Chimeric proteins for regulating cytokine receptor activity

Abstract

This invention relates to proteins and peptides comprising a single immunoglobulin variable domain (ISVD) fused with a cytokine, belonging to the field of immunology. In particular, the invention provides a chimeric protein comprising a single immunoglobulin variable domain (ISVD) fused with a cytokine, wherein the internal fusion site of the ISVD is linked to the cytokine and the internal fusion site is located within a loop or turn between two secondary structural elements. Fusion of the cytokine to the ISVD upon binding of the cytokine to a receptor or receptor subunit enables the regulation of cytokine receptor activity and / or downstream signaling.

Description

[Technical Field]

[0001] The present invention relates to proteins and polypeptides belonging to the field of immunology, comprising an immunoglobulin single variable domain (ISVD) fused with a cytokine, wherein the fusion is obtained by insertion of the cytokine into the ISVD at a fusion site in a loop or turn of the ISVD that is not a complementarity-determining region, and / or the cytokine is a circularly permuted variant of a wild-type cytokine. Binding of cytokine-ISVD chimeric proteins to cytokine receptors or receptor subunits enables modulation of cytokine receptor activity and / or (downstream) signaling. [Background technology]

[0002] Cytokines are small signaling proteins known to play a crucial role in the body's response to inflammation and immune attacks. Pro-inflammatory cytokines warn the immune system of the presence of potential infection or danger. However, uncontrolled cytokine production can lead to autoinflammatory disease states.

[0003] Cytokines are widely used as treatments for cancer, infections, or other diseases. Interleukin-2 (IL-2), the first cytokine discovered to have therapeutic benefits, was discovered in 1976 by Robert Gallo and Francis Ruscetti. This team demonstrated that this cytokine can dramatically stimulate the proliferation of T cells and natural killer (NK) cells, which are essential for the human immune response (Morgan DA, Ruscetti FW, Gallo R., “Selective in vitro growth of T lymphocytes from normal human bone marrows”, Science, 1976, 193(4257):1007-8). In 1988, Rosenberg SA's group published a preliminary report on the treatment of patients with metastatic melanoma using IL-2 (Rosenberg SA et al., “Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report”, N Engl J Med., 1988, 319(25):1676-80). IL-2 became the first cancer immunotherapy approved by the US FDA and is still used clinically 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 transmit their signals. Biological responses can vary between cytokine receptors and between cells, but generally include gene expression, changes in the cell cycle, and the release of mediators such as the cytokines themselves. Cytokine receptors generally function as oligomeric complexes consisting typically of 2 to 4 subunits, which may be the same or different. When cytokines bind to surface receptors, the cytokines induce receptor clustering or oligomerization (e.g., heterodimerization or heterotrimerization), followed by receptor activation and the generation of intracellular signals (downstream signaling) (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] Heterotrimeric receptors include, for example, the IL-2 receptor (IL-2R), which has 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, the β and γ combination forms a complex that binds to IL-2 with intermediate affinity (forming a heterodimer) mainly on memory T cells and NK cells, and all three receptor chains bind with high affinity (approximately 10) on activated T cells and regulatory T cells. -11It forms a complex (heterotrimer) that binds to IL-2 at M (Kd). The intermediate affinity and high affinity receptor forms are functional, and when IL-2 binds to them, it causes changes in cells (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 heterodimeric receptor is the IFN-alpha receptor (IFNAR), a substantially ubiquitous membrane receptor that binds to IFN-alpha. IFNAR comprises two subunits, IFNAR1 and IFNAR2, bound to the FERM domains of tyrosine protein kinases TYK2 and JAK1, respectively. The biological effects of type I IFN (e.g., IFN-alpha) include a different range of subtypes in different cells (van Boxel-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 heterodimer receptor. It consists of two distinct but structurally related immunoglobulin-like domains: IL-18Rα and IL-18Rβ, which are members of the IL-1 receptor family. Secreted mature IL-18 interacts with IL-18Rα. This complex heterodimerizes with the signaling IL-18Rβ coprotein, which promotes conformational changes of the receptor to enable 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 is solely involved in IL-18 binding. Considering the difference in affinity between IL-18Rα and the IL-18Rα / β complex regarding IL-18 binding, it is highly probable that IL-18Rα and the IL-18Rα / β complex may present different contact sites to IL-18. These differences may involve conformational changes, resulting in different orientations and a different number 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] Considering the above, for example, regulating the receptor-binding function of cytokines to modulate the response triggered by cytokines when they bind to receptors / receptor subunits would be useful for developing new cytokine-based therapies or improving existing ones. For example, inducing activation in a particular cell population (e.g., by producing IL-2 cytokines with enhanced affinity for specific receptor subunits) could be beneficial for treating certain pathological 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) describe an engineered fusion protein composed of circulating 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 sterically inhibits the interaction between the IL-2 of the fusion protein and the endogenous IL-2Rα subunit. When the interaction site of IL-2 with the endogenous IL-2Rα subunit is blocked, the IL-2 of the fusion protein becomes memory CD8 + It may be possible to retain the ability to signal via intermediate affinity IL-2R (subunits β and γ), which is constitutively expressed on T cells and NK cells. (Memory CD8) + T cells and NK cells have been shown to be necessary for protective anti-cancer immune responses.

[0010] However, there is a need for cytokines and methods for producing them that can generally regulate the receptor / receptor subunit binding functionality of cytokines, for example, globally, and therefore are not limited to specific cytokines and specific receptor subunits. [Overview of the project] [Means for solving the problem]

[0011] The present invention solves the above problems and provides a chimeric protein comprising an immunoglobulin single variable domain (ISVD) fused with a cytokine, wherein the internal fusion site of the ISVD is linked to the cytokine, the internal fusion site is located within a loop or turn between two secondary structural elements, and the cytokine is preferably a cyclically substituted cytokine. Preferably, in the chimeric protein, the internal fusion site of the ISVD is linked to the internal fusion site of the cytokine, and in both the ISVD and the cytokine, the internal fusion site is located within a loop or turn between two secondary structural elements. The chimeric protein provided by the present invention is more rigid compared to N-terminal / C-terminal end-to-end fusions (i.e., a "mega-body" type fusion (see, for example, International Publication No. 2019 / 086548)). Therefore, the chimeric protein of the present invention is less susceptible to proteasomal degradation or flexible migration compared to N-terminal / C-terminal end-to-end fusion proteins. In addition, since the ISVD present in the chimeric protein provided by the present invention can bind to its target, the chimeric protein may contain further target-binding sites that can be selected depending on the specific properties desired in the chimeric protein / polypeptide (extended half-life, labeling, specific localization, or any other functional requirements).

[0012] Furthermore, as described below, the chimeric proteins of the present invention can be linked to further parts having different functionalities (see polypeptides of the present invention).

[0013] Therefore, the chimeric proteins provided by the present invention enable directional modulation of cytokine receptor / receptor subunit binding functionality and may also have further advantages as described above.

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

[0015] As shown in the following examples, the chimeric proteins and / or polypeptides of the present invention can regulate the activity of cytokines contained in the chimeric proteins and / or proteins of the present invention (or the associated result of the binding of cytokines contained in the chimeric protein to at least one of its receptors or receptor subunits).

[0016] Further information is provided regarding nucleic acid molecules encoding the chimeric protein or polypeptide of the present invention, vectors containing the nucleic acid molecule of the present invention, host cells containing the chimeric protein and / or polypeptide of the present invention, or nucleic acid molecules or vectors encoding the chimeric protein of the present invention.

[0017] The present invention further provides a method for producing the chimeric protein and / or peptide of the present invention.

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

[0019] The present invention further provides a fusion protein comprising cytokines fused to an ISVD, either directly or by a linker, for regulating the binding affinity of the cytokines contained in the fusion protein to their receptors.

[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.

[0021] The drawings provided are schematic and not limiting. Some element dimensions in the drawings may be exaggerated and are not depicted to their actual dimensions for illustrative purposes only. [Brief explanation of the drawing]

