; and to reviews such as Presta 2006 (Adv. Drug Deliv. Rev., 58: 640), Levin and Weiss 2006 (Mol. Biosyst., 2: 49), Irving et al., 2001 (J. Immunol. Methods, 248: 31), Schmitz et al., 2000 (Placenta 21 Suppl., Supplement A: S106), Gonzales et al., 2005 (Tumour Biol., 26: 31), which describe techniques for protein engineering such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.

[0106] It is important to note that, as used herein, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise. Thus, for example, a reference to “a reagent” includes one or more such different reagents, and a reference to “the method” includes references to equivalent steps and methods known to those skilled in the art that can modify or replace the methods described herein.

[0107] Unless otherwise specified, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine many equivalent forms of the particular embodiments of the invention described herein using only conventional experimentation. Such equivalent forms are intended to be covered by the invention.

[0108] The term “and / or” as used herein includes the meaning of “and,” “or,” and “all or any other combination of elements connected by the term.”

[0109] Throughout this specification and the claims therein, unless the context otherwise requires, the word “comprise” and its variations such as “comprises” and “comprising” should be understood to imply inclusion of a whole or a group of steps of a statement, but not to exclude any other whole or a group of steps. When used herein, the term “comprise” may be replaced by the terms “containing” or “including”, or sometimes by the term “having”.

[0110] As used herein, “similar” is interchangeable with similar, analogous, comparable, corresponding and identical, and is intended to have the same or common features and / or to show comparable results in a quantifiable manner, i.e., with a variability of up to 20%, 10%, more preferably 5%, or even more preferably 1%, or less.

[0111] As used herein, the term "sequence" (e.g., in "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "V") refers to the sequence of an immunoglobulin, antibody, variable domain, or V sequence. HH In the terminology “sequence” or “protein sequence”, it should generally be understood to include the associated amino acid sequence as well as the nucleic acid or nucleotide sequence encoding that amino acid sequence, unless the context requires a more restrictive interpretation. An amino acid sequence should be interpreted as an unbranched sequence of a single amino acid or two or more amino acids, depending on the context. A nucleotide sequence should be interpreted as an unbranched sequence of three or more nucleotides.

[0112] As used herein, the terms “nucleotide sequence,” “DNA sequence,” or “one or more nucleic acid molecules” refer to a polymer of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Therefore, this term includes both double-stranded and single-stranded DNA, as well as RNA. It also includes known types of modifications, such as methylation, and “capping” substitution of one or more nucleotides in naturally occurring nucleotides with analogues.

[0113] "Nucleic acid constructs" refer to nucleic acid sequences that have been constructed to contain one or more functional units that do not exist naturally. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), plasmids (plasmids containing COS sequences from λ phage), and viral genomes containing non-natural nucleic acid sequences.

[0114] It should be understood that any reference to the amino acid sequences in this invention is intended to cover post-translational modifications of these sequences present in mammalian cells (such as CHO cells), including but not limited to N-glycosylation, O-glycosylation, deamidation, Asp isomerization / fragmentation, pyroglutamate formation, removal of C-terminal lysine, and Met / Trp oxidation.

[0115] When a nucleotide sequence or amino acid sequence is referred to as "containing" another nucleotide sequence or amino acid sequence, or as "consistently made up of" another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the first-mentioned nucleotide sequence or amino acid sequence, but more generally, this generally means that the first-mentioned nucleotide sequence or amino acid sequence contains within its sequence a segment of nucleotide or amino acid residues having the same nucleotide sequence or amino acid sequence as the latter sequence, regardless of how the first-mentioned sequence has actually been generated or obtained (which may be achieved, for example, by any suitable method described herein).

[0116] A coding sequence is a nucleotide sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into mRNA and / or translated into a polypeptide. The boundaries of a coding sequence are defined by a translation start codon at the 5' end and a translation stop codon at the 3' end. Coding sequences can include, but are not limited to, mRNA, cDNA, recombinant nucleotide sequences, or genomic DNA, and introns may also be present in some cases.

[0117] As used herein, "promoter region of a gene" refers to a functional DNA sequence unit that, when operably linked to a coding sequence and potentially placed under appropriate inducible conditions, is sufficient to promote transcription of said coding sequence. "Operably linked" means juxtaposed, wherein the components described thus are in a relationship that allows these components to function in their intended manner. A promoter sequence "operably linked" to a coding sequence is linked in such a manner that expression of the coding sequence is achieved under conditions compatible with the promoter sequence.

[0118] As used herein, “gene” includes both the promoter region and the coding sequence of a gene. It refers both to the genome sequence (including possible introns) and to cDNA derived from a splicing messenger that is operatively linked to the promoter sequence. The term “terminator” or “transcription termination signal” encompasses the control sequence, which is the DNA sequence at the end of a transcription unit that signals the 3' processing and polyadenylation of the primary transcript, as well as the termination of transcription. Terminators can originate from natural genes, from a variety of other plant genes, or from T-DNA. The terminator to be added can originate from, for example, the carmine synthase or octopine synthase gene, or alternatively, from another plant gene, or, less preferably, from any other eukaryotic gene.

[0119] The terms "gene construct," "chimeric gene," "chimeric construct," or "chimeric gene construct" refer to a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to or associated with a nucleic acid sequence encoding mRNA, such that the regulatory nucleic acid sequence can regulate the transcription or expression of the associated nucleic acid-coding sequence. The regulatory nucleic acid sequence of a chimeric gene is not operatively linked to any naturally occurring associated nucleic acid sequence. Specifically, as used herein, the term "gene fusion construct" refers to a gene construct encoding mRNA that is translated into a fusion protein as disclosed herein.

[0120] As used herein, the terms “vector,” “vector construct,” “expression vector,” or “gene transfer vector” mean a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked, and include any vector known to those skilled in the art, including any suitable type, including but not limited to plasmid vectors, granular vectors, bacteriophage vectors (such as λ phage), viral vectors (such as adenovirus, AAV, or baculovirus vectors), or artificial chromosome vectors (such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC)). Expression vectors comprise plasmids as well as viral vectors and typically contain the desired coding sequence and appropriate DNA sequence necessary for the expression of an operablely linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Expression vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., vectors having an origin of replication that functions in the host cell). Other vectors may integrate into the host cell’s genome upon introduction into the host cell, thereby replicating along with the host genome. As needed and depending on the specific host organism (e.g., bacterial cells, yeast cells), suitable vectors have regulatory sequences, such as promoters, enhancers, terminator sequences, etc. Cloning vectors are typically used to engineer and amplify a desired DNA fragment and may lack the functional sequences required to express the desired DNA fragment. The construction of expression vectors for use in transfecting prokaryotic cells is also well known in the art and can therefore be accomplished via standard techniques (see, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 4th edition, Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., *Current Protocols in Molecular Biology* (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terminology in the art).

[0121] “Host cell” can be prokaryotic or eukaryotic. Cells can be transiently or stably transfected. Such transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished by any technique known in the art, including but not limited to standard bacterial transformation, calcium phosphate coprecipitation, electroporation or liposome-mediated, DEAE-glucan-mediated, polycation-mediated, or virus-mediated transfection. For all standard techniques, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). In this context, recombinant host cells are those cells that have been genetically modified to contain the isolated DNA molecules, nucleic acid molecules, or expression constructs or vectors of the present invention. DNA can be introduced by any means known in the art suitable for a particular cell type, including but not limited to transformation, lipid transfection, electroporation, or virus-mediated transduction. DNA constructs capable of expressing the chimeric proteins of the present invention can be readily prepared using techniques known in the art, such as cloning, hybridization screening, and polymerase chain reaction (PCR). Standard techniques for cloning, DNA isolation, amplification, and purification; standard techniques for enzymatic reactions involving DNA ligases, DNA polymerases, restriction endonucleases, etc.; and various isolation techniques are those known and commonly used by those skilled in the art. Many standard techniques are described in Sambrook et al. (2012), Wu (ed.) (1993), and Ausubel et al. (2016). Representative host cells that can be used with the present invention include, but are not limited to, bacterial cells, yeast cells, insect cells, plant cells, and animal cells. The bacterial host cells suitable for use in this invention include Escherichia spp. cells, Bacillus spp. cells, Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, and Salmonella spp. cells.Animal host cells suitable for use in this invention include insect cells and mammalian cells (most particularly derived from Chinese hamsters (e.g., CHO)) and human cell lines such as HeLa. Yeast host cells suitable for use in this invention include species from the genera *Saccharomyceshia*, *Schizosaccharomyces*, *Kluyveromyces*, *Pichia* (e.g., *Pichia pastorisla*), *Hansenula* (e.g., *Hansenula polymorpha*), *Yarowia*, *Schwaniomyces*, *Schizosaccharomyces*, *Zygosaccharomyces*, etc. *Saccharomyces cerevisiae*, *S. carlsbergensis*, and *Kluyveromyces iactis* are the most commonly used yeast hosts and convenient fungal hosts. Host cells can be provided in the form of suspensions or flask cultures, tissue cultures, organ cultures, etc. Alternatively, host cells can also be transgenic animals.

[0122] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to refer to polymers containing amino acid residues, as well as their variants and synthetic analogs. Therefore, these terms apply to amino acid polymers where one or more amino acid residues are synthetic, non-naturally occurring amino acids, such as chemical analogs of corresponding naturally occurring amino acids, and to naturally occurring amino acid polymers, as described below. This term also includes post-translational modifications of polypeptides, such as glycosylation, phosphorylation, and acetylation. Based on the amino acid sequence and modifications, the atomic or molecular mass or weight of a polypeptide is expressed in (kilo) Daltons (kDa). “Recombinant polypeptide” means a polypeptide produced using recombinant technology (i.e., by expressing recombinant or synthetic polynucleotides). When a chimeric polypeptide or its bioactive portion is produced in a recombinant manner, the chimeric polypeptide or its bioactive portion is also preferably substantially free of culture medium, i.e., the culture medium constitutes less than about 20% of the volume of the protein preparation, more preferably less than about 10%, and most preferably less than about 5%. Conventionally, the amide bonds in the primary structure of a polypeptide are written in the order of the amino acids, with the amine terminus (N-terminus) of the polypeptide always on the left and the acid terminus (C-terminus) on the right. Any amino acid sequence containing post-translational modifications can be described as an amino acid sequence with modified sites (e.g., hydroxylation or glycosylation) that was originally translated using the symbols shown in Table 1 below, but which are not explicitly shown in the amino acid sequence. This definition includes any peptide or protein that can be expressed as a sequence modification such as a linker, crosslinker, cap, non-peptide bond, etc.

[0123] "Separated" means a substance that is substantially or essentially free of the components that normally accompany it in its natural state. For example, "separated polypeptide" refers to a polypeptide that has been purified from molecules flanking it in their natural state, such as the fusion proteins disclosed herein, which have been removed from molecules adjacent to the polypeptide present in the production host. Separated chimeras can be generated through amino acid chemical synthesis or through recombinant production. The expression "heterologous protein" can mean that the protein is not derived from the same species or strain used to display or express the protein.

[0124] The term “homology” of proteins encompasses peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions relative to the unmodified protein in question, and that have similar biological and functional activities to the unmodified protein from which it is derived.

[0125] "Amino acids" are those containing an amino group [a] (-NH4+). + 3) and carboxylic acids (-CO) -2) Organic compounds with functional groups and side chains (R groups) specific to each amino acid. For example, amino acids include those L-amino acids commonly found in naturally occurring proteins. In the context of this invention, "amino acid" also includes D-amino acids and non-natural, unusual, or unnatural amino acids, as described below. Amino acid residues will be represented according to standard three-letter or one-letter amino acid codes. Refer to Table A-2 on page 48 of WO 08 / 020079. Table 1 below lists examples of amino acids commonly found in proteins and represented by the genetic code. Other common amino acids (excluding those listed in Table 1 below) are described in the table on page 624 of Pure & Appl. Chem., Vol. 56, No. 5, pp. 595–624, 1984.

[0126] Table 1: Common Amino Acids (IUPAC)

[0127]

[0128] D-amino acids are also included in the definition of "amino acid". As used in this article, the term "D-amino acid" refers to an amino acid in which the α-carbon of the amino group has a D-configuration.

[0129] Unusual, unnatural, or non-natural amino acids are also included in the definition of “amino acid.” As used herein, the terms “unnatural amino acid,” “non-classical amino acid,” “non-natural amino acid,” or “novel amino acid” (or similar) refer to an amino acid that is not commonly found in naturally synthesized peptides and is one of the twenty amino acids known by the single-letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. An exemplary unnatural amino acid is described in Young et al., “Beyond the canonical 20 amino acids: expanding the genetic lexicon,” J. of Biological Chemistry, 285(15):11039-11044 (2010), the disclosure of which is incorporated herein by reference.

[0130] For the purpose of comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the corresponding nucleotides in the second nucleotide sequence] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], where each deletion, insertion, substitution, or addition of nucleotides in the second nucleotide sequence is considered a difference at a single nucleotide (position) compared to the first nucleotide sequence. Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0, with standard settings. Some other techniques, computer algorithms, and settings for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0967284, EP 1085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768. Typically, for the purpose of determining the "percentage of sequence identity" between two nucleotide sequences according to the calculation method outlined above, the nucleotide sequence with the largest number of nucleotides is designated as the "first" nucleotide sequence, and the other nucleotide sequence is designated as the "second" nucleotide sequence.

[0131] For the purpose of comparing two or more amino acid sequences, the percentage of “sequence identity” (also referred to herein as “amino acid identity”) between a first amino acid sequence and a second amino acid sequence can be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the corresponding amino acid residues in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], where each deletion, insertion, substitution, or addition of amino acid residues in the second amino acid sequence compared to the first amino acid sequence is considered a difference at a single amino acid residue (position), i.e., an “amino acid difference” as defined herein. Alternatively, the degree of sequence identity between two amino acid sequences can be calculated again using known computer algorithms (such as those described above for determining the degree of sequence identity of nucleotide sequences) with standard settings. Typically, to determine the percentage of “sequence identity” between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is designated as the “first” amino acid sequence, and the other amino acid sequence is designated as the “second” amino acid sequence.

[0132] Furthermore, in determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called “conserved” amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which have little or no effect on the 3D structure, function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art, for example according to WO 04 / 037999, GB 335768, WO 98 / 49185, WO 00 / 46383, and WO 01 / 09300; and the (preferred) types and / or combinations of such substitutions can be selected based on the relevant teachings from WO 04 / 037999 and WO 98 / 49185 and other references cited therein.

[0133] Such conservative substitutions are preferred in which one amino acid in the following groups (a)-(e) is replaced by another amino acid residue in the same group: (a) small aliphatic nonpolar or micropolar residues: Ala, Ser, Thr, Pro, and Gly; (b) negatively charged polar residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) positively charged polar residues: His, Arg, and Lys; (d) large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp. The particularly preferred conservative substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln, or Glu; Met is replaced by Leu, Tyr, or Ile; Phe is replaced by Met, Leu, or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp; and / or Phe is replaced by Val, Ile, or Leu.

[0134] If an amino acid sequence and a nucleic acid sequence have 100% sequence identity over their entire length (as defined herein), they are said to be “identical”. When comparing two amino acid sequences, the term “amino acid difference” refers to the insertion, deletion, or substitution of a single amino acid residue at a position in the first sequence compared to the second sequence; this is understood to mean that two amino acid sequences may contain one, two, or more such amino acid differences.

[0135] As used herein, "substitution" or "mutation" results from the substitution of one or more amino acids or nucleotides with different amino acids or nucleotides compared to the amino acid or nucleotide sequence of the parent protein or its fragments. It should be understood that proteins or their fragments can have conserved amino acid substitutions that have essentially no effect on the protein's activity.

[0136] The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. Wild-type genes are the most frequently observed genes in a population and are therefore arbitrarily engineered to be in a "normal" or "wild-type" form. In contrast, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits alterations (i.e., altered characteristics) in sequence, post-translational modifications, and / or functional properties compared to a wild-type gene or gene product. Notably, naturally occurring mutants can be isolated; these are identified by the fact that they possess altered characteristics when compared to a wild-type gene or gene product. Alternatively, variants may also include synthetic molecules; for example, chemokine ligand variants may be structurally and / or functionally similar to natural chemokines but may involve artificial small molecules or synthetic peptides or synthetic proteins. Variants with different functional properties may involve hyperagonists, hyperantagonists, and other functional differences, as known to those skilled in the art.

[0137] Protein domains are distinct functional and / or structural units within a protein. Typically, protein domains are responsible for specific functions or interactions, thus contributing to the overall function of the protein. Domains can exist in a variety of biological settings, where similar domains can be found in proteins with different functions. Secondary structure elements (SSEs) of proteins typically form spontaneously as intermediates before a protein folds into its three-dimensional tertiary structure. Two of the most common secondary structure elements of proteins are… Alpha (α) spiral and Beta (β) lamellae Although s-turns and ω-loops also exist, β-barrels are composed of tandemly repeated β-sheets that twist and coil to form closed loop structures in which the first chain bonds to the last chain (hydrogen bonds). In many β-barrels, the β-chains are arranged in an antiparallel manner. β-sheets consist of β-chains (also called β-chains) laterally linked by at least two or three backbone hydrogen bonds, forming typically twisted folds. β-chains are typically 3 to 10 amino acid segments of polypeptide chain with their backbone in an extended conformation. A "turn" is a type of irregular secondary structure in proteins that causes a change in the orientation of the polypeptide chain. When a protein chain needs to change orientation to connect two secondary structures... When connecting elements, turns typically occur. The most common is the β-turn, where the change of direction is performed in a space typically containing four residues. β-turns (β-bends, β-turns, sharp turns, or reverse turns) are very common motifs in proteins and polypeptides, primarily used to link β-chains. The polypeptide chain changes direction by 180° in a β-turn. A “loop” is an irregular structure that connects two secondary structural elements in a protein. They are typically located on the protein surface in solvent-exposed regions (Choi Y. et al., “How long is a piece of loop?”, Peer J., 2013, 1:e1). Generally, the number of amino acids in a loop is longer than that in a turn; see, for example, Milner-White and Poet, “Loops, bulges, turns and hairpins in proteins”, Trends in Biochemical Sciences, 1987, 12:189-192. For example, a ring with only 4 or 5 amino acid residues can also be called a turn when it has internal hydrogen bonds.

[0138] As used interchangeably herein, the terms "circular arrangement of proteins," "circularly arranged proteins," "circularly arranged proteins," or "circularly arranged proteins" refer to a molecule that, in its linear form, has ends joined together directly or via linkers to create a circular molecule (as an intermediate), and then the circular molecule is opened or cleaved at another location or position to produce a new molecule that, in its linear form, has ends (X1 and X2) different from those in the original molecule. Opening or cleaving the circular molecule at another location may include the removal of one or more nucleotides / amino acids from the original sequence. For example, when opening or cleaving a circular molecule, at least one, such as one, two, three, four, five, or more residues may be removed. Circularly arranged molecules include those whose structure is equivalent to a molecule that has been circulated and then opened, and / or for proteins, those molecules in which amino and carboxyl ends are joined together directly or via linkers, and new amino and carboxyl ends are formed at different positions within the protein sequence. Alternatively, cyclic molecules can also be synthesized de novo from a new linear form of the molecule (compared to the original molecule) without ever undergoing cyclization and opening steps. Cyclic molecules offer rearrangements within the molecule compared to the original wild-type molecule, but do not affect activity or functionality because the final (folded) molecule's fold or appearance is similar to or identical to the original molecule, the only difference being that the start and end points are at different positions. As mentioned above, in some cases, one or more nucleotides / amino acids of the original molecule are removed from it. Thus, cyclic molecules (which can be nucleic acid molecules or proteins) have normal ends that are usually fused with linkers, and contain a new end at another position. See Goldenbenberg et al. J. Mol. Biol. [Journal of Molecular Biology], 165: 407-413 (1983) and Pan et al. Gene [Genes] 125: 111-114 (1993), both of which are incorporated herein by reference. Circular arrangement is functionally equivalent to taking a straight-chain molecule, fusing the ends to form a cyclic molecule, and then cleaving the cyclic molecule at different positions to form new straight-chain molecules with different ends. Therefore, circular arrangement has the effect of essentially preserving the amino acid sequence and identity of the protein while generating new ends at different positions (see also Pastan et al. - EP 0 754 192 B1). A straightforward protein design for circular arrangement is one in which the ends of the original protein are close together and advantageously oriented; for example, when the ends are naturally close together, direct fusion of the ends with each other or the introduction of short linkers will have a relatively small effect. However, since linkers can be of any length, close proximity of the natural ends is not an absolute requirement. A specific circular arrangement of a molecule is specified by square brackets containing amino acid residues in which the peptide bonds are eliminated. Thus, for example, the name PRT[AA] X2-AA X1 The term "circular permutation" is specified for proteins with a circular permutation, where the open site (the location where the peptide bond is removed) appears between amino acid (AA) residues at positions X2 and X1 of the unpermutated or unmodified protein. Therefore, in the context of this invention, the term "circular permutation" (circularly permuted or circularly permutated) refers to the process of taking a protein or its homologous nucleic acid sequence and fusing the N-terminus and C-terminus (directly or via a linker, e.g., using a protein or recombinant DNA method) to form a circular molecule, and then cleaving (opening) the circular molecule at different positions to form new protein or homologous nucleic acid molecules with ends different from those in the original molecule. Thus, the circular permutation preserves the overall sequence (except for the linker (if introduced) and one or more amino acids (if any) removed), structure, and function of the protein, while generating new C-termini and N-termini at different positions, resulting in an improved orientation for fusion of the desired polypeptide fusion partner compared to the original molecule. As mentioned above, circularly permutated molecules can be synthesized de novo as linear molecules and never undergo cyclization and opening steps. Furthermore, in the context of this invention, the fusion of the N-terminus and C-terminus of a molecule (protein) can occur between the original N-terminus and C-terminus of the protein, or between the N-terminus and C-terminus resulting from the deletion of one or more residues (such as one, two, three, four, five, or more residues) from the original N-terminus, between the N-terminus and C-terminus resulting from the deletion of one or more residues (such as one, two, three, four, five, or more residues) from both the original N-terminus and C-terminus, or between the N-terminus and C-terminus resulting from the deletion of one or more residues (such as one, two, three, four, five, or more residues) from both the original N-terminus and C-terminus. Therefore, the different possibilities of N-terminal and C-terminal fusion of linear molecules may lead to the designation PRT[AA]. X2 -AA X1 Different versions of cyclically arranged proteins. Thus, for example, the names IFNA2a[D77-W76]V2 and IFNA2a[D77-W76]V4 represent two cyclically arranged proteins of IFNA2a, wherein the open site (the position where the peptide bond is eliminated) appears between amino acid residues at positions 77 and 76 of the unarranged or unmodified (but with different fused original N-termini and C-termini) proteins. Additionally, as described above, in the context of this invention, it is also possible to design cyclically arranged proteins by opening the cyclic molecule. NoThis occurs at two consecutive amino acid positions, resulting in the deletion of one or more amino acids from the protein. Therefore, in the context of this invention, the name IL-2 [F42-M39] refers to a cyclically arranged IL-2 cytokine, wherein the opening site (the position where the peptide bond is eliminated) appears between residues at positions 42 and 39 of the unarranged or unmodified IL-2. Residues 40 and 41 of the original protein have been deleted to generate a cyclically arranged protein. In the context of this invention, the opening of the cyclic molecule preferably occurs at an accessible or exposed site (preferably a β-turn or loop) of the protein, such that the folding (3D structure) of the cyclically arranged protein is preserved or similar to that of the wild-type protein. Therefore, in the context of this invention, the terms "cyclic arrangement of a protein" or "cyclically arranged protein" refer to a protein having a modified amino acid sequence in its amino acid sequence compared to the wild-type protein sequence, resulting in protein structures with different connectivity but generally similar three-dimensional (3D) shapes. The circular permutation of proteins is analogous to the mathematical concept of circular permutation, as the sequence of the first part (near the N-terminus) of the wild-type protein is associated with the sequence of the second part (near its C-terminus) of the resulting circularly permuted protein, as described, for example, in Bliven and Prlic (2012) (Circular permutation in proteins. PLOS Comput. Biol. [PLOS Computer] 8(3):e1002445). A circular permutation of a protein relative to its wild-type protein can be obtained by genetically or artificially engineering the protein sequence, whereby the N-terminus and C-terminus of the wild-type protein are “joined” (directly joined, through a linker and / or by removing one or more amino acids, as described above), and the protein sequence is interrupted at another site (where one or more amino acids may be removed, as described above) to produce novel N-terminus and C-terminus of said protein. The circularly arranged proteins (cytokines) of the present invention are the result of the connection of the N-terminus and C-terminus of the wild-type cytokine sequence and the cleavage or interruption of the sequence at accessible or exposed sites (preferably β-turns or loops) of the cytokine, thereby preserving or resembling the folding (3D structure) of the circularly arranged cytokine compared to the folding of the wild-type protein. The connection of the N-terminus and C-terminus in the circularly arranged cytokine may be the result of peptide bond linkage, the introduction of peptide linkers, or the deletion of peptide segments near the original N-terminus and C-terminus in the wild-type protein, followed by peptide bonds or remaining amino acids.The terms "circularly permuted" and "circular permutation" are well known in the art, see for example "CPSARST: Circular Permutation Search Aided by Ramachandran Sequential Transformation" ( http: / / 140.113.120.231 / ~lab / iSARST_2019 / srv / index.php?c=m2 ), Lo WC, Lyu PC. CPSARST: an efficient circular permutation search tool applied to the detection of novel protein structural relationships. Genome Biol. 2008 Jan 18; 9(1):R11, or "CPDB - the Circular Permutation Database" ( http: / / 10.life.nctu.edu.tw / cpdb / ). Circular permutation is performed to obtain a circularly permuted protein.

[0139] As used herein, the term "fused to" and may be used interchangeably herein as "linked to", "conjugated to", "ligated to", "linked to", or "bound to", particularly referring to "gene fusion", such as by recombinant DNA technology, and "chemical and / or enzymatic conjugation" resulting in stable covalent linkage.

[0140] The terms “chimeric polypeptide,” “chimeric protein,” “chimera,” “fusion polypeptide,” “fusion protein,” or “non-naturally occurring protein” are used interchangeably herein and refer to a protein comprising at least two separate and distinct polypeptide components, which may or may not be derived from the same protein, such as a protein comprising an immunoglobulin single variable domain (ISVD) fused to a cytokine. The term also refers to a non-naturally occurring molecule, meaning it is artificial. When referring to chimeric proteins (as defined herein), the terms “fused to” and other grammatical equivalents such as “covalently linked,” “linked,” “connected,” “attached,” “ligated,” “conjugated,” and “binded” refer to any chemical or recombination mechanism used to link two or more polypeptide components. As described herein, the fusion of two or more polypeptide components (e.g., ISVD and cytokine) can be a direct fusion of sequences, or it can be an indirect fusion, such as through intercalation of amino acid sequences or linker sequences or chemical linkers. The fusion of two polypeptides (e.g., ISVD and cytokine) as described herein can also refer to a non-covalent fusion obtained through chemical linking.

[0141] As used herein, the term "protein complex" or "complex" refers to a group of two or more associated macromolecules, at least one of which is a protein. As used herein, a protein complex typically refers to an association of macromolecules that can form under physiological conditions. The individual members of a protein complex are linked by non-covalent interactions. A protein complex can be a protein-protein complex consisting only of non-covalent interactions and is then referred to as a protein-protein complex; for example, non-covalent interactions of two proteins, three proteins, four proteins, etc. More specifically, a complex of a chimeric protein and a cytokine receptor, or a complex comprising a ligand protein of a cytokine or chemokine (such as a chimeric protein) and its specifically bound interacting element (such as a cytokine receptor capable of binding to a cytokine ligand). A protein complex comprising a chimeric protein fused with an ISVD of a cytokine (which binds to a chemokine receptor via its chemokine receptor-interacting region (its N-terminus), known to bind to the chemokine ligand), will be the complex formed herein.

[0142] As used herein, the terms “determine,” “measure,” “evaluate,” and “determine” are used interchangeably and include both quantitative and qualitative determinations.

[0143] The term "suitable conditions" refers to environmental factors such as temperature, motion, other components, and / or "buffer conditions," where "buffer conditions" specifically refers to the composition of the solution in which the measurement is performed. These components include buffer solutions and / or solutes, such as pH buffers, water, saline, physiological saline solutions, glycerol, preservatives, etc., the suitability of which is known to those skilled in the art to obtain optimal measurement performance.

[0144] In the context of this invention, the terms "specific," "specifically binding," or "specifically binding" refer to the number of different target molecules (such as antigens) that a particular binding unit can bind with a sufficiently high affinity (see below). "Specific," "specifically binding," or "specifically binding" may be used interchangeably herein with "selective," "selectively binding," or "selectively binding." Typically, a binding unit (such as binding ISVDs or cytokines) binds specifically to its designated target or receptor.

[0145] The specificity / selectivity of binding units can be determined based on affinity. Affinity represents the strength or stability of molecular interactions. Affinity is typically measured by K... D Alternatively, the dissociation constant may be given, in units of moles per liter (or M). Affinity can also be expressed as the association constant K. A It is equal to 1 / K D And the unit is (moles per liter). -1 (or M) -1 ).

[0146] "Affinity" is a measure of the strength of binding between a part of a target molecule and its binding site: K D The lower the value, the stronger the binding strength between the target molecule and the target region.

[0147] K D The value also characterizes the strength of molecular interactions in a thermodynamic sense, because it is expressed by the well-known relation DG=RT.ln(K). D (Equivalent to DG = -RT.ln(K)) A The value is related to the change in binding free energy (DG), where R is equal to the gas constant, T is equal to the absolute temperature, and ln represents the natural logarithm.

[0148] K D It can also be expressed as the dissociation rate constant of the complex (denoted as k). off ) and its association rate (denoted as k) on The ratio of (therefore K) D = k off / k on and K A = k on / k off Dissociation rate koff The unit is s -1 (Where s is the SI unit symbol for seconds). Association rate k on The unit is M -1 s -1 The association rate can reach 10. 2 M -1 s -1 To about 10 7 M -1 s -1 The variation between these values ​​approaches the diffusion-limited association rate constant of bimolecular interactions. The dissociation rate is determined by the relationship t... 1 / 2 = ln(2) / k off The half-life of the interaction with a given molecule is relevant. The dissociation rate can be as high as 10. -6 s -1 (Nearly irreversible complex, with multiple days t) 1 / 2 ) to 1 s -1 (t) 1 / 2 It varies between 0.69 s.

[0149] If the measurement process somehow affects the inherent binding affinity of the referred molecule, for example, by artifacts associated with a coating on a biosensor of a molecule, then the measured K... D It can correspond to apparent K D Furthermore, if a molecule contains more than one recognition site of another molecule or multiple molecules, the epigenetic K can be measured. D In such cases, the measured affinity may be affected by the affinity of the interaction between the two molecules.

[0150] dissociation constant (K) D () can be the actual or apparent dissociation constant that a technician clearly understands. Used to determine K D The method will be clear to a technician, and includes, for example, the techniques mentioned below. In this regard, it will also be clear that it is impossible to measure more than 10... -4 moles per liter or 10 -3 mol / L (e.g., 10) -2 The dissociation constant (mol / L). Optionally, as will be clear to those skilled in the art, the (actual or apparent) Ki D It can be based on the (actual or apparent) association constant (K) A ), through relation (K D = 1 / K A ) Calculation. K A = 1 / K D --> K A = [AB] / [A].[B].

[0151] The affinity of molecular interactions between two molecules can be measured using various techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technology (see, for example, Ober et al. 2001, Intern. Immunology [International Journal of Immunology] 13: 1551-1559). As used herein, the term “surface plasmon resonance” (SPR) refers to an optical phenomenon that allows the analysis of real-time, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and another molecule flows through the immobilized molecule under flowing conditions, thereby generating k on k off Measured value, and thus obtain K D (or K) A This can be achieved, for example, by using the well-known BIAcore value. ® The system was operated by BIAcore International AB (Cytiva lifesciences company, Uppsala, Sweden and Piscatave, New Jersey). For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51: 19-26), Jonsson et al. (1991 Biotechniques 11: 620-627), Johnson et al. (1995, J. Mol. Recognit. 8: 125-131), and Johnson et al. (1991, Anal. Biochem. 198: 268-277).

[0152] Another well-known biosensor technique for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, for example, Abdiche et al. 2008, Anal. Biochem. [Analytical Biochemistry] 377: 209-217). As used herein, the term “biolayer interferometry” or “BLI” refers to a label-free optical technique for analyzing the interference patterns of light reflected from two surfaces: an inner reference layer (reference beam) and an immobilized protein layer on the top of the biosensor (signal beam). Changes in the number of molecules bound to the top of the biosensor cause a shift in the interference pattern, which is reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the surface of the top of the biosensor. Because interactions can be measured in real time, association and dissociation rates, as well as affinity, can be determined. BLI can, for example, use the well-known Octet... ®The system (ForteBio, a division of Pall Life Sciences, Menlo Park, USA) was used.

[0153] Alternatively, KinExA can be used. ® The platform (Sapidyne Instruments Inc., Boise, USA) measures affinity using the Kinetic Exclusion Assay (KinExA) method (see, for example, Drake et al., “Characterizing high-affinity antigen / antibody complexes by kinetic- and equilibrium-based methods”, Anal. Biochem., 2004, 328: 35-43). As used herein, the term “KinExA” refers to a solution-based method that measures the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrium solution of the binding unit / target complex (such as an antibody / antigen complex) is passed through a chromatographic column carrying beads pre-coated with an antigen (or antibody), thereby binding the free antibody (or antigen) to the coated molecule. Detection of the thus captured antibody (or antigen) is performed using a fluorescently labeled protein of the bound antibody (or antigen).

[0154] Furthermore, GYROLAB ® Immunoassay systems provide a platform for automated bioanalysis and rapid sample turnaround (Fraley et al., “The Gyrolab™ immunoassay system: a platform for automated bioanalysis and rapid sample turnaround”, Bioanalysis 2013, 5: 1765-74).

[0155] The term “about” as used in the context of the parameters or ranges provided herein shall have the following meanings: Unless otherwise indicated, when the term “about” is applied to a particular value or range, that value or range is interpreted as being as accurate as the method used to measure it. If no error margin is specified in the application, the last decimal place of the numerical value indicates its accuracy. In the absence of other error margins, the maximum margin is determined by applying rounding conventions to the last decimal place; for example, for a pH value of about pH 2.7, the error margin is 2.65–2.74. However, specific margins shall apply to the following parameters: temperatures specified in °C without decimal places shall have an error margin of ±1°C (e.g., a temperature value of about 50°C means 50°C ± 1°C); times indicated in hours shall have an error margin of 0.1 hours, regardless of decimal places (e.g., a time value of about 1.0 hour means 1.0 hour ± 0.1 hours; a time value of about 0.5 hours means 0.5 hours ± 0.1 hours).

[0156] Methods for determining the spatial conformation of amino acids and proteins are known in the art and include, for example, X-ray crystallography and multidimensional nuclear magnetic resonance. The term "conformation" or "conformatory state" of a protein generally refers to the range of structures a protein can adopt at any given time. Those skilled in the art will recognize that the determinants of conformation or conformational state include the protein's primary structure, such as its amino acid sequence (including modified amino acids), and what is reflected in the protein's surrounding environment. Protein conformation or conformational state also involves structural features such as secondary structure (e.g., α-helices, β-sheets, β-barrels, etc.), tertiary structure (e.g., the three-dimensional folding of polypeptide chains), and quaternary structure (e.g., interactions of polypeptide chains with other protein subunits). Post-translational and other modifications to polypeptide chains, such as ligand binding, phosphorylation, sulfation, glycosylation, or attachment of hydrophobic groups, can affect protein conformation. Furthermore, environmental factors (such as pH, salt concentration, ionic strength, and osmotic pressure of the surrounding solution), as well as interactions with other proteins and cofactors, can influence protein conformation. The conformational state of a protein can be determined by functional assays targeting its activity or binding to another molecule, or by physical methods such as X-ray crystallography, NMR, or spin labeling. For a general discussion of protein conformation and conformational state, see Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological Macromolecules, WH Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, WH Freeman and Company, 1993.

[0157] According to this specification, “protein solubility” is a thermodynamic parameter defined as the concentration of a protein in a saturated solution in equilibrium with a solid phase (crystalline or amorphous) under a given set of conditions (see, for example, Kramer RM. et al., “Toward a molecular understanding of protein solubility: increased negative surface charge correlates with increased solubility”, Biophys J., 2012, 102(8):1907-15).

[0158] Finally, in the context of this invention, the terms "functional chimeric protein," "functional fusion protein," or "conformation-selective fusion protein" refer to a fusion protein that is functional in binding to its cytokine and / or ISVD targets (optionally in a conformation-selective manner), and / or in the activation / inactivation of cytokine receptors and / or ISVD targets (depending on the known characteristics of the ligand: agonist, antagonist, inverse agonist). A binding domain that binds to a specific conformation-selectively target protein refers to a binding domain that has a higher affinity for the target within a subset of conformations compared to other conformations the target may present. Those skilled in the art will recognize that a binding domain that binds to a specific conformation-selectively target will stabilize or maintain the target in that particular conformation. For example, an active-state conformation-selective binding domain will preferentially bind to the target in its active conformation and will not bind or bind to a lesser extent.

[0159] The chimeric protein of the present invention

[0160] In a first aspect, the present invention provides a chimeric protein comprising an immunoglobulin single variable domain (ISVD) fused to a cytokine. The chimeric protein of the present invention may also be referred to as a "fusion protein" or a "chimera".

[0161] The term "immunoglobulin single variable domain" (ISVD), which can be used interchangeably with "single variable domain," defines an immunoglobulin molecule in which an antigen-binding site is located on and formed by a single immunoglobulin domain. This distinguishes ISVDs from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, discFv), where two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V... H) and light chain variable structural domain (V L ) interact to form antigen-binding sites. In this case, V H and V L The complementary determinant regions (CDRs) of both will contribute to the formation of antigen binding sites, meaning that a total of 6 CDRs will participate in the formation of antigen binding sites.

[0162] Given the above definition, the antigen-binding domains of conventional 4-chain antibodies (such as IgG, IgM, IgA, IgD, or IgE molecules; known in the art) or Fab fragments, F(ab')2 fragments, Fv fragments such as disulfide-linked Fv or scFv fragments, or biantibodies derived from such conventional 4-chain antibodies (known in the art) are generally not considered ISVDs because in these cases, binding to the corresponding epitope of the antigen typically does not occur through a single immunoglobulin domain, but rather through a pair of related immunoglobulin domains such as light chain and heavy chain variable domains, i.e., through the V of the immunoglobulin domain that binds together to the epitope of the corresponding antigen. H -V L Regarding what happened.

[0163] In contrast, ISVDs are typically able to bind specifically to antigenic epitopes without pairing with additional immunoglobulin variable domains. The binding site of an ISVD consists of a single V... H A single V HH Or a single V L Domain formation.

[0164] In the context of this invention, ISVD can be a sequence of light chain variable structural domains (e.g., V...). L Sequences) or suitable fragments thereof; or heavy-chain variable domain sequences (e.g., V H Sequence or V HH (Sequence) or a suitable fragment thereof. The ISVD that can preferably be included in the chimeric protein of the present invention can be, for example, a heavy chain ISVD, such as V H V HH Including camel-derived V H Or humanized V HH Heavy chain ISVDs can be derived from conventional tetrachain antibodies or from heavy chain antibodies.

[0165] For example, an ISVD can be a single-domain antibody (or an amino acid sequence suitable for use as a single-domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb), or a Nanobody. ® ISVD (as defined in this document, and including but not limited to V) HH ); other single variable domains, or any suitable fragment thereof. Preferably, ISVD is VH , humanized V H , human V H , V HH , humanized V HH or camelized V H . More preferably, the ISVD is a Nanobody ® ISVD (such as V HH , including humanized V HH or camelized V H ) or a suitable fragment thereof. Nanobody® is a registered trademark of Ablynx N.V.

[0166] "V HH domain" (also known as V HH , V HH antibody fragment and V HH antibody) was initially described as the antigen-binding immunoglobulin variable domain of a "heavy chain antibody" (i.e., an "antibody without a light chain"), see Hamers-Casterman et al., Nature [自然], 363: 446-448, 1993. The term "V HH domain" was chosen to distinguish these variable domains from the heavy chain variable domains present in conventional 4-chain antibodies (referred to herein as "V H domain") and the light chain variable domains present in conventional 4-chain antibodies (referred to herein as "V L domain"). For additional information on V HH , refer to the review article by Muyldermans ("Single domain camel antibodies: current status [单结构域骆驼抗体:当前状态]", J Biotechnol.[生物技术杂志], 2001, 74: 277-302). V HH domains can be obtained from heavy chain only antibodies (HCAb) circulating in camelids, see, e.g., Muyldermans S., "A guide to: generation and design of nanobodies [纳米抗体的产生和设计指南]", FEBS J.[欧洲生物化学学会联合会杂志], 2021, 288(7): 2084-2102.

[0167] Typically, immunoglobulin production involves immunizing laboratory animals, fusing immunoglobulin-producing cells to create hybridomas, and screening for desired specificity. Alternatively, immunoglobulins can be generated by screening natural, immunoglobulin-producing, or synthetic libraries (e.g., via phage display).

[0168] Immunoglobulin sequences (such as V) HH The generation of antibodies has been extensively described in various publications, exemplified by WO 94 / 04678, Hamers-Casterman et al. 1993 (“Naturally occurring antibodies devoid of light chains”, Nature, 363: 446-448, 1993), and Muyldermans et al. 2001 (“Single domain camel antibodies: current status”, J Biotechnol., 2001, 74:277-302). In these methods, camels are immunized with the target antigen to induce an immune response against the target antigen. Further screening of the antibodies obtained from the immunization is then performed. HH V of the library binding (or not binding) to the target antigen HH .

[0169] In the context of this invention, immunoglobulin sequences from various sources may be used, including mouse, rat, rabbit, donkey, human, and camel immunoglobulin sequences. In the context of this invention, fully human sequences, humanized sequences, or chimeric sequences are also included. In the context of this invention, it also includes camel immunoglobulin sequences and humanized camel immunoglobulin sequences or camel-derived domain antibodies, for example, camel-derived dAbs as described by Ward et al. (Nature, 341: 544, 1989) (see, for example, WO 94 / 04678, and Davies and Riechmann, “'Camelising' human antibody fragments: NMR studies on VH domains”, Febs Lett., 339:285-290, 1994 and “Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains”). H Domains with improved protein stability [Monoantibody domains as small recognition units: Design and in vitro antigen screening of camel-derived human VH domains with improved protein stability], Prot. Eng., 1996, 9(6):531-537.

[0170] "Humanization V" HH "Contains V corresponding to natural occurrences" HH The amino acid sequence of the domain is the same as the amino acid sequence of the human body, but it has been "humanized," i.e., by using V, which is now present in conventional 4-chain antibodies from humans. H One or more amino acid residues at one or more corresponding positions in the domain (as shown above) replace the naturally occurring V. HH One or more amino acid residues in the amino acid sequence of a sequence (especially in a framework sequence). This can be done in a manner known per se, which will be clear to those skilled in the art, for example, based on further description herein and prior art (e.g., WO 2008 / 020079). Again, it should be noted that such humanized V HH It can be obtained in any suitable manner known in itself, and is therefore not strictly limited to the use of naturally occurring V. HHThe polypeptide is obtained by using peptides with structural domains as starting materials. Preferably, if the building unit of the present invention is V HH Then V HH It is humanized V HH .

[0171] "Camel Source V" H "Contains V corresponding to natural occurrences" H The amino acid sequence of the domain is a camel-derived amino acid sequence, but it has been "camel-derived," i.e., by using V of the heavy chain antibody. HH One or more amino acid residues appearing at one or more corresponding positions in the domain replace the naturally occurring V from a conventional 4-chain antibody. H One or more amino acid residues in the amino acid sequence of the domain. This can be done in a manner known per se, which will be clear to those skilled in the art, for example, based on further description herein and prior art (e.g., WO 2008 / 020079). Such "camelization" substitutions are typically inserted into the domain that forms and / or exists in V. H -V L At the amino acid sites of the interface, and / or at so-called cameloid marker residues, as defined herein (see, for example, WO 94 / 04678 and Davies and Riechmann 1994 and 1996, ibid.). In one embodiment, it is used as a source for generating or designing camel-derived V H V of the starting material or starting point H The sequence is the V of mammals. H Sequence, or human V H Sequences, such as V H 3. Sequence. However, it should be noted that such camel-derived V can be obtained in any suitable manner known per se. H And therefore not strictly limited to those already using naturally occurring V H Peptides obtained from peptides whose structural domains are used as starting materials.

[0172] The structure of an ISVD sequence can be considered to contain four frame regions (“FRs”), referred to in the art and herein as “Frame Region 1” (“FR1”); “Frame Region 2” (“FR2”); “Frame Region 3” (“FR3”); and “Frame Region 4” (“FR4”); these frame regions are interrupted by three complementarity determination regions (“CDRs”), referred to in the art and herein as “Complementarity Determination Region 1” (“CDR1”); “Complementarity Determination Region 2” (“CDR2”); and “Complementarity Determination Region 3” (“CDR3”).

[0173] Furthermore, as further described in paragraph q) on pages 58 and 59 of WO 2008 / 020079, the amino acid residues of ISVD are based on the sequence of proteins of immunological interest given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH, Bethesda, Maryland, Publication No. 91). H Domains are numbered using common designations, as in the article by Riechmann and Muyldermans, 2000 (J. Immunol. Methods [Journal of Immunological Methods] 240 (1-2): 185-195; see, for example, that publication). Figure 2 V from camelidae animals HH Structural domain. It should be noted (as in the art regarding V) H Domain and for V HH (As is well known about the domains), the total number of amino acid residues in each of these CDRs can vary and may not correspond to the total number of amino acid residues indicated by the Kabat number. That is, one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed for the Kabat number. This means that, generally, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. H Domain and V HH The total number of amino acid residues in the domain is typically in the range of 110 to 120, usually between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0174] In this application, the CDR sequence can also be described using AbM CDR annotations according to the Kabat numbering as described by Kontermann and Dübel (edited 2010, Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, FR1 contains amino acid residues at positions 1-25, CDR1 contains amino acid residues at positions 26-35, FR2 contains amino acids at positions 36-49, CDR2 contains amino acid residues at positions 50-58, FR3 contains amino acid residues at positions 59-94, CDR3 contains amino acid residues at positions 95-102, and FR4 contains amino acid residues at positions 103-113.

[0175] The determination of the CDR region can also be performed using different methods. In the CDR determination according to Kabat, ISVD FR1 contains amino acid residues at positions 1-30, ISVD CDR1 contains amino acid residues at positions 31-35, ISVD FR2 contains amino acid residues at positions 36-49, ISVD CDR2 contains amino acid residues at positions 50-65, ISVD FR3 contains amino acid residues at positions 66-94, ISVD CDR3 contains amino acid residues at positions 95-102, and ISVD FR4 contains amino acid residues at positions 103-113.

[0176] In such immunoglobulin sequences, the frame sequence can be any suitable frame sequence, and examples of suitable frame sequences will be clear to those skilled in the art, for example, based on standards manuals and further disclosures and the prior art mentioned herein.

[0177] The framework sequence is a suitable combination of immunoglobulin framework sequences or framework sequences derived from immunoglobulin framework sequences (e.g., through humanization or camelification). For example, the framework sequence could be derived from a light chain variable domain (e.g., V). L Sequence) and / or heavy-chain variable structural domains (e.g., V) H Sequence or V HH A frame sequence (sequence). In one respect, a frame sequence is derived from V. HH - A frame sequence of the sequence (where the frame sequence may optionally be partially or fully humanized) or a regular V sequence that has been camel-derived (as defined herein). H sequence.

[0178] In particular, the frame sequence present in the ISVD sequence mentioned in this invention may contain one or more marker residues (as defined herein), such that the ISVD sequence is a nanobody. ® ISVD, such as V HH Including humanized V HH Or camel-derived V H Some non-limiting examples of suitable combinations of such frame sequences will become clear as further disclosures in this paper.

[0179] However, it should be noted that, in the context of this invention, the source of the ISVD sequence or the source of the nucleotide sequence used to express it is not limited, nor is the manner in which the ISVD sequence or nucleotide sequence is generated or obtained (or has been generated or obtained). Therefore, the ISVD sequence can be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In a particular but non-limiting aspect, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, particularly partially or fully humanized V...). HH Immunoglobulin sequences, "camel-derived" (as defined herein) immunoglobulin sequences, and immunoglobulin sequences obtained by techniques such as affinity dematuration (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR transplantation, mosaicking, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques known to the technician for engineering immunoglobulin sequences; or any suitable combination of the foregoing.

[0180] Similarly, a nucleotide sequence can be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and can be, for example, a sequence isolated from a suitable naturally occurring template by PCR (e.g., DNA or RNA isolated from cells), a nucleotide sequence isolated from a library (especially an expression library), a nucleotide sequence prepared by introducing a mutation into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence prepared by PCR using overlapping primers, or a nucleotide sequence prepared using DNA synthesis techniques known per se.

[0181] For Nanobody ® A general description of ISVD is given with reference to this specification and the prior art cited herein. However, it should be noted in this regard that this specification and the prior art primarily describe the so-called "V". H Nanobody of "3 categories" ® ISVD (i.e., with V)H Three types of phylogenetic sequences, such as DP-47, DP-51, or DP-29, are highly sequence homologous to Nanobody sequences. ® (ISVD). However, it should be noted that this technology, in its broadest sense, can generally be used with any type of Nanobody® ISVD, and also, for example, with what is known as "V". H Nanobody of "4 categories" ® ISVD (i.e., with V) H Four types of holotype sequences, such as DP-78, are Nanobody sequences with high sequence homology. ® ISVD), for example, as described in WO 2007 / 118670.

[0182] In one embodiment, the ISVD contained in the chimeric molecule of the present invention is derived from the so-called "V" H Nanobody of "3 categories" ® ISVD, which is related to V H Nanobody sequences of three phylogenetic types (such as DP-47, DP-51, or DP-29) exhibit high sequence homology. ® ISVD.

[0183] Typically, Nanobody ® ISVD (especially V) HH Sequence, including (partially) humanized V HH Sequence and camel-derived V H The characteristics of a sequence can be the presence of one or more "marker residues" (as described herein) within one or more frame sequences (also as further described herein). Typically, Nanobody ® ISVD can be defined as an immunoglobulin sequence having the following (general) structure:

[0184] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

[0185] FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively, and one or more of these marker residues are further defined herein.