[0022] [Figure 1] Engineering principle of an antigen-binding chimeric protein constructed from a cyclically substituted variant of a scaffold protein inserted into the first β-turn connecting β-chains A and B of the ISVD. This scheme shows how an immunoglobulin single variable domain (ISVD) can be grafted onto a larger scaffold protein via two peptide bonds or two short linkers connecting the antigen-binding domain to the scaffold. Scissors indicate which exposed turns in the ISVD and scaffold need to be cut. Dashed lines show how the rest of the ISVD and scaffold must be linked by using peptide bonds or short peptide linkers to construct the antigen-binding chimeric protein. The CDR, framework residues, and β-turn region of the ISVD are defined according to IMGT (Lefranc MP, “Immunoglobulin and T Cell Receptor Genes: IMGT® and the Birth and Rise of Immunoinformatics”, Front Immunol., 2014, 5:22). [Figure 2]Engineering principle of an antigen-binding chimeric protein constructed from a cyclically substituted variant of IL-2, inserted into the first β-turn connecting β-chains A and B of ISVD. This scheme shows how ISVD can be grafted onto IL-2 via two peptide bonds with two short linkers connecting the antigen-binding domain to the cyclically substituted IL-2. Scissors indicate which exposed turns are cleaved in ISVD and the scaffold. Dashed lines show how the rest of ISVD and the scaffold are linked by peptide bonds or short peptide linkers to construct the antigen-binding chimeric protein. The CDR, framework residues, and β-turn regions of ISVD are defined according to IMGT. [Figure 3] Insertion sites on IL-2. This figure shows different sites where ISVD can be grafted onto IL-2 via two peptide bonds. The figure shows the start and end sites of the cyclically substituted IL-2 (see also Figure 4), as well as the reference numbers of the corresponding IL-2 "megabody protein" constructs. [Figure 4]Schematic diagrams of different IL-2 megabody proteins. This diagram shows the designs of different IL-2(K35E,C125S) "megabody proteins". The cyclically substituted amino acid (AA) sequence of IL-2(K35E,C125S) is given as a "collier-de-perle" starting at amino acid 1 and ending at amino acid 133. The small GG linker (white letters on a gray background) connects the C-terminus of IL-2(K35E,C125S) to the N-terminus of IL-2. Therefore, the first three amino acids of IL-2((K35E,C125S) are deleted and are shown as strikethrough in the figure. The point mutations K35E and C125S have a circular light gray background in the sequence. On this IL-2(K35E,C125S) sequence, the initiation of each IL-2(K35E,C125S)_ISVD207 "megabody protein" construct (NH3 +) and termination (COO-) are indicated. In some cases, additional glycine (G) is added between the GSG linker and IL-2(K35E,C125S) to make the linker between IL-2(K35E,C125S) and ISVD207 4 amino acids long instead of 3. To fully show how ISVD can be grafted with cyclically substituted IL-2(K35E,C125S), the insertion site of IL-2(K35E,C125S) for construct SA17667 is magnified below the figure as an example, and the cyclically substituted IL-2(K35E,C125S) is interrupted (in this construct, the amino acids between the scissors are deleted), and This shows how it fuses with ISVD207. Construct SA17667 begins at residues 1-12 of ISVD207, followed by a four-amino acid linker (GSGG) fused from amino acids 62 to 133 of IL-2(K35E,C125S), which is linked to amino acid 4 of IL-2(K35E,C125S) via a GG linker, terminating at amino acid L59, and connecting to residues 16-126 of ISVD207 via a four-amino acid linker (GGSG). The GSG linker between the cyclically substituted IL-2(K35E,C125S) and ISVD207 is given by a dotted circle, as is the additional glycine (G).In construct SA17667, the four amino acid linkers are located between IL-2 (K35E, C125S) and ISVD207, with one of the sites being cyclically substituted. [Figure 5] AlphaFold model of a 29kDa GFP-binding chimeric protein constructed from a cyclically substituted variant of IL-2 inserted into the first β-turn connecting β-chains A and B of anti-GFP ISVD. (A) A model of an antigen-binding chimeric protein produced by the fusion of anti-GFP ISVD (top) and a cyclically substituted variant of human IL-2 (bottom) via two peptide bonds or linkers connecting ISVD as a scaffold. (B) A cyclically substituted gene encoding cyclically substituted IL-2 (K35E, C125S) (bottom) was inserted into the first β-turn of anti-GFP ISVD (top, SEQ ID NO: 1) connecting β-chain A to β-chain B (β-turn AB). (C) Amino acid sequence of the resulting IL-2(K35E, C125S)[GS75-N71G]_ISVD207 antigen-binding chimeric megabody protein (SEQ ID NO: 11). Sequences derived from IL-2 (K35E, C125S) are shown in bold. Sequences derived from ISVD are underlined. Peptide linkers for connecting ISVD to IL-2 are underlined with dashed lines. Peptides that create cyclically substituted variants by linking the N-terminus and C-terminus of IL-2 are shown in italics. [Figure 6]AlphaFold model of a 29kDa GFP-binding chimeric protein constructed from a cyclically substituted variant of IL-2 inserted into the first β-turn connecting β-chains A and B of anti-GFP ISVD. (A) A model of an antigen-binding chimeric protein produced by the fusion of anti-GFP ISVD (upper right) and a cyclically substituted variant of human IL-2 (lower left) via two peptide bonds or linkers connecting ISVD as a scaffold. (B) A cyclically substituted gene encoding cyclically substituted IL-2 (K35E, C125S) (bottom) was inserted into the first β-turn of anti-GFP ISVD (top, SEQ ID NO: 1) connecting β-chain A to β-chain B (β-turn AB). (C) Amino acid sequence of the resulting IL-2(K35E, C125S)[GF42-M39G]_ISVD207 antigen-binding chimeric megabody protein (SEQ ID NO: 9). Sequences derived from IL-2 (K35E, C125S) are shown in bold. Sequences derived from ISVD are underlined. Peptide linkers for connecting ISVD to IL-2 are underlined with dashed lines. Peptides that create cyclically substituted variants by linking the N-terminus and C-terminus of IL-2 are shown in italics. [Figure 7]AlphaFold model of a 29kDa GFP-binding chimeric protein constructed from a cyclically substituted variant of IL-2 inserted into the first β-turn connecting β-chains A and B of anti-GFP ISVD. (A) A model of an antigen-binding chimeric protein produced by the fusion of anti-GFP ISVD (top left) and a cyclically substituted variant of human interleukin-2 (IL-2, bottom right) via two peptide bonds or linkers connecting ISVD as a scaffold. (B) A cyclically substituted gene encoding cyclically substituted IL-2 (K35E, C125S) (bottom) was inserted into the first β-turn of anti-GFP ISVD (top, SEQ ID NO: 1) connecting β-chain A to β-chain B (β-turn AB). (C) Amino acid sequence of the resulting IL-2(K35E, C125S)[GL85-P82G]_ISVD207 antigen-binding chimeric megabody protein (SEQ ID NO: 14). Sequences derived from IL-2 (K35E, C125S) are shown in bold. Sequences derived from ISVD are underlined. Peptide linkers for connecting ISVD to IL-2 are underlined with dashed lines. Peptides that create cyclically substituted variants by linking the N-terminus and C-terminus of IL-2 are shown in italics. [Figure 8]AlphaFold model of a 29kDa GFP-binding chimeric protein constructed from a cyclically substituted variant of IL-2 inserted into the first β-turn connecting β-chains A and B of anti-GFP ISVD. (A) A model of an antigen-binding chimeric protein produced by the fusion of anti-GFP ISVD (bottom) and a cyclically substituted variant of human IL-2 (top) via two peptide bonds or linkers connecting ISVD as a scaffold. (B) A cyclically substituted gene encoding cyclically substituted IL-2 (K35E, C125S) (bottom) was inserted into the first β-turn of anti-GFP ISVD (top, SEQ ID NO: 1) connecting β-chain A to β-chain B (β-turn AB). (C) Amino acid sequence of the resulting IL-2(K35E, C125S)[L132-I129]_ISVD207 antigen-binding chimeric megabody protein (SEQ ID NO: 18). Sequences derived from IL-2 (K35E, C125S) are shown in bold. Sequences derived from ISVD are underlined. Peptide linkers for connecting ISVD to IL-2 are underlined with dashed lines. Peptides that create cyclically substituted variants by linking the N-terminus and C-terminus of IL-2 are shown in italics. [Figure 9]Summary of the characteristics of different IL-2(K35E,C125S)_ISVD207 chimeric megabody proteins analyzed by yeast display and FACS. Display levels of megabody proteins on the surface of yeast cells were analyzed according to Uchanski et al. (2021). To demonstrate the functionality of ISVD207 in the chimeric protein, screening was performed by staining cells with GFP. Clones with a signal greater than 0.5% were retained for further analysis. The presence of IL-2 in these megabody proteins was confirmed by staining yeast cells with fluorescent mAbNARA1 or fluorescent mAb5111, respectively. To analyze whether the presented megabody proteins still bound to different IL-2 receptor moieties, cells were pre-incubated with fluorescently soluble domains CD25 or CD122 / CD132 heterodimers, respectively. For each binding experiment, a dot plot representation of the relative fluorescence intensity of individual EBY100 yeast cells transformed with different pCTCON2 derivatives encoding the IL-2(K35E,C125S)_ISVD207 megabody protein was created. Gating was established using negative and positive controls. The percentage of individual EBY100 clones entering the gate is given for each clone for different binding experiments. Constructs in which the relative fluorescence intensity of individual EBY100 yeast cells was higher than that of the negative control are marked with an asterisk. [Figure 10] This shows the phosphorylation of STAT5 in different immune cell subtypes upon treatment with IL-2-containing compounds. CD8+CD25- cells are evaluated in A and B, and CD4+CD25+ cells are evaluated in C and D. Treatment is performed in the absence (A and C) and in the presence (B and D) of human serum albumin (HSA). [Figure 10-1] Same as above. [Figure 11] This shows the proliferation of different immune cell subtypes upon treatment with IL-2-containing compounds. CD8+CD25- cells are evaluated in A and B, and CD4+CD25+ cells are evaluated in C and D. Treatment is performed in the absence of HSA (A and C) and in the presence of HSA (B and D). [Figure 11-1] Same as above. [Figure 12] Structure of the IL-2 receptor alpha, beta, and gamma 4-component complex using the AlphaFold model of the IL-2(K35E,C125S)[GS75-N71G]_ISVD207 megabody protein. [Figure 13] Structure of the IL-2 receptor alpha, beta, and gamma 4-component complex using the AlphaFold model of the IL-2(K35E,C125S)[GF42-M39G]_ISVD207 megabody protein. [Figure 14] Structure of the IL-2 receptor alpha, beta, and gamma 4-component complex using the AlphaFold model of IL-2(K35E,C125S)[L132-I129]_ISVD207 megabody protein (SA17678). [Figure 15] Structure of the IL-2 receptor alpha, beta, and gamma 4-component complex using the AlphaFold model of IL-2(K35E,C125S)[GL85-P82G]_ISVD207 megabody protein (SA17659). [Figure 16] This shows the production of IFNγ in a tetanus toxoid recall assay to investigate the functionality of anti-PD-L1-IL-2 compounds in donors D1688 (A and B) and ABL-0341-02 (C and D). [Figure 16-1] Same as above. [Figure 17] Structure of IFNA2a. This figure shows the structure of IFNA2a (PDB 1ITF), where IFNA2a is opened (between positions 76 and 77) to create new N-terminus and C-terminus, and where the N-terminus and C-terminus are linked together by a peptide linker to create a cyclically substituted variant of IFNA2a. [Figure 18]Flow cytometry analysis of expression levels of different circulating variants of IFNA2a compared to wild-type IFNA2a shows each construct presented separately on the surface of EBY100 yeast cells; flow cytometry analysis of binding of anti-human IFNA2a monoclonal antibodies to the same constructs shows each presented separately on the surface of EBY100 yeast cells. Top: Dot plot representation of the relative fluorescence intensity of individual EBY100 yeast cells in non-transformed 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), where each construct is fused to a mobile linker, 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 stained with phycoerythrin conjugate anti-mouse IgG-Fc to analyze display levels. Below: Dot plot representation of 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 construct fused to a mobile linker, 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 stained with phycoerythrin conjugate anti-mouse IgG-Fc to analyze the presence of IFNA2a. [Figure 19]Flow cytometry analysis of expression levels of different circulating variants of IFNA2a compared to wild-type IFNA2a shows each construct presented separately on the surface of EBY100 yeast cells; flow cytometry analysis of binding of IFNAR2 to the same construct shows each presented separately on the surface of EBY100 yeast cells. Top: Dot plot representation of the relative fluorescence intensity of individual EBY100 yeast cells in non-transformed 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), where each construct is fused to a mobile linker, Aga2p, ACP (SEQ ID NO: 32), and c-myc (SEQ ID NO: 33). Transformed and untransformed yeast cells were incubated with anti-c-myc monoclonal antibody and stained with phycoerythrin conjugate anti-mouse IgG-Fc to analyze display levels. Below: Dot plot representation of 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 construct is fused to Aga2p, ACP (SEQ ID NO: 32), and c-myc (SEQ ID NO: 33). Transformed and untransformed yeast cells were incubated with IFNAR2 (human IFN-alpha / beta R2 protein, His tagged) and stained with phycoerythrin conjugate anti-His antibody to analyze binding affinity. [Figure 20]AlphaFold model of a 31kDa HSA-binding chimeric protein constructed from a circulatingly substituted variant of interferon alpha-2a (IFNA2a) inserted into the first β-turn connecting β-chains A and B of anti-HSA ISVD. (A) A model of an antigen-binding chimeric protein produced by the fusion of anti-human serum albumin (HSA) ISVD (bottom) and a circulatingly substituted variant of human IFNA2a (top) via two peptide bonds or linkers connecting ISVD as a scaffold. (B) A circulatingly substituted gene encoding circulatingly substituted interferon alpha-2a (bottom) was inserted into the first β-turn of anti-HSA ISVD (top, SEQ ID NO: 55) connecting β-chain A to β-chain B (β-turn AB). (C) Amino acid sequence of the obtained IFNA2a[L9-T6]_ALB23002 antigen-binding chimeric protein (SEQ ID NO: 60). Sequences derived from IFNA2a are shown in bold. Sequences derived from ISVD are underlined. Peptide linkers for connecting ISVD to IFNA2a are underlined with a dashed line. Peptides that create cyclically substituted variants by linking the N-terminus and C-terminus of IFNA2a are shown in italics. [Figure 21]AlphaFold model of a 31kDa HSA-binding chimeric protein constructed from a cyclically substituted variant of interferon alpha-2a (IFNA2a) inserted into the first β-turn connecting β-chains A and B of anti-HSA ISVD. (A) A model of an antigen-binding chimeric protein created by the fusion of anti-HSA ISVD (left) and a cyclically substituted variant of IFNA2a (right) via two peptide bonds or linkers connecting ISVD as a scaffold. (B) A cyclically substituted gene encoding cyclically substituted interferon alpha-2a (bottom) was inserted into the first β-turn of anti-HSA ISVD (top, SEQ ID NO: 55) connecting β-chain A to β-chain B (β-turn AB). (C) Amino acid sequence of the obtained IFNA2a[S25-K23]_ALB23002 antigen-binding chimeric protein (SEQ ID NO: 61). Sequences derived from IFNA2a are shown in bold. Sequences derived from ISVD are underlined. The peptide linker for connecting ISVD to IFNA2a is underlined with a dashed line. The peptide that creates a cyclically substituted variant by linking the N-terminus and C-terminus of IFNA2a is shown in italics. [Figure 22]AlphaFold model of a 31kDa HSA-binding chimeric protein constructed from a circulatingly substituted variant of interferon alpha-2a inserted into the first β-turn connecting β-chains A and B of anti-HSA ISVD. (A) A model of an antigen-binding chimeric protein produced by the fusion of anti-HSA ISVD (left) and a circulatingly substituted variant of human interferon alpha-2a (IFNA2a) (right) via two peptide bonds or linkers connecting ISVD as a scaffold. (B) A circulatingly substituted gene encoding circulatingly substituted interferon alpha-2a (bottom) was inserted into the first β-turn of anti-HSA ISVD (top, SEQ ID NO: 55) connecting β-chain A to β-chain B (β-turn AB). (C) Amino acid sequence of the obtained Mb_IFNA2a[D32-L30]_ALB23002 antigen-binding chimeric protein (SEQ ID NO: 62). Sequences derived from IFNA2a are shown in bold. Sequences derived from ISVD are underlined. Peptide linkers for connecting ISVD to IFNA2a are underlined with a dashed line. Peptides that create cyclically substituted variants by linking the N-terminus and C-terminus of IFNA2a are shown in italics. [Figure 23]AlphaFold model of a 31kDa HSA-binding chimeric protein constructed from a circulatingly substituted variant of interferon alpha-2a inserted into the first β-turn connecting β-chains A and B of anti-HSA ISVD. (A) A model of an antigen-binding chimeric protein produced by the fusion of anti-HSA ISVD (right) and a circulatingly substituted variant of human interferon alpha-2a (left) via two peptide bonds or linkers connecting ISVD as a scaffold. (B) A circulatingly substituted gene encoding circulatingly substituted interferon alpha-2a (bottom) was inserted into the first β-turn of anti-HSA ISVD (top, SEQ ID NO: 55) connecting β-chain A to β-chain B (β-turn AB). (C) Amino acid sequence of the obtained Mb_IFNA2a[P109-T106]_ALB23002 antigen-binding chimeric protein (SEQ ID NO: 63). Sequences derived from IFNA2a are shown in bold. Sequences derived from ISVD are underlined. Peptide linkers for connecting ISVD to IFNA2a are underlined with a dashed line. Peptides that create cyclically substituted variants by linking the N-terminus and C-terminus of IFNA2a are shown in italics. [Figure 24] Structure of the human three-component complex IFNA2a-IFNAR, consistent with the AlphaFold model of the IFNA2a[L9-T6]_ALB23002 protein. [Figure 25] Structure of the human three-component complex IFNA2a-IFNAR, consistent with the AlphaFold model of the IFNA2a[P109-T6]_ALB23002 protein. [Figure 26] Structure of the human three-component complex IFNA2a-IFNAR, consistent with the AlphaFold model of the IFNA2a[S25-K23]_ALB23002 protein. [Figure 27] Structure of the human three-component complex IFNA2a-IFNAR, consistent with the AlphaFold model of the IFNA2a[D32-L30]_ALB23002 protein. [Figure 28]This shows the phosphorylation of STAT1 in A549 cells after treatment with an IFNA2a-containing compound. Treatment is performed in the absence (A) and in the presence (B) of HSA. [Figure 29] This shows the proliferation of RPMI 8226 (A and B) and NCI-H929 (C and D) cells after treatment with IFNA2a-containing compounds. Treatment is performed in the absence of HSA (A and C) and in the presence of HSA (B and D). [Figure 29-1] Same as above. [Figure 30] Design of a cyclically substituted variant of IL-18. This figure shows the location of the sites where the N-terminus and C-terminus are linked to each other when designing a cyclically substituted variant of IL-18 (PDB 3F62) by opening IL-18 (between positions 69 and 70) to create new N-terminus and C-terminus. [Figure 31] Flow cytometry analysis of expression levels of different circulating variants of IL-18 compared to wild-type IL-18 shows that each construct is presented separately on the surface of EBY100 yeast cells. The dot plot represents the relative fluorescence intensity of individual EBY100 yeast cells in non-transformed 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), with each construct fused to a mobile linker, Aga2p, ACP (SEQ ID NO: 32), and c-myc (SEQ ID NO: 33). Transformed and non-transformed yeast cells were incubated with anti-c-myc mAbs and colored with phycoerythrin conjugate anti-mouse IgG-Fc to analyze display levels. [Figure 32] A schematic diagram of gene fusion for obtaining the chimeric protein of the present invention. [Figure 33]mAb D044-3 binds to a circulating variant of IL-18. This is a flow cytometry analysis of mAbD044-3 binding to IL-18 and its circulating variant presented on the cell surface of yeast cells. Display levels of IL-18 and its circulating variant on the surface of yeast cells were analyzed as described by Uchanski et al. (2021). Proper folding of IL-18 or its circulating variant was confirmed by staining yeast cells with fluorescent mAbD044-3. Dot plots of relative fluorescence intensity are shown for individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding different IL-18 variants, respectively. Gating was performed using negative and positive controls. [Figure 33-1] Same as above. [Figure 34] IL18BP binds to several circulating variants of IL18. This is a flow cytometry analysis of the binding of IL18-BP presented on the cell surface of yeast cells to IL18 and its circulating variants. Display levels of IL-18 and its circulating variants on the surface of yeast cells were analyzed as described in Uchanski et al. (2021). Cells were pre-incubated with fluorescent IL18-BP to analyze whether the presented IL18 or its circulating variants still bind to IL18-BP. Dot plots of relative fluorescence intensity are shown for individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding different IL18 variants. Gating was performed using negative and positive controls. [Figure 34-1] Same as above. [Figure 35]AlphaFold model of a 33kD GFP-binding chimeric protein constructed from a cyclically substituted variant of IL18 inserted into the first β-turn connecting β-chains A and B of anti-GFP ISVD. (A) A model of an antigen-binding chimeric protein created by the fusion of anti-GFP ISVD (top) and a cyclically substituted variant of human IL18 (bottom) via two peptide bonds or linkers connecting ISVD as a scaffold. (B) The cyclically substituted gene encoding the cyclically substituted IL18 (bottom) was inserted into the first β-turn of anti-GFP ISVD (top, SEQ ID NO: 1) connecting β-chain A to β-chain B (β-turn AB). (C) Amino acid sequence of the obtained IL18_ISVD207 antigen-binding chimeric megabody protein (IL18[Y1-D157]_ISVD207_V1 megabody protein, SEQ ID NO: 230). Sequences derived from IL18 are shown in bold. Sequences derived from ISVD are underlined. The peptide linker for connecting ISVD to IL18 is underlined with a dashed line. The peptide that creates a cyclically substituted variant by linking the N-terminus and C-terminus of IL18 is shown in italics. [Figure 36]AlphaFold model of a 33kD GFP-binding chimeric protein constructed from a cyclically substituted variant of IL18 inserted into the first β-turn connecting β-chains A and B of anti-GFP ISVD. (A) A model of an antigen-binding chimeric protein created by the fusion of anti-GFP ISVD (top) and a cyclically substituted variant of human IL18 (bottom) via two peptide bonds or linkers connecting ISVD as a scaffold. (B) The cyclically substituted gene encoding the cyclically substituted IL18 (bottom) was inserted into the first β-turn of anti-GFP ISVD (top, SEQ ID NO: 1) connecting β-chain A to β-chain B (β-turn AB). (C) Amino acid sequence of the obtained IL18_ISVD207 antigen-binding chimeric megabody protein (IL18[K70-E69]_ISVD207_V2 megabody protein, SEQ ID NO: 233). Sequences derived from IL18 are shown in bold. Sequences derived from ISVD are underlined. The peptide linker for connecting ISVD to IL18 is underlined with a dashed line. The peptide that creates a cyclically substituted variant by linking the N-terminus and C-terminus of IL18 is shown in italics. [Figure 37]AlphaFold model of a 33kD GFP-binding chimeric protein constructed from a cyclically substituted variant of IL18 inserted into the first β-turn connecting β-chains A and B of anti-GFP ISVD. (A) A model of an antigen-binding chimeric protein created by the fusion of anti-GFP ISVD (top) and a cyclically substituted variant of human IL18 (bottom) via two peptide bonds or linkers connecting ISVD as a scaffold. (B) The cyclically substituted gene encoding the cyclically substituted IL18 (bottom) was inserted into the first β-turn of anti-GFP ISVD (top, SEQ ID NO: 1) connecting β-chain A to β-chain B (β-turn AB). (C) Amino acid sequence of the obtained IL18_ISVD207 antigen-binding chimeric megabody protein (IL18[P57-Q56]_ISVD207_V1 megabody protein, SEQ ID NO: 237). Sequences derived from IL18 are shown in bold. Sequences derived from ISVD are underlined. The peptide linker for connecting ISVD to IL18 is underlined with a dashed line. The peptide that creates a cyclically substituted variant by linking the N-terminus and C-terminus of IL18 is shown in italics. [Figure 38] mAb D044-3 binds to the IL18-ISVD megabody protein. This is a flow cytometry analysis of mAb D044-3 binding to the IL18_ISVD207 megabody protein displayed on the cell surface of yeast cells. Display levels of different proteins displayed on the surface of yeast cells were analyzed as described by Uchanski et al. (2021). Cells were pre-incubated with fluorescent mAb D044-3 to demonstrate the binding of mAb D044-3 to the displayed IL18_ISVD207 megabody protein. For each binding experiment, a dot plot representation of the relative fluorescence intensity of individual EBY100 yeast cells transformed with either a control protein or a pCTCON2 derivative encoding a different IL18_ISVD207 megabody protein was created. Gating was performed using negative and positive controls. [Figure 38-1] Same as above. [Figure 38-2] Same as above. [Figure 38-3] Same as above. [Figure 38-4] Same as above. [Figure 38-5] Same as above. [Figure 39] IL18BP binds to the IL18-ISVD megabody protein. This is a flow cytometry analysis of the binding of IL18-BP to the IL18_ISVD207 megabody protein displayed on the cell surface of yeast cells. The display levels of different proteins displayed on the surface of yeast cells were analyzed as described by Uchanski et al. (2021). Cells were pre-incubated with fluorescent IL18-BP to show the binding of IL18-BP to the displayed IL18_ISVD207 megabody protein. Variations in the binding of IL18-BP to different IL18_ISVD207 megabody proteins are observed. For each binding experiment, a dot plot representation of the relative fluorescence intensity of individual EBY100 yeast cells transformed with either a control protein or a pCTCON2 derivative encoding either a different IL18_ISVD207 megabody protein was created. Gating was set using negative and positive controls. [Figure 39-1] Same as above. [Figure 39-2] Same as above. [Figure 39-3] Same as above. [Figure 39-4] Same as above. [Figure 39-5] Same as above. [Figure 40]GFP binds to the IL18-ISVD megabody protein. This is a flow cytometry analysis of GFP binding to the IL18_ISVD207 megabody protein displayed on the cell surface of yeast cells. Display levels of different proteins displayed on the surface of yeast cells were analyzed as described in Uchanski et al. (2021). Cells were stained with GFP to demonstrate the functionality of ISVD207 in the chimeric protein. For each binding experiment, a dot plot representation of the relative fluorescence intensity of individual EBY100 yeast cells transformed with either a control protein or a pCTCON2 derivative encoding a different IL18_ISVD207 megabody protein was created. Gating was performed using negative and positive controls. [Figure 40-1] Same as above. [Figure 40-2] Same as above. [Figure 40-3] Same as above. [Figure 40-4] Same as above. [Figure 40-5] Same as above. [Figure 41] Experimental setup for collecting proteins displayed from yeast cell walls. IL18[K70-E69]V5b and IL18_ISVD207 megabody proteins, fused with acyl carrier proteins and several co-peptides displayed on the surface of yeast cells, can be released from the cell wall by labeling them with biotin using biotin-PEG3-coenzyme A and SFP synthase, and then adding DTT. [Figure 42] Biolayer interferometry (BLI) analysis of GFP binding to IL18_ISVD207 megabody protein. To analyze whether IL18_ISVD207 megabody protein, released from the cell wall and captured on an SA biosensor, binds to GFP, the mounted SA sensor was incubated with GFP. BLI sensorgrams were recorded for each construct and compared to IL18[K70-E69]V5b, which served as a negative control. [Figure 43]Biolayer Interferometry (BLI) analysis of GFP binding to IL18_ISVD207 IL18[K70-E69]_ISVD207_V1. Biolayer interferometry analysis using GFP was performed to demonstrate the functionality and affinity of ISVD207 in IL18_ISVD207 IL18[K70-E69]_ISVD207_V1. Binding and dissociation isotherms were recorded for each concentration, and the data were analyzed using Octet® Analysis Studio software. [Figure 44] Plasma PK profiles of IL-2-containing compounds after in vivo intravascular treatment of untreated female C57Bl / 6N mice. Results are expressed as mean ± SD. LOQ: Limit of quantification of the plasma PK assay. [Figure 45] Phosphorylation of STAT5 (pSTAT5) in different immune cells at two time points (24 hours and 48 hours) after in vivo intravascular treatment of untreated female C57Bl / 6N mice with an IL-2-containing compound. 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 shown as median (gray bar) and individual data (symbol). [Figure 46] Ki67 expression in different immune cells at two time points (48 hours and 72 hours) after in vivo intravascular treatment of untreated female C57Bl / 6N mice with an IL-2-containing compound. 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. The results are shown as the median (gray bar) and individual data (symbols): *: Statistically significant difference when comparing each treatment group to the vehicle group (****p<0.0001); #: Statistically significant difference when comparing the TP208 control group to TP207 and TP206 (#p<0.05;##p<0.01;###p<0.001;####p<0.0001); ns: No significant difference [Figure 47]Proliferation of different immune cells 72 hours after in vivo intravascular treatment of untreated female C57Bl / 6N mice with an IL-2-containing compound. 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 shown as median (gray bar) and individual data (symbol): *: Statistical significance of each treatment group compared to the vehicle group (****p<0.0001); #: Statistical significance of the TP208 control group compared to TP207 and TP206 (####p<0.0001); ns: No significant difference [Figure 48] Ratios of different immune cell populations 72 hours after in vivo intravascular treatment of untreated female C57Bl / 6N mice with IL-2-containing compounds. 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 shown as median (gray bar) and individual data (symbol): *: Statistically significant difference when comparing the TP208 control group with TP207 and TP206 (****p<0.0001); ns: No significant difference [Modes for carrying out the invention]

[0023] The present invention is described in relation to specific embodiments and with reference to certain drawings, but the invention is not limited thereto and is limited only by the claims. No reference numeral in the claims should be construed as limiting the scope. Naturally, it should be understood that not all aspects or advantages can necessarily be achieved according to any particular embodiment of the present invention. Therefore, for example, a person skilled in the art will recognize that the present invention can be embodied or practiced in a manner that achieves or optimizes one advantage or group of advantages taught herein without having to achieve other aspects or advantages that can be taught or proposed herein. The present invention, both in terms of configuration and operation, along with its features and advantages, can be best understood by referring to the following detailed description when read in conjunction with the accompanying drawings. The aspects and advantages of the present invention should be made clear and elucidated by referring to the embodiments described below. Throughout this specification, any reference to “one embodiment” or “a certain embodiment” means that a particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, the occurrence of the phrases “in one embodiment” or “in a particular embodiment” in various places throughout this specification does not necessarily all refer to the same embodiment, although they may refer to the same embodiment. Similarly, in the description of exemplary embodiments of the invention, it should be understood that various features of the invention may be summarized in a single embodiment, figure, or description thereof for the purpose of streamlining this disclosure and aiding in the understanding of one or more of the various inventive aspects. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than are expressly described in each claim. Rather, as reflected in the following claims, aspects of the invention are fewer than all the features of one disclosed embodiment described above.

[0024] definition Unless otherwise indicated or defined, all terms used have their ordinary meanings in the art and will be obvious to those skilled in the art. For example, see standard handbooks, e.g., Sambrook et al., 1989 (Molecular Cloning: A Laboratory Manual, 2 nd Ed., Vols. 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, 2 nd Ed., University of California Press, Berkeley, CA), Roitt et al., 2001 (Immunology, 6 th Ed.,Mosby / Elsevier,Edinburgh), Roitt et al.,2001 (Roitt's Essential Immunology,10 th Ed., Blackwell Publishing, UK) and Janeway et al., 2005 (Immunobiology, 6 th See reference to the ed., Garland Science Publishing / Churchill Livingstone, New York, and the general background art cited herein.

[0025] Unless otherwise indicated, all methods, processes, techniques and operations not described in detail herein may and may be carried out in ways known to those skilled in the art, as would be obvious to those skilled in the art. For example, see the standard handbooks and general background art referred to herein, as well as further references cited herein, and 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. A: S106), and Gonzales et al., 2005 (Tumour Biol., 26:31), which describe protein engineering techniques such as affinity maturation, and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.

[0026] Where used herein, the singular forms “a,” “an,” and “the” refer to multiple objects unless otherwise explicitly indicated by the context. For example, a reference to “reagent” includes one or more different such reagents, and a reference to “method” includes equivalent steps and methods known to those skilled in the art that can be modified or replaced by the methods described herein.

[0027] Unless otherwise indicated, the term “at least” preceding a set of elements should be understood to refer to all elements of that set. Those skilled in the art will be able to recognize or confirm, through mere conventional experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be incorporated into the invention.

[0028] The term "and / or" as used anywhere in this specification shall mean "and", "or", and "any combination of all or any other of the elements connected by said term".

[0029] Throughout this specification and the following claims, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are to be interpreted as including the recited integer or step or group of integers or steps but not excluding any other integer or step or group of integers or steps. As used herein, the term "comprising" may be replaced by the term "containing" or "including" or, when used herein, the term "having".

[0030] As used herein, "similar" is interchangeable with alike, analogous, comparable, corresponding, etc. and means having the same or common characteristics to show equivalent results, i.e., having variations of at most 20%, 10%, more preferably 5%, or even more preferably 1% or less.

[0031] As used herein, the term "array" (e.g., terms such as "immunoglobulin array", "antibody array", "variable domain array", "V HH array", or "protein array") shall generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding it, unless a more limited interpretation is required by the context. The amino acid sequence is to be interpreted as meaning a single amino acid or an unbranched sequence of two or more amino acids depending on the context. The nucleotide sequence is to be interpreted as meaning an unbranched sequence of three or more nucleotides.

[0032] When used herein, “nucleotide sequence,” “DNA sequence,” or “nucleic acid molecule” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of molecules. Therefore, this term includes double-stranded and single-stranded DNA, as well as RNA. It also includes known types of modifications, such as methylation and “cap” substitutions by one or more naturally occurring analogs of nucleotides.

[0033] A "nucleic acid construct" refers to a nucleic acid sequence constructed to contain one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phages), and viral genomes containing non-natural nucleic acid sequences.

[0034] Any reference to the amino acid sequences of the present invention is intended to encompass post-translational modifications of these sequences that occur in mammalian cells such as CHO cells, including but not limited to N-glycosylation, O-glycosylation, deamidation, Asp isomerization / fragmentation, pyroglutamate formation, C-terminal lysine removal, and Met / Trp oxidation.

[0035] When a nucleotide sequence or amino acid sequence is said to "contain" or "essentially consist of" another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence is incorporated into the first-referenced nucleotide sequence or amino acid sequence, but more generally, it means that the first-referenced nucleotide sequence or amino acid sequence contains within its sequence stretches of nucleotide or amino acid residues having identical nucleotide or amino acid sequences to the latter sequence, regardless of how the first-referenced sequence was actually produced or obtained (for example, by any preferred method described herein).

[0036] A "coding sequence" is a nucleotide sequence that, when controlled by appropriate regulatory sequences, is transcribed into mRNA and / or translated into a polypeptide. The boundaries of a coding sequence are determined by a translation start codon at the 5' end and a translation stop codon at the 3' end. Codextic sequences can include, but are not limited to, mRNA, cDNA, recombinant nucleotide sequences, or genomic DNA, although introns may be present under certain circumstances.

[0037] As used herein, “promoter region of a gene” refers to a functional DNA sequence unit that is operably ligated to a coding sequence and, if placed under appropriate induction conditions, is sufficient to promote the transcription of the coding sequence. “Operatably ligated” refers to a parallel relationship in which the components described in this way are made possible to function in the manner intended. A promoter sequence “operably ligated” to a coding sequence is ligated so that the expression of the coding sequence is achieved under conditions that are compatible with the promoter sequence.