[0186] Specifically, Nanobody ® ISVD can be an immunoglobulin sequence with the following (general) structure.

[0187] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

[0188] FR1 to FR4 refer to frame regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively, and the frame sequence is further defined in this paper.

[0189] More specifically, Nanobody ® ISVD can be an immunoglobulin sequence with the following (general) structure:

[0190] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

[0191] Wherein FR1 to FR4 refer to frame regions 1 to 4 respectively, and wherein CDR1 to CDR3 refer to complementarity-determining regions 1 to 3 respectively, and wherein:

[0192] According to the Kabat number, one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 are selected from the marker residues mentioned in Table 2 below.

[0193] Table 2: Nanobody ® Marker residues in ISVD (based on Kabat numbering)

[0194]

[0195] Therefore, Nanobody ® ISVD can be defined as an amino acid sequence having the following (general) structure:

[0196] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

[0197] FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively. One or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat number are selected from the marker residues mentioned in Table 2.

[0198] In a further preferred embodiment, the ISVD contained in the chimeric protein of the present invention is derived from ISVD, such as from heavy chain ISVD, preferably from nanobody. ®ISVDs, which have been further engineered / modified to include mutations that prevent / remove binding to pre-existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO2015 / 173325. For example, to prevent / remove binding to pre-existing antibodies / factors, the amino acid at position 11 (according to Kabat) may be Val or Leu, preferably Val; and / or the amino acid at position 89 (according to Kabat) may preferably be Val, Thr, or Leu, preferably Leu; and / or the amino acid at position 110 (according to Kabat) may preferably be Thr, Lys, or Gln, preferably Thr; and / or the amino acid at position 112 (according to Kabat) may be Ser, Lys, or Gln, preferably Ser; and / or the building blocks based on ISVDs may contain a C-terminal extension of 1-5 amino acids selected from any naturally occurring amino acids.

[0199] In one embodiment, the ISVD contained in the chimeric protein of the present invention specifically binds to its target (antigen), indicating that such interaction between the ISVD and its antigen is characterized by high specificity and / or high affinity, as defined herein.

[0200] In another embodiment, the ISVD contained in the chimeric protein of the present invention does not specifically bind to its target (antigen). In this particular embodiment, if the ISVD contained in the chimeric protein exhibits any interaction with its original target (antigen) or with any other protein, such interaction is characterized by low specificity and / or low affinity, as defined herein. Therefore, in this embodiment, the ISVD contained in the chimeric protein of the present invention may be derived from an ISVD (“ISVD precursor”). In this particular embodiment, an “ISVD precursor” is an ISVD that has been modified (e.g., by point mutation and / or by adding / removing amino acids in its sequence) to generate the ISVD contained in the chimeric protein of the present invention. For example, an “ISVD precursor” is modified so that it no longer specifically binds to any molecule (ISVD precursor target (antigen)) that the ISVD precursor specifically binds to.

[0201] Cytokines are a class of small proteins (5-20 kDa) that act as cell signaling molecules at picomolar or nanomolar concentrations to regulate inflammation and modulate cellular activities such as migration, growth, survival, and differentiation. Cytokines are a particularly large and diverse group of pro-inflammatory or anti-inflammatory factors, grouped into families based on structural homology of these factors or their receptors. Cytokines can include chemokines, interferons, interleukins, lymphokines, tumor necrosis factor, hormones, or growth factors. Interleukins (ILs) form a group of cytokines with complex immunomodulatory functions, including cell proliferation, maturation, migration, and adhesion, thus playing a crucial role in immune cell differentiation and activation. ILs can also have pro-inflammatory and anti-inflammatory effects and are under constant evolutionary pressure due to ongoing competition between the host immune system and the infecting organism; therefore, ILs have undergone significant evolution, resulting in minimal amino acid conservation among orthologous proteins, thus complicating the organization of their gene families. However, crystallographic data and identification of common structural motifs led to a classification into four main groups, including genes encoding IL1-like cytokines, class I helical cytokines (IL4-like, γ-chain, and IL6 / 12-like), class II helical cytokines (IL10-like and IL-28-like), and IL17-like cytokines, which are structurally independent of other IL subfamilies, and IL17F constitutes a cysteine ​​knot fold.

[0202] In the chimeric protein of the present invention, the fusion between ISVD and cytokines occurs at the internal fusion site of ISVD.

[0203] The term "internal fusion site" is defined herein as a location between two amino acids anywhere in a polypeptide sequence, more specifically in ISVDs and / or cytokines as used herein, and more specifically, "internal" means the fusion site is not at the N- or C-terminus of the protein. An internal fusion site can alternatively be defined as a location between two amino acids anywhere in a protein variant of the ISVD or cytokine, where a small number of amino acids are missing or added at the fusion site compared to the original protein sequence. The internal fusion site is a location to be cleaved so that another protein sequence can be inserted by creating a peptide bond between the cleaved protein sequence and the inserted protein sequence. In addition to actual cleavage, chimeric proteins with the aforementioned structure can also be obtained by designing gene fusions. Figure 32As shown, the chimeric protein of the present invention is thus obtained by translating a gene fusion of the chimeric protein corresponding to a protein sequence that begins with the N-terminal portion of the ISVD and ends at an internal fusion site (and / or a variant with a small number of amino acids added or deleted at the fusion site, such as adding or deleting 1 to 10 amino acids at the fusion site, such as adding or deleting 1 to 7 amino acids, or 1 to 5 amino acids, such as 1, 2, 3, 4, or 5 amino acids); which is then attached to the N-terminus of an insert protein, which is a cytokine or a circularly arranged variant of a cytokine in the present invention, the C-terminus of which is then attached to the remainder of the ISVD.

[0204] Therefore, an "internal fusion site" refers to a location in a polypeptide sequence where the original peptide bond between two amino acids present in an ISVD, or particularly a cytokine sequence, is broken, thus providing a point for the generation of two novel peptide bonds. Specifically, one peptide bond connects the amino acid sequence at the N-terminus of the internal fusion site to the N-terminus of the inserted protein sequence, and another peptide bond connects the C-terminus of the inserted protein sequence to the sequence at the C-terminus of the internal fusion site. Thus, in the context of this invention, an "internal fusion site" is a location within the sequence of an ISVD (and / or a cytokine, if circularly arranged), where a connection (fusion) with the cytokine (or ISVD) is established. Therefore, the internal fusion site can be used as a reference point in the amino acid sequence to divide its original protein sequence into the sequence at the N-terminus of the internal fusion site and the sequence at the C-terminus of the internal fusion site. The internal fusion site of an ISVD is preferably located at a loop or turn (preferably a β-turn) in a folded protein, more preferably between two β chains, and even more preferably between β chains A and B, as detailed below. The internal fusion sites of the cyclically arranged cytokines contained in the chimeric proteins of the present invention are located at corners or loops between two secondary elements of the cytokine, for example, between two β-chains or between two α-helices or between a β-chain and an α-helix. Preferably, the internal fusion sites of the cytokines are located in a position within the protein that would result in altered cytokine-receptor binding and / or altered downstream activity of the cytokine-receptor and / or altered oligomerization of the receptor / receptor subunit upon cytokine binding and / or functional alteration of cytokine-receptor / receptor subunit binding in chimeric molecules made by fusing ISVDs at said internal fusion sites of the cytokines. The terms “accessible site” and “exposed site” are used interchangeably herein and both refer to structurally accessible amino acid sites in a protein sequence, preferably located on or exposed to the protein surface. The term “internal fusion site” as used herein refers to an amino acid site of a protein, which is preferably also an accessible or exposed site. Exposed or accessible sites are preferably located in turns or loops between two secondary elements of a protein (e.g., two β-chains, or two α-helices, or β-chains and α-helices). Those skilled in the art will be able to identify those sites.

[0205] In the chimeric protein of the present invention in which cytokines are arranged in a circular pattern, the ISVD is linked to the circularly arranged cytokines at an internal fusion site of the ISVD, wherein the circularly arranged cytokine protein sequence is inserted as described above, wherein the N-terminus and C-terminus of the circularly arranged cytokine protein sequence are provided by cleaving the protein sequence at an internal fusion site as defined herein to provide N-terminus and C-terminus for forming peptide bonds, thereby producing the chimeric protein.

[0206] When cytokines are arranged in a circular pattern, the amino acids at the internal fusion site of the ISVD are linked to the amino acids at the internal fusion site of the cytokines. The internal fusion site is located in a loop or turn between two secondary structural elements of both the ISVD and the circularly arranged cytokines. For example, the internal fusion site of the ISVD can be located in a loop or turn between two secondary structural elements, such as in a β-turn. Similarly, the internal fusion site of a circularly arranged cytokine can be located in a loop or turn, thus providing a site for fusion with the ISVD protein sequence, which is also located at the internal fusion site of the ISVD. In one embodiment, the internal fusion site of the ISVD... No Located within any CDR of the ISVD. Therefore, in one embodiment, the inner fusion site of the ISVD is located in a loop or turn (such as a β turn). no CDR.

[0207] Therefore, the internal fusion site of ISVD is the location where the N-terminal amino acid at that site is linked at the C-terminus to the N-terminus of a cytokine (or cyclically arranged cytokine) protein, and wherein the C-terminal amino acid at the internal fusion site is linked to the C-terminus of the cytokine (or cyclically arranged cytokine) protein (see also...). Figure 32 (As an illustrative example). Preferably, in the chimeric protein of the present invention, the cytokines are cyclically arranged cytokines as described above. Therefore, if the cytokines are cyclically arranged, in the chimeric protein of the present invention, the original N-terminus and C-terminus of the cytokine protein sequence are linked to each other (because it is a cyclically arranged cytokine). For cyclic arrangement, the N-terminus and C-terminus of the cytokines can be linked to each other directly or through a linker, as described herein. In one embodiment, 0 to 10 amino acids are removed from the (original) N-terminal and / or C-terminal portions of the cytokines before linking the N-terminus and C-terminus of the cytokines to each other. Preferably, 0 to 7 amino acids are removed from the N-terminal and / or C-terminal portions of the cytokines before linking the N-terminus and C-terminus of the cytokines to each other, and even more preferably, 0 to 5 amino acids, such as 0, 1, 2, 3, or 4 amino acids, are removed from the N-terminal and / or C-terminal portions of the cytokines before linking the N-terminus and C-terminus of the cytokines to each other (directly or through a linker, as described herein).

[0208] In other embodiments, in the chimeric protein of the present invention, an internal chimeric fusion body in an ISVD as defined herein is obtained using non-circularly arranged cytokines. Thus, in this embodiment, the cytokines retain their original N-termini and C-termini, i.e., no new N-termini and C-termini are generated elsewhere in the cytokine sequence. In this embodiment, the cytokines are fused with amino acids of the ISVD located at the internal fusion site of the ISVD via their (original) N-termini and C-termini. Therefore, in this embodiment, the cytokines are not linked to amino acids of the ISVD's internal fusion site via amino acids located at the cytokines' internal fusion site. Similarly, 0 to 7 amino acids may be removed from the original N-termini and / or C-termini of the cytokines before fusing the cytokines with the amino acids located at the internal fusion site of the ISVD. Preferably, 0 to 7 amino acids are removed from the N-terminus and / or C-terminus of the cytokine before fusing the cytokine to the amino acids at the internal fusion site of the ISVD. More preferably, 0 to 5 amino acids, such as 0, 1, 2, 3, or 4 amino acids, are removed from the N-terminus and / or C-terminus of the cytokine before fusing the cytokine to the amino acids at the internal fusion site of the ISVD. Additionally, the peptide linker described herein may be added to the N-terminus and / or C-terminus of the cytokine before fusing the cytokine to the amino acids at the internal fusion site of the ISVD.

[0209] In one embodiment, the chimeric protein of the present invention is a continuous amino acid sequence. Therefore, in a preferred embodiment, in the chimeric protein of the present invention, the N-terminal sequence of the ISVD (located at the N-terminus of the internal fusion site) is linked to the original C-terminal portion of the cytokine (corresponding to the sequence located at the C-terminus of the internal fusion site of the cytokine) via a peptide linker, and the N-terminal portion of the original cytokine (corresponding to the sequence located at the N-terminus of the internal fusion site of the cytokine) is linked to the C-terminal sequence of the ISVD (located at the C-terminus of the internal fusion site of the ISVD) via a peptide linker and / or peptide bond to form a continuous amino acid sequence. Further details can be found in [link to further details]. Figure 2 and 32 .

[0210] Preferably, in the chimeric protein of the present invention, the tertiary structure of the ISVD and cytokines in the chimeric protein is maintained, except for the structure of the amino acids at the internal fusion sites connecting the ISVD and cytokines (if applicable). Therefore, in a preferred embodiment, the tertiary structure of the ISVD and cytokines in the chimeric protein is maintained, except for the structure of the amino acids at the internal fusion sites connecting the ISVD and cytokines (if applicable), compared to their tertiary structures when the ISVD and cytokines are not part of the chimeric protein. The tertiary structure may be partially maintained, such as maintaining at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% of the tertiary structure of the ISVD and / or cytokines, except for the structure of the amino acids at the internal fusion sites connecting the ISVD and cytokines (if applicable, i.e., if the cytokines are cyclically arranged and thus linked to the amino acids at the internal fusion sites of the ISVD and the cytokines, as described in detail above), compared to their tertiary structures when the ISVD and cytokines are not part of the chimeric protein.

[0211] The tertiary structure of a protein is its three-dimensional shape. Tertiary structure is primarily due to the interactions between the side chain groups of the amino acids that make up the protein. Side chain group interactions that contribute to tertiary structure include non-covalent interactions such as hydrogen bonding, ionic bonding, dipole-dipole interactions, and London dispersion forces. Equally important to tertiary structure are hydrophobic interactions, where amino acids with nonpolar hydrophobic side chain groups aggregate together inside the protein, leaving hydrophilic amino acids on the outside to interact with surrounding water molecules. Finally, disulfide bonds (covalent linkages between sulfur-containing side chains of cysteine) can also contribute to protein tertiary structure. Technologists are familiar with the techniques and methods used to determine the tertiary structure of proteins. For example, X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR), cryo-electron microscopy, or bipolar interferometry are tools that can be used to determine the tertiary structure of a given protein.

[0212] As explained in detail in WO 2019 / 086548, the manner in which ISVD and cytokines are fused in the chimeric proteins of this invention provides chimeras with more rigid, non-flexible connections. The classical linkage of polypeptide components (although typically linked in their native state) is carried out by directly or via peptide bonds linking their respective N-termini and C-termini to form a single, continuous polypeptide. These fusions are generally carried out via flexible linkers, or at least in a flexible manner, meaning that the fusion couples are not in a stable position or conformation relative to each other. As explained in WO 2019 / 086548… Figure 1As shown, fusion is readily achieved by linking proteins via N-terminus and C-terminus (a simple linear connection), but can be unstable, prone to degradation, and therefore unsuitable for therapeutic use in some cases. On the other hand, rigid chimeric / fusion proteins (with one or more fusion sites or connections within the primary topology of two or more proteins) as presented herein have at least one non-flexible fusion site (see [link to article]). Figure 1 As explained above, the chimeric protein of the present invention originates from the generation of a fusion between an ISVD and a cytokine, wherein the cytokine (preferably arranged in a circular pattern) disrupts the topology of the ISVD. Therefore, in one embodiment, the chimeric protein of the present invention is a continuous amino acid sequence, preferably obtained through gene fusion.

[0213] The embodiment provides a chimeric protein in which ISVD is fused with a cytokine (preferably circularly arranged) in such a way that the cytokine (preferably circularly arranged) “disrupts” the topology of the ISVD. Generally, the “topology” of a protein refers to the orientation of regular secondary structures relative to each other in three-dimensional space. Protein folding is primarily defined by the topology of the polypeptide chain (Orengo, C., Jones, D., and Thornton, J., “Protein superfamilies and domain superfolds”, Nature, 1994, 372:631-634). Therefore, at the most basic level, “primary topology” is defined as the sequence of secondary structural elements (SSEs) responsible for protein folding recognition motifs and thus for secondary and tertiary protein / domain folding. Thus, in terms of protein structure, the true or primary topology is the sequence of SSEs; that is, if one imagines being able to retain the N-terminus and C-terminus of a protein chain and pull them directly out, the topology will not change regardless of protein folding. Protein folding is then described as a tertiary topology, similar to the primary and tertiary structures of a protein (see also Martin AC., "The ups and downs of protein topology; rapid comparison of protein structure", Protein Eng. 2000, 13(12):829-37). Thus, by introducing cytokines (preferably arranged in a circular pattern), the ISVD contained in the chimeric protein of this invention is disrupted in its primary topology.

[0214] Novel chimeric proteins fuse in a unique manner to avoid the connection being a flexible, loose, fragile link / region within the chimeric protein structure. A convenient means of linking or fusing two polypeptides is to express them in a classically known manner as fusion proteins from recombinant nucleic acid molecules containing a first polynucleotide encoding a first polypeptide, which is operatively linked to a second polynucleotide encoding a second polypeptide. However, in the recombinant nucleic acid molecules of the present invention, the disruption of the ISVD topology by cytokines (preferably circularly arranged) is also reflected in the design of the gene fusion expressing the chimeric protein. Thus, in one embodiment, the chimeric protein is encoded by a chimeric gene formed by recombination of a portion of a gene encoding an ISVD and a portion of a gene encoding a cytokine, wherein the cytokine disrupts the primary topology of the encoded ISVD at one or more internal fusion sites of the ISVD via at least two or more direct fusions or fusions directly or through encoded peptide linkers. Thus, the polynucleotide encoding the polypeptide to be fused is fragmented and recombinated in such a manner to provide a chimeric protein that provides a rigid, non-flexible link, connection, or fusion between the proteins. The novel chimeric protein is prepared by fusing a cytokine with an ISVD in such a manner that the primary topology of the ISVD is disrupted. This means that the amino acid sequence of the antigen-binding domain is interrupted at the internal fusion site and linked to an amino acid in the cytokine. If the cytokine is circularly arranged, the amino acid sequence of the antigen-binding domain is interrupted at the internal fusion site and linked to an amino acid at the internal fusion site of the cytokine, which is also thus disrupted. In both the ISVD and the cytokine (if applicable), the internal fusion site is located in a loop or turn between two secondary structural elements, as described above.

[0215] Therefore, in the chimeric protein of the present invention in which ISVD is linked to cytokines, the amino acid of ISVD located at the N-terminus of the internal fusion site of ISVD is linked at its C-terminus to the N-terminus of the cytokine, and the C-terminus of the cytokine is linked to the amino acid present at the C-terminus of the internal fusion site of ISVD to form a continuous amino acid sequence. In embodiments in which the cytokines are arranged in a circular pattern (which is preferred, as explained above), such as, for example... Figure 1As shown in diagram 32, in the chimeric protein of the present invention, the N-terminus of the chimeric protein contains an ISVD amino acid sequence located at the N-terminus of the internal fusion site of the ISVD, followed by a (gene-fused) amino acid sequence of a circularly arranged cytokine sequence, followed by an ISVD amino acid sequence located at the C-terminus of the internal fusion site of the ISVD, wherein the circularly arranged cytokine sequence is provided as described herein, particularly wherein the circularly arranged cytokine is fused to the ISVD by linking the N-terminus of the amino acid at the C-terminus of the (cleaved) internal fusion site of the circularly arranged cytokine to the C-terminus of the amino acid at the N-terminus of the (cleaved) internal fusion site of the ISVD, and linking the amino acid at the N-terminus of the internal fusion site of the circularly arranged cytokine to the N-terminus of the amino acid at the C-terminus of the internal fusion site of the ISVD.

[0216] In a preferred embodiment, the N-terminal and C-terminal sequences before or after the internal fusion site of the ISVD, and / or the N-terminal and C-terminal sequences before or after the internal fusion site of a circularly arranged cytokine, respectively, correspond to at least a portion of the sequence of a loop or turn between two secondary structural elements of the corresponding internal fusion site (in the original sequence of the ISVD and / or the cytokine, if applicable). Thus, in this embodiment, the N-terminal and C-terminal sequences before or after the internal fusion site of the ISVD correspond to at least a portion of the sequence of a loop or turn (e.g., a β-turn) between two secondary structural elements (e.g., two β-chains) of the internal fusion site of the ISVD. Alternatively or additionally, the N-terminal and C-terminal sequences before or after the internal fusion site of the cytokine correspond to at least a portion of the sequence of a loop or turn between two secondary structural elements (e.g., two β-chains, or two α-helices, or one β-chain and one α-helice) of the internal fusion site of the cytokine. As described above, this correspondence can be “at least a portion of the sequence corresponding to a loop or turn”. This is because the chimeric protein may lack 0 to 10, preferably 0 to 5, more preferably 0 to 4, or even more preferably 0 to 3 (preferably consecutive) amino acid residues, such as 0, 1, 2, or 3, at the loops or turns of one or both proteins (i.e., ISVD and / or cytokines, if applicable). Additionally, as mentioned above, ISVD and cytokines (preferably arranged in a circular configuration) can be fused via peptide linkers. In other embodiments, the N-terminal and C-terminal amino acid sequences located at the internal fusion sites of ISVD and / or cytokines (if applicable) precisely correspond to the sequences at the loops or turns between the two secondary structural elements of the corresponding internal fusion sites in the original sequences of ISVD and / or cytokines (i.e., no missing amino acid residues at the loops or turns of one or both proteins in the chimeric protein).

[0217] In a further preferred embodiment, the N-terminus and C-terminus of the chimeric protein correspond to the N-terminus and C-terminus of the ISVD, respectively.

[0218] In one embodiment, the amino acid sequence (preferably a continuous amino acid sequence, as described above) of the chimeric protein of the present invention comprises:

[0219] (i) The N-terminal portion of the ISVD sequence; followed by

[0220] (ii) The sequence of cytokines; followed by

[0221] (iii) The remainder of the ISVD sequence (i.e., the C-terminal portion of the ISVD).

[0222] As described above, one or more amino acids from the N-terminus and / or C-terminus of the sequence located at the internal fusion site of the cytokine can be removed. Alternatively, a peptide linker can be added to the N-terminus and / or C-terminus of the sequence located at the internal fusion site of the cytokine.

[0223] In another embodiment, when the cytokines are arranged in a circular pattern, the amino acid sequence of the chimeric protein of the present invention (preferably a continuous amino acid sequence, as described above) comprises:

[0224] (i) The N-terminal portion of the ISVD; followed by

[0225] (ii) The sequence located at the C-terminus of the internal fusion site for obtaining circularly arranged cytokines; and

[0226] (iii) The remainder of the sequence located at the N-terminus of the internal fusion site for obtaining the circular arrangement of cytokines; followed by

[0227] (iv) The sequence of the ISVD located at the C-terminus of the internal fusion site of the ISVD (i.e., the C-terminal portion of the ISVD).

[0228] For further details, please refer to Figure 1 and 32 Starting from its N-terminus, the amino acid sequence of the chimeric protein first contains the N-terminal amino acid of ISVD (e.g., Figure 1The sequence begins with the β-chain A of the chimeric protein, followed by the C-terminus of the amino acid at the internal fusion site of the ISVD, which is linked to the N-terminus of the cyclically arranged cytokine (prepared by cleaving the sequence at the internal fusion site located at the turn or loop to form a novel N-terminus and C-terminus of the cyclically arranged cytokine compared to the original cytokine). The amino acid sequence of the chimeric protein then continues with the (remaining) sequence of the cyclically arranged cytokine, ending at its C-terminus (which corresponds to the amino acid at the N-terminus of the cytokine's internal fusion site, to design the cyclically arranged cytokine), and finally with the internal fusion site of the ISVD (located at the turn or loop, in this case at the β-turn, by…). Figure 1 The black line in the diagram indicates that the N-terminus of the amino acid located at the C-terminus is connected to the rest of the sequence of the ISVD (the C-terminal portion of the ISVD).

[0229] Therefore, as described herein, in the chimeric protein of the present invention, when the cytokines are arranged in a circular pattern, the primary amino acid sequence of the circularly arranged cytokines is inserted into the primary sequence of the ISVD (or the amino acid sequence of the circularly arranged cytokines interrupts the primary sequence (amino acid sequence) of the ISVD).

[0230] In some embodiments of the invention, fusion may be direct fusion or fusion via a linker peptide, the fusion site being designed to produce a rigid, non-flexible fusion protein. In addition to the location of the selected internal fusion site, the length and type of the linker peptide also contribute to the rigidity of the resulting chimeric protein. In the context of the invention, the polypeptides constituting the chimeric protein (see, for example, (i) to (iii) or (i) to (iv) above) are fused directly (by linking via peptide bonds) or indirectly to each other, whereby indirect coupling is achieved by assembling two polypeptides via short peptide linkers. Preferred "linker molecules," "linkers," or "short polypeptide linkers" are peptides of up to ten amino acids in length, more likely four amino acids, typically only three or four amino acids in length, but preferably only two or even more preferably only a single amino acid, to provide the desired rigidity for the connection of the fusion body at an accessible site. Non-limiting examples of suitable adapter sequences are described in Table A-1 and the Examples section. These sequences may be randomized, and adapters have been successfully selected to maintain a fixed distance between domains and / or maintain the independent function of the fusion partner (e.g., antigen binding and / or cytokine receptor binding), if this is desired by the chimeric protein of the present invention. Non-limiting examples of such adapters are GSGG (SEQ ID NO.: 120), GGSG (SEQ ID NO.: 121), or GSG (SEQ ID NO.: 5).