[0038] As used herein, “gene” includes both the promoter region and the coding sequence of a gene. This refers to both the genomic sequence (including possible introns) and the cDNA derived from a splicing messenger operably ligated to the promoter sequence. The terms “terminator” or “transcription termination signal” encompass regulatory sequences, which are DNA sequences at the ends of transcription units that signal the 3' processing and polyadenylation of the primary transcript and the termination of transcription. Terminators may be derived from native genes, various other plant genes, or T-DNA. The attached terminator may be derived, for example, from a nopalin synthase or octopin synthase gene, or alternatively from another plant gene, or more preferably from any other eukaryotic gene.

[0039] "Genetic construct," "chimeric gene," "chimeric construct," or "chimeric gene construct" means a recombinant nucleic acid sequence to which a promoter or regulatory nucleic acid sequence is operably ligated, or in which the regulatory nucleic acid sequence is associated with a nucleic acid sequence encoding mRNA so as to be able to regulate the transcription or expression of the relevant nucleic acid coding sequence. The regulatory nucleic acid sequence of a chimeric gene is not operably ligated to a relevant nucleic acid sequence as found in nature. In particular, the term "gene fusion construct" as used herein refers to a gene construct encoding mRNA that is translated into the fusion protein of the present invention disclosed herein.

[0040] The terms “vector,” “vector construct,” “expression vector,” or “gene transfer vector,” as used herein, are intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is ligated, and include, but are not limited to, any vector known to those skilled in the art, including any preferred type such as plasmid vectors, cosmid vectors, phage vectors such as lambda phages, viral vectors such as adenovirus vectors, AAV or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). Expression vectors, including plasmids and viral vectors, generally contain a desired coding sequence and a suitable DNA sequence necessary for the expression of the operably ligated coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Expression vectors can autonomously replicate in the host cell to which they are introduced (e.g., a vector having an origin of replication that functions within the host cell). Other vectors, when introduced into a host cell, are integrated into the host cell's genome and can thereby replicate together with the host genome. Suitable vectors may, as desired, contain regulatory sequences such as promoters, enhancers, and terminator sequences from a specific host organism (e.g., bacterial cells, yeast cells). Cloning vectors are generally used to manipulate and amplify certain desired DNA fragments and may lack the functional sequences necessary for the expression of 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 achieved through standard techniques (for example, Sambrook, et al. Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and for definitions of technical terms, see Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016)).

[0041] The “host cell” may be either a prokaryote or a eukaryote. The cell may be transfected transiently or stably. Such transfection of expression vectors into prokaryotic and eukaryotic cells may be achieved by any technique known in the art, including, but not limited to, standard bacterial transformation, calcium phosphate coprecipitation, electroporation, or liposome-mediated, DEAE-dextran-mediated, polycation-mediated, or viral-mediated transfection. For all standard techniques, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., 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). Recombinant host cells in this context are genetically modified to contain the isolated DNA molecule, nucleic acid molecule, or expression construct or vector of the present invention. DNA can be introduced by any means known in the art that is appropriate for a particular type of cell, including but not limited to transformation, lipofection, electroporation, or virus-mediated introduction. DNA constructs that can enable the expression of the chimeric protein of the present invention can be readily prepared by technically known techniques such as cloning, hybridization screening, and polymerase chain reaction (PCR). Standard techniques for cloning for enzymatic reactions, including DNA ligases, DNA polymerases, and restriction endonucleases, as well as for DNA isolation, amplification, and purification, and various separation techniques are known and commonly used by those skilled in the art. Several standard techniques are described in Sambrook et al. (2012), Wu (ed.) (1993), and Ausubel et al. (2016).Representative host cells that can be used in the present invention include, but are not limited to, bacterial cells, yeast cells, insect cells, plant cells, and animal cells. Suitable bacterial host cells for use in the present 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. Suitable animal host cells for use in the present invention include insect cells and mammalian cells (most particularly derived from Chinese hamsters (e.g., CHO)), as well as human cell lines such as HeLa. Suitable yeast host cells for use in the present invention include species such as Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g., Pichia pastoris), Hansenula (e.g., Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, and Zygosaccharomyces. Saccharomyces cerevisiae, specifically S. carlsbergensis and K. iactis, are the most commonly used yeast hosts and are favorable fungal hosts. Host cells can be supplied in suspension or flask culture, tissue culture, or organ culture. Alternatively, host cells may also be transgenic animals.

[0042] The terms “protein,” “polypeptide,” and “peptide” are used more interchangeably herein to refer to polymers of amino acid residues, as well as their variants and synthetic analogs. Therefore, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-natural amino acids, such as chemical analogs of the corresponding natural amino acids, as well as to natural 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 mass or molecular mass or atomic weight or molecular weight of a polypeptide is expressed in (kilo)daltons (kDa). “Recombinant polypeptide” means a polypeptide produced using recombinant technology, i.e., by the expression of recombinant or synthetic polynucleotides. When a chimeric polypeptide or its biologically active portion is produced by recombinant synthesis, it also preferably contains substantially no culture medium; i.e., the culture medium accounts for less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. By convention, the amide bond in the primary structure of a polypeptide is the order in which the amino acids are written, 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-translationally modified amino acids may be written as the first translated amino acid sequence, using the symbols shown in Table 1 below, along with the modified position, e.g., hydroxylation or glycosylation, but these modifications shall not be explicitly indicated in the amino acid sequence. Any peptide or protein that can be expressed as a modified sequence, such as by linking, cross-linking and terminal capping, or non-peptidyl linking, is included in this definition.

[0043] "Isolated" usually means a substance that does not substantially or essentially contain its associated components in its natural state. For example, "isolated polypeptide" refers to a polypeptide purified from a neighboring molecule in its natural state, such as a fusion protein disclosed herein that has been removed from a molecule present in a production host adjacent to the polypeptide. Isolated chimeras can be produced by amino acid chemosynthesis or by recombinant production. The expression "heterogeneous protein" may mean that a protein does not originate from the same species or strain used to express or represent the protein.

[0044] Protein homologues include peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions in relation to the unmodified protein in question, and that possess similar biological and functional activity to the unmodified protein from which they are derived.

[0045] "Amino acid" is amino[a](-NH) + 3) and carboxylate (-CO - 2) Organic compounds containing functional groups and side chains (R groups) specific to each amino acid. For example, amino acids include L-amino acids commonly found in naturally occurring proteins. In relation to the present invention, "amino acids" also include D-amino acids and non-natural, abnormal, or unnatural amino acids, as described below. Amino acid residues are indicated according to standard three-letter or one-letter amino acid codes. See Table A-2 on page 48 of International Publication No. 08 / 020079. Examples of amino acids commonly found in proteins and represented in genetic codes are listed in Table 1 below. Other common amino acids (other than those listed in Table 1 below) are listed in the table on page 624 of Pure & Appl. Chem., Vol. 56, No. 5, pp. 595-624, 1984.

[0046] [Table 1]

[0047] D-amino acids are also included in the definition of "amino acids." As used herein, the term "D-amino acid" refers to an amino acid in which the stereoisomerized carbon α with respect to the amino group has a D-configuration.

[0048] Unusual, unnatural, or non-natural amino acids are also included in the definition of “amino acids.” As used herein, the terms “unnatural amino acids,” “non-standard amino acids,” “unnatural amino acids,” or “novel amino acids” (etc.) refer to 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, rather than one of the 20 amino acids commonly found in naturally synthesized peptides. Exemplary unnatural amino acids are found 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.

[0049] 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 nucleotides at the corresponding positions 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 compared to the first nucleotide sequence is considered a difference at a single nucleotide (position)). 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 using standard settings. Several other techniques, computer algorithms, and settings for determining the degree of sequence identity are described, for example, in International Publication 04 / 037999, European Patent No. 0967284, European Patent No. 1085089, International Publication 00 / 55318, International Publication 00 / 78972, International Publication 98 / 49185, and British Patent No. 2357768. Typically, for the purpose of determining the percentage of “sequence identity” between two nucleotide sequences according to the calculation methods outlined above herein, the nucleotide sequence containing the largest number of nucleotides is considered the “first” nucleotide sequence, and the other nucleotide sequence is considered the “second” nucleotide sequence.

[0050] To compare 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 amino acid residue at the corresponding position 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 an amino acid residue in the second amino acid sequence is considered a difference at a single amino acid residue (position) compared to the first amino acid sequence, i.e., an “amino acid difference” as defined herein. Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms, such as those described above for determining the degree of sequence identity of nucleotide sequences, and similarly using standard settings. Typically, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above in this specification, the amino acid sequence containing the largest number of amino acid residues is considered the "first" amino acid sequence, and the other amino acid sequence is considered the "second" amino acid sequence.

[0051] When determining the degree of sequence identity between two amino acid sequences, those skilled in the art can also take into account so-called “conservative” 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 with 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 conservative amino acid substitutions are well known in the art, for example, by International Publication No. 04 / 037999, British Patent No. 335768, International Publication No. 98 / 49185, International Publication No. 00 / 46383 and International Publication No. 01 / 09300, and (preferred) types and / or combinations of such substitutions can be selected based on appropriate teachings from International Publication No. 04 / 037999 and International Publication No. 98 / 49185 and from further references cited therein.

[0052] Such conservative substitutions are preferably those in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0053] Amino acid sequences and nucleic acid sequences are said to be "exactly the same" if they have 100% sequence identity (as defined herein) over their entire length. 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 the position of the first sequence being compared to the second sequence, and it will be understood that the two amino acid sequences may contain one, two, or more such amino acid differences.

[0054] As used herein, “substitution” or “mutation” results from the substitution of one or more amino acids or nucleotides by different amino acids or nucleotides compared to the amino acid sequence or nucleotide sequence of the parent protein or fragment thereof. It is understood that a protein or fragment thereof may have conservative amino acid substitutions that do not substantially affect the protein’s activity.

[0055] 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 in a population, and therefore the "normal" or "wild-type" form of the gene is arbitrarily designed. In contrast, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits modifications in sequence, post-translational modifications, and / or functional properties (i.e., altered properties) compared to a wild-type gene or gene product. It should be noted that naturally occurring mutants can be isolated (these are identified by the fact that they have altered properties compared to a wild-type gene or gene product). Alternatively, variants may also include synthetic molecules; for example, chemokine ligand variants may be small molecules or artificially created synthetic peptides or proteins that are structurally and / or functionally similar to natural chemokines. The variants having different functional properties may relate to superagonists, superantagonists, among other functional differences, as is known to those skilled in the art.

[0056] A "protein domain" is a distinct functional and / or structural unit in a protein. Typically, protein domains are involved in specific functions or interactions and contribute to the overall role of the protein. Domains can exist in a variety of biological contexts, and similar domains may be found in proteins with different functions. Protein secondary structure elements (SSEs) typically form spontaneously as intermediates before a protein folds into its three-dimensional tertiary structure. The two most common secondary structure elements of proteins are the alpha (α) helix and the beta (β) sheet, but s-turns and omega loops also occur. A beta barrel is a beta sheet composed of tandem repeats that twist and coil, forming a closed toroidal structure where the first chain is bonded (hydrogen bonded) to the last chain. The beta chains in many beta barrels are arranged antiparallel. A beta sheet consists of beta chains (also called β chains) that are laterally connected by at least two or three skeletal hydrogen bonds, and generally forms a twisted, pleated sheet. A β-chain is a stretch of a polypeptide chain, typically 3–10 amino acids long, that has a backbone in its elongated conformation. A "turn" is a type of irregular secondary structure in a protein that causes a change in the orientation of a polypeptide chain. Turns generally occur when a protein chain needs to change direction to connect two other elements of a secondary structure. The most common is the beta-turn, in which case the change in direction is generally carried out in a space of four residues. Beta-turns (β-turns, β-bends, tight turns, or reverse turns) are a very common motif in proteins and polypeptides and primarily serve to connect β-chains. A polypeptide chain undergoes a 180° change in direction during a beta-turn. A "loop" is an irregular structure that connects two secondary structure elements in a protein. They are generally located on the surface of the protein in solvent-exposed regions of the protein (Choi Y. et al., “How long is a piece of loop?”, Peer J., 2013, 1:e1).Generally, loops have a longer number of amino acids than turns (see, for example, Milner-White and Poet, “Loops, bulges, turns and hairpins in proteins”, Trends in Biochemical Sciences, 1987, 12:189-192). For example, loops with only four or five amino acid residues may also be called turns if they have internal hydrogen bonds.

[0057] The terms “circularly permuted protein,” “circularly permuted protein,” or “circularly permuted protein,” as used interchangeably herein, refer to a molecule that, in its linear form, has ends linked either directly or via a linker to form a cyclic molecule (as an intermediate), and subsequently, at another location or position, opens or cleaves the cyclic molecule to produce a novel molecule that, in its linear form, has ends (X1 and X2) different from the ends in the original molecule. The ring-opening or cleavage of the cyclic molecule at another location may involve the removal of one or more nucleotides / amino acids from the original sequence. For example, when ring-opening or cleaving a cyclic molecule, at least one, e.g., one, two, three, four, five or more residues may be removed. Examples of cyclically substituted molecules include those whose structure is equivalent to a molecule that has been cyclized and then unfolded, and / or, in the case of proteins, molecules in which the amino and carboxyl terms are joined to each other, directly or via a linker, and the new amino and carboxyl terms are formed at different locations within the protein sequence. Alternatively, cyclically substituted molecules can be de novo synthesized starting from a new linear form of the molecule (compared to the original molecule) and never undergo the cyclization and ring-opening steps. Cyclically substituted molecules offer intramolecular reconfiguration compared to the original wild-type molecule, as the folding or appearance of the final (folded) molecule is similar to or identical to the original molecule, without affecting activity or functionality, with the only difference being that the start and end points are in different positions. As mentioned above, in some cases, one or more nucleotides / amino acids of the original molecule are removed from the original molecule. Thus, cyclically substituted molecules, which may be nucleic acid molecules or proteins, have their normal ends fused, often with a linker, and contain new ends at different positions. See Goldenberg, et al. J.Mol.Biol., 165:407-413 (1983) and Pan et al. Gene 125:111-114 (1993) (both incorporated herein by reference).Cyclic substitution is functionally equivalent to taking a linear molecule, fusing its ends to form a cyclic molecule, and then cleaving the cyclic molecule at different positions to form a new linear molecule with different ends. Therefore, cyclic substitution has the effect of generating new ends at different positions while essentially preserving the amino acid sequence and identity of the protein (see also Pastan et al. - European Patent No. 0754192B1). Proteins for which cyclic substitution is easy to design are those where the ends of the original protein are close together and conveniently oriented; for example, if the ends are naturally located close to each other, direct fusion of the ends or introduction of a short linker has relatively little effect. However, since the linker can be of any length, proximity of the natural ends is not an absolute requirement. Specific cyclic substitutions of a molecule are indicated by square brackets containing the amino acid residues between which the peptide bond is excluded. Thus, for example, PRT[AA. X2 -AA X1The term "PRT" refers to a circulatingly permuted protein in which a ring-opening site (a position where a peptide bond is excluded) is created between amino acid (AA) residues at positions X2 and X1 of an unsubstituted or unmodified protein. Therefore, in relation to the present invention, the terms "circularly permuted," "circularly permuted," or "circularly permuted" refer to the process of taking a protein or its homologous nucleic acid sequence, fusing its N-terminus and C-terminus (directly or via a linker, for example using a protein or recombinant DNA method) to form a circular molecule, and then cleaving (opening the ring) the circular molecule at different positions to form a new protein or homologous nucleic acid molecule having different ends than those in the original molecule. Thus, circular substitution generates new C-terminus and N-terminus at different positions while preserving the overall sequence (except for the linker, if introduced, and one or more amino acids removed, if present) of the protein, thereby resulting in an improved orientation for fusing a desired polypeptide fusion partner compared to the original molecule. As described above, cyclically substituted molecules can be de novo synthesized as linear molecules without ever undergoing cyclization and ring-opening steps. In addition, in relation to the present 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 created after the deletion of one or more residues, for example, one, two, three, four, five or more residues from the original N-terminus, or between the N-terminus and C-terminus created after the deletion of one or more residues, for example, one, two, three, four, five or more residues from both the N-terminus and the C-terminus. Therefore, the different possibilities for the fusion of the N-terminus and C-terminus of linear molecules are PRT[AA X2 -AA X1This can result in different versions of the cyclically substituted protein, indicated as ]Vn. For example, the names IFNA2a[D77-W76]V2 and IFNA2a[D77-W76]V4 indicate both cyclically substituted IFNA2a proteins, where a ring-opening site (a position where a peptide bond is excluded) occurs between amino acid residues at positions 77 and 76 of the unsubstituted or unmodified protein, but the original N-terminus and C-terminus are fused differently. In addition, as mentioned above, in relation to the present invention, the design of a cyclically substituted protein by ring-opening of a cyclic molecule cannot occur at two consecutive amino acid positions, resulting in the deletion of one or more amino acids from the protein. Therefore, in relation to the present invention, the name IL-2[F42-M39] indicates a cyclically substituted IL-2 cytokine, where the ring-opening site (a position where a peptide bond is excluded) occurs between residues at positions 42 and 39 of the unsubstituted or unmodified IL-2. Restless residues 40 and 41 of the original protein are deleted to produce the cyclically substituted protein. In relation to the present invention, ring-opening of a 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 permuted protein is maintained or similar to that of the wild-type protein. Therefore, in relation to the present invention, the terms “circular permutation of a protein” or “circularly permuted protein” refer to a protein having an altered order of amino acids in its amino acid sequence compared to the wild-type protein sequence, resulting in a protein structure having a different binding affinity but an overall similar three-dimensional (3D) shape. Circular permutation of a protein is similar to the mathematical concept of cyclic permutation in that the sequence of a first portion of the wild-type protein (adjacent to the N-terminus) relates to the sequence of a second portion of the resulting circularly permuted protein (near its C-terminus), as described, for example, in Bliven and Prlic (2012) (Circular permutation in proteins. PLOS Comput. Biol. 8(3):e1002445).The circular substitution of the protein compared to the wild-type protein is obtained by genetic engineering or artificial engineering of the protein sequence, thereby "linking" the N-terminus and C-terminus of the wild-type protein (directly by a linker and / or with one or more amino acids removed as described above), and the protein sequence is interrupted at another location (where one or more amino acids can be removed as described above), creating novel N-terminus and C-terminus of the protein. The circularly substituted protein (cytokine) of the present invention is the result of linked N-terminus and C-terminus of the wild-type cytokine sequence, as well as cleavage or interruption of the sequence at an accessible or exposed site (preferably a β-turn or loop) of the cytokine, thereby the folding (3D structure) of the circularly substituted cytokine is similar to, or comparable to, the folding of the wild-type protein. The linkage of the N-terminus and C-terminus in the circularly substituted cytokine may be the result of a peptide bond, or the introduction of a peptide linker, or the deletion of a peptide stretch near the original N-terminus and C-terminus in the wild-type protein, followed by the deletion of a peptide bond or the 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 circularly permuted proteins.

[0058] As used herein, the term “fused” is used interchangeably with “connected to,” “conjugated,” “ligated to,” “linked,” or “joined,” and specifically refers to “gene fusion” by recombinant DNA technology, as well as “chemical and / or enzymatic conjugation” that results in a stable covalent bond.

[0059] The terms “chimeric polypeptide,” “chimeric protein,” “chimera,” “fusion polypeptide,” “fusion protein,” or “protein not found in nature” are used interchangeably herein and refer to a protein containing at least two distinct polypeptide components, which may or may not originate from the same protein, e.g., an immunoglobulin single variable domain (ISVD) fused with a cytokine. The term also refers to a molecule not found in nature, meaning that it is artificial. The term “fused,” and other grammatical equivalents such as “covalently bonded,” “linked,” “attached,” “ligated,” “conjugated,” and “joined,” refer to any chemical or recombination mechanism for linking two or more polypeptide components when referring to a chimeric protein (as defined herein). The fusion of two or more polypeptide components, e.g., ISVD and cytokine, as described herein, may be a direct fusion of sequences or an indirect fusion with an intervening amino acid sequence or linker sequence, or a chemical linker. The fusion of two polypeptides, such as ISVD and a cytokine, as described herein may also refer to non-covalent fusions obtained by chemical bonding.

[0060] As used herein, the terms “protein complex” or “complex” refer to a group of two or more related macromolecules, thereby at least one of which is a protein. As used herein, a protein complex typically refers to an association of macromolecules that can be formed under physiological conditions. Individual members of a protein complex are linked by non-covalent interactions. A protein complex can be a non-covalent interaction of only proteins, and is therefore called a protein-protein complex; for example, a non-covalent interaction of two proteins, three proteins, four proteins, etc. More specifically, a complex of a chimeric protein with a cytokine receptor, or a complex of a ligand protein (such as a chimeric protein) with a cytokine or chemokine and its specifically bound interactor (e.g., a cytokine receptor capable of binding to a cytokine ligand). A protein complex of a chimeric protein containing an ISVD fused with a cytokine binds to its chemokine receptor by its chemokine receptor interacting region (its N-terminus) and to a chemokine receptor known to bind to the chemokine ligand, thereby forming the complex as used herein.

[0061] As used herein, the terms “determine,” “measure,” “evaluate,” and “assay” are used interchangeably and include both quantitative and qualitative determinations.

[0062] The term “preferred conditions” refers, in particular, to environmental factors such as temperature, migration, other components, and / or “buffer conditions,” where “buffer conditions” specifically refers to the composition of the solution in which the assay is performed. The composition may include buffer solutions and / or solutes, such as pH buffers, water, saline solutions, physiological saline, glycerol, and preservatives, which are recognized by those skilled in the art as suitable for obtaining optimal assay performance.

[0063] In relation to the present invention, the terms “specificity,” “specifically binding,” or “specific binding” refer to the number of different target molecules, such as antigens, to which a particular binding unit can bind with sufficiently high affinity (see below). “Specificity,” “specifically binding,” or “specific binding” are used herein interchangeably with “selectivity,” “selectively binding,” or “selective binding.” Generally, binding units, such as bound ISVDs or cytokines, bind specifically to their designated targets or receptors.

[0064] The specificity / selectivity of bonding units can be determined based on affinity. Affinity indicates the strength or stability of molecular interactions. Affinity is generally expressed in units of mol / liter (or M), such as K. D Alternatively, it is given by the dissociation constant. Affinity is given by the binding constant K. A It can also be expressed as, this is 1 / K D Equivalent to (mol / liter) -1 (or M -1 It is expressed in units of ).

[0065] "Affinity" is a measure of the binding strength between a part of a molecule and a binding site on a target molecule. D The smaller the value, the stronger the binding strength between the target molecule and the targeting site.

[0066] K D The value is the well-known relationship DG=RT.ln(K D )(Equivalently, DG = -RT.ln(K A Because it relates to the change in the free energy (DG) of the bond, it also characterizes the strength of molecular interactions in a thermodynamic sense, where R is equal to the gas constant, T is equal to the absolute temperature, and ln represents the natural logarithm.

[0067] K D is, k off The dissociation rate constant of the complex shown is k on It can also be expressed as a ratio to the bonding rate shown (K D =k off / k on and K A =kon / k off (So ​​that it becomes so). Offrate k off is, in units of s -1 It has (where s is the SI unit representation of seconds). On rate k on The unit is M -1 s -1 It has. The on rate is 10 2 M -1 s -1 ~about 10 7 M -1 s -1 It can vary between and approaches the diffusion-limited coupling rate constant of bimolecular interactions. The off-rate is t 1 / 2 =ln(2) / k off The relationship relates to the half-life of a given molecular interaction. The off-rate is 10 -6 s -1 (Multiple days t 1 / 2 (A nearly irreversible complex having ~1s) -1 (t 1 / 2 It can vary between 0.69s.

[0068] If the measurement process, for example, involves artifacts related to the coating on a biosensor of a single molecule, which in some way affects the intrinsic binding affinity of the implied molecule, then the measured K D is the apparent K D This may correspond to [something]. Also, if one molecule contains two or more recognition sites for one or more other molecules, the apparent K D This can also be measured. In such situations, the measured affinity may be influenced by the affinity of the interaction between the two molecules.

[0069] Dissociation constant (K D ) can be the actual dissociation constant or the apparent dissociation constant, as will be obvious to those skilled in the art. D Methods for determining this are obvious to those skilled in the art and include, for example, the techniques mentioned below. In this regard, 10 -4 moles / liter or 10 -3 moles / liter greater than (for example, 10 -2It will also be apparent that it may be impossible to measure the dissociation constant (moles / liter). Optionally, as will also be apparent to those skilled in the art, (actual or apparent) K D is the (actual or apparent) coupling constant (K A Based on ), the relation (K D = 1 / K A ) can be calculated by K. A = 1 / K D →K A =[AB] / [A]·[B].

[0070] The affinity of molecular interactions between two molecules can be measured by various techniques known on their own, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, e.g., Ober et al. 2001, Intern. Immunology 13:1551-1559). The term "surface plasmon resonance" (SPR), as used herein, refers to an optical phenomenon that enables real-time analysis of biospecific interactions by detecting changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule passes over the immobilized molecule under flow conditions, k on , k off Measured value, therefore K D (or K A Surface plasmon resonance yields a value. Surface plasmon resonance can be performed, for example, using the well-known BIAcore® system (BIAcore International AB, a Cytiva lifesciences company, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Jonsson et al. (1993, Ann. Biol. Clin. 51:19-26), Jonsson et al. (1991 Biotechniques 11:620-627), Johnsson et al. (1995, J. Mol. Recognit. 8:125-131), and Johnsson et al. (1991, Anal. Biochem. 198:268-277).

[0071] Another well-known biosensor technique for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, e.g., Abdiche et al. 2008, Anal. Biochem. 377:209-217). As used herein, the terms “biolayer interferometry” or “BLI” refer to a label-free optical technique that analyzes the interference patterns of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized proteins on a biosensor chip (signal beam). A change in the number of molecules bound to the tip of the biosensor causes a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the tip surface of the biosensor. Because interactions can be measured in real time, association and dissociation rates, as well as affinity, can be determined. BLI can be performed, for example, using the well-known Octet® Systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0072] Alternatively, affinity can be measured using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA) by the KinExA® equilibrium exclusion 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 for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrium solution of binding unit / target complexes, such as antibody / antigen complexes, is passed through a column containing beads pre-coated with an antigen (or antibody) to bind the free antibody (or antigen) to these coated molecules. Detection of the thus captured antibody (or antigen) is achieved by a fluorescently labeled protein conjugated to the antibody (or antigen).

[0073] Furthermore, the GYROLAB® immunoassay system provides 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).

[0074] The term "approximately" as used in relation to parameters or ranges of parameters provided herein shall have the following meanings: Unless otherwise indicated, when the term "approximately" is applied to a particular value or range, that value or range shall be interpreted as being as accurate as the method used to measure it. If a tolerance is not specified at the time of application, the last decimal place of the number indicates the degree of precision. If no other tolerance is given, the maximum tolerance is determined by applying the convention of rounding to the last decimal place; for example, for a pH value of approximately pH 2.7, the tolerance is 2.65 to 2.74. However, special tolerances shall apply to the following parameters: Temperatures specified in °C without decimal places shall have a tolerance of ±1 °C (for example, a temperature value of approximately 50 °C means 50 °C ± 1 °C); periods expressed in hours shall have a tolerance of 0.1 hours regardless of decimal places (for example, a time value of approximately 1.0 hour means 1.0 hour ± 0.1 hours; a time value of approximately 0.5 hours means 0.5 hours ± 0.1 hours).

[0075] 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 “conformational state” of a protein generally refers to the range of structures that a protein can adopt at any given time. Those skilled in the art will recognize that the determinants of conformation or conformational conditions include the primary structure of the protein, reflected in the amino acid sequence (including modified amino acids), and the environment surrounding the protein. The conformation or conformational state of a protein is also related to structural features such as the protein's secondary structure (e.g., α-helix, β-sheet, β-barrel), tertiary structure (e.g., three-dimensional folding of the polypeptide chain), and quaternary structure (e.g., interactions between the polypeptide chain and other protein subunits). Post-translational and other modifications to the polypeptide chain, such as ligand binding, phosphorylation, sulfation, glycosylation, or linking of hydrophobic groups, can, among other things, affect the conformation of a protein. Furthermore, environmental factors, particularly pH, salt concentration, ionic strength, and osmotic pressure of the surrounding solution, as well as interactions with other proteins and cofactors, can affect protein conformation. The conformational state of a protein can be determined by functional assays for activity or binding to other molecules, or by physical methods such as X-ray crystallography, NMR, or spin labeling. General discussions of protein conformation and conformational states are referenced in 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.

[0076] According to this specification, "protein solubility" is a thermodynamic parameter defined as the concentration of protein in a saturated solution in equilibrium with either a crystalline or amorphous solid phase under given 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).

[0077] Finally, the terms “functional chimeric protein,” “functional fusion protein,” or “conformation-selective fusion protein” as used in this invention refer to a fusion protein that is functional in its binding to cytokines and / or ISVD targets and / or functional in the activation / inactivation of cytokine receptors and / or ISVD targets in an arbitrarily conformation-selective manner (depending on the known characteristics of the ligand: agonist, antagonist, or inverse agonist). A binding domain that selectively binds to a particular conformation of a target protein refers to a binding domain that binds to the target with higher affinity in a subset of conformations than in other conformations the target may be expected to have. Those skilled in the art will recognize that a binding domain that selectively binds to a particular conformation of a target stabilizes or retains the target in that particular conformation. For example, an active conformation-selective binding domain preferentially binds to the target in the active conformation, or does not bind to it, or binds to it to a lower degree.

[0078] The chimeric protein of the present invention In a first aspect, the present invention provides a chimeric protein comprising an immunoglobulin single variable domain (ISVD) fused with a cytokine. The chimeric protein of the present invention may also be called a “fusion protein” or “chimera”.

[0079] The term "Immunoglobulin Single Variable Domain" (ISVD), used interchangeably with "Single Variable Domain," defines an immunoglobulin molecule in which the antigen-binding site resides on a single immunoglobulin domain, thereby forming the ISVD. This distinguishes ISVDs from "conventional" immunoglobulins (e.g., monoclonal antibodies) or their fragments (e.g., Fab, Fab', F(ab')2, scFv, di-scFv) where two immunoglobulin domains (particularly two variable domains) interact to form the antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V) is present. H ) and light chain variable domain (V L ) interact to form an antigen-binding site. In this case, V H and V L Both complementarity-determining regions (CDRs) contribute to the antigen-binding site, meaning that a total of six CDRs are involved in the formation of the antigen-binding site.

[0080] In light of the above definition, conventional four-chain antibodies (e.g., IgG, IgM, IgA, IgD, or IgE molecules known in the art), or Fv fragments such as Fab fragments, F(ab')2 fragments, disulfide-linked Fv fragments, or scFv fragments, or diabodies derived from such conventional four-chain antibodies (all known in the art) are not usually considered ISVDs, because in these cases, binding to each epitope of the antigen is usually not by one (single) immunoglobulin domain, but by a pair of (related) immunoglobulin domains such as light chain and heavy chain variable domains, i.e., the V of immunoglobulin domains that co-bind to each epitope of the antigen. H -V L This is because it occurs in pairs.

[0081] In contrast, ISVDs are generally able to specifically bind to antigen epitopes without the need for additional immunoglobulin variable domains. The binding site of ISVDs is a single V H , single V HH , or a single V L It is formed by domains.

[0082] In connection with the present invention, an ISVD can be a light chain variable domain sequence (e.g., a V L sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a V H sequence or a V HH sequence) or a suitable fragment thereof. Preferably, an ISVD that can be included in the chimeric protein of the present invention is, for example, a heavy chain ISVD containing camelized V H or humanized V HH , such as V H , V HH . The heavy chain ISVD can be derived from a conventional four-chain antibody or a heavy chain antibody.

[0083] 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), a Nanobody® ISVD (as defined herein, including but not limited to V HH ); another single variable domain, or any suitable fragment of any of these. Preferably, an ISVD is V H , humanized V H , human V H , V HH , humanized V HH or camelized V H . More preferably, an ISVD is a Nanobody® ISVD (e.g., V HH containing humanized V H or camelized V HH ), or a suitable fragment thereof. Nanobody® is a registered trademark of Ablynx N.V.

[0084] The "V HH domain" refers to V HH , V HH antibody fragments, and V HHIt is also known as an antibody and was originally 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 363;446-448, 1993). HH The term "domain" refers to these variable domains as the heavy chain variable domains present in conventional four-chain antibodies (referred to as "V" in this specification). H (referred to as "domain") and the light chain variable domain present in conventional 4-chain antibodies (referred to as "V" in this specification). L It is chosen to distinguish it from what is called a "domain." HH For further explanation, please refer to the review article by Muyldermans ("Single domain camel antibodies: current status", J Biotechnol., 2001, 74:277-302). HH The domain can be obtained from heavy chain-only antibodies (HCAb) circulating in camelids (see, for example, Muyldermans S., “A guide to: generation and design of nanobodies”, FEBS J., 2021, 288(7):2084-2102).

[0085] Typically, immunoglobulin production involves immunizing experimental animals, fusing immunoglobulin-producing cells to create hybridomas, and screening for desired specificity. Alternatively, immunoglobulins can be produced by screening naive, immuno-, or synthetic libraries, such as phage display.

[0086] V HHsThe generation of immunoglobulin sequences such as V is widely described in various published literature, including, as examples, International Publication No. 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, camelids are immunized with the target antigen to induce an immune response to the target antigen. The V obtained from the immunization is V HHs The repertoire is further V HHs The drug is screened to determine whether it binds to (or does not bind to) the target antigen.

[0087] In connection with the present invention, immunoglobulin sequences of different origins may be used, including immunoglobulin sequences from mice, rats, rabbits, donkeys, humans, and camelids. In connection with the present invention, fully human sequences, humanized sequences, or chimeric sequences are also included. In connection with the present invention, this also includes immunoglobulin sequences of camelids and immunoglobulin sequences of humanized camelids, or camelized domain antibodies, such as camelized dAbs as described by Ward et al. (Nature, 341:544, 1989) (see, for example, International Publication No. 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 with improved protein stability”, Prot.Eng., 1996, 9(6):531-537).

[0088] "Humanization V" HH " is a naturally occurring V HH This corresponds to the amino acid sequence of the domain, i.e., the naturally occurring V HH One or more amino acid residues in the amino acid sequence (and especially in the framework sequence) of the sequence are extracted from conventional human-derived 4-chain antibodies. H The amino acid sequence is “humanized” by substitution with one or more amino acid residues present at corresponding positions within the domain (e.g., as shown above). This can be done by methods known in themselves and will be apparent to those skilled in the art, for example, based on further description herein and the prior art (e.g., International Publication No. 2008 / 020079). In this case as well, such humanized V HH V can be obtained by any preferred method known in itself and is therefore naturally occurring. HHIt should be noted that the present invention is not strictly limited to polypeptides obtained using a polypeptide containing a domain as a starting material. Preferably, the components of the present invention are V HH If V HH Humanized V HH That is the case.

[0089] "Camelization V" H " is a naturally occurring V H It corresponds to the amino acid sequence of the domain, but it has been "camelized," meaning it is a naturally occurring V derived from conventional quadruple-chain antibodies. H One or more amino acid residues in the amino acid sequence of the domain are used in the V of the heavy chain antibody. HH The amino acid sequence is “camelized” by substitution of one or more amino acid residues at corresponding positions in the domain. This can be done by methods known in themselves and will be apparent to those skilled in the art, for example, based on further description herein and the prior art (e.g., International Publication No. 2008 / 020079). Such “camelized” substitutions are typically V as defined herein. H -V L It forms an interface and / or a so-called Camelidae (Camellidae) Hallmark residue and / or is inserted into the position of an amino acid present therein (see, for example, International Publication No. 94 / 04678 and Davies and Riechmann, 1994 and 1996, cited above). In one embodiment, Camelized V H V is used as a starting material or starting point for generating or designing. H The sequence is derived from mammalian V H Sequence, or human-derived V H Arrays, for example V H It has 3 sequences. However, such camelid V H V can be obtained in any suitable form known by itself and therefore exists naturally as an initiating material. H Please note that this is not strictly limited to polypeptides obtained using polypeptides containing domains.

[0090] The structure of an ISVD sequence can be considered to consist of four framework regions ("FRs"), which are referred to in the art and herein as "framework region 1" ("FR1"), "framework region 2" ("FR2"), "framework region 3" ("FR3"), and "framework region 4" ("FR4"), respectively. These framework regions are interrupted by three complementarity-determining regions ("CDRs"), which are referred to in the art and herein as "complementarity-determining region 1" ("CDR1"), "complementarity-determining region 2" ("CDR2"), and "complementarity-determining region 3" ("CDR3"), respectively.

[0091] As further described in paragraph q) on pages 58 and 59 of International Publication No. 2008 / 020079, the amino acid residues of ISVD are V, as designated by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). H Following the general domain numbering, see the article by Riechmann and Muyldermans, 2000 (J. Immunol. Methods, 240(1-2):185-195; see, for example, Figure 2 in this published document) for V derived from camelids. HH The numbers are also assigned so that they apply to the domain. V H Domain and V HH It should be noted that, as is well known in the art, the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering. That is, one or more positions indicated by Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than permitted by Kabat numbering. This generally means that Kabat numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence. HDomain and V HH The total number of amino acid residues in a domain is typically in the range of 110 to 120, and often 112 to 115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein.

[0092] In this application, the CDR sequences were determined according to the Kabat numbering with AbM CDR annotations described in Kontermann and Duebel (Eds. 2010, Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, FR1 contains amino acid residues from positions 1 to 25, CDR1 contains amino acid residues from positions 26 to 35, FR2 contains amino acids from positions 36 to 49, CDR2 contains amino acid residues from positions 50 to 58, FR3 contains amino acid residues from positions 59 to 94, CDR3 contains amino acid residues from positions 95 to 102, and FR4 contains amino acid residues from positions 103 to 113.

[0093] The determination of the CDR region can also be performed according to different methods. In CDR determination according to Kabat, FR1 of ISVD contains amino acid residues from positions 1 to 30, CDR1 of ISVD contains amino acid residues from positions 31 to 35, FR2 of ISVD contains amino acids from positions 36 to 49, CDR2 of ISVD contains amino acid residues from positions 50 to 65, FR3 of ISVD contains amino acid residues from positions 66 to 94, CDR3 of ISVD contains amino acid residues from positions 95 to 102, and FR4 of ISVD contains amino acid residues from positions 103 to 113.

[0094] In such immunoglobulin sequences, the framework region can be any suitable framework sequence, and examples of suitable framework sequences will be apparent to those skilled in the art, for example, based on standard handbooks and further disclosures and prior art referred to herein.

[0095] The framework sequence is a preferred combination of an immunoglobulin framework sequence or a framework sequence derived from an immunoglobulin framework sequence, for example, by humanization or camelization. For example, the framework sequence is a light chain variable domain (e.g., V L - Sequence) and / or heavy chain variable domain (e.g., V H - Array or V HH It may be a framework array derived from the array. In one embodiment, the framework array is V HH - A framework sequence derived from a sequence (in which the framework sequence may be optionally partially or completely humanized), or a camelized (as defined herein) conventional V H It is one of the arrays.

[0096] In particular, the framework sequence present in the ISVD sequence referred to in this invention is, for example, a humanized V HH or Camelization V H V including HH It may include one or more hallmark residues (as defined herein) to form Nanobody® ISVD such as the above. Several non-limiting examples of preferred combinations of such framework sequences will become apparent from the disclosure herein.

[0097] However, it should be noted that in relation to the present invention, the origin of the ISVD sequence or the origin of the nucleotide sequence used to express it is not limited, nor is the method by which the ISVD sequence or nucleotide sequence is generated or obtained. Therefore, the ISVD sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In certain, but non-limiting, embodiments, the ISVD sequence may be a "humanized" immunoglobulin sequence (as defined herein) (e.g., a partially or fully humanized mouse or rabbit immunoglobulin sequence, in particular a partially or fully humanized V HHImmunoglobulin sequences include, but are not limited to, any preferred combination of the foregoing, including, naturally occurring sequences (from any preferred species), synthetic or semi-synthetic sequences.

[0098] Similarly, nucleotide sequences may be naturally occurring nucleotide sequences or synthetic or semi-synthetic sequences, and may also be, for example, sequences isolated by PCR from a suitable naturally occurring template, such as DNA or RNA isolated from cells, nucleotide sequences isolated from a library (and in particular an expression library), nucleotide sequences prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable method known to be known, such as mismatch PCR), nucleotide sequences prepared by PCR using overlap primers, or nucleotide sequences prepared using a known method for DNA synthesis.

[0099] For a general description of Nanobody® ISVD, please refer to this specification and the prior art cited herein. However, in this regard, this description and the prior art are so-called "V H "3 classes", that is, V HPlease note that this primarily describes Nanobody® ISVDs of three classes of human germline sequences (Nanobody® ISVDs having a high degree of sequence homology to DP-47, DP-51, or DP-29, etc.). However, in its broadest sense, this technology can generally use any type of Nanobody® ISVD, and please note that, for example, as described in International Publication No. 2007 / 118670, Nanobody® ISVDs belonging to the so-called "VH4 class," i.e., Nanobody® ISVDs having a high degree of sequence homology to VH4 class human germline sequences such as DP-78, can also be used.

[0100] In one embodiment, it should be noted that the ISVD contained in the chimeric molecule of the present invention is derived from Nanobody®ISVD belonging to the so-called "VH3 class," that is, Nanobody®ISVD having a high degree of sequence homology to human germline sequences of the VH3 class such as DP-47, DP-51, or DP-29.

[0101] Generally, Nanobody® ISVD (especially (partially) humanized V HH Sequence and camelized V H V containing an array HH The sequence may be characterized by the presence of one or more "Hallmark residues" (as further described herein) in one or more framework sequences (as described herein).

[0102] Generally, Nanobody® ISVD has the following (typical) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 It can be defined as an immunoglobulin sequence having the following characteristics: Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively, and one or more Hallmark residues are as further defined herein.

[0103] In particular, Nanobody(registered trademark) ISVD has the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 It can be defined as an immunoglobulin sequence having the following characteristics: Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively, and the framework sequence is as further defined herein.

[0104] More specifically, Nanobody® ISVD has the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 It can be defined as an immunoglobulin sequence having the following characteristics: Here, FR1 to FR4 refer to framework domains 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determination domains 1 to 3, respectively. One or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to Kabat numbering are selected from the Hallmark residues listed in Table 2 below.

[0105] [Table 2]

[0106] [Table 3]

[0107] Therefore, Nanobody(registered trademark) ISVD has the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 It can be defined as an amino acid sequence having the following characteristics: Here, FR1 to FR4 refer to framework regions 1 to 4, respectively; CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively; and one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to Kabat numbering are selected from the Hallmark residues mentioned in Table 2.