[0231] In embodiments involving the use of rigid linkers, these are generally known to exhibit unique conformations by employing an α-helical structure or by containing multiple proline residues. In many cases, they are more efficient at separating functional domains than flexible linkers, which may also be suitable, preferably short in length of only 1-4 amino acids.

[0232] Therefore, in a preferred embodiment, the ISVD and the cytokine are fused via at least one (preferably two) peptide linkers as defined above. In another preferred embodiment, the ISVD sequence located at the N-terminus of the internal fusion site of the ISVD is linked to a circularly arranged cytokine sequence via a peptide linker, and / or the N-terminal portion of the original cytokine is linked to a sequence located at the C-terminus of the internal fusion site of the ISVD via a peptide linker.

[0233] As also mentioned above, fusion between ISVD and cytokines can be achieved by first removing some amino acids from the internal fusion site of the ISVD and / or cytokines, provided that they are cyclic (or removing them from the N-terminus and / or C-terminus of the cytokines, provided that they are not cyclic).

[0234] Therefore, in one embodiment, the sequence at the N-terminus of the internal fusion site of the ISVD and the sequence of the (circular) cytokine, as well as the sequence at the C-terminus of the internal fusion site of the ISVD, are linked to each other (directly or via a linker, as defined above) by first removing 0 to 10, preferably 0 to 5, more preferably 0 to 3, such as 0, 1, 2, or 3 (continuous) amino acids from the N-terminal sequence of the (circular) cytokine, and then linking the N-terminal sequence of the internal fusion site of the ISVD to the adaptive N-terminus of the (circular) cytokine, and then to the sequence at the C-terminus of the internal fusion site of the ISVD, optionally via a peptide linker, as described above. Similarly, in another embodiment, the sequence at the N-terminus of the internal fusion site of the ISVD and the (circularly arranged) cytokine sequence and / or the sequence at the C-terminus of the internal fusion site of the ISVD are linked to each other (directly or via a linker, as defined above) by first removing 0 to 10, preferably 0 to 5, more preferably 0 to 3, such as 0, 1, 2, or 3 (continuous) amino acids from the sequence at the internal fusion site of the ISVD, and linking the sequence at the N-terminus of the internal fusion site of the ISVD to the (circularly arranged) cytokine, and then to the sequence located at the C-terminus of the internal fusion site of the ISVD, optionally via a peptide linker, as described above.

[0235] In one embodiment, the internal fusion site of the ISVD is located in an exposed region of the domain fold. This exposed region is identified as a less fixed amino acid segment, primarily located on the surface of the protein and at the edges of its structure. In a preferred embodiment, the internal fusion site is located in an exposed loop between two β-chains in the ISVD, for example, in an exposed turn, such as a β-turn as defined by IMGT, see below. More preferably, the internal fusion site of the ISVD is located at a β-turn, such as an exposed β-turn.

[0236] As mentioned above, the internal fusion sites of ISVDs are contained within loops or turns defined by IMGT (Lefranc MP, "Immunoglobulin and T Cell Receptor Genes: IMGT"). ® and the Birth and Rise of Immunoinformatics [Immunoglobulin and T-cell receptor gene: IMGT] ® [With the birth and rise of immunoinformatics], Front Immunol. [Fronts in Immunology], 2014, 5:22), preferably contained in the β-turn. Preferably, the cytokine (preferably circularly arranged) is inserted or fused to the amino acid of the internal fusion site of the ISVD, wherein the internal fusion site is located at:

[0237] a. The first β-turn connecting β-chains A and B of the ISVD; or

[0238] b. The β-turn connecting the β-chains C and C' of the ISVD; or

[0239] c. The β-turn connecting the β-chain C” and D of ISVD; or

[0240] d. The β-turn of the β-chains D and E connecting ISVD; or

[0241] e. The β-turn connecting the β chains E and F of ISVD.

[0242] In a preferred embodiment, the internal fusion site in the ISVD is located in the exposed region of the AB β-turn, which connects the A and B β-chains of the ISVD. Alternatively, the internal fusion site in the ISVD is located in the exposed region defined by the CC' β-turn, which connects the C and C' β-chains of the ISVD. Another embodiment includes internal fusion sites in the C”D β-turn or the EF β-turn. In practice, these are surface loops connecting β-chains A and B, C and C', C” and D, or E and F, respectively, forming a typical sandwich β-chain to provide immunoglobulin folding.

[0243] In the ISVD contained in the chimeric protein of the present invention, the CDR relates to the exposed region (loop or turn) between two secondary elements (see, for example...). Figure 1 In the case of ISVD, disruption of those sites used for fusion of ISVD with cytokines may result in loss of antigen-binding ability. In this case, as defined above, the ISVD contained in the chimeric protein of the present invention may no longer have the ability to specifically bind its antigen. If it is desirable to retain antigen-binding ability, CDR will not be the most suitable internal fusion site for ISVD.

[0244] Therefore, in one embodiment, the internal fusion site is located in an exposed region, loop, or turn, such that the CDR of the ISVD retains its ability to bind to the epitope of the target protein. Thus, in a preferred embodiment, the ISVD contained in the chimeric protein of the present invention is a functional ISVD, i.e., an ISVD that specifically binds to its antigen.

[0245] When arranged in a ring, the internal fusion site of the cytokine contained in the chimeric protein of the present invention is located in a turn or loop between two secondary elements of the cytokine, for example, in a β-turn, or between two β-chains, or between two α-helices, or in a loop between a β-chain and an α-helix. Preferably, the internal fusion site of the cytokine is located in a position within the protein that would result in altered cytokine-receptor binding or altered downstream activity of the cytokine-receptor and / or altered oligomerization of the receptor / receptor subunit upon cytokine binding and / or functional alteration of cytokine-receptor / receptor subunit binding of a chimeric molecule made by fusing ISVD at the internal fusion site of the cytokine. Preferably, the internal fusion site of the cytokine is located in an exposed region of a folded domain. The exposed region is identified as a less fixed amino acid segment that is primarily located on the surface and edges of the protein structure. In a preferred embodiment, the internal fusion site is an exposed loop or turn between two β-chains or between two α-helices in the cytokine. More preferably, the internal fusion site of the cytokine is a β-turn, such as an exposed β-turn. In another embodiment, the internal fusion site of the cytokine is located in a loop or turn between a β-chain and an α-helix.

[0246] In one embodiment, the chimeric protein (having two peptide bonds or two short linkers) is obtained by linking an ISVD to a cytokine via interrupting the primary topology of the ISVD at an internal fusion site located at an AB β turn in the sequence of the ISVD, by fusing with a looped cytokine at an internal fusion site located in an exposed region (turn or loop, as defined above) of its sequence (where the exposed or accessible site is not the original N-terminus or C-terminus of the cytokine, as explained above).

[0247] In another embodiment, the chimeric protein (having two peptide bonds or two short linkers) is obtained by linking an ISVD to a cytokine in a manner that interrupts the primary topology of the ISVD at an internal fusion site located at the AB β turn in the sequence of the ISVD by fusing with a non-circularly arranged cytokine, i.e., the fusion site of the ISVD fuses with the cytokine via the (original) N-terminus and C-terminus of the cytokine (through peptide linkers and / or by deleting one or more amino acids from the N-terminus and / or C-terminus of the cytokine, as explained above).

[0248] In one embodiment, the ISVD and cytokines contained in the chimeric protein are further linked via disulfide bonds. The disulfide bonds may be formed by cysteine ​​residues located within the ISVD, preferably near a turn or loop, particularly near the AB β turn, at the end of β-chain A, and / or at the end of β-chain G. In one embodiment, the ISVD and cytokines are further linked via disulfide bonds to improve the rigidity of the chimeric protein.

[0249] As described above, the cytokines present in the chimeric protein of the present invention are preferably cyclically arranged cytokines. In one embodiment, the N-terminus and C-terminus of the cytokines (i.e., the original N-terminus and C-terminus before cyclic arrangement) are linked together to generate cyclically arranged cytokines directly or via peptide linkers (such as GG or any other peptide linkers), as defined above and in Table A-1. Alternatively, in another embodiment, the cyclically arranged cytokines contained in the chimeric protein of the present invention are generated by linking the (original) N-terminus and C-terminus of the cytokines together by first removing 0 to 10, preferably 0 to 5, more preferably 0 to 3, such as 0, 1, 2, or 3 (continuous) amino acids from the N-terminus and / or C-terminus of the cytokines, and then linking the N-terminus and C-terminus (i.e., the original N-terminus and C-terminus of the cytokines before cyclic arrangement) directly or via peptide linkers, as defined above and in Table A-1.

[0250] In another embodiment, as described above, the cytokines present in the chimeric protein of the present invention are not arranged in a circular pattern as described herein. In this embodiment, the N-termini and C-termini of the cytokines (i.e., the original N-termini and C-termini, although one or more amino acids of the N-termini and / or C-termini may be removed, as explained above) are adjacent to each other. No It connects and is used to fuse cytokines (directly or via a linker, as explained above) to amino acids at the internal fusion site of the ISVD.

[0251] In a preferred embodiment, the cytokines included in the chimeric protein of the present invention are any type of cytokine, such as interleukins, chemokines, interferons, colony-stimulating factors (CSF), transforming growth factors, or tumor necrosis factors. Cytokines comprise a highly diverse superfamily of ligands, such as superfamilies of cytokines having conserved core domains or motifs based on or containing β-chains, thereby revealing internal fusion sites at their exposed regions, which are located in β-turns or loops interconnecting these β-chains. Therefore, the cytokines included in the chimeric protein of the present invention can be selected from:

[0252] -Interleukin-subfamily:

[0253] oIL-1 family

[0254] oIL-2 family

[0255] oIL-6 family

[0256] oIL-10 family

[0257] oIL-12 family

[0258] oIL-17 family

[0259] -Chemokine-Subfamily

[0260] oC

[0261] oCC

[0262] oCXC

[0263] oCX3C

[0264] - Colony-stimulating factor (CSF)

[0265] - Interferon

[0266] oI type IFN

[0267] oII type IFN

[0268] oIII type IFN

[0269] Transforming growth factor (TGF)

[0270] oα type

[0271] oβ type

[0272] -Tumor necrosis factor (TNF)

[0273] In a preferred embodiment, the cytokine is selected from interleukins or interferons. More preferably, the cytokine is IL-2, IFNA2a, or IL18.

[0274] Interleukin-2 (IL-2) (e.g., gene ID: 3558) is a member of the cytokine family (“IL-2 family”), each member of which has four α-helical bundles; the family also includes IL-4, IL-7, IL-9, IL-15, and IL-21. It is a 15.5–16 kDa tetra-α-helical bundle cytokine (see [link to relevant documentation]). Figure 2 This cytokine exerts its effects by binding to various IL-2 receptors (IL-2Rs), particularly monomeric, dimeric, and trimeric IL-2Rs (see, for example, Arenas-Ramirez, N. et al., “Interleukin-2: biology, design and application”, 2015, Trends in Immunology, 36(12):763-777). Figure 12-15 and Figure 1 The monomeric IL-2R containing IL-2α (CD25) is usually associated with the cell membrane, but it also exists in a soluble form and at a concentration of approximately 10. -8 M's low K d Binding to IL-2. The interaction of IL-2 with CD25 alone does not induce signaling. Instead, both the dimer and trimer IL-2R lead to downstream signaling upon binding to IL-2. The dimer IL-2R comprises IL-2Rβ (CD122) and IL-2Rγ [better referred to as the common γ chain (γ c ) or CD132], while the trimer IL-2R contains CD25, CD122 and γ c Considering only IL-2R with signal transduction capabilities, the dimer IL-2R can be described as having low affinity (K). d Approximately 10 -9 M)IL-2R, while the trimer IL-2R can be called high affinity (K)IL-2R. d Approximately 10 -11 M)IL-2R. At the molecular level, the affinity of a single trimer IL-2R for binding IL-2 is about 10-100 times that of a single dimer IL-2R (from Arenas-Ramirez, N. et al., “Interleukin-2:biology, design and application”, 2015, Trends in Immunology, 36(12):763-777).

[0275] Human interferon alpha-2 (IFNA2a) (e.g., gene ID: 3440) is a cytokine belonging to the type I IFN family. The mature protein is composed of 165 amino acids. The secondary structure of IFNA2a consists of five α-helices A through E from the N-terminus to the C-terminus. Helices A, B, C, and E are organized into a bundle containing a long loop (AB loop) between helices A and B, and two disulfide bonds that link helice E to the AB loop and helice C to the N-terminus. The type I IFN receptor (IFNAR) consists of two subunits, IFNAR1 and IFNAR2, see, for example... Figure 24-27 .

[0276] Interleukin-18 (IL-18) (e.g., gene ID: 3606) belongs to the IL-1 superfamily and folds into a fully β-sheet molecule; see, for example... Figure 30 The “IL-1 receptor-type interleukins” superfamily or “IL-1 family” interleukins, used interchangeably in this document, include, for example, interleukins IL-1α, IL-1β, IL-1Ra, IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-36Ra, IL-37, and IL-38. These cytokines are interconnected through origin, receptor structure, and signal transduction pathways. The receptors of IL-1 superfamily interleukins share a similar architecture, consisting of three lg-like domains in the extracellular domain and an intracellular Toll / IL-1 R (TIR) ​​domain, also found in Toll-like receptors. Initiation of cytokine signaling requires two receptors: a primary specific receptor and a co-receptor, which can be shared in some cases. The primary receptor is responsible for binding specific cytokines, while the co-receptor itself does not bind cytokines but associates with a pre-assembled binary complex derived from the cytokine and the primary receptor. Cytokines bind to their corresponding receptors to form a signal transduction ternary complex, which leads to the dimerization of the TIR domains of the two receptors. This initiates intracellular signal transduction by activating mitogen-activated protein kinase (MAPK) and activating nuclear factor-κB light chain enhancer (NF-κB) in B cells. Signal transduction induces inflammatory responses, such as induction of cyclooxygenase type 2, increased expression of adhesion molecules, and nitric oxide synthesis.

[0277] IL-18 first binds to the IL-18α receptor, forming a low-affinity complex. Upon binding to the IL-18β receptor, a higher-affinity heterotrimeric complex is formed, which initiates the signal transduction process.

[0278] The three-dimensional structures of several interleukin cytokines in the IL-1 superfamily have been determined, and it has been demonstrated that, despite limited sequence similarity, these cytokines employ a conserved characteristic β-trefoil fold containing 12 antiparallel β-chains arranged in a triplet symmetry pattern, see, for example... Figure 30 The β-barrel core motif is wrapped in varying amounts of helices in each cytokine structure. The stacking of Cα atoms for each human cytokine reveals a conserved hydrophobic core with remarkable flexibility in the loop regions. Surface residues and loops between the three chains do not appear to be critical to overall stability and exhibit significant divergence among cytokines, consistent with their low sequence similarity and partly explaining their unique recognition by their respective receptors (involving specific loops). For example, human IL-18 shares 65% sequence identity with mouse IL-18, but only 15% and 18% with human IL-1α and human IL-1β, respectively. However, IL-18 shows striking similarity in its three-dimensional structure to other IL-1 cytokines. Thus, this IL-1-like receptor, interleukin, provides an example of a superfamily within the cytokine family with a conserved core domain based on s-chains interconnected by flexible β-turns or loops, some of which are involved in receptor recognition, while others may be involved in linking to folded scaffold proteins as presented herein to obtain novel, expanded fusion ligands.

[0279] In one embodiment, the cytokine included in the chimeric protein of the present invention is not erythropoietin (EPO), such as human EPO (hEPO). In another embodiment, the cytokine is not a granulocyte colony-stimulating factor, such as human granulocyte colony-stimulating factor (hGCSF). In yet another embodiment, the cytokine is neither hEPO nor hGCSF.

[0280] As described below, in the chimeric protein of the present invention, the cytokines contained therein may be functional (because they retain their receptor-binding functionality in a similar manner compared to cytokines not fused with ISVD) or non-functional (because they do not retain their receptor-binding functionality in a similar manner compared to cytokines not fused with ISVD, as described above in the context of ISVD). Furthermore, the receptor-binding functionality of the cytokines contained in the chimeric protein of the present invention can be enhanced by their fusion with ISVD. adjust As described in detail below. Furthermore, compared to the oligomerization of cytokine receptors / receptor subunits upon binding when the chimeric protein does not contain cytokines (i.e., if the cytokine is not fused to the ISVD by two peptide bonds), the oligomerization of cytokine receptors / receptor subunits upon binding when cytokines are included in the chimeric protein may be affected / altered. Additionally, cytokine signaling can be modulated using cytokines contained in the chimeric protein of this invention.

[0281] Therefore, compared to cytokines not fused with ISVD, the cytokines contained in the chimeric protein of the present invention may... No Its receptor-binding functionality is preserved in a similar manner. This means that cytokines included in the chimeric protein of the present invention can bind to their receptors with better specificity and / or higher affinity compared to cytokines not fused with ISVD. It also means that cytokines included in the chimeric protein of the present invention can bind to their receptors with lower specificity and / or lower affinity compared to cytokines not fused with ISVD. It also means that the downstream signaling of the cytokine receptor when bound by cytokines present in the chimeric protein of the present invention may differ from that when cytokines not fused with ISVD bind. It also means that the oligomerization of receptor / receptor subunits when cytokines bound by cytokines present in the chimeric protein of the present invention may be affected (e.g., it may differ from the oligomerization of receptor / receptor subunits when cytokines not fused with ISVD bind).

[0282] Therefore, by fusing cytokines with ISVD in the chimeric protein of the present invention, the receptor-binding functionality of cytokines contained in the chimeric protein of the present invention can be modulated (e.g., improved or worsened, or simply altered), as described in detail below.

[0283] In one embodiment, if the cytokine is an interleukin and it is arranged in a circular pattern, the internal fusion site of the cytokine may be a β-turn of the interleukin β-barrel core motif, as described above.

[0284] In one embodiment, the chimeric protein comprises an anti-GFP ISVD, preferably comprising an ISVD containing a sequence as defined in SEQ ID NO.: 1 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 1.

[0285] In another embodiment, the chimeric protein comprises an ISVD that acts as a half-life extension portion or has half-life extension properties.

[0286] As used herein, the term "half-life" can generally be defined as described in paragraph o) on page 57 of WO 2008 / 020079, and as mentioned therein, refers to the time taken for the serum concentration of a compound or polypeptide in vivo to decrease by 50%, for example, due to degradation of the sequence or compound and / or clearance or chelation of the sequence or compound by natural mechanisms. The in vivo half-life of the protein-based carrier building blocks and / or molecules of the present invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and can be, for example, generally described as in paragraph o) on page 57 of WO 2008 / 020079. As also mentioned in paragraph o) on page 57 of WO 2008 / 020079, half-life can be measured using t 1 / 2 -α、t 1 / 2 The half-life is expressed using parameters such as -β and the area under the curve (AUC). In this regard, it should be noted that, as used herein, the term "half-life" specifically refers to t... 1 / 2 -β or terminal half-life (where t) 1 / 2 -α and / or AUC or both may be disregarded). See, for example, standard manuals such as Kenneth, A et al.: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). Also see “Pharmacokinetics”, M. Gibaldi and D. Perron, by Marcel Dekker, 2nd revised edition (1982). Similarly, the term “increased half-life” or “increased half-life” is also defined as in paragraph o) on page 57 of WO 2008 / 020079, and specifically refers to t 1 / 2 -β increases, and there is or is no t 1 / 2 -Increases in α and / or AUC or both.

[0287] In vivo half-life can be prolonged by increasing the hydrodynamic radius (size) or reducing molecule clearance. In vivo half-life extension components (such as binding units that can bind, for example, serum albumin) increase the half-life of the molecules they are attached to by binding, for example, serum albumin. Albumin is the most abundant plasma protein, is highly soluble, very stable, and has a particularly long circulating half-life due to its size and as a direct result of its interaction with the FcRn-mediated recycling pathway, see, for example, Sleep D. et al., “Albumin as a versatile platform for drug half-life extension”, BiochimBiophys Acta, 2013, 1830(12):5526-34.

[0288] For example, WO 2004 / 041865 describes an ISVD that binds to serum albumin (and particularly HSA), which can be used to increase the half-life of the chimeric protein or polypeptide of the present invention.

[0289] International application WO 2006 / 122787 (the contents of which are incorporated herein by reference) describes a number of ISVDs targeting (human) serum albumin. These ISVDs include those known as Alb-1 (SEQ ID NO: 52 in WO 2006 / 122787) and its humanized variants (such as Alb-8 (SEQ ID NO: 62 in WO 2006 / 122787)). Similarly, these can be used to extend the half-life of therapeutic proteins and peptides, as well as other entities or portions (such as the chimeric proteins or peptides of this invention).

[0290] WO 2012 / 175400 (the contents of which are incorporated herein by reference) describes a further improved version of Alb-1, called Alb-23.

[0291] In one embodiment, the chimeric protein or polypeptide of the present invention comprises a serum albumin-binding moiety selected from the following: Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10 (described in WO 2006 / 122787), and Alb-23. In one embodiment, the serum albumin-binding moiety is Alb-8 or Alb-23 or a variant thereof, as shown on pages 7-9 of WO 2012 / 175400. In one embodiment, the serum albumin-binding portion is selected from albumin binders described in WO 2012 / 175741, WO 2015 / 173325, WO 2017 / 080850, WO 2017 / 085172, WO 2018 / 104444, WO 2018 / 134235 and WO 2018 / 134234, the contents of which are incorporated herein by reference. Some serum albumin binders are also shown in Table 3 below.

[0292] In one embodiment, the chimeric protein or polypeptide of the present invention comprises the serum albumin-binding moiety Alb23 (SEQ ID NO: 123) as defined in Table 3 below. In a preferred embodiment, the molecule of the present invention comprises the serum albumin-binding moiety Alb23002 (SEQ ID NO.: 55) as defined in Table 3 below. In another preferred embodiment, the molecule of the present invention comprises the serum albumin-binding moiety Alb23002(E1D) (SEQ ID NO.: 137) as defined in Table 3 below.

[0293] Table 3: Serum albumin-bound ISVD sequences (“ID” refers to SEQ ID NO as used herein)

[0294]

[0295] In one embodiment, the molecule of the present invention comprises the HLE portion as described in item A below:

[0296] A.ISVD, which is combined with HSA and contains

[0297] i. CDR1 as the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence that differs from SEQ ID NO: 138 by 2 or 1 amino acid;

[0298] ii. CDR2 as the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence differing from SEQ ID NO: 139 by 2 or 1 amino acids; and

[0299] iii. CDR3 as the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that differs from SEQ ID NO: 140 by 2 or 1 amino acids.

[0300] In one embodiment, the ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 138, CDR2 as the amino acid sequence of SEQ ID NO: 139, and CDR3 as the amino acid sequence of SEQ ID NO: 140.

[0301] Instances of such ISVDs that bind to HSA have one or more or all of the frame regions (other than the CDRs as defined in item A above) indicated for construct ALB23002 (SEQ ID NO: 55) as shown in Tables 4 and 5. In one embodiment, it is an ISVD containing the complete amino acid sequence of construct ALB23002 (SEQ ID NO: 55) or composed of it.

[0302] Table 4: Sequence of CDRs and frame annotations based on AbM CDRs (“ID” refers to the given SEQ ID NO)

[0303]

[0304] Table 5: CDR sequences based on Kabat CDR and frame annotations (“ID” refers to the given SEQ ID NO)

[0305]

[0306] Project A' can also be described using the Kabat CDR definition as follows:

[0307] A'.ISVD, which combines with HSA and contains

[0308] i. CDR1 as the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence that differs from SEQ ID NO: 146 by 2 or 1 amino acids;

[0309] ii. CDR2 as the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence differing from SEQ ID NO: 148 by 2 or 1 amino acid; and

[0310] iii. CDR3 as the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that differs from SEQ ID NO: 140 by 2 or 1 amino acids.

[0311] In one embodiment, the ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 146, CDR2 as the amino acid sequence of SEQ ID NO: 148, and CDR3 as the amino acid sequence of SEQ ID NO: 140.

[0312] Instances of such ISVDs that bind to HSA have one or more or all of the frame regions (other than the CDRs as defined in item A' above) as indicated in Table 5 for construct ALB23002. In one embodiment, it is an ISVD containing the complete amino acid sequence of construct ALB23002 (SEQ ID NO: 55, see also Table 5) or composed thereof.

[0313] Similarly, in another embodiment, the amino acid sequence of the ISVD bound to HSA may have more than 90%, such as more than 95% or more than 99%, sequence identity with SEQ ID NO: 55, wherein the CDR is as defined in item A or A' above. In one embodiment, the ISVD bound to HSA comprises or is composed of the amino acid sequence of SEQ ID NO: 55.

[0314] When such an ISVD that binds to HSA has a difference of 2 or 1 amino acid in at least one CDR relative to the corresponding reference CDR sequence (item A or A' above), the ISVD has at least half or at least the same binding affinity to HSA compared to the construct ALB23002 (SEQ ID NO: 55), wherein the binding affinity is measured using the same method (such as SPR).