[0108] In further preferred embodiments, the ISVD base contained in the chimeric protein of the present invention is derived from ISVDs such as heavy chain ISVD, preferably Nanobody® ISVD, which has been further manipulated / modified to include mutations that prevent / remove binding of existing antibodies / factors. Examples of such mutations are described, for example, in International Publication No. 2012 / 175741 and International Publication No. 2015 / 173325. For example, to prevent / remove the binding of 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 be Val, Thr, or Leu, preferably Leu; and / or the amino acid at position 110 (according to Kabat) may 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 ISVD-based component may include a C-terminal extension of 1 to 5 amino acids selected from any native amino acids.

[0109] In one embodiment, the ISVD contained in the chimeric protein of the present invention specifically binds to its target (antigen), and such interaction between ISVD and the antigen is characterized by high specificity and / or high affinity as defined herein.

[0110] 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 interaction with its original target (antigen) or 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 ISVD ("ISVD precursor"). In this particular embodiment, the "ISVD precursor" is an ISVD that has been modified (e.g., by point mutation and / or addition / deletion of amino acids to its sequence) to produce the ISVD contained in the chimeric protein of the present invention. For example, the "ISVD precursor" is modified so that it no longer specifically binds to any molecule (ISVD precursor target (antigen)) to which the ISVD precursor specifically binds.

[0111] 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 control cellular activities such as migration, growth, survival, and differentiation. Cytokines are a very large and diverse group of pro-inflammatory or anti-inflammatory factors that are grouped into families based on their structural homology or the structural homology of their receptors. Examples of cytokines include chemokines, interferons, interleukins, lymphocytes, tumor necrosis factors, hormones, or growth factors. Interleukins (ILs) form a group of cytokines with complex immunomodulatory functions, including cell proliferation, maturation, migration, and adhesion, and play a crucial role in the differentiation and activation of immune cells. ILs can also have pro-inflammatory and anti-inflammatory effects and are under constant pressure to evolve due to ongoing competition between the host immune system and infectious organisms. As a result, ILs have undergone significant evolution, which has led to little amino acid conservation between orthologous proteins and complicates the organization of gene families. However, based on crystallographic analysis data and the identification of common structural motifs, these cytokines can be classified into four main groups: genes encoding IL1-like cytokines, class I helical cytokines (IL4-like, Y-chain, and IL6 / 12-like), class II helical cytokines (IL10-like and IL-28-like), and IL17-like cytokines (structurally unrelated to other IL subfamilies, with IL17F constituting a cysteine ​​knotfold).

[0112] In the chimeric protein of the present invention, fusion between ISVD and cytokine occurs at the internal fusion site of ISVD.

[0113] In this specification, “internal fusion site” refers to a position between two amino acids located at any location in a polypeptide sequence, more specifically in an ISVD and / or cytokine as used herein, and more specifically, to an internal fusion site that is not the N-terminus or C-terminus of a protein. An internal fusion site may instead be defined as a position between two amino acids located at any location in the protein variant of the ISVD or cytokine, where a small number of amino acids are deleted or added compared to the original protein sequence. The internal fusion site is a position that needs 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 of the structure can also be obtained by designing gene fusions. Therefore, as shown in Figure 32, the chimeric protein of the present invention is obtained by translation of a gene fusion of the chimeric protein, which corresponds to a protein sequence that starts at the N-terminal portion of the ISVD ending at an internal fusion site (and / or a variant in which few amino acids are added or deleted at the fusion site, e.g., 1 to 10 amino acids, e.g., 1 to 7, or 1 to 5 amino acids are added or deleted at the fusion site, or a variant in which 1, 2, 3, 4, or 5 amino acids are added or deleted at the fusion site) (attached to the N-terminus of the inserted protein, which is the cytokine or a cyclically substituted variant of the cytokine in the present invention, and then the C-terminus is attached to the rest of the ISVD).

[0114] Therefore, “internal fusion site” means a point where the original peptide bond between two amino acids present in the ISVD or particularly in the cytokine sequence is interrupted, and two new peptide bonds are formed, specifically a point where one peptide bond is formed connecting the amino acid sequence located at the N-terminus of the internal fusion site to the N-terminus of the inserted protein sequence, and which includes one peptide bond connecting the C-terminus of the inserted protein sequence to the sequence located at the C-terminus of the internal fusion site. Thus, in relation to the present invention, “internal fusion site” is a location in the sequence of the ISVD (and / or cytokine, if cyclically substituted) where a link (fusion) to the cytokine (or ISVD) is established. Thus, the internal fusion site can be used as a reference point for the amino acid sequence that divides the original protein sequence at the sequence located at the N-terminus of the internal fusion site and the sequence located at the C-terminus of the internal fusion site of the protein. The internal fusion site of the ISVD is preferably located in a loop or turn in the folded protein, preferably in a beta turn, more preferably between two beta chains, and even more preferably between beta chains A and B (see below for details). The internal fusion sites of circulatingly substituted cytokines contained in the chimeric protein of the present invention are located within a turn or loop between two secondary elements of the cytokine, for example, between two β-chains or between two α-helices, or within a turn or loop between a β-chain and an α-helix. Preferably, the internal fusion sites of the cytokine are located within the protein to provide the modified cytokine receptor / receptor subunit binding functionality to the chimeric molecule created by cytokine binding and / or the modification of the receptor / receptor subunit oligomerization at cytokine binding and / or fusion of ISVD at the internal fusion site of the cytokine. The terms “accessible site” or “exposed site” are used interchangeably herein and all refer to amino acid sites of a protein sequence that are structurally accessible, preferably located on the surface of a protein, or exposed on the surface. “Internal fusion site” as used herein is preferably an amino acid site of a protein that is also an accessible site or an exposed site.The exposed or accessible sites are preferably located within a turn or loop between two secondary elements in the protein (e.g., two β-chains, or two α-helices, or a β-chain and an α-helices). Those skilled in the art will be able to determine these sites.

[0115] In the chimeric protein of the present invention in which cytokines are cyclically substituted, ISVD is linked to the cyclically substituted cytokine at an internal fusion site of ISVD, the cyclically substituted cytokine protein sequence is inserted as described above, and the N-terminus and C-terminus of the cyclically substituted cytokine protein sequence are provided by cleavage of the protein sequence at an internal fusion site as defined herein, providing the N-terminus and C-terminus for the formation of peptide bonds to create a chimeric protein.

[0116] When cytokines undergo cyclic substitution, amino acids at the internal fusion site of ISVD are linked to amino acids at the internal fusion site of the cytokine. The internal fusion site is located in a loop or turn between two secondary structural elements of both ISVD and the cyclically substituted cytokine. For example, the internal fusion site of ISVD may be located in a loop or turn between two secondary structural elements, e.g., a beta turn. For example, the internal fusion site of the cyclically substituted cytokine may be located in a loop or turn, thereby providing a location for fusion to the ISVD protein sequence, as well as the location of the ISVD internal fusion site. In one embodiment, the internal fusion site of ISVD is not located in any of the CDRs of ISVD. Therefore, in one embodiment, the loop or turn (e.g., a beta turn) in which the internal fusion site of ISVD is located is not a CDR.

[0117] Therefore, the internal fusion site of the ISVD is the position where the amino acid located at the N-terminus of the site is connected at the C-terminus that terminates at the N-terminus of the cytokine (or circulatingly substituted cytokine) protein, and the amino acid located at the C-terminus of the internal fusion site is connected at the C-terminus of the cytokine (or circulatingly substituted cytokine) protein (see also Figure 32 as an example for clarity). Preferably, in the chimeric protein of the present invention, the cytokine is a circulatingly substituted cytokine as described above. Therefore, when the cytokine is circulatingly substituted in the chimeric protein of the present invention, the original N-terminus and C-terminus of the cytokine protein sequence are linked together (because it is a circulatingly substituted cytokine). To circulatingly substituted, the N-terminus and C-terminus of the cytokine may be linked together directly or by a linker, as described herein. In one embodiment, 0 to 10 amino acids from the (original) N-terminus and / or C-terminus of the cytokine are removed before the N-terminus and C-terminus of the cytokine are linked together. Preferably, 0 to 7 amino acids from the N-terminus and / or C-terminus of the cytokine are removed before the N-terminus and C-terminus of the cytokine are linked together; more preferably, 0 to 5 amino acids, e.g., 0, 1, 2, 3, or 4 amino acids, from the N-terminus and / or C-terminus of the cytokine are removed before the N-terminus and C-terminus of the cytokine are linked together (directly or by a linker as described herein).

[0118] In other embodiments, in the chimeric protein of the present invention, the internal chimeric fusion in the ISVD as defined herein is obtained using a cytokine that has not undergone cyclic substitution. In this embodiment, the cytokine retains its original N-terminus and C-terminus, i.e., no new N-terminus and C-terminus are created anywhere else in the cytokine sequence. In this embodiment, the cytokine is fused to the amino acids of the ISVD located at the internal fusion site of the ISVD via the (original) N-terminus and C-terminus of the cytokine. In this embodiment, the cytokine is not linked to the amino acids of the internal fusion site of the ISVD via the amino acids located at the internal fusion site of the cytokine. As above, 0 to 7 amino acid sequences from the original N-terminus and / or C-terminus of the cytokine may be removed before fusing the cytokine to the amino acids located at the internal fusion site of the ISVD. Preferably, 0 to 7 amino acids from the N-terminus and / or C-terminus of the cytokine are removed before fusing the cytokine to the amino acids at the internal fusion site of the ISVD, and more preferably, 0 to 5 amino acids from the N-terminus and / or C-terminus of the cytokine, e.g., 0, 1, 2, 3, or 4 amino acids, are removed before fusing the cytokine to the amino acids located at the internal fusion site of the ISVD. In addition, peptide linkers as 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.

[0119] In one embodiment, the chimeric protein of the present invention has a continuous amino acid sequence. Therefore, in a preferred embodiment, in the chimeric protein of the present invention, the N-terminal sequence of ISVD located at the N-terminus of the internal fusion site is linked via a peptide linker to the original C-terminal portion of the cytokine corresponding to the sequence located at the C-terminus of the cytokine's internal fusion site, and the N-terminal portion of the original cytokine corresponding to the sequence located at the N-terminus of the cytokine's internal fusion site is linked via a peptide linker and / or peptide bond to the C-terminal sequence of ISVD located at the C-terminus of the ISVD's internal fusion site to form a continuous amino acid sequence. For details, please refer to Figures 2 and 32.

[0120] Preferably, in the chimeric protein of the present invention, the tertiary structure of ISVD and cytokines in the chimeric protein is maintained, except where applicable, the amino acid structure at the internal fusion site linking ISVD and cytokines. Thus, in preferred embodiments, the tertiary structure of ISVD and cytokines in the chimeric protein is maintained, except where applicable, the amino acid structure at the internal fusion site linking ISVD and cytokines, compared to the tertiary structure of ISVD and cytokines when ISVD and cytokines are not part of the chimeric protein. The tertiary structure can be partially maintained, for example, when applicable (i.e., the cytokine is cyclically substituted and therefore linked to the amino acids at the internal fusion site of ISVD and the amino acids at the internal fusion site of cytokines, as described in detail above), at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% of the tertiary structure of ISVD and / or cytokines is maintained, compared to the tertiary structure of ISVD and cytokines when ISVD and cytokines are not part of the chimeric protein, except where applicable, the amino acid structure at the internal fusion site linking ISVD and cytokines.

[0121] The tertiary structure of a protein is its three-dimensional shape. This three-dimensional structure is primarily due to the interactions between the side chain groups of the amino acids that make up the protein. These side chain group interactions contribute to the tertiary structure and include non-covalent interactions such as hydrogen bonds, ionic bonds, dipole interactions, and London dispersion forces. Also important for the tertiary structure are hydrophobic interactions, where amino acids with nonpolar, hydrophobic side chain groups cluster together inside the protein, while hydrophilic amino acids on the outside continue to interact with surrounding water molecules. Finally, disulfide bonds (covalent bonds between sulfur-containing side chains of cysteine) can also contribute to the protein's tertiary structure. Those skilled in the art are familiar with the techniques and methodologies for establishing the tertiary structure of proteins. For example, X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR), cryo-electron microscopy, or two-plane polarization interferometry are tools that can be used to confirm the tertiary structure of a given protein.

[0122] As described in detail in International Publication No. 2019 / 086548, the method by which ISVD and cytokines are fused in the chimeric protein of the present invention provides a chimera having a more rigid, immobile junction. Classical junctions of polypeptide components are typically linked in their native state, but are achieved by linking their respective N-terminuses and C-terminuses directly or via peptide bonds to form a single continuous polypeptide. These fusions are often achieved via a mobile linker or at least linked in a mobile manner, meaning that the fusion partners are not in a stable position or conformation relative to each other. By linking proteins via the N-terminuses and C-terminuses, as shown in Figure 1 of International Publication No. 2019 / 086548, fusion is easy but unstable, easily degraded, and in some cases unsuitable for therapeutic use, due to simple linear linking. On the other hand, rigid chimeric / fusion proteins, such as those presented herein, which have one or more fusion sites or junctions within the primary topology of two or more proteins, have at least one immobile fusion site (see Figure 1). As described above, the chimeric protein of the present invention originates from the formation of a fusion between ISVD and a cytokine, where the cytokine (preferably cyclically substituted) disrupts the topology of ISVD. Therefore, in one embodiment, the chimeric protein of the present invention is preferably a continuous amino acid sequence obtained by gene fusion.

[0123] One embodiment provides a chimeric protein in which ISVD is fused with a cytokine (preferably circulatingly substituted) in such a way that the cytokine (preferably circulatingly substituted) “disrupts” the topology of ISVD. Generally, the “topology” of a protein refers to the regular orientation of secondary structures relative to each other in three-dimensional space. Protein folding is primarily defined by polypeptide chain topology (Orengo, C., Jones, D. & Thornton, J., “Protein superfamilles and domain superfolds”, Nature, 1994, 372:631-634). Thus, at the most basic level, the “primary topology” is defined as the sequence of secondary structural elements (SSEs) involved in protein folding recognition motifs, and therefore secondary and tertiary protein / domain folding. Therefore, with respect to protein structure, the true or primary topology is the sequence of SSEs, i.e., if we imagine that the N-terminus and C-terminus of the protein chain are held and can be pulled straight out, then the topology does not change regardless of how the protein folds. Therefore, protein folding is described as a tertiary topology, similar to the primary and tertiary structures of proteins (see also Martin AC., “The ups and downs of protein topology; rapid comparison of protein structure”, Protein Eng. 2000, 13(12):829-37). Accordingly, the ISVD contained in the chimeric protein of the present invention is disrupted in its primary topology by introducing cytokines (preferably cyclically replaced).

[0124] Novel chimeric proteins are fused in a unique manner to avoid the junction being a mobile, loose, and weak link / region within the chimeric protein structure. A convenient means of linking or fusing two polypeptides, in classical known methods, is to express them as a fusion protein from a recombinant nucleic acid molecule containing a first polynucleotide encoding a first polypeptide operably linked to a second polypeptide encoding a second polypeptide. However, in the recombinant nucleic acid molecule of the present invention, the disruption of the ISVD topology by cytokines (preferably cyclically substituted) is also reflected in the design of the gene fusion from which the chimeric protein is expressed. Thus, in one embodiment, the chimeric protein is encoded by a chimeric gene formed by rejoining a portion of the gene encoding ISVD with a portion of the gene encoding cytokines, the cytokines disrupting the primary topology of the encoded ISVD at one or more internal fusion sites of the ISVD, either directly or via at least two direct fusions or fusions made by encoded peptide linkers. Therefore, the polynucleotide encoding the polypeptide to be fused is fragmented and recombined in such a manner to provide a chimeric protein resulting in a rigid, immobile link, connection, or fusion between the proteins. Novel chimeric proteins are created by fusing a cytokine with an ISVD in such a way that the primary topology of the ISVD is interrupted, i.e., the amino acid sequence of the antigen-binding domain is interrupted at the internal fusion site and bound to an amino acid in the cytokine. If the cytokine is cyclically substituted, the amino acid sequence of the antigen-binding domain is interrupted at the internal fusion site and bound to an amino acid at the internal fusion site of the cytokine, and thus its sequence is also interrupted. In both the ISVD and the cytokine (if applicable), the internal fusion site is located in a loop or turn between the two secondary structural elements, as described above.

[0125] Therefore, in the chimeric protein of the present invention in which ISVD is bound to a cytokine, the amino acid of ISVD located at the N-terminus of the internal fusion site of ISVD is linked to the N-terminus of the cytokine at its C-terminus, 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, forming a continuous amino acid sequence. In embodiments in which the cytokine is cyclically substituted (which is preferred as described above), for example, as shown in Figure 1 or Figure 32, the N-terminus of the chimeric protein of the present invention comprises an ISVD amino acid sequence located at the N-terminus of the ISVD's internal fusion site, followed by the (gene-fused) amino acid sequence of the cyclically substituted cytokine sequence, followed by the ISVD amino acid sequence located at the C-terminus of the ISVD's internal fusion site, where the cyclically substituted cytokine sequence is provided as described herein, and in particular the cyclically substituted cytokine is fused to ISVD by ligating the N-terminus of the amino acid located at the C-terminus of the (cleaved) internal fusion site of the cyclically substituted cytokine to the C-terminus of the amino acid located at the N-terminus of the (cleaved) internal fusion site of ISVD, and by ligating the N-terminus of the amino acid located at the N-terminus of the C-terminus of the ISVD's internal fusion site.

[0126] In one 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 the circulatingly substituted cytokine, correspond to at least a portion of the sequence of loops or turns between the two secondary structural elements of each internal fusion site (if applicable, in the original sequence of the ISVD and / or the cytokine). 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 loops or turns (e.g., β-turns) between the two secondary structural elements (e.g., two β-chains) of the internal fusion site of the ISVD. Alternatively or in addition, 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 loops or turns between the two secondary structural elements (e.g., two β-chains, or two α-helices, or one β-chain and one α-helices) of the internal fusion site of the cytokine. As stated above, correspondence can be "to at least a portion of the sequence of loops or turns." This is because 0 to 10, preferably 0 to 5, more preferably 0 to 4, and even more preferably 0 to 3 (preferably consecutive) amino acid residues, for example, 0, 1, 2, or 3, may be missing in the loop or turn of one or both proteins (i.e., ISVD and / or cytokine, if applicable) in the chimeric protein. In addition, ISVD and cytokine (preferably cyclically substituted) may be fused via a peptide linker as described above. In other embodiments, the amino acid sequences located at the N-terminus and C-terminus of the internal fusion site of ISVD and / or cytokine (if applicable) correspond precisely to the sequences of the loop or turn between the two secondary structural elements of each internal fusion site in the original sequence of ISVD and / or cytokine (i.e., without missing amino acid residues in the loop or turn of one or both proteins in the chimeric protein).

[0127] In addition, in a further preferred embodiment, the N-terminus and C-terminus of the chimeric protein correspond to the N-terminus and C-terminus of ISVD, respectively.

[0128] In one embodiment, the amino acid sequence of the chimeric protein of the present invention (preferably a continuous amino acid sequence as described above) is: (i) The N-terminal portion of the ISVD sequence; followed by (ii) cytokine sequence; followed by (iii) The remainder of the ISVD sequence (i.e., the C-terminal portion of ISVD) Includes.

[0129] As described above, one or more amino acids may be removed from the sequences located at the N-terminus and / or C-terminus of the cytokine's internal fusion site. In addition, peptide linkers may be added to the sequences located at the N-terminus and / or C-terminus of the cytokine's internal fusion site.

[0130] In another embodiment, when cytokines are circulatingly substituted, the amino acid sequence of the chimeric protein of the present invention (preferably a continuous amino acid sequence as described above) is: (i) The N-terminus of ISVD; followed by (ii) Sequences located at the C-terminus of the internal fusion site used to obtain circulatingly substituted cytokines; and (iii) the remainder of the sequence located at the N-terminus of the internal fusion site used to obtain the circulatingly substituted cytokine; followed by (iv) The sequence of ISVD located at the C-terminus of the internal fusion site of ISVD (i.e., the C-terminal portion of ISVD) Includes.

[0131] For details, please refer to Figures 1 and 32. The amino acid sequence of the chimeric protein, starting from its N-terminus, first includes the N-terminal amino acid of ISVD (e.g., β-chain A in Figure 1), followed by the C-terminus of the amino acid at the internal fusion site of ISVD, which is ligated to the N-terminus of the circulatingly substituted cytokine (the circulatingly substituted cytokine is created by cleaving the sequence at the internal fusion site located in a turn or loop and comparing it to the original cytokine to form a new N-terminus and C-terminus of the circulatingly substituted cytokine). The amino acid sequence of the chimeric protein then continues with the (remaining) sequence of the circulatingly substituted cytokine, ending at its C-terminus (corresponding to the amino acid that was located at the N-terminus of the cytokine's internal fusion site in order to design the circulatingly substituted cytokine), and finally ligated to the N-terminus of the amino acid located at the C-terminus of the internal fusion site of ISVD (located in a turn or loop, in this case at the beta turn represented by the black line in Figure 1) and the remainder of the ISVD sequence (the C-terminal portion of ISVD).

[0132] Therefore, in the chimeric protein of the present invention, if a cytokine is circulatingly substituted, the primary amino acid sequence of the circulatingly substituted cytokine is inserted into the primary sequence of ISVD (or the amino acid sequence of the circulatingly substituted cytokine interrupts the primary sequence (amino acid sequence) of ISVD).