[0315] In one embodiment, when such an ISVD binding to HSA has a C-terminal position, it exhibits C-terminal extension, such as a C-terminal alanine, cysteine, or glycine extension. In one embodiment, such an ISVD is selected from SEQ ID Nos: 124, 125, 127, 129, 130, 131, 132, 133, 134, and 55 (see Table 3 above). In another embodiment, the ISVD binding to HSA has a position other than the C-terminal position (i.e., not a C-terminal ISVD of the molecule of the present invention). In one embodiment, such an ISVD is selected from SEQ ID Nos: 55, 122, 123, 136, 128, and 137 (see Table 3 above).

[0316] In one embodiment, the one or more other groups, residues, portions, or binding units that provide the molecule with an increased half-life are peptides that can bind to HSA.

[0317] Specifically, "serum albumin-binding polypeptide or binding domain" can be any suitable serum albumin-binding peptide that can increase the half-life of the molecule (preferably T). 1 / 2 β, as defined above (compared to the same molecule that does not have a serum albumin-binding peptide or binding domain).

[0318] In particular, polypeptide sequences suitable for prolonging serum half-life are polypeptide sequences that can bind to serum proteins with long serum half-lives (such as serum albumin, transferrin, IgG, etc., especially human serum albumin (HSA)).

[0319] Peptide sequences capable of binding to serum albumin have been previously described, and in particular can be serum albumin-binding peptides as described in WO 2008 / 068280 (and especially WO 2009 / 127691 and WO 2011 / 095545) (the contents of which are incorporated herein by reference).

[0320] In another embodiment, the chimeric protein of the present invention comprises an ISVD that acts as a half-life extension portion or has half-life extension properties, such as an anti-HSA ISVD, as described above, preferably comprising an ISVD containing a sequence as defined in SEQ ID NO.: 55, or a sequence as defined in SEQ ID NO: 154, or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 55 or SEQ ID NO: 154.

[0321] In alternative embodiments, the chimeric protein of the present invention comprises ISVD, which includes the CDR1, 2 and 3 sequences of SEQ ID NO: 154, as defined for CDR1 in SEQ ID NO: 151, for CDR2 in SEQ ID NO: 152 and for CDR3 in SEQ ID NO: 153 (disclosed as an HSA binder in WO 2019 / 016237 A1).

[0322] In one embodiment, the chimeric protein comprises a cytokine, which is IL-2, preferably comprising a cytokine having a sequence (IL-2) as defined in SEQ ID NO.: 2 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 2.

[0323] In another embodiment, the chimeric protein comprises a cytokine containing a sequence as defined in SEQ ID NO.: 3 (IL-2(K35E,C125S)) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 3.

[0324] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 4 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[S75-Q74]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 4.

[0325] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise sequences as defined in SEQ ID NO.: 172 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[L17-L14]) or sequences having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 172.

[0326] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 173 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[P34-Y31]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 173.

[0327] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 174 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[F42-M39]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 174.

[0328] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 175 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[M46-F42]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 175.

[0329] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 176 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[E62-L59]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 176.

[0330] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 177 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[S75-N71]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 177.

[0331] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 178 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[N77-S75]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 178.

[0332] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 179 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[F78-Q74]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 179.

[0333] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 180 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[L85-P82]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 180.

[0334] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 181 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[T101-G98]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 181.

[0335] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 182 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[T102-E100]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 182.

[0336] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 183 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[F103-S99]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 183.

[0337] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 184 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[L132-I129]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 184.

[0338] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 185 (IL-2 (K35E,C125S) without the first four amino acids) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 185.

[0339] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 186 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[K35-K32]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 186.

[0340] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 187 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[I92-I89]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 187.

[0341] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 188 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[L96-V93]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 188.

[0342] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 189 (circularly arranged IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[S4-T133]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO.: 189.

[0343] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 262 (circularly arranged IL-2, referred to as IL-2[L132-I129]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 262.

[0344] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 264 (circularly arranged IL-2, referred to as IL-2[F42-M39]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 264.

[0345] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 268 (circularly arranged IL-2, referred to as IL-2[S75-N71]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 268.

[0346] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 270 (circularly arranged IL-2, referred to as IL-2[T102-E100]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 270.

[0347] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 272 (circularly arranged IL-2, referred to as IL-2[F103-S99]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 272.

[0348] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 274 (circularly arranged IL-2, referred to as IL-2[L85-P82]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 274.

[0349] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 190 (IL-2 in TP072) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 190.

[0350] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 191 (IL-2 in TP075) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 191.

[0351] In another embodiment, the chimeric protein comprises a cytokine, which is IFNA2a, preferably comprising a sequence as defined in SEQ ID NO.: 56 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 56.

[0352] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 58 (circularly arranged IFNA2a, referred to as IFNA2a[D77-W76]V2) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 58.

[0353] In another embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 59 (circularly arranged IFNA2a, referred to as IFNA2a[D77-W76]V4) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 59.

[0354] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 192 (IFNA2a in TP093) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 192.

[0355] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 193 (IFNA2a in TP095) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 193.

[0356] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 194 (IFNA2a in TP098) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 194.

[0357] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 195 (IFNA2a in TP109) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 195.

[0358] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 197 (IFNA2a in TP089) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 197.

[0359] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 199 (IFNA2a in TP090) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 199.

[0360] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 201 (IFNA2a in TP091) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 201.

[0361] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 203 (IFNA2a in TP092) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 203.

[0362] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 205 (IFNA2a in TP095) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 205.

[0363] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 207 (IFNA2a in TP096) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 207.

[0364] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 209 (IFNA2a in TP097) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 209.

[0365] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 211 (IFNA2a in TP099) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 211.

[0366] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 213 (IFNA2a in TP100) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 213.

[0367] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 215 (IFNA2a in TP101) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 215.

[0368] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 217 (IFNA2a in TP102) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 217.

[0369] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 220 (IFNA2a in TP104) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 220.

[0370] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 222 (IFNA2a in TP105) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 222.

[0371] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 225 (IFNA2a in TP107) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 225.

[0372] In one embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 227 (IFNA2a in TP108) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 227.

[0373] In one embodiment, the chimeric protein comprises a cytokine, IL-18, preferably a cytokine comprising a sequence as defined in SEQ ID NO.:64 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.:64.

[0374] In another embodiment, the chimeric protein comprises a cytokine, IL-18, preferably a cytokine comprising a sequence as defined in SEQ ID NO.:66 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.:66.

[0375] In another embodiment, the chimeric protein comprises a cytokine, IL-18, preferably a cytokine comprising a sequence as defined in SEQ ID NO.:68 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.:68.

[0376] In another embodiment, the chimeric protein comprises a cytokine, IL-18, preferably a cytokine comprising a sequence as defined in SEQ ID NO.:70 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.:70.

[0377] In another embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 244 (circularly arranged IL-18, referred to as IL18[K79-N78]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 244.

[0378] In another embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 245 (circularly arranged IL-18, referred to as IL18[Q56-S55]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 245.

[0379] In another embodiment, the circularly arranged cytokines contained in the chimeric protein of the present invention comprise a sequence as defined in SEQ ID NO.: 246 (circularly arranged IL-18, referred to as IL18[P57-Q56]) or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity with SEQ ID NO.: 246.

[0380] In one embodiment, the chimeric protein of the present invention is selected from proteins comprising or composed of the following sequences: sequences as defined in SEQ ID NO.: 7-25, 36-54, 60-63, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218-219, 221, 223-224, 226, 230-237, 261, 263, 265-267, 269, 271, or 273, or sequences related to SEQ ID NO.: The sequences 7-25, 36-54, 60-63, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218-219, 221, 223-224, 226, 230-237, 261, 263, 265-267, 269, 271 or 273 have at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity.

[0381] In another embodiment, the chimeric protein of the present invention comprises an ISVD fused to a cytokine (preferably circularly arranged), wherein the cytokine comprises or consists of the following sequences: as defined in SEQ ID NO.: 4, 58, 59, 66, 68, 70, 172-195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 220, 222, 225, 227, 244-246, 262, 264, 268, 270, 272, or 274, or sequences associated with SEQ ID NO.: 4, 58, 59, 66, 68, 70, 172-195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 220, 222, 225, 227, 244-246, 262, 264, 268, 270, 272 or 274 have a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 97% identity.

[0382] The polypeptide of the present invention

[0383] The present invention further provides polypeptides comprising the chimeric protein of the present invention. In addition to the chimeric protein, the polypeptides of the present invention may also comprise other groups, residues, portions, or binding units.

[0384] For example, such additional groups, residues, portions, or binding units may be one or more additional immunoglobulins to form a (fusion) protein or (fusion) polypeptide (the polypeptide of the present invention). In a preferred but non-limiting aspect, one or more additional groups, residues, portions, or binding units are ISVDs. Even more preferably, one or more additional groups, residues, portions, or binding units are selected from the group consisting of: domain antibodies, ISVDs suitable for use as domain antibodies, single-domain antibodies, ISVDs suitable for use as single-domain antibodies, "dAb", ISVDs suitable for use as dAbs, V HH Humanized V HH Camel source V H or Nanobody ® V HH Alternatively, such groups, residues, portions, or binding units may be, for example, chemical groups, residues, or portions that may or may not have biological and / or pharmacological activity. For example, but not limited to, such groups may be linked to one or more domains of the polypeptide of the present invention to provide a “derivative” of the polypeptide of the present invention, as further described herein. The polypeptide of the present invention may also include additional groups having certain functional groups, such as labels, toxins, one or more linkers, binding sequences, etc. These additional functional groups include both amino acid-based groups and non-amino acid-based groups.

[0385] Therefore, in one embodiment, the polypeptide of the present invention may further comprise (in addition to the chimeric protein of the present invention) one or more ISVDs. Preferably, the ISVD may be an HLE ISVD, a targeted ISVD, or a therapeutic ISVD. Thus, one or more ISVDs that may be further included in the polypeptide of the present invention (in addition to the ISVDs included in the chimeric protein) may form a “multivalent” or “multispecific” polypeptide or construct.

[0386] A polypeptide containing two or more ISVDs (such as the ISVD contained in a chimeric protein and one or more additional ISVDs) will be referred to herein as a “multivalent polypeptide” or “multivalent construct”, and these may provide certain advantages compared to their corresponding monovalent polypeptides. Generally, a protein or polypeptide containing a single ISVD (such as the chimeric protein of the present invention) will be referred herein as a “monovalent” protein or polypeptide or “monovalent construct”.

[0387] As described herein, the multivalent peptides of the present invention may be, for example, but not limited to, multispecific (such as bispecific or trispecific) or multicomplementary (such as bicomplementary) constructs (or constructs that are both multicomplementary and multispecific), and may be, for example, constructs comprising at least two binding domains or binding units each targeting different epitopes on the same subunit, constructs comprising at least two binding domains or binding units each having different biological functions (e.g., a binding domain that can block or inhibit receptor-ligand interactions, and a binding domain that does not block or inhibit receptor-ligand interactions), or constructs comprising at least two binding domains or binding units each targeting different targets.

[0388] For a general description of multivalent and multispecific polypeptides containing one or more ISVDs and their preparation, see also Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology, 74 (2001), 277-302; and, for example, WO 96 / 34103, WO 99 / 23221, WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO 2009 / 068627.

[0389] It should be understood that the terms “peptide construct” and “peptide” are used interchangeably in this document (unless the context clearly specifies otherwise).

[0390] The polypeptides of the present invention can generally be prepared by a method comprising at least one of the following steps: directly or suitably linking the chimeric protein of the present invention to one or more additional groups, residues, portions or binding units, as described herein.

[0391] The polypeptides of the present invention can also be prepared by a method generally comprising at least the following steps: providing a nucleic acid encoding the polypeptide of the present invention, expressing the nucleic acid in a suitable manner, and recovering the expressed polypeptide of the present invention. Such methods can be carried out in ways known per se, which will be apparent to those skilled in the art, for example based on the methods and techniques further described herein.

[0392] It should be understood that the order of the chimeric protein and other groups, residues, portions, or binding units (if present) in the polypeptide of the present invention, such as the order of a first domain (e.g., the chimeric protein of the present invention), a second binding domain (e.g., an ISVD binding HSA), a third binding domain (e.g., a domain binding to a therapeutically relevant target), etc. (i.e., the orientation or configuration of the chimeric protein and other groups, residues, portions, or binding units (if present)), can be selected according to the needs of those skilled in the art and the relative affinities, which may depend on the position of the chimeric protein and other groups, residues, portions, or binding units (if present) in the polypeptide. Whether the polypeptide includes one or more linkers to interconnect the chimeric protein and optionally other groups, residues, portions, or binding units is a matter of design choice. However, some orientations with or without linkers may provide preferred binding characteristics compared to other orientations. The present invention covers all different possible orientations.

[0393] For example, the sequence of the polypeptide of the present invention may include one or more ISVDs (linked together directly or via a linker as defined herein), followed by the chimeric protein of the present invention, which is linked directly or via a linker as defined herein to the one or more ISVDs. In this embodiment, the one or more ISVDs will be located in the N-terminal region of the polypeptide, and the chimeric protein will be located in the C-terminal region of the polypeptide.

[0394] For example, the sequence of the polypeptide of the present invention may include the chimeric protein of the present invention, followed by one or more ISVDs (directly or via adapters) that are directly or via adapters to the chimeric protein of the present invention. In this embodiment, one or more ISVDs will be located in the C-terminal region of the polypeptide, and the chimeric protein will be located in the N-terminal region of the polypeptide.

[0395] For example, the sequence of the polypeptide of the present invention may include one or more ISVDs (directly or via adapters) that are directly or via adapters linked to the chimeric protein of the present invention, followed by another one or more ISVDs (directly or via adapters). Thus, in this embodiment, the chimeric protein of the present invention will have one or more ISVDs side-attached to the N-terminal and C-terminal regions of the polypeptide.

[0396] The use of linkers to connect two or more (multi)peptides is well known in the art. One commonly used class of peptide linkers is called “Gly-Ser” or “GS” linkers. These are linkers that are essentially composed of glycine (G) and serine (S) residues and typically contain one or more repeats of a peptide motif, such as the GGGGS (SEQ ID NO: 155) motif (e.g., shown as (Gly-Gly-Gly-Gly-Ser)). n , where n can be 1, 2, 3, 4, 5, 6, 7 or greater). Some common examples of such GS connectors are 9GS connectors (e.g., GGGGSGGGS, SEQ ID NO: 156), 15GS connectors (n = 3), and 35GS connectors (n = 7). See, for example, Chen et al., Adv. Drug Deliv. Rev., 2013, 65(10):1357-1369; and Klein et al., Protein Eng. Des. Sel., 2014, 27(10): 325-330. In certain but non-limiting embodiments, the connectors are selected from the group consisting of connectors of GSG, GSGG, GGSG, 3A, 3GS, 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, and 35GS, see Table A-1.

[0397] Table A-1: ​​Connector Sequences (“ID” refers to SEQ ID NO as used herein)

[0398]

[0399] For example, the polypeptides of the present invention may additionally comprise groups, residues, portions, or binding units that provide the chimeric protein / polypeptide of the present invention with an increased (in vivo) half-life compared to a corresponding chimeric protein / polypeptide lacking one or more of said other groups, residues, portions, or binding units (“(in vivo) half-life extended portion” or “half-life extended (HLE) portion”). Therefore, in this embodiment, if the ISVD contained in the chimeric molecule of the present invention is an HLE portion, then the (multivalent and multispecific) polypeptide of the present invention may comprise at least two HLE portions: the ISVD contained in the chimeric molecule and at least one additional HLE portion. The HLE portions contained in the polypeptide of the present invention may be the same or different.

[0400] As used herein, the term "half-life" is defined in the context of chimeric proteins of the present invention and is equally applicable to this embodiment. In one embodiment, an additional binding unit (other than the chimeric protein) contained in the polypeptide of the present invention is an ISVD, such as an ISVD that binds HSA. ISVDs that bind HSA are defined in the context of chimeric proteins of the present invention and are equally applicable to this embodiment.

[0401] Additional HLE portions that may be included in the polypeptides of the present invention are HLE portions such as polyethylene glycol or ELNN polypeptides, which increase the size of the molecules to which they are attached, thus bypassing renal clearance and thereby increasing the half-life of those molecules.

[0402] The types of HLE groups, residues, portions, or binding units are generally unrestricted and may be selected, for example, from the group consisting of: polyethylene glycol (PEG) molecules, ELNN polypeptides or fragments thereof as described above, serum proteins or fragments thereof, binding units that can bind to serum proteins (such as ISVDs that bind to HSA, as described above), Fc portions, and small proteins or peptides that can bind to serum proteins.

[0403] The polypeptides of the present invention may additionally comprise (in addition to chimeric proteins) one or more targeting moieties. As defined herein, a “targeting moiety” is any group, residue, portion, or binding unit that can be directed by its binding to a target. “(Specific) binding”, “can (specifically) bind” to a specific antigenic determinant, epitope, antigen, or protein, or a specific non-protein molecule such as nucleic acid (such as DNA or RNA) or glycan (or at least a portion, fragment, or epitope thereof), an amino acid sequence that “has an affinity” for and / or “has specificity” for the aforementioned substances (e.g., ISVD, antibody, antigen-binding domain, or fragment, such as V...). HH Domain or V H / V L A domain (or generally an antigen-binding protein or polypeptide or fragment thereof) is referred to as “pair” or “target” of the antigenic determinant, epitope, antigen, protein, or non-protein molecule. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined by any suitable means known per se, including, for example, Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays, and various variants thereof known in the art; as well as other techniques mentioned herein.

[0404] Furthermore, the polypeptides of the present invention may additionally comprise one or more therapeutic moieties. As defined herein, a “therapeutic moiety” is any group, residue, portion, or binding unit capable of exerting therapeutic activity in animals and / or humans. The therapeutic moieties may also be in the form of a precursor, which is then activated to exert its therapeutic activity. A non-limiting example of a therapeutic moieties that may be present in the polypeptides of the present invention is a programmed death-ligand 1 (PD-L1) binding molecule.

[0405] The nucleic acid of the present invention

[0406] The present invention further provides a nucleic acid molecule encoding the chimeric protein and / or the polypeptide of the present invention.

[0407] Nucleic acids can be used to transform / transfect host cells or host organisms, for example, for the expression and / or production of polypeptides. Suitable (non-human) hosts or host cells for production purposes will be clear to those skilled in the art and can be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, prokaryotic or eukaryotic organism. This invention also covers hosts or host cells comprising nucleic acids encoding the chimeric proteins and / or polypeptides of this invention.

[0408] Nucleic acid can be, for example, DNA, RNA, or a hybrid thereof, and may also contain (e.g., chemically modified) nucleotides, such as PNA. It can be single-stranded or double-stranded. In one embodiment, it is in the form of double-stranded DNA. For example, the nucleotide sequence of the present invention can be genomic DNA or cDNA.

[0409] The nucleic acids of the present invention can be prepared or obtained in a manner known per se, and / or isolated from suitable natural sources. Nucleotide sequences encoding naturally occurring (poly)peptides can, for example, be subjected to site-directed mutagenesis to provide nucleic acid molecules encoding polypeptides with sequence variations. Furthermore, it will be apparent to those skilled in the art that, in order to prepare nucleic acids, several nucleotide sequences (such as at least one nucleotide sequence encoding a target moiety) and nucleic acids, for example, encoding one or more adapters, can be linked together in a suitable manner.

[0410] The techniques used to generate nucleic acids will be clear to a technician and may include, for example, but not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more portions thereof); introducing mutations that result in the expression of truncated expression products; introducing one or more restriction enzyme sites (e.g., to form boxes and / or regions that can be easily digested and / or linked using appropriate restriction enzymes) and / or introducing mutations through PCR reactions using one or more “mismatched” primers.

[0411] In another embodiment, a chimeric gene having at least a promoter, a nucleic acid molecule encoding a chimeric protein, and a 3' terminal region containing a transcription termination signal is described. Another embodiment relates to an expression cassette encoding the chimeric protein of the present invention or containing a nucleic acid molecule or chimeric gene encoding a chimeric protein. In some embodiments, the expression cassette is used in a generic form as an immune library containing a large set of ISVDs to select the most suitable binder for the target (if an ISVD binding to the target is desired).

[0412] The carrier of the present invention

[0413] The present invention further provides vectors comprising the nucleic acid molecules of the present invention. Vectors, as used herein, are suitable mediators for carrying genetic material into cells. Vectors include naked nucleic acids (such as plasmids or mRNA) or nucleic acids embedded in larger structures (such as liposomes or viral vectors).

[0414] In some embodiments, the vector comprises at least one nucleic acid optionally linked to one or more regulatory elements, such as, for example, one or more suitable promoters, enhancers, terminators, etc. In one embodiment, the vector is an expression vector, i.e., a vector suitable for expressing a encoded polypeptide or construct under appropriate conditions, such as when the vector is introduced into a cell (e.g., human). DNA-based vectors include the presence of elements for transcription (e.g., promoters and poly-A signals) and translation (e.g., Kozak sequences).

[0415] Therefore, another embodiment relates to a vector comprising the expression cassette or nucleic acid molecule encoding the chimeric protein and / or polypeptide of the present invention. In a particular embodiment, the vector for expression in *E. coli* or *Saccharomyces cerevisiae* allows for the generation and purification of the chimeric protein and / or polypeptide.

[0416] In one embodiment, within the vector, the at least one nucleic acid and the regulatory element are "operably linked" to each other, which generally means that they are in a functional relationship. For example, a promoter is considered "operably linked" to a coding sequence (where the coding sequence should be understood as being "under the control" of the promoter) if it is capable of initiating or otherwise controlling / regulating transcription and / or expression of the coding sequence. Typically, when two nucleotide sequences are operably linked, the two nucleotide sequences will be in the same orientation and usually in the same reading frame. The two nucleotide sequences are also usually substantially contiguous, but this may not be necessary.

[0417] In one embodiment, any regulatory elements of the vector enable it to provide its intended biological function in the intended host cell or host organism.

[0418] For example, a promoter, enhancer, or terminator should be "operable" in the intended host cell or host organism, meaning, for example, that the promoter should be able to initiate or otherwise control / regulate the transcription and / or expression of a nucleotide sequence (e.g., a coding sequence) that is operably linked to it.

[0419] The invention, providing the aforementioned vector, further encompasses options for high-throughput cloning in a universal fusion vector. The universal vector is preferably specifically designed for surface display in yeast, bacteriophages, bacteria, or viruses. Furthermore, the vector can be used to select and screen immune libraries containing such universal vectors or expression cassettes with a large set of different ISVDs, wherein the same N-terminus of conserved ISVDs and cytokines is fused with the remaining ISVD sequence provided by the library. Thus, differentially expressed sequences in the library constructed for screening novel chimeric proteins for specific targets are provided by differences in the ISVD sequences, and more specifically, by differences in the CDR regions of the ISVD library.

[0420] The host cell of the present invention

[0421] Alternative embodiments involve host cells containing the chimeric proteins and / or polypeptides of the present invention, or nucleic acid molecules, expression cassettes, or vectors encoding the chimeric proteins of the present invention.

[0422] Suitable host cells or host organisms are clear to those skilled in the art and are, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, *Saccharomyces cerevisiae*, *Escherichia coli*, or *Komagataella phaffii* (*Pichia pastoris*, see Bernauer L. et al., “Komagataella phaffii asemerging model organism in fundamental research”, *Frontiers in Microbiology*, 2021, 11:1-16). In one embodiment, the host is *Pichia pastoris*. In another embodiment, the host is *Escherichia coli*. In a preferred embodiment, the host is *Saccharomyces cerevisiae*. Of course, cell-free systems can also be used to generate the protein-based vector building blocks and / or molecules of the present invention, as commented, for example, in Gregorio NE, Levine MZ, Oza JP, “A user's guide to cell-free protein synthesis”, Methods Protoc., 2019, 2(1):24.

[0423] Another embodiment discloses the use of the host cell or membrane formulations or proteins isolated therefrom for ligand screening, drug screening, protein capture and purification, or biophysical research.

[0424] Methods for generating the chimeric proteins and / or polypeptides of the present invention

[0425] Another embodiment of the present invention relates to a method for generating the chimeric protein and / or polypeptide of the present invention, wherein the method includes the following steps:

[0426] (i) Select ISVD and cytokines as described above in this instruction manual;

[0427] (ii) Design a gene construct encoding the protein sequence of the ISVD, wherein the protein sequence is interrupted by the sequence of the cytokine at at least one internal fusion site (such as two internal fusion sites), the cytokine preferably being circularly arranged, wherein the internal fusion site is located at a loop or turn between two secondary structural elements, and preferably at one of the following β-turns in the ISVD (according to IMGT classification):

[0428] i. In the first β-turn of the β-chains A and B connecting the ISVD; or

[0429] ii. In the β-turn of the β-chains C and C' connecting the ISVD; or

[0430] iii. In the β-turn of the β-chain C” and D connecting the ISVD; or

[0431] iv. In the β-turn of the β-chains D and E connecting the ISVD; or

[0432] v. In the β-turn of the β-chains E and F connecting this ISVD;

[0433] (iii) Introduce the gene fusion construct into an expression system to obtain the chimeric proteins and / or polypeptides of the present invention, as described above in this specification.

[0434] Optionally, the method of the present invention includes step (iv): recovering the obtained chimeric protein and / or polypeptide, and optionally purifying it.

[0435] The above method may further include, after step (i), selecting one or more additional groups, residues, portions, or binding units (other than the chimeric protein of the present invention) that may be included in the polypeptide. If this is the case, step (ii) should further include the design of a gene construct that includes one or more additional groups, residues, portions, or binding units in addition to the chimeric protein.

[0436] To generate / obtain the chimeric proteins and / or peptides of the present invention, host cells or host organisms or cell-free systems are typically maintained, sustained, and / or cultured under conditions that optimally express / produce the (desired) chimeric proteins and / or peptides of the present invention. Suitable conditions will be apparent to those skilled in the art and generally depend on the host cells / host organisms or cell-free systems used and the regulatory elements controlling the expression of the chimeric proteins and / or peptides of the present invention.

[0437] Suitable host cells or host organisms for production purposes will be clear to those skilled in the art and can be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, *Saccharomyces cerevisiae*, *Escherichia coli*, or *Pichia pastoris*. In one embodiment, the host is *Pichia pastoris*. In another embodiment, the host is *Escherichia coli*. In a preferred embodiment, the host is *Saccharomyces cerevisiae*.

[0438] In one embodiment, the method of the present invention further includes step (v): screening for chimeric proteins that bind to at least one of a cytokine receptor or receptor subunit with increased or decreased affinity compared to binding to wild-type cytokines. In another embodiment, the method of the present invention further includes step (v): screening for chimeric proteins in which the cytokines contained in these chimeric proteins exhibit modified cytokine signaling compared to cytokines not fused with ISVD, or screening for chimeric proteins that, when the cytokines contained in these chimeric proteins bind to at least one of their receptors or receptor subunits, affect the oligomerization of the receptor or receptor subunit.

[0439] Methods and uses of chimeric proteins of the present invention

[0440] This invention further provides a method for modulating cytokine activity by fusing cytokines with ISVDs (directly or via a linker, as described herein) to form fusion proteins. In a preferred embodiment, a cytokine (or a circularly arranged variant of a cytokine) is inserted into an ISVD as described herein (e.g., as a “macro antibody” type fusion as described herein). The inventors have surprisingly discovered that by fusing cytokines with ISVDs (directly or via a linker, as described herein) as an internal fusion, the activity of the cytokine in binding to its receptor or receptor subunit is altered or modified (or the downstream consequences of the cytokine binding to at least one of its receptors or receptor subunits are altered, such as modified cytokine signaling and / or oligomerization of modified or affected receptors or receptor subunits upon cytokine binding compared to cytokines not fused with ISVD). Therefore, the inventors have surprisingly discovered that by fusing cytokines with ISVDs (directly or via a linker, as described herein), the receptor-binding functionality of the cytokine may be altered compared to the receptor-binding functionality of the cytokine not fused with ISVD. For example, the binding affinity of a cytokine to at least one of its receptors or receptor subunits can be modulated (increased or decreased) by fusing a cytokine to an ISVD in a fusion protein. Alternatively, downstream signaling generated by the interaction of a cytokine with at least one of its receptors or receptor subunits can be modulated by fusing a cytokine to an ISVD. Furthermore, the potency of the cytokine / receptor interaction, and the potency of the cytokine bound to the ISVD, can also be modulated (increased or decreased) in this manner. Additionally, the oligomerization of the receptor or receptor subunit when a cytokine fused to an ISVD binds is also affected (modified, modulated) compared to the oligomerization of the receptor or receptor subunit when a cytokine is not fused to an ISVD. Therefore, the present invention provides a method for modulating the potency of a cytokine receptor, wherein the method includes the step of fusing a cytokine to an ISVD (directly or through a connector, as described herein), or a method for modulating downstream signaling generated by the interaction of a cytokine with at least one of its receptors or receptor subunits, or a method for modulating (influencing) the oligomerization of the receptor or receptor subunit when a cytokine fused to an ISVD binds.

[0441] In a preferred embodiment, the method of the present invention for modulating cytokine activity includes the step of fusing a cytokine with an ISVD to form a chimeric protein, as described herein. As illustrated in the examples, by fusing a cytokine with an ISVD to form a chimeric protein, as described herein, the activity of the cytokine (or the downstream consequence of the cytokine binding to at least one of its receptor or receptor subunit / receptor or receptor subunit oligomerization, as described above) can be modulated. Furthermore, in addition to wild-type cytokines, the chimeric protein of the present invention may comprise a circularly arranged cytokine variant, such as a circularly arranged cytokine mutant with different characteristics (e.g., as illustrated herein using a circularly arranged IL-2 (K35E), which may also generate modulation of cytokine receptor efficacy.

[0442] The method of the present invention may further include the step of screening for fusion or chimeric proteins in which the cytokines exhibit different receptor-binding functionality compared to that of cytokines not fused with ISVD, as described above.

[0443] For example, the screening step may include screening for fusion or chimeric proteins in which the cytokines exhibit different (increased or decreased) binding affinity to at least one of their receptors or receptor subunits compared to the binding affinity of cytokines not fused with ISVD.

[0444] For example, the screening step may include screening for fusion or chimeric proteins in which the cytokines exhibit different (increased or decreased) cytokine / receptor interactions compared to the efficacy of cytokines not fused with ISVD.

[0445] For example, the screening step may include screening for fusion or chimeric proteins in which the cytokines generate different (increased or decreased) downstream signaling when interacting with the same receptor or receptor subunit, compared to downstream signaling generated by the interaction of cytokines not fused with ISVD with at least one of their receptors or receptor subunits.

[0446] For example, the screening step may include screening for fusion or chimeric proteins that contain cytokines that generate different receptor or receptor subunit oligomerizations when bound to cytokines fused to ISVD, compared to receptor or receptor subunit oligomerizations when cytokines that are not fused to ISVD.

[0447] Without being bound by theory, it appears that the fusion of a cytokine with an ISVD (optionally producing a chimeric protein as described herein) can modify (e.g., through steric hindrance and / or conformational changes in the cytokine ligand induced by its fusion with an ISVD) the binding between the cytokine and its receptor present in the chimeric protein. Therefore, a different downstream effect (or signaling) triggered by the binding of a cytokine to its receptor or receptor subunit can be obtained compared to the binding of a wild-type cytokine (not fused with an ISVD, e.g., not part of the fusion or chimeric protein) to at least one of its receptor or receptor subunit.

[0448] Therefore, the present invention provides a method for modulating the function of a cytokine receptor when interacting with a cytokine (fusion protein) fused with an ISVD as described herein (directly or via a linker, as described herein), or when interacting with a cyclically arranged cytokine contained in a chimeric protein of the present invention. The method includes contacting a cytokine (fused with an ISVD, as described herein) with at least one of a cytokine receptor or a receptor subunit to modulate the function of the cytokine receptor (e.g., modulating downstream signaling and / or receptor / receptor subunit oligomerization resulting from the binding of the cytokine to its receptor or at least one of its receptor or receptor subunits).

[0449] The present invention further includes a method for regulating cytokine signaling, the method comprising the following steps:

[0450] - Provides the chimeric protein of the present invention; and

[0451] - Screen for chimeric proteins or peptides in which the cytokines contained in the chimeric protein or peptide exhibit modified cytokine signaling compared to cytokines not fused with ISVD.

[0452] Therefore, the present invention provides the use of the ISVD-fused cytokine, chimeric protein, or polypeptide of the present invention for modulating the activity of cytokines contained in chimeric proteins. For example, the present invention provides the use of the ISVD-fused cytokine, chimeric protein, or polypeptide of the present invention for modulating the binding affinity of cytokines to at least one of their receptors or receptor subunits. Additionally, the ISVD-fused cytokine, chimeric protein, or polypeptide of the present invention can be used to modulate downstream signaling generated by the interaction of cytokines with at least one of their receptors or receptor subunits. Furthermore, the present invention provides the use of the ISVD-fused cytokine, chimeric protein, or polypeptide of the present invention for modulating the efficacy of cytokine / receptor interactions and for modulating the potency of cytokines present in chimeric proteins. Therefore, the present invention provides the use of the ISVD-fused cytokine, chimeric protein, or polypeptide of the present invention for modulating the efficacy and / or functionality of cytokine receptors when interacting with the chimeric proteins of the present invention.

[0453] Therefore, the present invention also provides the use of the cytokine, chimeric protein, or polypeptide fused with ISVD of the present invention for regulating the binding affinity of the cytokine to its receptor and / or for altering or modifying cytokine signaling and / or for influencing, altering or modifying receptor oligomerization when the cytokine binds to at least one of its receptor or receptor subunit.

[0454] The present invention further provides chimeric proteins or polypeptides for medical use. For example, the chimeric proteins or polypeptides of the present invention can be used to treat cancer and / or inflammatory diseases. Therefore, the present invention provides chimeric proteins or polypeptides for treating cancer and / or inflammatory diseases. Cancer can be a solid tumor and / or a liquid tumor. Example

[0455] generally

[0456] We have designed antigen-binding chimeric proteins (also referred to as Megabody in this paper). ® Protein, Megabody ® Protein constructs or Megabody ® These chimeric proteins (constructors) are built from antigen-binding domains that are transplanted onto scaffold proteins, particularly onto cytokines, via two short polypeptide bonds linking the antigen-binding domains to cytokines. Depending on their properties, these antigen-binding chimeric proteins can be used for various applications.

[0457] For example, one of the antigen-binding chimeras proposed in this paper is called the IL-2 macroantibody protein, and it is constructed from ISVDs transplanted onto interleukin-2 (IL-2). The topological structure of the IL-2 molecule was investigated, and different sites were selected for ISVD transplantation.

[0458] Example 1: Design and generation of a 29 kDa antigen-binding chimeric protein constructed from a circularly arranged variant of IL-2 inserted into the first β-turn of the β-chains A and B that link anti-GFP ISVD.

[0459] As a proof of concept for obtaining IL-2 macro antibody protein ( Figure 2 ),according to Figure 1 ISVD was transplanted onto a circularly arranged IL-2 variant protein by linking ISVD to two peptide bonds of a scaffold to construct a macro antibody protein.

[0460] The topology of IL-2 (PDB: 2B5I) was examined to pinpoint several sites on which ISVDs could be transplanted, thus selecting 22 distinct sites. Figure 3 ).

[0461] To design IL-2 macroantibody molecules, a circularly arranged variant of IL-2 is required. In 2020, Lopes et al. disclosed a fusion protein with a circularly arranged version of IL-2 ("ALKS 4230: a novel engineered IL-2 fusion protein with an improved cellular selectivity profile for cancer immunotherapy", Journal for ImmunoTherapy of Cancer, 2020, 8:e000673), demonstrating that this version of IL-2 is well-folded. Wang et al. (“Site-specific mutation of the human interleukin-2 gene: structure-function analysis of the cysteine ​​residues”, Science, 1984, 224(4656):1431-3) demonstrated that a point mutation in IL-2 produces a more stable and supersecreting IL-2 variant that remains biologically active both in vitro and in vivo. Based on these findings, 26 different versions of the IL-2 (K35E, C125S)_ISVD207 macroantibody protein were developed. Figure 4 ).

[0462] like Figure 2 The non-limiting examples illustrated herein illustrate that the 29 kDa macroantibody protein described herein is derived from... Figure 1This is a chimeric polypeptide formed by linking portions of an immunoglobulin monovariable domain and a portion of a scaffold protein. The ISVD used here is the anti-GFP ISVD as depicted in SEQ ID NO: 1. The scaffold protein is a variant of IL-2 (SEQ ID NO: 3). All parts are linked to each other via peptide bonds from the amino terminus to the carboxyl terminus in the following given order: β-chain A of anti-GFP ISVD (residues 1-12 of SEQ ID NO: 1), short peptide linker (SEQ ID NO: 5 or SEQ ID NO: 120), C-terminal portion of IL-2 (K35E,C125S) (SEQ ID NO: 3, from amino acid position X2 to amino acid position 133), peptide linker connecting the C-terminal portion of IL-2 (K35E,C125S) to the N-terminal portion to produce a circular arrangement variant of the scaffold protein (SEQ ID NO: 6), N-terminal portion of IL-2 (K35E,C125S) from residue 4 to amino acid position X1 (SEQ ID NO: 3), short peptide linker (SEQ ID NO: 5 or SEQ ID NO: 121), followed by β-chains B to G of anti-GFP ISVD (SEQ ID NO: 120, ... Residues 16-126 of 1), where X1 and X2 are selected insertion sites on the cytokine sequence for scaffold construction. In several cases, an additional glycine (G) is inserted between the short peptide linker (SEQ ID NO: 5) and the cyclically arranged IL-2 (K35E, C125S) to create a 4-amino acid linker between ISVD and IL-2 (K35E, C125S) (SEQ ID NO: 120 or SEQ ID NO: 121), such as... Figure 4 As shown in (SEQ ID NO: 7-23). ​​An α-sheet model of some (non-limiting) examples of the IL-2(K35E,C125S)_ISVD207 macroantibody protein is shown in... Figure 5-8 middle.

[0463] according to Figure 1Two additional constructs were generated in which IL-2(K35E,C125S) was inserted into the first β-turn of the β-chains A and B connecting the anti-GFP ISVD: IL-2(K35E,C125S)_ISVD207 macro antibody protein (SA17521) (SEQ ID NO: 24), wherein all parts are linked to each other by peptide bonds from the amino terminus to the carboxyl terminus in the following given order: anti-GFP ISVD β-chain A (residues 1-12 of SEQ ID NO: 1), short peptide linker (SEQ ID NO: 120), IL-2(K35E,C125S) from amino acid 4 to amino acid 133, short peptide linker (SEQ ID NO: 5), and anti-GFP ISVD β-chains B to G (residues 16-126 of SEQ ID NO: 1); and IL-2(K35E,C125S)_ISVD207 macro antibody protein (SA17653) (SEQ ID NO: 24). SEQ ID NO: 25), in which all parts are linked together by peptide bonds from the amino terminus to the carboxyl terminus in the following given order: anti-GFP ISVD β chain A (residues 1-12 of SEQ ID NO: 1), short peptide linker (SEQ ID NO: 5), IL-2 (K35E, C125S) from amino acid at position 5 to amino acid at position 133, short peptide linker (SEQ ID NO: 5), and anti-GFP ISVD β chains B to G (residues 16-126 of SEQ ID NO: 1).

[0464] To demonstrate that IL-2 macroantibody proteins can be expressed as well-folded functional proteins, we designed several IL-2(K35E,C125S)_ISVD207 macroantibody proteins and displayed them on yeast surfaces (Boder, ET and Wittrup, KD, “Yeast surface display for screening combinatorial polypeptide libraries”, Nat Biotechnol, 1997, 15:553-557), and examined the specific binding of the homoantigen (GFP) to yeast cells displaying the macroantibody protein by flow cytometry. To display the IL-2(K35E,C125S)_ISVD207 macroantibody protein in yeast, we constructed an open reading frame (OPF) encoding the IL-2(K35E,C125S)_ISVD207 macroantibody protein fused with numerous accessory peptides and proteins. This macroantibody protein, from its N-terminus to its C-terminus, is the appS4 leader sequence (SEQ ID NO: 31) that guides extracellular secretion in yeast (Rakestraw et al., “Directed evolution of a secretory leader for the improved expression of heterologous proteins and full-length antibodies in Saccharomyces cerevisiae”, Biotechnol.Bioeng.[Biotechnology and Bioengineering], 2009, 103:1192-1201), IL-2(K35E,C125S)_ISVD207 macro antibody protein composed of the β chain A of anti-GFP ISVD (residues 1-12 of SEQ ID NO: 1), a short peptide linker (SEQ ID NO: 5 or SEQ ID NO: 120), the C-terminal portion of IL-2(K35E,C125S) (SEQ ID NO: 3, from amino acid position X2 to amino acid position 133), a peptide linker connecting the C-terminal portion of IL-2(K35E,C125S) to the N-terminal portion to produce a circular arrangement variant of the scaffold protein (SEQ ID NO: 6), the N-terminal portion of IL-2(K35E,C125S) (from residue 4 to amino acid position X1, SEQ ID NO: 3), a short peptide linker (SEQ ID NO: 5 or SEQ ID NO: 121), followed by anti-GFP The ISVD consists of the β chain B to G (residues 16-126 of SEQ ID NO: 1), a flexible peptide linker, Aga2p (the adhesion subunit of the yeast lectin protein Aga2p, which attaches to the yeast cell wall via a disulfide bond with the Aga1p protein), an acyl carrier protein for orthogonal fluorescent staining of the fusion protein for display (Johnsson N. et al., “Protein chemistry on the surface of living cells”, Chembiochem: a European journal of chemical biology, 2005, 6:47-52) (SEQ ID NO: 32), followed by a cMyc tag (SEQ ID NO: 33).

[0465] After each transcriptional control by the galactose-inducible GAL1 / 10 promoter, all different open reading frames of the IL-2(K35E,C125S)_ISVD207 macro antibody protein construct were cloned into the pCTCON2 vector using standard cloning techniques (Chao G. et al., “Isolating and engineering human antibodies using yeast surface display”, Nat Protoc., 2006, 1: 755-768).

[0466] Each construct was introduced into yeast strain EBY100 (Saccharomyces cerevisiae), and the growth conditions were changed from glucose-rich medium to galactose-rich medium, allowing the EBY100 clones carrying the corresponding plasmids to grow and be induced overnight to trigger the expression and secretion of the IL-2(K35E,C125S)_ISVD207-Aga2p-ACP fusion protein. For orthogonal staining of ACP, cells were incubated for 1 h in the presence of a fluorescently labeled CoA analog (coA-647, 2 µM) and a catalytic amount of SFP synthase (1 µM).

[0467] To analyze the functionality of IL-2(K35E,C125S)_ISVD207 macroantibody proteins with different displays, we examined their binding to the homoantigen (GFP) by flow cytometry. Yeast cells stained with orthogonal chromatography were incubated for 1 h in the presence of 100 nM GFP (Scholz, O. et al., “Quantitative analysis of gene expression with animproved green fluorescent protein”, European journal of biochemistry / FEBS, 2000, 267:1565-1570). After washing these cells, we observed detectable amounts of GFP bound to IL-2(K35E,C125S)_ISVD207 macroantibody proteins with different displays, which may have been linearly correlated with the expression level of IL-2(K35E,C125S)_ISVD207 macroantibody proteins on the yeast surface. In fact, two-dimensional flow cytometry analysis confirmed that GFP (high GFP fluorescence level) only bound to yeast cells with a significant macroantibody protein display level (high CoA647 fluorescence level) (data not shown). GFP did not bind to EBY100 yeast cells stained in the same manner but not expressing macroantibody proteins, nor to EBY100 yeast cells expressing only circularly arranged IL-2 (K35E, C125S) [S75-Q74] (SEQ ID NO: 4). As a positive control, cYgjk_ISVD207 macroantibody protein (SEQ ID NO: 34), fused with many accessory peptides and proteins, was expressed and displayed on the surface of yeast cells. Figure 1 This macroantibody protein is a chimeric polypeptide composed of a portion of anti-GFP ISVD and a portion of YgjK (an 86 kDa periplasmic protein of E. coli (PDB 3W7S)), forming a 100 kDa macroantibody protein that is proven to bind GFP. Figure 9).

[0468] From these experiments, we concluded that 16 different versions of the Mb_IL-2(K35E,C125S)_ISVD207 macroantibody protein can be expressed on yeast surfaces as well-folded and functional antigen-binding (GFP-binding) chimeric proteins. Figure 9 ).

[0469] Example 2: Binding of a specific IL-2 monoclonal antibody to the IL-2_ISVD207 macroantibody protein.

[0470] To confirm the correct folding of IL-2(K35E,C125S) within the IL-2(K35E,C125S)_ISVD207 macroantibody protein displayed on the surface of yeast cells, yeast cells expressing these IL-2(K35E,C125S)_ISVD207 macroantibody proteins were incubated for 1 h in the presence of monoclonal antibody mAb5111-human Fc at a final concentration of 2 µg / ml. After washing three times, the cells were incubated for 1 h in the presence of 2 µg / ml anti-human IgG Fc (a phycoerythrin-conjugated AffiniPure F(ab)2 fragment specific to goat anti-human IgG Fcy fragment, Jackson Immuno Research), washed three times, and analyzed by flow cytometry. We observed detectable levels of fluorescence binding to yeast cells displaying the specific IL-2(K35E,C125S)_ISVD207 macroantibody protein, thus confirming that IL-2(K35E,C125S) is well folded in certain IL-2(K35E,C125S) macroantibody protein constructs. Figure 9 The mAb5111 epitope on IL-2 is known (PDB 5UTZ). In some constructs, the mAb5111-human Fc epitope is absent and / or inaccessible in the macroantibody protein due to the fact that ISVD207 is inserted in or near the epitope. In fact, the mAb5111-human Fc antibody cannot bind to any IL-2(K35E,C125S)_ISVD207 macroantibody protein construct where ISVD207 is inserted near the epitope (AA16-31; AA70-AA86). In parallel, the expression of the same construct was tracked and confirmed by incubating the clones at a final concentration of 4 µg / ml for 1 h in the presence of mouse anti-Myc monoclonal antibody (Roche / #11 667 149 001), followed by washing three times and incubating with the anti-human IgG Fc antibody (a phycoerythrin-conjugated AffiniPure F(ab)2 fragment specific to goat anti-human IgG Fcy fragment, Jackson Biosciences) as described above. Cells were analyzed using flow cytometry. Figure 9).

[0471] Binding of the monoclonal antibody NARA1 was only confirmed on wild-type IL-2 because the binding epitope is disrupted in IL-2(K35E,C125S) due to the K35E mutation. Since all IL-2(K35E,C125S)_ISVD207 macroantibody proteins carry this mutation, binding was not observed in flow cytometry. Figure 9 ).

[0472] Example 3: Binding of CD25 or CD122 / CD132 to IL-2(K35E,C125S)_ISVD207 macroantibody protein.

[0473] Since the binding of the monoclonal antibody mAb5111-human Fc to the IL-2(K35E,C125S)_ISVD207 macroantibody protein was confirmed for a defined clone, we examined whether we could confirm the binding of CD25 or CD122 / CD132 to the IL-2(K35E,C125S)_ISVD207 macroantibody protein displayed on the surface of yeast cells. Yeast cells expressing and displaying different IL-2(K35E,C125S)_ISVD207 macroantibody proteins were incubated for 1 h at a final concentration of 4 µg / ml in the presence of His-tagged CD25 protein (human IL-2 RαAcrobiosystems #ILA-H52H9). In parallel experiments, yeast cells from the same batch expressing and displaying different IL-2 (K35E, C125S)_ISVD207 macroantibody proteins were incubated for 1 h at a final concentration of 4 µg / ml in the presence of His-tagged CD122 / CD132 proteins (human IL-2 Rβ and IL-2 Rγ heterodimers, His-tagged and Twin strep-tagged, Acrobiosystems #ILG-H5283). All cells were washed three times and incubated for 1 h in the presence of mouse anti-His-PE antibody (miltenyibiotec / #130-120-718; 1 / 50 dilution), washed three times, and analyzed by flow cytometry. We observed detectable levels of fluorescence binding to yeast cells displaying the specific IL-2(K35E,C125S)_ISVD207 macroantibody protein, thus confirming that CD25 or CD122 / CD132 can bind to certain IL-2(K35E,C125S) macroantibody protein constructs. Figure 9This provides evidence of the well-folded IL-2(K35E,C125S) domain within the IL-2(K35E,C125S)_ISVD207 macroantibody protein. In constructs where ISVD207 is inserted near the binding site of CD122 / 132, almost no fluorescence is observed, while in other constructs, CD122 / 132 binding is confirmed. In fact, as an example, we observed that CD122 / CD132 almost completely fails to bind to the IL-2(K35E,C125S)[L132-I129]_ISVD207 macroantibody protein, while CD122 / CD132 binding is observed at the IL-2(K35E,C125S)[N77-S75]_ISVD207 macroantibody protein. Expression of all constructs was tracked and confirmed as follows: Clones were incubated for 1 h at a final concentration of 4 µg / ml in the presence of mouse anti-Myc monoclonal antibody (Roche / #11 667 149 001), followed by three washes and incubation in the presence of anti-mouse IgG Fc (phycoerythrin-conjugated AffiniPure goat anti-mouse IgG Fcγ-specific, Jackson Immunological Research). After three washes, cell expression was analyzed by flow cytometry. Figure 9 ).

[0474] Example 4: Binding of IL-2 macroantibody protein to ISVD target

[0475] To further characterize the macroantibody protein, a similar IL-2 macroantibody protein was designed using anti-HSA ISVD or anti-PD-L1 ISVD instead of anti-GFP ISVD, and expressed in Pichia pastoris or CHO EBNALT85, followed by purification according to standard protocols. For purification purposes, a FLAG3HIS6 tag (SEQ ID NO: 35) was fused to the macroantibody protein at its C-terminus.

[0476] HSA combination

[0477] Human serum albumin (HSA) (Sigma-Aldrich, A8763) was biotinylated using NHS-LC-Biotin (Thermo Fisher Scientific, 21336) with an average labeling degree of 1. Following the blocking step, the biotinylated HSA was captured at a concentration of 0.5 µg / mL on an MSD GOLD 96-well Small Spot Streptavidin SECTOR Plate (MSD, L45SA-1). Subsequently, 25 µL of a mixture (23 dilutions, 1 / 3 dilution factor) of 1 nM test compound and fixed concentrations of HSA ranging from 1.13 pM to 10 µM were added to the plate. The mixture was incubated at room temperature (RT) for 2 hours to reach equilibration. Samples were incubated with biotinylated HSA for 10 minutes, followed by washing with 3 x 150 µL PBS + 0.05% Tween-20. During the final assay step, 25 µL of sulfonated anti-VHH antibody was added at a concentration of 2 µg / mL and incubated for 1 hour, followed by a final wash with 150 µL 1x PBS + 0.05% Tween-20. Plates were read on an MSD QuickPlex SQ120 reader after adding 150 µL of MSD read buffer. Data were analyzed using a four-parameter logistic (4PL) fit in a GraphPad Prism 9.