[0133] In some embodiments of the present invention, the fusion may be a direct fusion or a fusion made by a linker peptide, the fusion site being designed to result in a rigid, immobile fusion protein. In addition to the arrangement of the selected internal fusion site, the length and type of the linker peptide contribute to the rigidity of the resulting chimeric protein. In relation to the present invention, the polypeptides constituting the chimeric protein (see, for example, (i)-(iii) or (i)-(iv) above) are fused to each other directly, by linkage via peptide bonds, or indirectly, in indirect coupling, by linkage via a short peptide linker. To provide the desired rigidity at the fusion junction at an accessible site, a preferred “linker molecule,” “linker,” or “short polypeptide linker” is a peptide having a length of up to 10 amino acids, more likely 4 amino acids, typically 3 or 4 amino acids, but preferably only 2, or even more preferably only 1 amino acid. Non-limiting examples of suitable linker sequences are described in Table A-1 and the Examples section, which can be randomized, and the linkers have been successfully selected to maintain fixed distances between structural domains and / or to maintain independent functions of the fusion partner (e.g., antigen binding and / or cytokine receptor binding) (if this is desirable for the chimeric protein of the present invention). Non-limiting examples of such linkers are GSGG (SEQ ID NO: 120), GGSG (SEQ ID NO: 121), or GSG (SEQ ID NO: 5).

[0134] In embodiments relating to the use of rigid linkers, they are generally known to exhibit specific conformations by employing an α-helical structure or by containing multiple proline residues. Under various circumstances, they isolate functional domains more efficiently than mobile linkers, which may be suitable even with a short length of as few as 1 to 4 amino acids.

[0135] Therefore, in one preferred embodiment, the ISVD and 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 the circulatingly substituted cytokine sequence via a peptide linker, and / or the N-terminus portion of the original cytokine is linked to the sequence located at the C-terminus of the internal fusion site of the ISVD via a peptide linker.

[0136] As described above, fusion between ISVD and cytokines can occur by first removing several amino acids from the internal fusion site of ISVD and / or cytokine (if it is circulatingly substituted) (or from the N-terminus and / or C-terminus of the cytokine if the cytokine is not circulatingly substituted).

[0137] Therefore, in one embodiment, the sequence located at the N-terminus of the ISVD internal fusion site, the (circularly substituted) cytokine sequence, and the sequence located at the C-terminus of the ISVD internal fusion site are linked to each other (directly or by a linker, as defined above) by first removing 0 to 10, preferably 0 to 5, more preferably 0 to 3, for example, 0, 1, 2, or 3 (consecutive) amino acids from the N-terminus of the (consecutively substituted) cytokine sequence, and the N-terminus of the ISVD internal fusion site relative to the adopted N-terminus of the (circularly substituted) cytokine is linked to the sequence located at the C-terminus of the ISVD internal fusion site via a peptide linker, as described above, at an optional choice. Similarly, in another embodiment, the sequence located at the N-terminus of the ISVD internal fusion site and the sequence of the (circularly substituted) cytokine and / or the sequence located at the C-terminus of the ISVD internal fusion site are linked together (directly or by linker as defined above) by first removing 0 to 10, preferably 0 to 5, more preferably 0 to 3, for example 0, 1, 2, or 3 (consecutive) amino acids from the sequence at the ISVD internal fusion site, and the sequence at the N-terminus of the ISVD internal fusion site for the (circularly substituted) cytokine is optionally linked to the sequence located at the C-terminus of the ISVD internal fusion site via a peptide linker as described above.

[0138] In one embodiment, the internal fusion site of the ISVD is located in an exposed region of domain folding. The exposed region is identified as a less fixed amino acid stretch, mainly located on the surface and structural edges of the protein. In a preferred embodiment, the internal fusion site is located in an exposed turn, such as a β-turn defined by IMGT, within an exposed loop between two β-chains in the ISVD (see below). More preferably, the internal fusion site of the ISVD is located in a β-turn, such as an exposed β-turn.

[0139] As described above, the internal fusion site of ISVD is contained within a loop or turn, preferably a beta turn, as defined by IMGT (Lefranc MP, “Immunoglobulin and T Cell Receptor Genes: IMGT® and the Birth and Rise of Immunoinformatics”, Front Immunol., 2014, 5:22). Preferably, cytokines (preferably circulatingly substituted) are inserted into or fused to amino acids of the internal fusion site of ISVD, and the internal fusion site is a. Located in the first β-turn connecting the β-chains A and BB of ISVD; or b. Located in the β-turns connecting the β-chain C and C' of ISVD; or c. Located in the β-turn connecting the β-chains C'' and D of ISVD; or d. Located in the β-turn connecting the β-chains D and E of ISVD; or e. It is located in the β-turn connecting the β-chains E and F of ISVD.

[0140] In a preferred embodiment, the internal fusion site within the ISVD is located in the exposed region of the ABβ turn connecting the A and B β chains of the ISVD. Alternatively, the internal fusion site within the ISVD is located in the exposed region defined by the CC'β turn connecting the C and C' β chains of the ISVD. Another embodiment includes an internal fusion site in the C''Dβ turn, or in the EFβ turn. In fact, these are surface loops that connect β chains A and B, C and C', C'' and D, or E and F, respectively, forming a typical sandwich β chain and providing immunoglobulin folding.

[0141] In the ISVD contained in the chimeric protein of the present invention, the CDR relates to the exposed region (loop or turn) between the two secondary elements (see, for example, Figure 1). In the case of ISVD, disruption of these sites for fusing the ISVD to the cytokine can result in a loss of antigen-binding ability. In this case, the ISVD contained in the chimeric protein of the present invention may no longer have the ability to specifically bind to its antigen as defined above. If it is desirable to retain antigen-binding ability, the CDR would not be the most suitable internal fusion site for the ISVD.

[0142] Therefore, in one embodiment, the internal fusion site is located in an exposed region, loop, or turn so that the CDR of the ISVD retains the 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.

[0143] The internal fusion site of the cytokine in the chimeric protein of the present invention, when cyclically substituted, is located in a turn or loop between two secondary elements of the cytokine, for example, in a β-turn, or in a loop between two β-chains, or between two α-helices, or between one β-chain and one α-helices. Preferably, the internal fusion site of the cytokine is located in a protein position that provides modified cytokine receptor binding or downstream activity of the modified cytokine receptor and / or oligomerization of the modified receptor / receptor subunit upon cytokine binding and / or binding functionality of the modified cytokine receptor / receptor subunit to a chimeric molecule created 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 domain folding. The exposed region is identified as a less fixed amino acid stretch located mainly on the surface and structural edges of the protein. In preferred embodiments, 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 cytokine's internal fusion site is located in a loop or turn between one β chain and one α helix.

[0144] In one embodiment, a chimeric protein (having two peptide bonds or two short linkers) is obtained by linking the ISVD to a cytokine by fusing the ISVD with a cytokine that has been circulatingly substituted at its internal fusion site located in an exposed region of its sequence (a turn or loop as defined above) via a disruption of the primary topology of the ISVD at an internal fusion site in its sequence located in an AB beta turn (the exposed or accessible site is not the original N-terminus or C-terminus of the cytokine as described above).

[0145] In another embodiment, a chimeric protein (having two peptide bonds or two short linkers) is obtained by fused with a non-circularly substituted cytokine via a disruption of the primary topology of ISVD at an internal fusion site in its sequence located in an AB beta turn, i.e., by fusion of the fusion site of ISVD with the cytokine through the (original) N-terminus and C-terminus of the cytokine (using one or more amino acids deleted from the peptide linker and / or the N-terminus and / or C-terminus of the cytokine, as described above).

[0146] In one embodiment, the ISVD and cytokines contained in the chimeric protein are further connected via disulfide bonds. Disulfide bonds can be formed by cysteine ​​residues located within the ISVD, preferably near a turn or loop, preferably near an AB beta turn, at the terminus of the β chain A, and / or at the terminus of the β chain G. In one embodiment, the ISVD and cytokines are further connected via disulfide bonds to improve the rigidity of the chimeric protein.

[0147] As described above, the cytokines present in the chimeric protein of the present invention are preferably circulatingly substituted cytokines. In one embodiment, the N-terminus and C-terminus of a cytokine (i.e., the original N-terminus and C-terminus before circulating substitution) are linked to each other directly or via a peptide linker such as GG or any other peptide linker as defined above and in Table A-1 to generate a circulatingly substituted cytokine. In addition, in another embodiment, the circulatingly substituted cytokines contained in the chimeric protein of the present invention are generated by first linking the (original) N-terminus and C-terminus of a cytokine by removing 0 to 10, preferably 0 to 5, more preferably 0 to 3, for example, 0, 1, 2, or 3 (consecutive) amino acids from the N-terminus and / or C-terminus of the cytokine, and then linking the N-terminus and C-terminus (i.e., the original N-terminus and C-terminus of the cytokine before circulating substitution) directly or via a peptide linker as defined above and in Table A-1.

[0148] In another embodiment, as described above, the cytokines present in the chimeric protein of the present invention are not cyclically substituted as described herein. In this embodiment, the N-terminus and C-terminus of the cytokine (i.e., the original N-terminus and C-terminus, but from which one or more amino acids may be removed as described above) are not linked to each other and are used to fuse the cytokine to amino acids at the internal fusion site of the ISVD (directly or by a linker as described above).

[0149] In preferred embodiments, the cytokines included in the chimeric protein of the present invention are any type of cytokine, such as interleukins, chemokines, interferons, colony-stimulating factors (CSFs), transforming growth factors, or tumor necrosis factors. The cytokines comprise a very diverse superfamily of ligands, such as cytokine superfamilies having conserved core domains or motifs that are β-chain based or contain β-chains, clearly indicating internal fusion sites in their exposed regions located in β-turns or loops that interconnect these β-chains. Therefore, the cytokines included in the chimeric protein of the present invention may be selected from the following: - Interleukin-subfamily: o IL-1 family o IL-2 family o IL-6 family o IL-10 family o IL-12 family o IL-17 family - Chemokine subfamily o C oCC o CXC o CX3C - Colony-stimulating factor (CSF) - Interferon o Type I IFN o Type II IFN o Type III IFN - Transformation Growth Factor (TGF) o Alpha type o Beta type - Tumor necrosis factor (TNF)

[0150] In preferred embodiments, the cytokine is selected from interleukins or interferons. More preferably, the cytokine is IL-2, IFNA2a, or IL18.

[0151] 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 alpha-helix bundles; this family also includes IL-4, IL-7, IL-9, IL-15, and IL-21. It is a 15.5–16 kDa four-alpha-helix bundle cytokine (see Figure 2) that exerts its effects through binding to various IL-2 receptors (IL-2Rs), particularly monomeric, dimeric, and trimeric IL-2Rs (see, for example, Figures 12–15 and Figure 1 in Arenas-Ramirez, N., et al., "Interleukin-2: biology, design and application", 2015, Trends in Immunology, 36(12):763–777). The monomer IL-2R, which contains IL-2α(CD25), is usually cell membrane-bound, but it also exists in a soluble form and is attached to IL-2 by approximately 10 -8 M's low K d They bind via [a specific linkage]. The interaction between IL-2 and CD25 alone does not induce a signal. Conversely, both the dimer and trimer IL-2R produce downstream signals when they bind to IL-2. The dimer IL-2R binds to IL-2Rβ(CD122) and IL-2Rγ[common γ chain (γ c ) or better known as CD132], while the trimer IL-2R contains CD25, CD122, and γ c It includes. Considering only IL-2R with signal transduction capability, dimeric IL-2R has low affinity (approximately 10 -9 M's K d ) Called IL-2R, the trimer IL-2R has high affinity (approximately 10%). -11 M's K d It can be called IL-2R. At the molecular level, a single trimer IL-2R binds to IL-2R with approximately 10 to 100 times higher affinity than a single dimer IL-2R (according to Arenas-Ramirez, N., et al., “Interleukin-2: biology, design and application”, 2015, Trends in Immunology, 36(12):763-777).

[0152] Human interferon alpha-2 (IFNA2a) (e.g., gene ID: 3440) is a cytokine belonging to the type I IFN family. The mature protein consists of 165 amino acids. The secondary structure of IFNA2a consists of five α-helices: A to E, from the N-terminus to the C-terminus. Helices A, B, C, and E are organized as a bundle having a long loop between helices A and B (AB loop) and two disulfide bonds connecting helix E to the AB loop and helix C to the N-terminus. The type I IFN receptor (IFNAR) consists of two subunits, IFNAR1 and IFNAR2 (see, for example, Figures 24-27).

[0153] Interleukin-18 (IL-18) (e.g., gene ID: 3606) belongs to the IL-1 superfamily and folds as an all-beta pleated sheet molecule (see, for example, Figure 30). The “IL-1 receptor interleukin” superfamily or “IL-1 family” interleukins, when used interchangeably herein, 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 related to each other by origin, receptor structure, and signaling pathway. The receptors of IL-1 superfamily interleukins share a similar structure consisting of three lg-like domains in their ectodomain and an intracellular Toll / IL-1R (TIR) ​​domain, which is also found in Toll-like receptors. The initiation of cytokine signaling requires two receptors: a primary specific receptor and a co-receptor (which may be shared in some cases). The primary receptor is involved in specific cytokine binding, but the co-receptor itself does not bind to cytokines; instead, it associates with a pre-assembled two-component complex from the cytokine and the primary receptor. The binding of each cytokine to its respective receptor results in a three-component signaling complex, leading to the dimerization of the TIR domains of the two receptors. This initiates intracellular signaling by activating mitogen-activated protein kinase (MAPK) and nuclear factor kappa light chain-activated B-cell enhancing factor (NF-κB). This signaling induces inflammatory responses, including the induction of cyclooxygenase type 2, increased expression of adhesion molecules, and nitric oxide synthesis.

[0154] IL-18 first binds to the IL-18α receptor, forming a lower-affinity complex. Upon binding to the IL-18β receptor, a higher-affinity heterotrimeric complex is formed, initiating the signaling process.

[0155] The three-dimensional structures of several interleukin cytokines in the IL-1 superfamily have been determined, and despite limited sequence similarity, these cytokines demonstrate the adoption of a conserved signature β-trefoil fold consisting of 12 antiparallel β-chains arranged in a triple-symmetric pattern (see, for example, Figure 30). The β-barrel core motif packs varying amounts of helices within each cytokine structure. The superposition of each Cα atom in human cytokines reveals a conserved hydrophobic core with significant mobility in the loop region. Inter-triple-chain surface residues and inter-triple-chain loops do not appear to be important for overall stability and differ significantly among cytokines, consistent with their low sequence similarity and partially explaining their unique recognition by their respective receptors (with specific loops). For example, human IL-18 shares 65% sequence identity with mouse IL-18, but only 15% and 18% identity with human IL-1α and human IL-1β, respectively. Nevertheless, IL-18 exhibits remarkable similarities to other IL-1 cytokines in its three-dimensional structure. Thus, this IL-1-like receptor interleukin provides an example of a superfamily within cytokines having conserved s-chain-based structural core domains interconnected by mobile β-turns or loops, some of which are involved in receptor recognition, while others are involved in attachment to folded scaffold proteins, such as those presented herein, to obtain novel expanded fusion ligands.

[0156] In one embodiment, the cytokine contained 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.

[0157] As described below, in the chimeric protein of the present invention, the cytokines contained therein may be functional (in that they retain their receptor-binding functionality in a similar manner to cytokines not fused to ISVD) or non-functional (in that they do not retain their receptor-binding functionality in a similar manner to cytokines not fused to ISVD, as described above in relation to ISVD). In addition, the receptor-binding functionality of cytokines contained in the chimeric protein of the present invention may be regulated by their fusion to ISVD, as described in detail below. Furthermore, oligomerization of cytokine receptors / receptor subunits may be affected / altered upon binding of cytokines contained in the chimeric protein compared to oligomerization of cytokine receptors / receptor subunits upon binding when cytokines are not contained in the chimeric protein (i.e., when cytokines are not fused to ISVD via two peptide bonds). In addition, cytokine signaling may be regulated by cytokines contained in the chimeric protein of the present invention.

[0158] Therefore, cytokines contained in the chimeric protein of the present invention may not retain their receptor-binding functionality in a similar manner to cytokines not fused to ISVD. This means that cytokines contained in the chimeric protein of the present invention may bind to their receptor with better specificity and / or higher affinity compared to cytokines not fused to ISVD. This also means that cytokines contained in the chimeric protein of the present invention may bind to their receptor with lower specificity and / or lower affinity compared to cytokines not fused to ISVD. This also means that downstream signaling of the cytokine receptor upon binding of cytokines present in the chimeric protein of the present invention may differ from downstream signaling of the cytokine receptor upon binding of cytokines not fused to ISVD. This also means that oligomerization of the receptor / receptor subunit upon binding of cytokines present in the chimeric protein of the present invention may be affected (for example, differ from oligomerization of the receptor / receptor subunit upon binding of cytokines not fused to ISVD).

[0159] Therefore, by fusing cytokines to ISVD in the chimeric protein of the present invention, the receptor binding functionality of the cytokines contained in the chimeric protein of the present invention can be adjusted (e.g., improved or worsened, or simply altered) as described in detail below.

[0160] In one embodiment, if the cytokine is an interleukin and is circulatingly displaced, the internal fusion site of the cytokine may be a β-turn of the interleukin β-barrel core motif, as described above.

[0161] In one embodiment, the chimeric protein includes an anti-GFP ISVD, preferably a sequence defined by SEQ ID NO: 1, or an ISVD 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: 1.

[0162] In another embodiment, the chimeric protein includes an ISVD that acts as a half-life extension portion or has half-life extension properties.

[0163] As used herein, the term “half-life” may generally be defined as described in paragraph o) on page 57 of International Publication No. 2008 / 020079, as mentioned therein, and refers to the time required for the serum concentration of a compound or polypeptide to decrease by 50% in vivo due to, for example, the degradation of the sequence or compound by natural mechanisms and / or the clearance or sequestration of the sequence or compound. The in vivo half-life of the protein-based carrier components and / or molecules of the present invention can be determined by any method known in itself, such as pharmacokinetic analysis. Preferred techniques will be apparent to those skilled in the art and may be, for example, as generally described in paragraph o) on page 57 of International Publication No. 2008 / 020079. As also mentioned in paragraph o) on page 57 of International Publication No. 2008 / 020079, half-life is t 1 / 2 -alpha, t 1 / 2 -It can be expressed using parameters such as beta and area under the curve (AUC). In this regard, the term "half-life" as used herein is particularly t 1 / 2 -Beta or terminal half-life (where t 1 / 2-Note that this refers to alpha and / or AUC (or both), which may not be taken into consideration. For example, see standard handbooks, e.g., Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinetic analysis: A Practical Approach (1996). See also “Pharmacokinetics”, M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982). Similarly, the terms “increased half-life” or “extended half-life” are also defined in paragraph o) on page 57 of the International Publication No. 2008 / 020079 pamphlet, in particular t 1 / 2 - Whether or not alpha and / or AUC or both, t 1 / 2 - This refers to an increase in beta.

[0164] The (in vivo) half-life can be extended by increasing the hydrodynamic radius (size) or decreasing the molecular clearance. For example, (in vivo) half-life extending portions, such as binding units that can bind to serum albumin, increase the half-life of the molecule to which they are bound, for example, by binding to serum albumin. Albumin is the most abundant plasma protein, is highly soluble, highly stable, and has an unusually long circulating half-life as a direct result of its size and interaction with the FcRn-mediated recycling pathway (see, for example, Sleep D. et al., “Albumin as a versatile platform for drug half-life extension”, Biochim Biophys Acta, 2013, 1830(12):5526-34).

[0165] For example, International Publication No. 2004 / 041865 describes serum albumin-conjugating ISVD (particularly for HSA) which can be used to increase the half-life of the chimeric protein or polypeptide of the present invention.

[0166] International Publication No. 2006 / 122787, the International Application whose contents are incorporated herein by reference, describes several ISVDs for (human) serum albumin. These ISVDs include an ISVD called Alb-1 (SEQ ID NO: 52 in International Publication No. 2006 / 122787) and its humanized variant, such as Alb-8 (SEQ ID NO: 62 in International Publication No. 2006 / 122787). These, too, can be used to extend the half-lives of therapeutic proteins and polypeptides, as well as other entities or parts of the present invention, such as chimeric proteins or polypeptides.

[0167] International Publication No. 2012 / 175400, whose contents are incorporated herein by reference, describes a further improved version of Alb-1 called Alb-23.

[0168] In one embodiment, the chimeric protein or polypeptide of the present invention comprises a serum albumin binding moiety selected from Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10 (described in WO 2006 / 122787 pamphlet) 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 pamphlet. In one embodiment, the serum albumin binding moiety is selected from albumin binders described in WO 2012 / 175741 pamphlet, WO 2015 / 173325 pamphlet, WO 2017 / 080850 pamphlet, WO 2017 / 085172 pamphlet, WO 2018 / 104444 pamphlet, WO 2018 / 134235 pamphlet and WO 2018 / 134234 pamphlet (the contents of which are incorporated herein by reference). Some serum albumin binders are also shown in Table 3 below.

[0169] In one embodiment, the chimeric protein or polypeptide of the present invention comprises a 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 a 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 a serum albumin binding moiety Alb23002(E1D) (SEQ ID NO: 137) as defined in Table 3 below.

[0170]

Table 4

[0171]

Table 5

[0172]

Table 6

[0173] In one embodiment, the molecule of the present invention includes an HLE moiety as described in item A below: A.HSA is linked to ISVD containing the following: i. CDR1 is an amino acid sequence of SEQ ID NO: 138, or an amino acid sequence that differs from SEQ ID NO: 138 by two or one amino acid; ii. CDR2, which is the amino acid sequence of SEQ ID NO: 139, or an amino acid sequence that differs from SEQ ID NO: 139 by two or one amino acid; and iii. CDR3 is the amino acid sequence of SEQ ID NO: 140, or an amino acid sequence having two or one amino acid differences from SEQ ID NO: 140.

[0174] In one embodiment, ISVD includes CDR1, which is the amino acid sequence of SEQ ID NO: 138; CDR2, which is the amino acid sequence of SEQ ID NO: 139; and CDR3, which is the amino acid sequence of SEQ ID NO: 140.

[0175] Examples of such ISVDs that bind to HSA have one or more or all of the framework regions shown for construct ALB23002 (SEQ ID NO: 55) in Tables 4 and 5 (in addition to the CDR defined in the previous section A). In one embodiment, this is an ISVD that includes or consists of the complete amino acid sequence of construct ALB23002 (SEQ ID NO: 55).

[0176] [Table 7]

[0177] [Table 8]

[0178] Item A' can also be described using the definition in Kabat CDR as follows: Join to A'.HSA and include the following ISVD i. CDR1 that is the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence having two or one amino acid differences from SEQ ID NO: 146; ii. CDR2 that is the amino acid sequence of SEQ ID NO: 148, or an amino acid sequence having two or one amino acid differences from SEQ ID NO: 148; and iii. CDR3 that is the amino acid sequence of SEQ ID NO: 140, or an amino acid sequence having two or one amino acid differences from SEQ ID NO: 140.

[0179] In one embodiment, the ISVD comprises CDR1 that is the amino acid sequence of SEQ ID NO: 146, CDR2 that is the amino acid sequence of SEQ ID NO: 148, and CDR3 that is the amino acid sequence of SEQ ID NO: 140.

[0180] Examples of such ISVDs that bind to HSA have one or more or all of the framework regions shown for construct ALB23002 in Table 5 (in addition to the CDRs defined in previous item A’). In one embodiment, this is an ISVD that comprises or consists of the complete amino acid sequence of construct ALB23002 (SEQ ID NO: 55, see also Table 5).

[0181] In another embodiment, the amino acid sequence of the ISVD that binds to HSA may also have more than 90%, such as more than 95% or more than 99% sequence identity with SEQ ID NO: 55, and the CDRs are as defined in previous item A or A’. In one embodiment, the ISVD that binds to HSA comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0182] When such ISVD binding to HSA has two or one amino acid differences in at least one CDR compared to the corresponding reference CDR sequence (item A or A’ above), the ISVD has at least half the binding affinity for HSA, or at least the same binding affinity, compared to construct ALB23002 (SEQ ID NO: 55), and the binding affinity is measured using the same method such as SPR.

[0183] In one embodiment, if such an ISVD that binds to HSA has a C-terminal position, it exhibits a C-terminal elongation such as a C-terminal alanine, cysteine, or glycine elongation. 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 that binds to HSA has a position other than the C-terminal position (i.e., it is not the 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).

[0184] In one embodiment, the one or more other groups, residues, moieties, or binding units that result in an increased half-life for the molecule are peptides that can bind to the HSA.

[0185] In particular, the "serum-albumin-binding polypeptide or binding domain" has a shorter half-life (preferably T as defined above) compared to the same molecule without the serum-albumin-binding peptide or binding domain. 1 / 2 This could be any suitable serum-albumin-binding peptide that can increase β).

[0186] Specifically, polypeptide sequences suitable for extending the serum half-life are polypeptide sequences that can bind to serum proteins with long serum half-lives, such as serum albumin, transferrin, and IgG, and especially human serum albumin (HSA).

[0187] Polypeptide sequences capable of binding to serum albumin have been previously described, and in particular may be serum albumin-binding peptides described in International Publication No. 2008 / 068280 (particularly International Publication No. 2009 / 127691 and International Publication No. 2011 / 095545), the contents of which are incorporated herein by reference.

[0188] In another embodiment, the chimeric protein of the present invention includes 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 a sequence defined by SEQ ID NO: 55 or SEQ ID NO: 154, or an ISVD that has at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO: 55 or SEQ ID NO: 154.

[0189] In alternative embodiments, the chimeric protein of the present invention comprises an ISVD containing the sequences of CDR1, 2, and 3 as defined by SEQ ID NO: 151 for CDR1, SEQ ID NO: 152 for CDR2, and SEQ ID NO: 154 for CDR3 (disclosed as an HSA binder in International Publication No. 2019 / 016237A1).

[0190] In one embodiment, the chimeric protein includes a cytokine that is IL-2, preferably a sequence defined by SEQ ID NO: 2 (IL-2), or a cytokine that has at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO: 2.

[0191] In another embodiment, the chimeric protein includes a cytokine comprising the sequence 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%, and even more preferably at least 97% identity with SEQ ID NO: 3.

[0192] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 4 (circulatingly substituted IL-2(K35E,C125S) called 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.

[0193] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by Sequence ID No. 172 (circulatingly substituted IL-2(K35E,C125S) called IL-2(K35E,C125S)[L17-L14]), 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 Sequence ID No. 172.

[0194] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes a sequence defined by Sequence ID No. 173 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 173.

[0195] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 174 (circulatingly substituted IL-2(K35E,C125S) called 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.

[0196] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes a sequence defined by Sequence ID No. 175 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 175.

[0197] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by Sequence ID No. 176 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 176.

[0198] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by Sequence ID No. 177 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 177.

[0199] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by Sequence ID No. 178 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 178.

[0200] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by Sequence ID No. 179 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 179.

[0201] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by Sequence ID No. 180 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 180.

[0202] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes a sequence defined by Sequence ID No. 181 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 181.

[0203] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by Sequence ID No. 182 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 182.

[0204] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes a sequence defined by Sequence ID No. 183 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 183.

[0205] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by Sequence ID No. 184 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 184.

[0206] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 185 (IL-2(K35E,C125S) which does not contain the first four amino acids, or a sequence which has at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with SEQ ID NO: 185.

[0207] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by Sequence ID No. 186 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 186.

[0208] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by Sequence ID No. 187 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 187.

[0209] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes a sequence defined by Sequence ID No. 188 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 188.

[0210] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by Sequence ID No. 189 (circulatingly substituted IL-2(K35E,C125S) called 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 Sequence ID No. 189.

[0211] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes a circulatingly substituted IL-2 called IL-2[L132-I129], as defined by SEQ ID NO: 262, 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: 262.

[0212] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes a circulatingly substituted IL-2 called IL-2[F42-M39], as defined by SEQ ID NO: 264, 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: 264.

[0213] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes a circulatingly substituted IL-2 called IL-2[S75-N71], as defined by SEQ ID NO: 268, 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: 268.

[0214] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes a circulatingly substituted IL-2 called IL-2[T102-E100], as defined by SEQ ID NO: 270, 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: 270.

[0215] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes a circulatingly substituted IL-2 called IL-2[F103-S99], as defined by SEQ ID NO: 272, 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: 272.

[0216] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes a circulatingly substituted IL-2 called IL-2[L85-P82], as defined by SEQ ID NO: 274, 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: 274.

[0217] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 190 (IL-2 in TP072), 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: 190.

[0218] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 191 (IL-2 in TP075), 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: 191.

[0219] In another embodiment, the cytokine includes an IFNA2a cytokine, preferably a sequence defined by SEQ ID NO: 56, or a cytokine 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: 56.

[0220] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 58 (circulatingly substituted IFNA2a called IFNA2a[D77-W76]V2), 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: 58.

[0221] In another embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 59 (a circulatingly substituted IFNA2a called IFNA2a[D77-W76]V4), 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: 59.

[0222] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 192 (IFNA2a in TP093), 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: 192.

[0223] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence 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%, and even more preferably at least 97% identity with SEQ ID NO: 193.

[0224] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 194 (IFNA2a in TP098), 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: 194.

[0225] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 195 (IFNA2a in TP109), 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: 195.

[0226] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 197 (IFNA2a in TP089), 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: 197.

[0227] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 199 (IFNA2a in TP090), 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: 199.

[0228] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 201 (IFNA2a in TP091), 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: 201.

[0229] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 203 (IFNA2a in TP092), 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: 203.

[0230] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 205 (IFNA2a in TP095), 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: 205.

[0231] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 207 (IFNA2a in TP096), 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: 207.

[0232] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 209 (IFNA2a in TP097), 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: 209.

[0233] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 211 (IFNA2a in TP099), 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: 211.

[0234] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 213 (IFNA2a in TP100), 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: 213.

[0235] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 215 (IFNA2a in TP101), 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: 215.

[0236] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 217 (IFNA2a in TP102), 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: 217.

[0237] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 220 (IFNA2a in TP104), 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: 220.

[0238] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 222 (IFNA2a in TP105), 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: 222.

[0239] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 225 (IFNA2a in TP107), 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: 225.

[0240] In one embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes the sequence defined by SEQ ID NO: 227 (IFNA2a in TP108), 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: 227.

[0241] In one embodiment, the chimeric protein includes a cytokine that is IL-18, preferably a sequence defined by SEQ ID NO: 64, or a cytokine 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: 64.

[0242] In another embodiment, the cytokine includes IL-18, preferably a sequence defined by SEQ ID NO: 66, 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: 66.

[0243] In another embodiment, the cytokine includes IL-18, preferably a sequence defined by SEQ ID NO: 68, 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: 68.

[0244] In another embodiment, the cytokine includes IL-18, preferably a sequence defined by SEQ ID NO: 70, 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: 70.

[0245] In another embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes a circulatingly substituted IL-18 called IL18[K79-N78], as defined by SEQ ID NO: 244, 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: 244.

[0246] In another embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes a circulatingly substituted IL-18 called IL18[Q56-S55], as defined by SEQ ID NO: 245, 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: 245.

[0247] In another embodiment, the circulatingly substituted cytokine contained in the chimeric protein of the present invention includes a circulatingly substituted IL-18 called IL18[P57-Q56], as defined by SEQ ID NO: 246, 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: 246.

[0248] In one embodiment, the chimeric protein of the present invention is a sequence defined by SEQ ID NOs: 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 SEQ ID NOs: 7-25, 36-54, 60-63, 196, 198, 2 A protein is selected from a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with 00, 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 a protein comprising such a sequence.

[0249] In another embodiment, the chimeric protein of the present invention comprises ISVD fused to a cytokine (preferably circulatingly substituted), wherein the cytokine is a sequence defined by SEQ ID NOs: 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 a sequence The sequence contains or consists of sequences having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identity with numbers 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.

[0250] The polypeptide of the present invention The present invention further provides polypeptides comprising the chimeric protein of the present invention. In addition to the chimeric protein, the polypeptide of the present invention may comprise further groups, residues, parts, or binding units.

[0251] For example, such further groups, residues, moieties, or binding units may be one or more further immunoglobulins to form a (fusion) protein or (fusion) polypeptide (the polypeptide of the present invention). In a preferred but non-limiting embodiment, one or more other groups, residues, moieties, or binding units may be ISVDs. More preferably, one or more other groups, residues, moieties, or binding units may be 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 dAb, V HH Humanization V HH Camelization V H , or Nanobody(registered trademark)V HH Selected from the group consisting of the following. Alternatively, such groups, residues, parts, or binding units may be chemical groups, residues, or parts that are biologically and / or pharmacologically active or not by themselves, for example. For example, but not limited to, such groups may be linked to one or more domains in the polypeptide of the present invention to provide “derivatives” of the polypeptide of the present invention, as further described herein. The polypeptide of the present invention may also include additional groups having specific functional groups such as labels, toxins, one or more linkers, and binding sequences. These additional functional groups include both amino acid-based and non-amino acid-based groups.

[0252] Therefore, in one embodiment, the polypeptide of the present invention may further comprise one or more ISVDs (in addition to the chimeric protein of the present invention). Preferably, the ISVD may be an HLE ISVD, a targeted ISVD, or a therapeutic ISVD. Thus, one or more ISVDs that may further comprise the polypeptide of the present invention (in addition to those contained in the chimeric protein) may form a "polyvalent" or "multispecific" polypeptide or construct.

[0253] Polypeptides containing two or more ISVDs (such as an ISVD contained in a chimeric protein and one or more further ISVDs) are referred to herein as “polyvalent polypeptides” or “polyvalent constructs,” and these may offer certain advantages compared to the corresponding monovalent polypeptides. Generally, proteins or polypeptides containing a single ISVD (such as the chimeric protein of the present invention) are referred to herein as “monovalent” proteins or polypeptides or “monovalent constructs.”

[0254] As described herein, the polyvalent polypeptides of the present invention may be, for example, multispecific (such as bispecific or triplicate) or multiparatopic (such as biparatopic) constructs (or both multiparatopic and multispecific), for example, constructs comprising at least two binding domains or binding units, each directed to a different epitope on the same subunit; constructs comprising at least two binding domains or binding units, each having a different biological function (e.g., one binding domain capable of blocking or inhibiting receptor-ligand interactions, and one binding domain that does not block or inhibit receptor-ligand interactions); or constructs comprising at least two binding domains or binding units, each directed to a different target.

[0255] For a general description of polyvalent and multispecific polypeptides containing one or more ISVDs and their preparations, see Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology 74(2001), 277-302; and, for example, International Publication Nos. 96 / 34103, 99 / 23221, 04 / 041862, 2006 / 122786, 2008 / 020079, 2008 / 142164, or 2009 / 068627.

[0256] It should be understood that the terms “polypeptide construct” and “polypeptide” can be used interchangeably herein (unless otherwise explicitly indicated in the context).

[0257] The polypeptides of the present invention can generally be prepared by a method comprising at least one step of suitably linking the chimeric protein of the present invention to one or more further groups, residues, parts or binding units, either directly or via one or more suitable linkers, as described herein.

[0258] The polypeptides of the present invention can also be prepared by a method generally comprising at least the steps of 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 will be obvious to those skilled in the art and can be carried out in methods known in themselves, for example, based on the methods and techniques further described herein.

[0259] It should be understood that the order of the chimeric protein in the polypeptide of the present invention and, if present, the order of further groups, residues, moieties or binding units, for example, the order of the first domain (the chimeric protein of the present invention), the second binding domain (e.g., the HSA-binding ISVD), and the third binding domain (e.g., the domain that binds to a therapeutically relevant target) in the polypeptide (i.e., the orientation or arrangement of the chimeric protein and, if present, the further groups, residues, moieties or binding units), can be selected according to the needs of those skilled in the art, and according to relative affinity which may depend on the position of the chimeric protein and, if present, the further groups, residues, moieties or binding units in the polypeptide. Whether the polypeptide contains one or more linkers for optionally interconnecting the chimeric protein and further groups, residues, moieties or binding units is a matter of design choice. However, with or without linkers, some arrangements may result in preferred binding properties compared to others. All different possible orientations are encompassed in the present invention.

[0260] For example, the polypeptide sequence of the present invention may comprise one or more ISVDs linked directly or by a linker, as defined herein, followed by a chimeric protein of the present invention, as defined herein, linked directly to one or more ISVDs or by a linker. In this embodiment, one or more ISVDs are located in the N-terminal region of the polypeptide, while the chimeric protein is located in the C-terminal region of the polypeptide.

[0261] For example, the polypeptide of the present invention may comprise the chimeric protein of the present invention and one or more ISVDs that are linked together directly or by a linker, as defined herein, directly linked to the chimeric protein of the present invention, or linked by a linker. In this embodiment, one or more ISVDs are located in the C-terminal region of the polypeptide, while the chimeric protein is located in the N-terminal region of the polypeptide.

[0262] For example, the polypeptide sequence of the present invention may include one or more ISVDs that are directly linked together or linked together by a linker, directly linked to or linked together by a linker, and subsequently one or more further ISVDs that are directly linked together or linked together by a linker. Thus, in this embodiment, the chimeric protein of the present invention is flanked by one or more ISVDs at the N-terminal and C-terminal regions of the polypeptide.

[0263] The use of linkers to link two or more (poly)peptides is well known in the art. A frequently used class of single-peptide linkers is known as "Gly-Ser" or "GS" linkers. These are linkers that essentially consist of a glycine (G) residue and a serine (S) residue, and are usually the GGGGS (SEQ ID NO: 155) motif (e.g., formula (Gly-Gly-Gly-Gly-Ser)). nThe formula includes one or more repeating peptide motifs, such as (where n can be 1, 2, 3, 4, 5, 6, 7 or more). Some commonly used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 156), the 15GS linker (n=3), and the 35GS linker (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 linker is selected from the group consisting of GSG, GSGG, GGSG, 3A, 3GS, 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, and 35GS linkers (see Table A-1).

[0264] [Table 9]

[0265] For example, the polypeptide of the present invention may additionally contain groups, residues, parts, or binding units that provide the chimeric protein / polypeptide of the present invention having an increased (in vivo) half-life compared to the corresponding chimeric protein / polypeptide that does not contain the one or more other groups, residues, parts, or binding units ("(in vivo) half-life extension part" or "half-life extension (HLE) part"). Therefore, in this embodiment, if the ISVD contained in the chimeric molecule of the present invention is the HLE part, the (polyvalent and multispecific) polypeptide of the present invention contains at least two HLE parts: the ISVD contained in the chimeric molecule and at least one further HLE part. The HLE parts contained in the polypeptide of the present invention may be the same or different.

[0266] The term “half-life” as used herein has been defined in relation to the chimeric protein of the present invention and is similarly applicable to this embodiment. In one embodiment, further binding units included in the polypeptide of the present invention (in addition to the chimeric protein) are ISVDs, such as HSA-bound ISVDs. HSA-bound ISVDs have already been defined in relation to the chimeric protein of the present invention and are similarly applicable to this embodiment.

[0267] Further HLE moieties that may be included in the polypeptide of the present invention are HLE moieties such as polyethylene glycol or ELNN polypeptide, which increase the size of the molecule to which they are bound, thus bypassing renal clearance and thus increasing the half-life of that molecule.

[0268] The types of HLE groups, residues, moieties, or binding units are not generally limited and may be selected from the group consisting of, for example, 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 HSA-binding ISVD, Fc moieties, and small proteins or peptides that can bind to serum proteins.

[0269] The polypeptides of the present invention may additionally comprise one or more targeting moieties (in addition to the chimeric protein). “Targeting moiety” as defined herein is any group, residue, part, or binding unit that can be directed to a target by its binding. It is an amino acid sequence (ISVD, antibody, antigen-binding domain or fragment, e.g., V) that “(specifically) binds,” “(specifically) can bind,” “has affinity,” and / or “has specificity” to a particular antigenic determinant, epitope, antigen or protein, or a particular non-protein molecule, such as nucleic acids (DNA or RNA, etc.) or glycans (or at least a part thereof, fragment or epitope). HH Domain or V H / V LA domain, or more generally, an antigen-binding protein or polypeptide or a fragment thereof, is said to be "directed to" or "directed to" the antigenic determinant, epitope, antigen, protein, or non-protein molecule. The specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined by any suitable method known on its own, including, for example, scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIAs), enzyme immunoassays (EIAs), and sandwich competitive assays, as well as various variants thereof known on their own in the art, and other techniques referred to herein.

[0270] Furthermore, the polypeptide of the present invention may additionally contain one or more therapeutic moieties. “Therapeutic moiety” as defined herein is any group, residue, part, or binding unit capable of exerting therapeutic activity in animal and / or human bodies. Therapeutic moieties may also be in the form of precursors, which are subsequently activated to exert their therapeutic activity. A non-limiting example of a therapeutic moiety that may be present in the polypeptide of the present invention is a programmed cell death ligand 1 (PD-L1) binding molecule.

[0271] The nucleic acid of the present invention The present invention further provides nucleic acid molecules encoding the chimeric protein and / or polypeptide of the present invention.

[0272] Nucleic acids can be used to transform / transfect host cells or host organisms for, for example, polypeptide expression and / or production. Suitable (non-human) hosts or host cells for production purposes will be obvious to those skilled in the art and may be, for example, any suitable fungal, prokaryotic, or eukaryotic cell or cell line, or any suitable fungal, prokaryotic, or eukaryotic organism. Hosts or host cells containing nucleic acids encoding the chimeric proteins and / or polypeptides of the present invention are also included in the present invention.

[0273] Nucleic acids can be, for example, DNA, RNA, or hybrids thereof, and may also include (e.g., chemically) modified nucleotides such as PNA. They can be single-stranded or double-stranded. In one embodiment, this is in the form of double-stranded DNA. For example, the nucleotide sequence of the present invention may be genomic DNA or cDNA.

[0274] The nucleic acids of the present invention can be prepared or obtained by methods known in themselves, and / or isolated from suitable natural sources. Nucleotide sequences encoding naturally occurring (poly)peptides can be subjected to site-directed mutagenesis, for example, to provide nucleic acid molecules encoding polypeptides with sequence mutations. Also, as will be apparent to those skilled in the art, several nucleotide sequences, for example, at least one nucleotide sequence encoding a targeting moiety and nucleic acids encoding, for example, one or more linkers, can be linked and integrated in a suitable manner to prepare nucleic acids.

[0275] Techniques for generating nucleic acids are obvious to those skilled in the art and may include, for example, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring sequences and / or synthetic sequences (or two or more portions thereof); introducing mutations resulting in the expression of cleavage-type expression products; introducing one or more restriction sites (e.g., to create cassettes and / or regions that can be readily digested and / or ligated using a suitable restriction enzyme); and introducing mutations by PCR reactions using one or more "mismatch" primers.

[0276] In another embodiment, the chimeric gene is described together with at least a promoter, the nucleic acid molecule encoding the chimeric protein, and a 3' terminal region containing a transcription termination signal. In another embodiment, the present invention relates to an expression cassette comprising a chimeric protein encoding the present invention, or a chimeric gene encoding a nucleic acid molecule or a chimeric protein. The expression cassette is a particular embodiment applied in a common format as an immunolibrary containing a large set of ISVDs for selecting the most suitable conjugate for the target (if ISVD binding to the target is desired).

[0277] The vector of the present invention The present invention further provides vectors comprising nucleic acid molecules of the present invention. When used herein, vectors are suitable vehicles for delivering genetic material to cells. Vectors include naked nucleic acids such as plasmids or mRNA, or nucleic acids embedded in larger structures such as liposomes or viral vectors.

[0278] In some embodiments, the vector comprises at least one nucleic acid optionally linked to one or more regulatory elements, such as 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 polypeptide or construct encoded under suitable conditions when the vector (e.g., human) is introduced into a cell. DNA-based vectors include the presence of elements for transcription (e.g., promoters and polyA signals) and translation (e.g., Kozak sequences).