[0478] GFP binding

[0479] Binding to green fluorescent protein (GFP) (Sino Biological, 13105-S07E) was detected by surface plasmon resonance (SPR) (Cytiva, Biacore 8K+, #2626160). In short, anti-FLAG M2 monoclonal antibody (mAb) (Sigma-Aldrich, F3165) was immobilized on a CM5 sensor (Cytiva, BR100399) using standard amine coupling chemistry. Different concentrations (2 to 60 nM) of the test compound were injected at a flow rate of 10 μL / min for 180 seconds. Subsequently, 250, 100, 40, 16, 6.4, 2.6, and 1 nM GFP were injected at a flow rate of 30 μL / min for 2 minutes to allow binding to the anti-GFP ISVD / macro antibody protein, followed by 10 minutes of run buffer (Stenofan, BR100669, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20, pH 7.4) to allow separation from the anti-GFP instrument. The chip was regenerated at 45 µL / min using two 30-second pulses of 10 mM glycine (pH 1.5). Binding data were collected at 25°C and analyzed using Insight Evaluation software version 3.0.12 provided by the manufacturer (Stenofan) according to a 1:1 binding fit model.

[0480] PD-L1 binding

[0481] Binding to human PD-L1 (human PD-L1(CD274)-hFc) was detected using surface plasmon resonance (SPR) (Stopfan, Biacore 8K+, #2743662). In short, the anti-human Fc binder was immobilized on a CM5 sensor (Stopfan, BR100399) using standard amine coupling chemistry. Human PD-L1-hFc was injected at a flow rate of 10 μL / min at a rate of 1 µg / mL for 180 seconds. The test compounds at concentrations of 25, 8.33, 2.78, 0.93, 0.31, 0.10, 0.034, 0.011, 0.0038, 0.0013, and 0.00042 nM were then injected at a flow rate of 30 μL / min for 2 minutes to allow binding to the target. This was followed by injection of run buffer (Stenofan, BR100669, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20, pH 7.4) for 10 minutes to allow separation from the target. The chip was regenerated using two 30-second pulses of 0.85% H3PO4 at 30 µL / min. Binding data were collected at 25°C and analyzed using InsightEvaluation software version 3.0.12 provided by the manufacturer (Stenofan) according to a 1:1 binding fit model.

[0482] The results are summarized in Tables 6a to 6c, which show the kinetic parameters of the interaction between the IL-2 macroantigen protein and the multivalent protein construct (a polypeptide containing the macroantigen protein), i.e., the macroantigen protein fused with ISVD, where ISVD targets human serum albumin (HSA) (ISVD = ALB23002; Table 6a), GFP (ISVD = ISVD207; Table 6b), and / or PD-L1 (ISVD = ISVD10F11; Table 6c). Additionally, the fold differences between the different compounds and their corresponding references were calculated.

[0483] Table 6a: Affinity of IL-2(K35E, C125S)_ALB23002 macroantibody protein to HSA

[0484]

[0485] Table 6b. Kinetic parameters of the interaction between IL-2(K35E, C125S)_ISVD207 macroantibody protein and GFP.

[0486]

[0487] Table 6c. Kinetic parameters of the interaction between IL-2(K35E, C125S)_ISVD10F11 macroantibody protein and multivalent protein constructs and PD-L1.

[0488]

[0489] Data show that the ISVD portion maintains its binding to the target when formatted in macroantibody constructs. For ISVD-HSA interaction, the maximum fold change compared to the reference ISVD is 3. For ISVD-GFP interaction, the maximum fold change is 2, and for ISVD-PD-L1 interaction, the maximum fold change is even lower, further highlighting that the ISVD is fully functional when formatted as a macroantibody construct.

[0490] Example 5: IL-2 macroantibody protein induces STAT5 phosphorylation in different T cell subsets

[0491] STAT5 phosphorylation induced by IL-2 macroantibody protein was characterized in different T cell subsets to demonstrate differential signal transduction profiles based on their interactions with IL-2 receptors (IL-2Rα, IL-2Rβ, IL-2Rγ). STAT5 phosphorylation in CD8+ T cells, CD25-CD4+ T cells, and CD25+FoxP3+CD4+ Treg cells was determined by flow cytometry. Briefly, PBMCs isolated from healthy donors within 4 hours of blood collection were removed from cryogenic storage and thawed in culture medium (RPMI 1640, Glutamax, 25 mM Hepes, Gibco 72400-021, supplemented with 10% heat-inactivated FBS, Sigma F7524, 1 mM sodium pyruvate, Gibco 11360-039, MEM non-essential amino acids, Gibco 11140-035, and 1x penicillin / streptomycin, Life Technologies 15140). PBMCs were incubated overnight at 37°C with 5,000,000 cells / mL in culture medium under a 5% CO2 atmosphere. PBMCs were washed once with D-PBS (Gibco 14190) and then stained in the dark at RT for 15 min with ZombieNIR fixative (Biolegend, 423105). After the washing step with culture medium, 300,000 PBMCs were seeded in 250 µL of culture medium in each well of a 96-well U-bottom deep-well plate (Thermo Scientific 260251) and incubated at 37°C with a 5% CO2 atmosphere for at least 30 min. Then, an equal volume of the test compound with or without human serum albumin (HSA, final concentration 30 µM, CSL Behring 2160-679) was added, and the cells were incubated at 37°C for 15 min. Next, cells were fixed by adding 500 µL / well of pre-warmed (37°C) fixation buffer I (BD Biosciences 557870) and incubating at 37°C for 15 min. After washing twice with FACS buffer (D-PBS, Gibco 14190, supplemented with 2% heat-inactivated FBS, Sigma F7524 and 0.05% sodium azide, Acrosorganics 19038), pre-cooled (to -20°C) Perm Buffer III (BD Biosciences 558050) was slowly added to the cell pellet, and the pellet was then incubated on ice for 30 min.After two washing steps with FACS buffer, cells were incubated at RT with a mixture of human Fc blocker (BD Pharmingen 564220, 12.5 µg / mL), anti-human CD3-PE-Cy7 (Biolegend 344816), anti-human CD4-Brilliant Violet 421 (Biolegend 344632), anti-human CD8-Brilliant Violet 510 (Biolegend 344732), anti-human CD25-PE (BD Bioscience 557138), anti-human FoxP3-Alexa Fluor 647 (BD Bioscience 560045), and anti-human pSTAT5 (pY694)-Alexa Fluor488 (BD Bioscience 612598) for 60 min. Cells were analyzed using a MACS Quant flow cytometer (Miltenyi Biotec) after two washes with FACS buffer. Mean fluorescence intensity (MFI) of pSTAT5-Alexa Fluor 488 staining was determined after gating different T cell subsets. Results for IL-2 (K35E, C125S) macroantibody protein with ISVD targeting HSA and controls are shown in Tables 7a and 7b.

[0492]

[0493] The IL-2 (K35E, C125S) macroantibody protein with HSA-targeting ISVD induces phosphorylation of STAT5 (pSTAT5) in primary T cell subsets, presenting different profiles. For compounds TP027 and TP028, there is at least a 100-fold difference in potency against cells expressing IL-2Rβγ (CD8+ and CD4+CD25-) versus cells expressing IL-2Rαβγ (CD4+CD25+). This difference is even greater for wild-type IL-2 (TP027), suggesting that K35E and / or C125S affect the interaction with the IL-2 receptor (primarily IL-2Rα). For reference compound 1 (an engineered IL-2 with IL-2Rβγ bias), this ratio is less than 3, suggesting that IL-2Rα is not involved in downstream signaling (Klein et al. 2013: Blood 122:2278). For reference compound 2, the interaction with cells that primarily express ILRβγ but not IL-2Rα is affected, as expected based on literature (Peterson et al. 2018, J Autoimmun [Journal of Autoimmunology] 95:1-14).

[0494] For the 12 different insertion sites explored in the IL-2 (K35E, C125S) macroantibody protein with HSA-binding ISVDs, different profiles were observed in terms of absolute potency and potency ratios against cells expressing IL-2Rβγ versus those expressing IL-2Rαβγ. Compounds TP056 and TP065 showed lower potency against IL-2Rαβγ-expressing cells compared to TP027 and TP028, with highly similar potency against both IL-2Rβγ-expressing and IL-2Rαβγ-expressing cells, resulting in ratios of 2 and 4, respectively. This is similar to the profile of reference compound 1. At the other end of the spectrum are compounds TP063 and TP064, which showed predominantly lower potency against IL-2Rβγ-expressing cells compared to TP027 and TP028. Compared to TP028, they maintained good potency against cells expressing IL-2Rαβγ, resulting in an increased potency ratio against IL-2Rβγ-expressing cells to those expressing IL-2Rαβγ. This is similar to the profile of reference compound 2, which has an IL-2Rαβγ bias. Overall, different functional profiles were observed, suggesting that the insertion site of ISVDs into cytokines can modulate the interaction between cytokines and their receptors, leading to differential signaling.

[0495] Binding of ISVDs to their target HSA can reduce potency, albeit in a compound-dependent manner (Table 7b). In cells expressing IL-2Rβγ, the potency of TP057 was reduced to as low as 1 / 10. In cells expressing IL-2Rαβγ, the potency of TP019 was reduced to less than 1 / 100. As expected, the potency of the reference compound, which did not contain HSA-bound ISVDs, was not affected by the presence of HSA in the assay.

[0496] To investigate the roles of K35E and C125S mutations in IL-2, wild-type IL-2 was formatted in a macroantibody protein and its functionality was tested (PBMC-pSTAT5). Results are summarized in Tables 8a and 8b. Figure 10 middle.

[0497]

[0498] Similar to the IL-2 (K35E, C125S) macroantibody protein of ISVDs targeting HSA, the IL-2 macroantibody protein of ISVDs targeting HSA also induces STAT5 phosphorylation in primary T cell subsets, presenting a different profile. Differences in EC50 values ​​and calculated fold changes from previous experiments can be attributed to differences between PBMC donors, with the overall trend and ranking being the same. For compound TP027, there was a 61-fold difference in potency against cells expressing IL-2Rβγ compared to those expressing IL-2Rαβγ. For reference compound 1, this ratio was less than 3, indicating that IL-2Rα is not involved in downstream signaling. For reference compound 2, the interaction with cells primarily expressing ILRβγ but not IL-2Rα was affected, as expected based on the literature.

[0499] For eight different insertion sites explored in the IL-2 macroantibody protein with HSA-binding ISVDs, different profiles were observed in terms of absolute potency and potency ratios against cells expressing IL-2Rbg versus those expressing IL-2Rαβγ. Compounds TP115 and TP119 showed lower potency against IL-2Rαβγ-expressing cells compared to TP027 and TP121, with highly similar potency against both IL-2Rβγ-expressing and IL-2Rαβγ-expressing cells, resulting in a ratio of 2. This is similar to the profile of reference compound 1. At the other end of the spectrum are compounds TP118 and TP072, which showed primarily lower potency against IL-2Rβγ-expressing cells compared to the reference. They still exhibited good potency against IL-2Rαβγ-expressing cells, resulting in a higher fold difference in potency against IL-2Rβγ-expressing cells versus those expressing IL-2Rαβγ. This is similar to the profile of reference compound 2 with IL-2Rαβγ bias. Similarly, for the IL-2 macroantibody protein, different functional profiles were detected, suggesting that the location of ISVD insertion into cytokines can regulate the interaction between cytokines and their receptors. Overall, for macroantibody proteins containing either IL-2 (K35E, C125S) (Tables 7a and 7b) or IL-2 (Tables 8a and 8b), the compound ranking based on the fold difference in potency against cells expressing IL-2Rβγ versus those expressing IL-2Rαβγ was similar.

[0500] Binding of ISVDs to their target HSA can reduce potency, albeit in a compound-dependent manner. The greatest effect was observed with TP075, showing a reduction in potency to 1 / 40th against both IL-2Rβγ and IL-2Rαβγ-expressing cells. In addition to the macroantibody protein, the N-terminal to C-terminal fused ISVD-cytokine construct (TP121) also showed lower potency in the presence of HSA. As expected, the potency of the reference compound, which did not contain HSA-bound ISVDs, was unaffected by the presence of HSA in the assay.

[0501] To assess the impact of ISVDs used in the macroantibody form, the IL-2 (K35E,C125S) macroantibody protein was formatted with a GFP-bound ISVD (ISVD207) and compared with an HSA-bound ISVD-IL-2 (K35E,C125S) macroantibody protein. The data are shown in Table 9.

[0502]

[0503] Similar to the IL-2 (K35E, C125S) macroantibody protein with an ISVD targeting HSA, the IL-2 (K35E, C125S) macroantibody protein with an ISVD (ISVD207) targeting GFP also induces STAT5 phosphorylation in primary T cell subsets, presenting a different profile. For compounds TP027 and TP028, there was at least a 30-fold difference in potency against cells expressing IL-2Rβγ compared to those expressing IL-2Rαβγ. This difference was even greater with wild-type IL-2, suggesting that K35E and / or C125S affect interaction with the IL-2 receptor (primarily IL-2Rα). For reference compound 1, this ratio was less than 3.

[0504] Three distinct sites within IL-2 were explored for insertion of ISVDs targeting HSA or GFP. Different profiles were observed for the three explored sites in terms of absolute potency and potency ratios between cells expressing IL-2Rβγ and those expressing IL-2Rαβγ. Compounds TP031 and TP019 showed lower fold differences between cells expressing IL-2Rβγ and those expressing IL-2Rαβγ compared to TP028, while TP030 and TP018 showed higher fold differences between cells expressing IL-2Rβγ and those expressing IL-2Rαβγ compared to TP028.

[0505] Example 6: Activity of IL-2 macroantibody protein in PBMC proliferation assay

[0506] In a PBMC proliferation assay with Ki67 readout, the stimulation of CD4+ and CD8+ T cell proliferation by the IL-2 macroantibody protein was characterized. Additionally, CD25 was added to differentiate between IL-2Rα-positive and negative populations. Ki67 is a nuclear protein associated with cell proliferation. PBMCs isolated from healthy donors were removed from cryogenic storage and thawed in thawing medium (RPMI 1640 medium, GlutaMAX™ supplement, HEPES (Life Technologies-Gibco, 72400-021), supplemented with 10% heat-inactivated FBS (Sigma F9665) and 1% penicillin / streptomycin (Life Technologies, 15140)). PBMCs were seeded at 300,000 cells / well in 100 µL of medium (CTS™ OpTmizer™ T-cell expansion SFM and OpTmizer™ T-cell expansion supplement (LifeScience & Technology Corporation-Gibberellic A.S., A10221-01-A10484-02), 2 mM L-glutamine (LifeScience & Technology Corporation-Gibberellic A.S., A2916801), 5% CTS™ immune cell SR (LifeScience & Technology Corporation-Gibberellic A.S., A25961-01), and 1% penicillin / streptomycin (LifeScience & Technology Corporation, 15140)) in 96-well U-shaped plates (Costar, 3799). An equal volume of the test compound with or without human serum albumin (HSA, final concentration 30 µM, CSL Behring 2160-679) was then added, and the cells were incubated at 37°C in a 5% CO2 atmosphere for 6 days. Following incubation, Ki67 expression in CD4+ and CD8+ T cells was determined by flow cytometry using CD25 as an additional marker. Cells were transferred to V-plates (Greiner, 651180) and washed with D-PBS (Gibberco, 14190), then stained with ZombieAqua fixative live / dead stain (Baijin, 423102) for 15 min under RT.After washing with FACS buffer (D-PBS supplemented with 2% heat-inactivated FBS and 0.05% sodium azide (Acrosorganics 19038), cells were incubated at RT with human Fc blocking agent (BD Pharmingen, 564220, 12.5 µg / mL) for 15 min, and then stained at 4°C with a mixture of anti-human CD3-APC-H7 (BD Pharmingen, 560176 / 560275), anti-human CD8-PerCP / Cy5.5 (Baijin, 344709 / 344710), and anti-human CD25-Alexa Fluor 647 (Baijin, 356127 / 356128) for 30 min. After two washes with FACS buffer, cells were fixed and permeabilized for 1 hour at RT with 1x Fix / Perm buffer from the FoxP3 / transcription factor staining buffer kit (eBioscience, 00-5523-00). After two washes with 1x Perm buffer provided in the buffer kit, cells were stained at RT for 45 min with a mixture of anti-human CD4-FITC (Baijin, 344604) and anti-human Ki67-Brilliant Violet 421 (Baijin, 350505 / 350506). After two washes with 1x Perm buffer, cells were resuspended in FACS buffer and analyzed using a MACSQuant flow cytometer (Mitengene Biosciences). The percentage of Ki67-positive cells was analyzed in gated CD4+ and CD8+ T cells. Results are shown below. Figure 11 In Tables 10a and 10b.

[0507]

[0508] As expected based on pSTAT5 data (Table 7), the IL-2 (K35E, C125S) macroantibody protein with ISVDs targeting HSA was able to drive the proliferation of different immune cell populations (CD8+CD25-, CD4+CD25-, and CD4+CD25+). For compound TP027, there was a 160-fold difference in potency against cells expressing IL-2Rβγ (CD8+CD25- and CD4+CD25-) compared to cells expressing IL-2Rαβγ (CD4+CD25+). For reference compound 1, this ratio was less than 3. A full-dose response curve for reference compound 2 against CD8+CD25- and CD4+CD25- T cells was not obtained, but this compound maintained its potency against CD4+CD25+ T cells compared to wild-type human IL-2 (TP027).

[0509] Different insertion sites explored in the IL-2 (K35E, C125S) macroantibody protein with HSA-binding ISVDs yielded different profiles in terms of absolute potency and potency ratios against cells expressing IL-2Rβγ versus those expressing IL-2Rαβγ. Compounds TP056 and TP065 showed lower potency against IL-2Rαβγ-expressing cells compared to TP027, with highly similar potency against both IL-2Rβγ-expressing and IL-2Rαβγ-expressing cells, resulting in ratios of 0.2 and 0.3, respectively. This is consistent with the pSTAT5 data (Table 7). At the other end of the spectrum is compound TP064, which showed predominantly lower potency against IL-2Rβγ-expressing cells compared to TP027. However, this protein still exhibited good potency against IL-2Rαβγ-expressing cells. This is consistent with the pSTAT5 data (Table 7) and is similar to the profile of reference compound 2, which has an IL-2Rαβγ bias. Overall, different functional profiles were detected, suggesting that the location of ISVD insertion into cytokines can modulate the interaction between cytokines and their receptors.

[0510] When ISVD binds to its target, the compound’s bias toward IL-2Rβγ or IL-2Rαβγ remains unchanged. In contrast to observations made in the pSTAT5 assay, no effect of HSA binding was observed in the proliferation assay. This is hypothesized to be due to the nature of the characterization assay, where, unlike the 15-min incubation in the pSTAT5 assay, the proliferation assay is run over a 6-day period, allowing for the achievement of equilibrium conditions.

[0511] To investigate the role of K35E and C125S mutations in IL-2 in PBMC proliferation assays, wild-type IL-2 was formatted in macroantibody proteins. Results are summarized in Tables 11a and 11b. Figure 11 middle.

[0512]

[0513] Similar to IL-2 (K35E, C125S) macroantibody proteins with ISVDs targeting HSA, IL-2 macroantibody proteins with ISVDs targeting HSA can also drive the proliferation of different immune cell populations (CD8+CD25-, CD4+CD25-, and CD4+CD25+). For compound TP027, there was a 50-fold difference in potency against cells expressing IL-2Rβγ (CD8+CD25- and CD4+CD25-) compared to cells expressing IL-2Rαβγ (CD4+CD25+).

[0514] For the eight different insertion sites explored in the IL-2 macroantibody protein with HSA-binding ISVD, different profiles were observed in terms of absolute potency and potency ratio against cells expressing IL-2Rβγ versus those expressing IL-2Rαβγ. Compounds TP115 and TP119 showed lower potency against cells expressing IL-2Rαβγ compared to TP027, with highly similar potency against both IL-2Rβγ and IL-2Rαβγ-expressing cells, resulting in a ratio of 0.5. This is consistent with pSTAT5 data (Table 8) and similar to the profile of reference compound 1. The α-sheet model of TP115 and TP119 (…) Figure 12 (SA17669) and (SA17658) show that ISVD localizes towards the IL-2Rα protein, thereby interfering with the IL-2-IL-2Rα interaction. At the other end of the spectrum are compounds TP118 and TP072, which, compared to the reference TP027, primarily show lower potency against cells expressing IL-2Rβγ. They still exhibit good potency against cells expressing IL-2Rαβγ, resulting in a higher fold difference in potency against cells expressing IL-2Rβγ compared to those expressing IL-2Rαβγ. This is consistent with the pSTAT5 data (Table 8) and similar to the profile of reference compound 2. The α-sheet model of TP118 (…) Figure 14 (SA17678) shows that ISVD localizes towards the IL-2Rγ protein, thereby interfering with IL-2-IL-2Rγ interactions while retaining binding to IL-2Rα. The α-sheet model of TP116 ( Figure 15(SA17659) shows that ISVD localizes towards the IL-2Rβ protein, thereby interfering with IL-2-IL-2Rβ interactions. Typically, different functional profiles are detected for IL-2 macroantibody proteins, suggesting that the location of ISVD insertion into cytokines can modulate the interaction between cytokines and their receptors. Furthermore, for macroantibody proteins containing either IL-2 (K35E, C125S) (Tables 10a and 10b) or IL-2 (Tables 11a and 11b), the compound ranking based on the fold difference in potency against cells expressing IL-2Rβγ versus those expressing IL-2Rαβγ is similar.

[0515] When ISVD binds to its target, the bias of the compound toward the IL-2Rβγ or IL-2Rαβγ receptor complex remains unchanged. In contrast to observations made in the pSTAT5 assay, no effect of HSA binding was observed in the PBMC proliferation assay. This is hypothesized to be due to the nature of the characterization assay, where, unlike the 15-min incubation in the pSTAT5 assay, the PBMC proliferation assay is run over a 6-day period, allowing for equilibrium conditions to be achieved.

[0516] Example 7: Binding of IL-2 macroantibody protein to IL-2 receptor

[0517] IL-2Rα binding

[0518] Binding to human IL-2Rα (ACRO Biosystems, ILA-H5251) was detected using surface plasmon resonance (SPR) (Stopfan, Biacore 8K+, #2743662). In short, the anti-human Fc binder was immobilized on a CM5 sensor (Stopfan, BR100399) using standard amine coupling chemistry. Human IL-2Rα was injected at a flow rate of 10 μL / min for 180 seconds at concentrations of 0.75, 1, or 10 µg / mL. Subsequently, the test compound was injected as a 7-point dilution series at a flow rate of 30 μL / min, starting at a concentration of 500 or 100 nM (dilution factor 2.5), for 2 minutes to allow binding to the target, followed by 10 minutes of run buffer (Stenofan, BR100669, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20, pH 7.4) to allow separation from the target. The chip was regenerated using two 30-second pulses of 0.85% H3PO4 at 30 µL / min. Binding data were collected at 25°C and analyzed using Insight Evaluation software version 3.0.12 provided by the manufacturer (Stenofan) according to a 1:1 binding fit model.

[0519] IL-2Rβ binding

[0520] Binding to human IL-2Rβ (ACRO Biosystems, ILB-H5253) was detected using surface plasmon resonance (SPR) (Stopfan, Biacore 8K+, #2743662). In short, the anti-human Fc binder was immobilized on a CM5 sensor (Stopfan, BR100399) using standard amine coupling chemistry. Human IL-2Rβ was injected at a flow rate of 10 μL / min for 180 seconds at concentrations of 5, 10, or 20 µg / mL. Subsequently, the test compound was injected as a 7-point dilution series at a flow rate of 30 μL / min, starting at a concentration of 500 or 250 nM (dilution factor 2.5), for 2 minutes to allow binding to the target, followed by 10 minutes of run buffer (Stenofan, BR100669, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20, pH 7.4) to allow separation from the target. The chip was regenerated using two 30-second pulses of 0.85% H3PO4 at 30 µL / min. Binding data were collected at 25°C and analyzed using Insight Evaluation software version 3.0.12 provided by the manufacturer (Stenofan) according to a 1:1 binding fit model.

[0521] IL-2Rβ / γ binding

[0522] The binding of human IL-2Rβ / γ (self-made human IL-2RB(ECD)(T191C)-THR-zipper) was detected by surface plasmon resonance (SPR) (Stopfan, Biacore 8K+, #2626160). In short, as described in the kit protocol, Strep-Tactin®XT (Iba Life Sciences, Twin-Strep-tag Capture Kit, 2-4370-000) was immobilized on a CM5 sensor (Stopfan, BR100399). Human IL-2Rβ / γ was injected at a flow rate of 10 μL / min at 0.75 µg / mL for 180 seconds. Subsequently, the test compound was injected as a 7-point dilution series at a flow rate of 30 μL / min, starting at a concentration of 250 or 150 nM (dilution factor 2.5), for 2 minutes to allow binding to the target, followed by 10 minutes of run buffer (Stenofan, BR100669, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20, pH 7.4) to allow separation from the target. The chip was regenerated using three 70-second pulses of 3M GuHCl at 30 µL / min. Binding data were collected at 25°C and analyzed using InsightEvaluation software version 3.0.12 provided by the manufacturer (Stenofan) according to a 1:1 binding fit model.

[0523] The results are summarized in Tables 12a, 12b, and 12c.

[0524] Table 12a: Kinetic parameters of the interaction between the IL-2 macroantibody protein and IL-2 receptor α. Additionally, the fold change compared to the reference TP027 was calculated.

[0525]

[0526] Table 12b: Kinetic parameters of the interaction between the IL-2 macroantibody protein and IL-2 receptor β. Additionally, the fold change compared to the reference TP027 was calculated.

[0527]

[0528] Table 12c: Kinetic parameters of the interaction between the IL-2 macroantibody protein and the IL-2 receptor β / γ. Additionally, the fold change compared to the reference TP027 was calculated.

[0529]

[0530] As expected by pSTAT5 and proliferation assays, the IL-2 macroantibody protein exhibited a distinct binding profile to cytokine receptors. Compounds TP115, TP116, and TP119 showed significantly reduced affinity for IL-2Rα. Compound TP116 also showed reduced affinity for IL-2Rβ, although it could still bind to the IL-2Rβ / γ heterodimer. A similar trend was observed for compound TP118. Differential binding to the IL-2Rβ / γ heterodimer was less pronounced in the current assays. Overall, these distinct binding profiles suggest that the location of ISVD insertion into cytokines can modulate the interaction between cytokines and their receptors.

[0531] Example 8: Activity of IL-2 macroantibody protein in tetanus toxoid (TT) recall assay

[0532] Primary T cell activation of the multivalent construct (i.e., the macroantibody protein fused with ISVD) was tested in an autologous tetanus toxoid recall assay (monitored via IFNγ production). Briefly, PBMCs isolated from healthy donors were retrieved from cryogenic storage and thawed in medium (RPMI 1640 medium, GlutaMAX™ supplement, 25 mM HEPES (LifeScience & Technology Corporation - Gibco, 72400-021), supplemented with 10% heat-inactivated FBS (Sigma F9665) and 1% penicillin / streptomycin (LifeScience & Technology Corporation, 15140)). Monocytes were isolated by negative magnetic separation according to the supplier's instructions for the EasySep Human Monocyte Isolation Kit (Stemcell Technologies, 19359), and cultured in human Mo-DC differentiation medium (Medtronic Biotechnology, 130-094-812) for 7 days, with an equal volume of fresh medium added midway through day 3. Seven days later, monocytes differentiated into immature dendritic cells (iDCs). iDCs were harvested and frozen in liquid nitrogen for later use. PBMCs from the same donor were thawed in assay medium (RPMI 1640 medium, GlutaMAX™ supplement, 25 mM HEPES (LifeScience-Gibberellic Acid, 72400-021), supplemented with 10% heat-inactivated human AB serum (BioIVT, SM-612-HSI) and 1% penicillin / streptomycin) and cultured for 7 days at 37°C in a 5% CO2 atmosphere in assay medium supplemented with 0.5 µg / mL tetanus toxoid (TT) (Calbiochem, 582231). After 7 days, TT-specific T cells were enriched. Cells were harvested and frozen in liquid nitrogen for later use. For characterization assays, iDCs were thawed in assay medium and seeded at 5000 cells / well in 96-well U-type plates (Corning, 3799) with assay medium supplemented with 0.5 µg / mL TT, and incubated at 37°C for 4 hours in a 5% CO2 atmosphere. Autologous TT-rich T cells were thawed in assay medium, and 100,000 cells / well were added to the iDCs, along with a series of dilutions of the multivalent construct. After 3 days of co-culture, the cell supernatant was harvested, and IFNγ concentrations were determined by ELISA. Results are shown in... Figure 16 And in Table 13.

[0533]

[0534] All tested compounds containing IL-2 alone, anti-PD-L1 ISVD alone, or combinations of both in different forms induced dose-dependent IFNγ production in the TT recall assay. Optimal potency was observed for the IL-2-only construct. Although potency decreased when IL-2 was combined with anti-PD-L1 ISVD, efficacy increased and higher levels of IFNγ production were detected, demonstrating the additive effect of combining IL-2 with anti-PD-L1 ISVD on primary T cell function. This was also observed for combination therapy, N-terminal to C-terminal ISVD-cytokine fusions, and for macroantibody protein and ISVD macroantibody fusions (multivalent constructs), suggesting that both IL-2 and anti-PD-L1 ISVD are functional in their different forms. Compared to anti-PD-L1-IL-2 macroantibody protein (TP048), ALB-IL-2 macroantibody protein (TP021) behaved similarly to IL-2 alone, with better potency but lower efficacy.

[0535] Example 9: Design of a cyclic variant of interferon α-2a (IFNA2a).

[0536] To design the IFNA2a_NbALB23 macroantibody protein, a well-folded, circularly arranged version of interferon α-2a (IFNA2a) is required. Therefore, the structure of IFNA2a (PBD: 1ITF, 3S9D) was examined, and a theoretical construct was designed, in which the folded circular arrangement is as follows: Figure 17 A site was depicted, and a peptide linker was introduced to ligate the C-terminus of IFNA2a to its N-terminus. Finally, two constructs were created and cloned into a yeast display vector containing all the accessory proteins described above to enable the display of circular arrangement variants on the surface of EBY100 yeast cells. In one construct, the last five amino acids of IFNA2a were deleted, and a three-amino acid peptide linker (SEQ ID NO: 57) was introduced to ligate the C-terminus (residue 160) of IFNA2a to the N-terminus of IFNA2a, resulting in a circular arrangement variant of IFNA2a, designated IFNA2a[D77-W76]V2 (SEQ ID NO: 58). In another construct, the last two amino acids of IFNA2a were deleted, and a three-amino acid peptide linker (SEQ ID NO: 57) was introduced to attach the C-terminus (residue 163) of IFNA2a to its N-terminus. This cyclic variant of IFNA2a was named IFNA2[D77-W76]V4 (SEQ ID NO: 59). Wild-type IFNA2a (SEQ ID NO: 56) was cloned in parallel and displayed on the surface of yeast cells.

[0537] Example 10: Binding of a specific anti-IFNA2a monoclonal antibody to interferon α-2a and its circular variants.

[0538] The circular arrangement of IFNA2a was expressed and displayed on the surface of EBY100 yeast cells. To analyze the folding of IFNA2a[D77-W76]V2 (SEQ ID NO: 58) and IFNA2[D77-W76]V4 (SEQ ID NO: 59) and compare them with IFNA2a (SEQ ID NO: 56), yeast cells expressing different constructs were incubated for 1 h in the presence of monoclonal antibody mAb93452 (human IFN-α2 / IFNA2a antibody, R&D Systems: MAB93452) at a final concentration of 2.5 µg / ml. EBY100 yeast cells not expressing any constructs were also incubated for 1 h in the presence of monoclonal antibody mAb93452 (human IFN-α2 / IFNA2a antibody, R&D Systems: MAB93452) as a negative control. After washing three times, all cells were incubated for 1 h in the presence of 2 µg / ml anti-mouse IgG Fc (phycoerythrin-conjugated AffiniPure goat anti-mouse IgG Fcγ-specific, Jackson Immunological Research, Inc.), washed three times, and analyzed by flow cytometry. Further fluorescence was observed after incubation with monoclonal mAb93452, binding to yeast cells displaying IFNA2a (SEQ ID NO: 56), IFNA2[D77-W76]V2 (SEQ ID NO: 58), or IFNA2[D77-W76]V4 (SEQ ID NO: 59). Figure 18 For EBY100 yeast cells that did not express any constructs, no significant fluorescence shift was observed. Expression of all constructs was tracked and confirmed by incubating clones for 1 h at a final concentration of 4 µg / ml in the presence of mouse anti-Myc monoclonal antibody (Roche / #11667 149 001), followed by three washes and incubation in the presence of anti-mouse IgG Fc (phycoerythrin-conjugated AffiniPure goat anti-mouse IgG Fcγ-specific, Jackson Immunological Research). After three washes, cell expression was analyzed by flow cytometry. Figure 18 ).

[0539] Example 11: Binding of IFNAR2 to interferon α-2a and its cyclic variants.

[0540] The circular arrangement of IFNA2a was expressed and displayed on the surface of EBY100 yeast cells. To analyze the folding of IFNA2a[D77-W76]V2 (SEQ ID NO: 58) and IFNA2[D77-W76]V4 (SEQ ID NO: 59) and compare them with IFNA2a (SEQ ID NO: 56), yeast cells expressing different constructs were incubated for 1 h in the presence of his-tagged IFNAR2 (human IFN-α / β R2 protein, His tag, Acro Biosystems) at a final concentration of 4 µg / ml. EBY100 yeast cells not expressing any constructs were also incubated for 1 h in the presence of IFNAR2 (human IFN-α / β R2 protein, His tag, Acro Biosystems) as a negative control. After washing three times, all cells were incubated for 1 h in the presence of mouse anti-His antibody-PE (Medini Biotech, Inc. / #130-120-718; 1 / 50 dilution), washed three times, and analyzed by flow cytometry. After incubation with IFNAR2, increased fluorescence was observed in yeast cells displaying IFNA2a (SEQ ID NO: 56), IFNA2a[D77-W76]V2 (SEQ ID NO: 58), or IFNA2[D77-W76]V4 (SEQ ID NO: 59). Figure 19 For EBY100 yeast cells that did not express any constructs, a smaller fluorescence shift was observed. Expression of all constructs was tracked and confirmed by incubating clones for 1 h at a final concentration of 4 µg / ml in the presence of mouse anti-Myc monoclonal antibody (Roche / #11 667 149 001), followed by three washes and incubation in the presence of anti-mouse-IgG-Fc-PE (phycoerythrin-conjugated AffiniPure goat anti-mouse IgG Fcγ specific, Jackson Immunological Research). After three washes, cells were analyzed by flow cytometry. Figure 19 ).

[0541] Example 12: Design of a 31 kDa antigen-binding chimeric protein constructed from a circularly arranged variant of interferon α-2a inserted into the first β-turn of the β-chains A and B of the anti-HSA ISVD chain.

[0542] Based on the successful design of the IL-2(K35E,C125S)_ISVD207 macroantibody protein and the knowledge that we can prepare a circular variant of IFNA2a, we also designed an ISVD molecule transplanted onto interferon α-2a (IFNA2a).

[0543] Check interferon alpha-2a (IFNA2a PDB 1ITF, 3S9D, Figure 17 The topology of IFNA2_ALB23 was determined, and different sites were selected for grafting ISVD ALB23002 onto it. This resulted in twenty-three different versions of the IFNA2_ALB23 macroantibody protein. The 31 kDa macroantibody protein described herein was developed based on... Figure 1 A chimeric polypeptide formed by linking a single-domain immunoglobulin portion and a scaffold protein portion. Here, the immunoglobulin domain used is anti-HSAISVD as shown in SEQ ID NO: 55. The scaffold protein is IFNA2a (SEQ ID NO: 56). All parts are linked to each other via peptide bonds from the amino terminus to the carboxyl terminus in the following given order: β-chain A of anti-HSA ISVD (residues 1-12 of SEQ ID NO: 55), short peptide linker (SEQ ID NO: 5), C-terminal portion of IFNA2a (SEQ ID NO: 56, from amino acid position X2 to amino acid position 165), peptide linker connecting the C-terminal portion of IFNA2a to its N-terminus (SEQ ID NO: 56, from amino acid position 1 to amino acid position X1) to produce a circular arrangement variant of the scaffold protein (SEQ ID NO: 57), N-terminal portion of IFNA2, short peptide linker (SEQ ID NO: 5), followed by β-chains B to G of anti-HSA ISVD (residues 16-126 of SEQ ID NO: 55). An α-sheet model of some (non-limiting) examples of the IFNA2a_ALB23002 macroantibody protein is shown in... Figure 20-23 (SEQ IDNO: 60-63)

[0544] Example 13: Binding of IFNA2a macro antibody protein to IFNα receptor IFNAR2 and ISVD target human serum albumin (HSA)

[0545] IFNAR2 binding

[0546] Binding to human IFNAR2 (Sinochem Biosciences, 10359-H02H) was detected by surface plasmon resonance (SPR) (Stopfan, Biacore 8K+, #2626160). In short, the anti-human Fc binder was immobilized on a CM5 sensor (Stopfan, BR100399) using standard amine coupling chemistry. Human IFNAR2 was injected at a flow rate of 10 μL / min for 180 seconds at a concentration of 1 or 20 µg / mL. Subsequently, the test compound was injected as a 7-point dilution series at a flow rate of 30 μL / min, with initial concentrations of 500, 100, 50, or 25 nM (dilution factor 2.5), for 2 minutes to allow binding to the target. This was followed by 10 minutes of run buffer (Stenofan, BR100669, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20, pH 7.4) to allow separation from the target. The chip was regenerated using two 30-second pulses of 0.85% H3PO4 at 30 µL / min. Binding data were collected at 25°C and analyzed using InsightEvaluation software version 3.0.12 provided by the manufacturer (Stenofan) according to a 1:1 binding fit model.

[0547] HSA combination

[0548] Binding to human serum albumin (HSA) (Sigma-Aldrich, A8763) was detected by surface plasmon resonance (SPR) (Stopvan, Biacore 8K+, #2626160). In short, HSA was immobilized on a C1 sensor (Stopvan, BR100535) using standard amine coupling chemistry. The test compound was then injected as a 9-point dilution series at a flow rate of 30 μL / min, starting at 2500 nM (dilution factor 2.5), for 2 minutes to allow binding to the target, followed by 10 minutes of run buffer (Stopvan, BR100669, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20, pH 7.4) to allow separation from the target. The chip was regenerated using two 30-second pulses of 10 mM glycine (pH 1.5) at 30 µL / min. Combined data were collected at 25°C and analyzed using Insight Evaluation software version 3.0.12 provided by the manufacturer (Stopfan) based on a 1:1 combined fit model.

[0549] The macroantibody construct was designed to modulate the interaction of IFNA2a with its high-affinity receptor IFNAR2, while maintaining the interaction of the ISVD with its target. A systematic analysis of IFNA2a cytokines produced affinity (KD) profiles for IFNAR2, where compounds with higher (cluster C) or lower (cluster B) affinity compared to the reference compound TP088 were grouped into cluster A. Compounds with ISVD insertion sites in the same region of the IFNA2a cytokine spectrum showed similar affinity. Data are shown in Table 14. TP093 (cluster B, Figure 24 ) and TP109 (cluster C, Figure 25 The α-sheet model of ISVD shows that it is localized towards the low-affinity receptor IFNAR1 and away from the high-affinity receptor IFNAR2. Combined with kinetics, the effect on IFNAR2 affinity is indeed limited; for TP093 and TP109, the affinity decreases to 1 / 5 and increases to 10-fold, respectively. On the other hand, TP095 (cluster B, Figure 26 ) and TP098 (cluster B, Figure 27 The α-sheet model showed that ISVD localizes towards the high-affinity receptor IFNAR2. For TP095, a decrease in affinity for IFNAR2 was observed to 1 / 70, while for TP098, affinity could not be determined due to binding too low. Furthermore, different macroantibody proteins can have similar functional outcomes (i.e., the same clustering) despite different mechanisms. For compounds where ISVD targets IFNAR1 (e.g., TP093), the regulation of cytokine activity may be a result of inhibiting heterodimerization of the receptor complex. For other compounds where ISVD targets IFNAR2 (e.g., TP095), this effect may be a direct result of the interaction with IFNAR2.

[0550]

[0551] For ISVD-HSA interaction, the expected KD is 1-10 nM. Binding of ISVD to its target HSA is not hindered by its formatting as an IFNA2a macroantibody protein. For all macroantibody proteins, the binding affinity to HSA differs by up to 3.5-fold compared to the reference compound TP113.

[0552] Example 14: Activity of IFNA2a macro antibody protein in STAT1 phosphorylation assay

[0553] STAT1 signaling in A549 cells (human lung cancer, ATCC CCL-185) induced by macroantibody proteins was characterized to demonstrate their differential signaling profile. STAT1 phosphorylation was determined by flow cytometry. Briefly, A549 cells were incubated at 37°C in a 5% CO2 atmosphere in Ham's F-12K (Kaighn's) medium supplemented with 10% heat-inactivated FBS; Life Sciences Corporation - Gibco 21127 and Sigma F9665, respectively. On the day of assay, A549 cells were harvested, washed with D-PBS (Gibco 14190), and then stained in the dark at RT with ZombieNIR fixative live / dead stain (Baijin, 423105) for 15 min. After washing with culture medium, 150,000 cells were seeded in 75 µl of culture medium in each well of a 96-well U-shaped plate (Costar 3799) and incubated at 37°C in a 5% CO2 atmosphere for at least 30 min. Then, an equal volume of macroantibody protein or a combination of macroantibody protein and human serum albumin (HSA, final concentration 30 µM, CSL Behring 2160-679) was added, and the cells were incubated at 37°C for 15 min. Next, the cells were fixed by adding 150 µl / well of pre-warmed (37°C) fixation buffer I (BD Biosciences 557870) and incubating at 37°C for 15 min. After washing twice with FACS buffer (D-PBS, Gibco 14190, supplemented with 2% heat-inactivated FBS, Sigma F7524 and 0.05% sodium azide, Acros organics 19038), pre-cooled (-20°C) Perm Buffer III (BD Biosciences 558050) was slowly added to the cell pellet, followed by incubation on ice for 30 min. After two washes with FACS buffer, cells were incubated at 4°C with human Fc blocker (BD Pharmingen 564220, 12.5 µg / mL) for 15 min, followed by the addition of PE-conjugated anti-human STAT1 (pY701) (BD Biosciences 562069) and incubation at RT in the dark for 60 min. Cells were analyzed using a MACS Quant flow cytometer (Miltenyi Biotec) after two washes with FACS buffer. Median fluorescence intensity (MFI) of pSTAT1-PE staining was determined after gating live cells. Results are shown in... Figure 28 And in Table 15.

[0554]

[0555] Systematic analysis of IFNA2a cytokines yielded a range of potencies, with some compounds showing better (cluster C) and others worse (cluster B) potency compared to a reference compound. Compounds with potency similar to TP088 were grouped into cluster A. Compounds with ISVD insertion sites in the same region of the IFNA2a cytokine spectrum showed similar functionality. Functionality was correlated with binding data (Table 14). HSA could further modulate functionality in a compound-dependent manner.

[0556] Example 15: Antiproliferative activity of IFNA2a macro antibody protein against RPMI 8226 and NCI-H929 cells

[0557] The antiproliferative effects of macroantibody protein on RPMI 8226 (human B lymphocyte line from plasmacytoma, ATCC CCL-155) and NCI-H929 cells (human myeloma cell line, DSMZ ACC 163) were characterized. Cells were cultured in media specific to RPMI 8226 cells (RPMI 1640, Glutamax, 25 mM Hepes, Gibco 72400-021, supplemented with 10% heat-inactivated FBS, Sigma F9665, 1 mM sodium pyruvate, Gibco 11360-039, and 1% penicillin / streptomycin, Gibco 15140-122) and media specific to NCI-H929 cells (RPMI 1640, Glutamax, 25 mM Hepes, Gibco 72400-021, supplemented with 10% heat-inactivated FBS, Sigma F9665, 1 mM sodium pyruvate, Gibco 11360-039, 50 µM β-mercaptoethanol, Gibco 21985-023, and 1% penicillin / streptomycin, Gibco 15140-122). Cells were grown in (15140-122). On the day of assay, cells were harvested and seeded at 5,000 cells / well in 40 µl of medium in 384-well flat-bottomed clear white TC-treated plates (Corning, 3765). An equal volume of the compound or a combination of the compound and human serum albumin (HSA, final concentration 30 µM, CSL Behring 2160-679) was then added, and the cells were incubated at 37°C in a 5% CO2 atmosphere for 3 days. Next, the 40 µl supernatant was removed and replaced with 40 µl of CellTiter-Glo reagent (Promega, G7570), then resuspended, shaken, and incubated for 10 min. Finally, luminescence was measured using an EnVision device (PerkinElmer). Results are shown in [Figure number missing]. Figure 29 And in Table 16.

[0558]

[0559] Systematic analysis of IFNA2a cytokines yielded a range of potencies, with some exhibiting better (cluster C) or worse (cluster B) potency compared to the reference compound TP088. Compounds with similar potency to TP088 were grouped into cluster A. ...

Claims

1. A chimeric protein comprising an immunoglobulin single variable domain (ISVD) fused to a cytokine, wherein an internal fusion site of the ISVD is linked to the cytokine, wherein the internal fusion site is located in a loop or turn between two secondary structural elements in the ISVD, and wherein the cytokine is a cyclically arranged cytokine.

2. The chimeric protein of claim 1, wherein the internal fusion site of the ISVD is connected to the internal fusion site of the cytokine, and wherein in the cytokine, the internal fusion site is located in a loop or turn between two secondary structural elements.

3. The chimeric protein according to any one of claims 1 to 2, wherein the ISVD and the cytokine are fused through at least one, preferably two, peptide linkers.

4. The chimeric protein according to any one of claims 1 to 3, wherein the internal fusion site is a loop or turn between two β-chains in the ISVD, and / or a loop or turn between two β-chains or two α-helices or between one β-chain and one α-helix in the cytokine.

5. The chimeric protein according to any one of claims 1 to 4, wherein the ISVD is V H or V HH Preferably, the ISVD is V HH More preferably, humanized V HH Or camel-derived V H .

6. The chimeric protein according to any one of claims 1 to 5, wherein the cytokine is an interleukin or interferon, preferably wherein the interleukin is interleukin-2 (IL-2) or interleukin-18 (IL-18) and / or wherein the interferon is interferon (IFN) α2a (IFNA2a).

7. The chimeric protein according to any one of claims 1 to 6, wherein the cytokine fuses with the ISVD at an internal fusion site, the internal fusion site being located in one of the following corners of the ISVD according to IMGT classification: a. In the first β-turn of the β-chains A and B connecting the ISVD; or b. In the β-turn of the β-chains C and C' connecting the ISVD; or c. In the β-turn of the β-chain C” and D connecting the ISVD; or d. In the β-turn of the β-chains D and E connecting this ISVD; or e. In the β-turn of the β-chains E and F connecting the ISVD.

8. The chimeric protein according to any one of claims 1 to 7, wherein the cytokine is an interleukin, and wherein the internal fusion site of the cytokine is an exposed β-turn of the interleukin β-barrel core motif.

9. The chimeric protein according to any one of claims 1 to 8, wherein the chimeric protein comprises an ISVD, the ISVD comprising a sequence as defined in SEQ ID NO.: 1 or 55, or a sequence having at least 80% identity with SEQ ID NO.: 1 or 55.

10. The chimeric protein according to any one of claims 1 to 8, wherein the chimeric protein comprises a cytokine comprising a sequence as defined in SEQ ID NO.: 4, 58, 59, 64, 66, 68, 70, 172-195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 220, 222, 225, 227, 244-246, 262, 264, 268, 270, 272, or 274, or a sequence associated with SEQ ID NO.: 4, 58, 59, 64, 66, 68, 70, 172-195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 220, 222, 225, 227, 244-246, 262, 264, 268, 270, 272, or 274 are sequences with at least 80% identity.

11. The chimeric protein according to any one of claims 1 to 10, wherein the chimeric protein comprises or is composed of the following sequences: The sequence defined in SEQ ID NO.: 7-25, 36-54, 60-63, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218-219, 221, 223-224, 226, 230-237, 261, 263, 265-267, 269, 271 or 273, or the sequence with SEQ ID NO.: Sequences 7-25, 36-54, 60-63, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218-219, 221, 223-224, 226, 230-237, 261, 263, 265-267, 269, 271, or 273 have at least 80% identity.

12. A polypeptide comprising a chimeric protein according to any one of claims 1 to 11, optionally wherein the polypeptide further comprises one or more additional groups, residues, portions or binding units, preferably wherein the polypeptide further comprises one or more ISVDs.

13. A nucleic acid molecule encoding a chimeric protein according to any one of claims 1 to 11 or a polypeptide according to claim 12.

14. A method for altering and / or modifying cytokine signaling and / or for influencing, altering and / or modifying receptor oligomerization when a cytokine binds to at least one of its receptor or receptor subunit by fusing a cytokine with an ISVD, wherein an internal fusion site of the ISVD is used for inserting a cytokine or a cyclically arranged cytokine, wherein the internal fusion site is located in a loop or turn between two secondary structural elements in the ISVD.

15. A method for regulating cytokine signaling, the method comprising the following steps: - Provide a chimeric protein according to any one of claims 1 to 11 or a polypeptide according to claim 12; as well as - Screen for chimeric proteins or peptides in which the cytokines contained in the chimeric protein or peptide exhibit modified cytokine signaling compared to cytokines not fused with the ISVD.

16. The method of claim 15, wherein the screening is performed by testing chimeric proteins or peptides containing the cytokine in a functional assay of the cytokine to identify chimeric proteins or peptides having modified cytokine activity.

17. The chimeric protein according to any one of claims 1 to 11 or the polypeptide according to claim 12, wherein the chimeric protein or the polypeptide is intended for use in medicine.

18. The chimeric protein according to any one of claims 1 to 11 or the polypeptide according to claim 12, wherein the chimeric protein or the polypeptide is used in the treatment of cancer and / or the treatment of inflammatory diseases, preferably wherein the cancer is a solid tumor and / or a liquid tumor.

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