[0279] 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 Escherichia coli (E. coli) or S. cerevisiae (S. cerevisiae) allows for the production and purification of chimeric proteins and / or polypeptides.

[0280] In one embodiment, within a vector, the at least one nucleic acid and the regulatory element are “operably linked” to each other, which generally means they are functionally related to each other. For example, a promoter is considered “operably linked” to a coding sequence if the promoter can initiate or otherwise control / regulate the transcription and / or expression of the coding sequence (where the coding sequence should be understood as being “under the control” of the promoter). Generally, when two nucleotide sequences are operably linked, they are oriented in the same direction and usually within the same reading frame. They are usually contiguous in nature, but this is also not always necessary.

[0281] In one embodiment, each regulatory element of the vector is capable of providing its intended biological function in the intended host cell or host organism.

[0282] For example, a promoter, enhancer, or terminator must be "operable" in the intended host cell or host organism, meaning, for example, that the promoter must be able to initiate or otherwise control / regulate the transcription and / or expression of the nucleotide sequence, such as a coding sequence, to which it is operably linked.

[0283] The present invention, providing the aforementioned vector, further encompasses options for high-throughput cloning in generic fusion vectors. The generic vector is preferably specifically suited for surface display in yeast, phages, bacteria, or viruses. Furthermore, the vector finds use in the selection and screening of immunological libraries containing such generic vectors or expression cassettes having a large set of different ISVDs, where the same N-terminus of a conserved ISVD and cytokines are fused with the remaining ISVD sequences provided by the library. Thus, differential sequences within the library constructed for screening novel chimeric proteins against a specific target are provided by the ISVD sequences, more specifically by differences in the CDR region of the ISVD library.

[0284] Host cells of the present invention Alternative embodiments relate to host cells comprising the chimeric protein and / or polypeptide of the present invention, or nucleic acid molecules, expression cassettes, or vectors encoding the chimeric protein of the present invention.

[0285] Suitable host cells or host organisms will be apparent to those skilled in the art, and include, for example, any suitable fungal, prokaryotic, or eukaryotic cell or cell line, or any suitable fungal, prokaryotic, or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, S. cerevisiae, Escherichia coli, or Komagataella phaffii (Pichia pastoris, see Bernauer L., et al. "Komagataella phaffii as emerging model organism in fundamental research", Front. Microbiol., 2021, 11:1-16). In one embodiment, the host is Komagataella phaffii (Pichia pastoris). In another embodiment, the host is Escherichia coli. In preferred embodiments, the host is S. cerevisiae. Naturally, cell-free systems may be used to produce the protein-based carrier components and / or molecules of the present invention, as reviewed, for example, in Gregorio NE, Levine MZ, Oza JP, “A user's guide to cell-free protein synthesis”, Methods Protoc., 2019, 2(1):24.

[0286] Another embodiment discloses the use of the host cells or isolated membrane preparations thereof, or proteins isolated therefrom, for ligand screening, drug screening, protein capture and purification, or biophysical studies.

[0287] Method for producing chimeric proteins and / or polypeptides according to the present invention Another embodiment of the present invention is a method for producing the chimeric protein and / or polypeptide of the present invention, wherein the method is (i) the step of selecting ISVD and cytokines as described herein; (ii) A step of designing a gene construct that encodes a protein sequence of ISVD interrupted by at least one internal fusion site, for example, two internal fusion sites, by a sequence of cytokines (preferably circulatingly substituted), wherein the internal fusion site is located in a loop or turn between two secondary structural elements, preferably in one of the following β-turns in ISVD (according to the IMGT classification): i. During the first β-turn connecting β-chains A and B of ISVD; or ii. In the β-turns connecting the β-chain C and C' of ISVD; or iii. During the β-turns connecting the β-chain C'' and D of ISVD; or iv. During the β-turn connecting β-chains D and E of ISVD; or The process of designing the β-turns that connect β-chains E and F of v.ISVD; (iii) A method comprising the step of introducing the gene construct into an expression system in order to obtain the chimeric protein and / or polypeptide of the present invention, as described above herein.

[0288] Optionally, the method of the present invention comprises (iv) a step of recovering the obtained chimeric protein and / or polypeptide and optionally purifying it.

[0289] The above method may further include, after step (i), a step of selecting one or more additional groups, residues, moieties or binding units that may be included in the polypeptide in addition to the chimeric protein of the present invention. In this case, step (ii) should further include designing a gene construct that includes one or more additional groups, residues, moieties or binding units in addition to the chimeric protein.

[0290] To produce / obtain the chimeric protein and / or polypeptide of the present invention, host cells or host organisms or cell-free systems can generally be maintained and / or cultured under conditions that allow for optimal expression / production of the (desired) chimeric protein and / or polypeptide of the present invention. Preferred conditions will be apparent to those skilled in the art and will typically depend on the host cells / host organisms or cell-free systems used, as well as the regulatory elements that control the expression of the chimeric protein and / or polypeptide of the present invention.

[0291] Suitable host cells or host organisms for the production purpose will be apparent to those skilled in the art and may be, for example, any suitable fungal, prokaryotic, or eukaryotic cells or cell lines, or any suitable fungal, prokaryotic, or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, S. cerevisiae, Escherichia coli, or Komagataella phaffii (Pichia pastoris). In one embodiment, the host is Komagataella phaffii (Pichia pastoris). In another embodiment, the host is Escherichia coli. In a preferred embodiment, the host is S. cerevisiae.

[0292] In one embodiment, the method of the present invention further comprises (v) screening for chimeric proteins that bind to at least one cytokine receptor or receptor subunit having increased or decreased affinity compared to binding of wild-type cytokines. In another embodiment, the method of the present invention further comprises (v) screening for chimeric proteins in which the cytokines contained therein exhibit modified cytokine signaling compared to cytokines not fused to ISVD, or screening for chimeric proteins in which the cytokines contained therein affect the oligomerization of the receptor or receptor subunit upon binding of the cytokines contained therein to at least one of its receptor or receptor subunits.

[0293] Method and Use of Chimeric Proteins of the Present Invention The present invention further provides a method for regulating cytokine activity by fusing cytokines to ISVDs (directly or by linkers, as described above) to form fusion proteins. In preferred embodiments, cytokines (or cyclically substituted variants of cytokines) are inserted into ISVDs as described herein (for example, as “megabody” fusions, as described herein). The inventors have surprisingly found that fusing cytokines to ISVDs (directly or by linkers, as described herein) as internal fusions results in a modified or altered cytokine that binds to its receptor or receptor subunit (or alters the downstream outcomes of the cytokine’s binding to at least one of its receptor or receptor subunit, resulting in, for example, altered cytokine signaling and / or oligomerization of an altered or affected receptor or receptor subunit compared to cytokines not fused to ISVDs). Thus, the inventors have surprisingly found that fusing cytokines to ISVDs (directly or by linkers, as described herein) can alter the receptor-binding functionality of cytokines compared to cytokines not fused to ISVDs. For example, the binding affinity of a cytokine to at least one of its receptors or receptor subunits can be regulated (increased or decreased) by fusing the cytokine to ISVD in a fusion protein. In addition, downstream signaling generated by the interaction between the cytokine and at least one of its receptors or receptor subunits can also be regulated by fusing the cytokine to ISVD. Furthermore, the effectiveness of cytokine / receptor interactions can be regulated (increased or decreased) in this way, as can the potency of cytokines that bind to ISVD.In addition, oligomerization of receptors or receptor subunits can be affected (modified and regulated) when cytokines fused to ISVDs bind, compared to oligomerization of receptors or receptor subunits when cytokines not fused to ISVDs bind. Accordingly, the present invention provides a method for modulating the efficacy of cytokine receptors, comprising the step of fusion of a cytokine to an ISVD (directly or by a linker 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 (modifying, influencing) oligomerization of receptors or receptor subunits when cytokines fused to ISVDs bind.

[0294] In preferred embodiments, a method for regulating cytokine activity according to the present invention includes the step of fusing the cytokine to an ISVD to form a chimeric protein, as described herein. By fusing the cytokine to an ISVD to form a chimeric protein, as shown in the examples, the activity of the cytokine (or, as described herein, the downstream consequence of the binding of the cytokine to at least one of its receptors or receptor subunits / oligomerization of the receptor or receptor subunit) can be regulated. Furthermore, in addition to wild-type cytokines, the chimeric proteins of the present invention may include circulatingly substituted cytokine variants having different properties, such as circulatingly substituted cytokine mutants (e.g., using circulatingly substituted IL-2(K35E), as exemplified herein), which can also generate regulation of cytokine receptor efficacy.

[0295] As described above, the method of the present invention may further include a step of screening for fusion or chimeric proteins in which cytokines contained within the ISVD exhibit different receptor binding functionalities compared to the receptor binding functionalities of cytokines not fused to the ISVD.

[0296] For example, the screening step may include screening for fusion or chimeric proteins in which the cytokines contained therein 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 to the ISVD.

[0297] For example, the screening process may include screening for fusion or chimeric proteins in which the cytokines contained within exhibit different (increased or decreased) efficacy of cytokine / receptor interactions compared to the efficacy of cytokines not fused to ISVD.

[0298] For example, the screening step may include screening for fusion or chimeric proteins that produce different (increased or decreased) downstream signaling when the cytokines contained therein interact with one of the receptors or receptor subunits, compared to downstream signaling produced by the interaction of cytokines that are not fused to ISVD with the same receptor or receptor subunit.

[0299] For example, the screening process may include screening for fusion or chimeric proteins in which the cytokines contained therein produce oligomerization of a different receptor or receptor subunit when bound to a cytokine fused to an ISVD, compared to oligomerization of the receptor or receptor subunit when bound to a cytokine not fused to an ISVD.

[0300] While we do not wish to be bound by theory, the fusion of cytokines to ISVDs (optionally resulting in the chimeric proteins described herein) appears to alter the binding between the chimeric protein and its receptor (e.g., by steric impairment and / or conformational changes to the cytokine ligand induced by the fusion of the cytokine to the ISVD). As a result, different downstream effects (or signaling) may be obtained by the binding of a cytokine to at least one of its receptors or receptor subunits compared to the binding of a wild-type cytokine (not fused to an ISVD, e.g., not being part of a fused or chimeric protein) to at least one of its receptors or receptor subunits.

[0301] Accordingly, the present invention provides a method for modulating the functionality of a cytokine receptor in interaction with a cytokine fused to an ISVD (fusion protein) as described herein (directly or by a linker as described herein), or in interaction with a circulatingly substituted cytokine contained in the chimeric protein of the present invention. The method comprises contacting a cytokine (fused to an ISVD as described herein) with at least one of the cytokine's receptor or receptor subunit to modulate the functionality of the cytokine receptor (e.g., modulating downstream signaling and / or oligomerization of the receptor / receptor subunit generated by the binding of the cytokine to at least one of its receptor or receptor subunit).

[0302] The present invention relates to a method for regulating cytokine signaling, - A step of providing the chimeric protein of the present invention; - A step of screening chimeric proteins or polypeptides, wherein the cytokines contained therein exhibit modified cytokine signaling compared to cytokines not fused to ISVD, This further includes methods that include [specific methods].

[0303] Accordingly, the present invention provides the use of the ISVD-fused cytokine, chimeric protein, or polypeptide of the present invention for regulating 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 regulating the binding affinity of a cytokine to at least one of its receptors or receptor subunits. In addition, the ISVD-fused cytokine, chimeric protein, or polypeptide of the present invention can be used to regulate downstream signaling generated by the interaction of a cytokine with at least one of its 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 regulating the effectiveness of cytokine / receptor interactions and for regulating the potency of cytokines present in chimeric proteins. Accordingly, the present invention provides the use of the ISVD-fused cytokine, chimeric protein, or polypeptide of the present invention for regulating the effectiveness and / or functionality of cytokine receptors in interaction with the chimeric protein of the present invention.

[0304] Accordingly, the present invention also provides the use of cytokines, chimeric proteins, or polypeptides fused to the ISVD of the present invention for modifying the binding affinity of the cytokine to its receptor, and / or altering or modifying cytokine signaling, and / or altering or modifying the oligomerization of the receptor when the cytokine binds to at least one of its receptor or receptor subunits.

[0305] The present invention further provides chimeric proteins or polypeptides for use in pharmaceuticals. For example, the chimeric proteins or polypeptides of the present invention may be used in the treatment of cancer and / or inflammatory diseases. Therefore, the present invention provides chimeric proteins or polypeptides for use in the treatment of cancer and / or inflammatory diseases. Cancer can be a solid tumor and / or a humoral tumor. [Examples]

[0306] Overview The inventors have designed antigen-binding chimeric proteins (hereinafter also referred to as megabody® proteins, megabody® protein constructs, or megabody® constructs) constructed from scaffold proteins, particularly antigen-binding domains grafted onto cytokines via two short polypeptide links connecting the antigen-binding domain to the cytokine. Depending on the properties of the chimeric proteins, these antigen-binding chimeric proteins may serve different purposes.

[0307] As an example, one of the antigen-binding chimeras presented herein is called the IL-2 megabody protein and is constructed from ISVD grafted onto interleukin-2 (IL-2). The topology of the IL-2 molecule was investigated, and different sites were selected for grafting ISVD.

[0308] Example 1: Design and generation of a 29kDa antigen-binding chimeric protein constructed from a cyclically substituted variant of IL-2 inserted during the first β-turn linking β-chains A and B of anti-GFP ISVD. As a proof-of-concept for obtaining the IL-2 megabody protein (Figure 2), the megabody protein was constructed by grafting ISVD onto a circulatingly substituted IL-2 variant protein via two peptide bonds connecting ISVD to a scaffold as shown in Figure 1.

[0309] By examining the topology of IL-2 (PDB:2B5I), we identified several sites where ISVDs could be grafted, leading to a selection of 22 different sites (Figure 3).

[0310] A cyclically substituted variant of IL-2 is required to enable the design of IL-2 megabody molecules. In 2020, a fusion protein with a cyclically substituted version of IL-2 was published by 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), demonstrating that this version of IL-2 folds well. Wang et Mark ("Site-specific mutagenesis of the human interleukin-2 gene: structure-function analysis of the cysteine ​​residues", Science, 1984, 224(4656):1431-3) demonstrated that a single point mutation in IL-2 creates a more stable, over-secreted IL-2 variant that remains biologically active in vitro and in vivo. Based on these findings, we created 26 different versions of the IL-2(K35E,C125S)_ISVD207 megabody protein (Figure 4).

[0311] As schematically illustrated by the non-limiting example in Figure 2, the 29 kDa megabody proteins described herein are chimeric polypeptides ligated from a portion of an immunoglobulin monovariate domain and a portion of a scaffold protein ligated according to Figure 1. Here, ISVD is anti-GFP ISVD as shown in SEQ ID NO: 1. The scaffold protein is a variant of IL-2 (SEQ ID NO: 3). All parts were linked together by 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 120), C-terminal portion of IL-2(K35E,C125S) (amino acid positions X2 to 133 of SEQ ID NO: 3), peptide linker to connect the C-terminus of IL-2(K35E,C125S) to the N-terminus (SEQ ID NO: 6) to generate a cyclically substituted variant of the scaffold protein, N-terminal portion of IL-2(K35E,C125S) starting from residue 4 to amino acid position X1 (SEQ ID NO: 3), short peptide linker (SEQ ID NO: 5 or 121), followed by β-chains B-G of anti-GFP ISVD (residues 16-126 of SEQ ID NO: 1) (wherein X1 and X2 are positions on the cytokine sequence selected to create insertion sites into the scaffold). In some cases, an additional glycine (G) was inserted between a short peptide linker (SEQ ID NO: 5) and a cyclically substituted IL-2(K35E,C125S) to create a 4-amino acid linker (SEQ ID NO: 120 or 121) between ISVD and IL-2(K35E,C125S) as shown in Figure 4 (SEQ ID NOs: 7-23). ​​Alpha-fold models of some (non-restrictive) examples of the IL-2(K35E,C125S)_ISVD207 megabody protein are shown in Figures 5-8.

[0312] According to Figure 1, two additional constructs were created, where IL-2(K35E,C125S) was inserted during the first β-turn linking β-chains A and B of anti-GFP ISVD: IL-2(K35E,C125S)_ISVD207 megabody protein (SA17521) (SEQ ID NO: 24), where all parts were linked together by 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: 120), IL-2(K35E,C125S) from amino acid 4 to amino acid 133, short peptide linker (SEQ ID NO: 5), and anti-GFP The β-chains B-G of ISVD (residues 16-126 of SEQ ID NO: 1), and IL-2(K35E,C125S)_ISVD207 megabody protein (SA17653) (SEQ ID NO: 25), where all parts are linked together by 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), a short peptide linker (SEQ ID NO: 5), IL-2(K35E,C125S) starting from amino acid 5 to amino acid 133, a short peptide linker (SEQ ID NO: 5), and β-chains B-G of anti-GFP ISVD (residues 16-126 of SEQ ID NO: 1).

[0313] To demonstrate that IL-2 megabody proteins can be expressed as well-folded, functional proteins, we designed several IL-2(K35E,C125S)_ISVD207 megabody proteins and presented them on the surface of yeast (Boder, ET, and Wittrup, KD, “Yeast surface display for screening combinatorial polypeptide libraries”, Nat Biotechnol, 1997, 15:553-557). We then examined the specific binding of the alloantigen (GFP) to yeast cells presenting these megabody proteins by flow cytometry. To present the IL-2(K35E,C125S)_ISVD207 megabody protein on yeast, we constructed an open reading frame encoding the IL-2(K35E,C125S)_ISVD207 megabody protein fused to numerous accessory peptides and proteins from the amino terminus to the carboxyl terminus using standard methods: appS4 reader sequence for inducing extracellular secretion in yeast (SEQ ID NO: 31) (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.).,2009,103:1192-1201), the IL-2(K35E,C125S)_ISVD207 megabody protein consists 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 120), the C-terminal portion of IL-2(K35E,C125S) (SEQ ID NO: 3, amino acid position X2 to amino acid position 133), a peptide linker to connect the C-terminus of IL-2(K35E,C125S) to the N-terminal portion (SEQ ID NO: 6) to generate a cyclically substituted variant of the scaffold protein, the N-terminal portion of IL-2(K35E,C125S) (starting from residue 4 of SEQ ID NO: 3 to position X1), a short peptide linker (SEQ ID NO: 5 or 121), and then anti-GFP The ISVD consists of β-chain B-G (residues 16-126 of SEQ ID NO: 1), a mobile peptide linker, Aga2p, the adhesion subunit of the yeast agglutinin protein (Aga2p binds to the yeast cell wall via a disulfide bond to the Aga1P protein), an acyl carrier protein for orthogonal fluorescence staining of the presented fusion protein (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).

[0314] Using standard cloning techniques, all different open reading frames of the IL-2(K35E,C125S)_ISVD207 megabody protein construct were cloned into the pCTCON2 vector each time under transcriptional control of the galactose-inducible GAL1 / 10 promoter (Chao G., et al., “Isolating and engineering human antibody using yeast surface display”, Nat Protoc., 2006, 1:755-768).

[0315] Each construct was introduced into the yeast strain EBY100 (S. cerevisiae), and EBY100 clones of each construct carrying the corresponding plasmid were grown. The growth conditions were changed from a glucose-rich medium to a galactose-rich medium and induced overnight to stimulate expression and secretion of the IL-2(K35E,C125S)_ISVD207-Aga2p-ACP fusion protein. For orthogonal staining of ACP, cells were incubated for 1 hour in the presence of a fluorescently labeled CoA analog (coA-647, 2 μM) and a catalytic amount of SFP synthase (1 μM).

[0316] To analyze the functionality of differently presented IL-2(K35E,C125S)_ISVD207 megabody proteins, the inventors examined their binding to a congener antigen (GFP) by flow cytometry. Orthogonally stained yeast cells were incubated for 1 hour in the presence of 100 nM GFP (Scholz, O. et al., “Quantitative analysis of gene expression with an improved green fluorescent protein”, European journal of biochemistry / FEBS, 2000, 267:1565-1570). After washing these cells, the inventors observed detectable levels of GFP bound to differently presented IL-2(K35E,C125S)_ISVD207 megabody proteins that could linearly correlate with the expression levels of IL-2(K35E,C125S)_ISVD207 megabody proteins on the surface of the yeast. In fact, two-dimensional flow cytometry analysis confirmed that GFP (high GFP fluorescence level) binds only to yeast cells with significant megabody protein presentation levels (high CoA647 fluorescence level) (data not shown). GFP does not bind to EBY100 yeast cells stained in the same way but not expressing megabody proteins, nor to EBY100 yeast cells expressing only circulating IL-2 (K35E, C125S) [S75-Q74] (SEQ ID NO: 4). As a positive control, cYgjk_ISVD207 megabody protein (SEQ ID NO: 34), fused to numerous accessory peptides and proteins, was expressed and presented on the surface of yeast cells. This megabody protein is a chimeric polypeptide that forms a 100 kDa megabody protein that has been shown to bind to GFP by linking a portion of anti-GFP ISVD with a portion of YgjK, an 86 kDa periplasmic protein from Escherichia coli (PDB 3W7S), as shown in Figure 1 (Figure 9).

[0317] From these experiments, we concluded that 16 different versions of the Mb_IL-2(K35E,C125S)_ISVD207 megabody protein can be fully folded on the surface of yeast and expressed as a functional antigen-binding (GFP-binding) chimeric protein (Figure 9).

[0318] Example 2: Binding of a specific IL-2 monoclonal antibody to the IL-2_ISVD207 megabody protein. To confirm the proper folding of IL-2(K35E,C125S) within the IL-2(K35E,C125S)_ISVD207 megabody protein presented on the surface of yeast cells, yeast cells expressing these IL-2(K35E,C125S)_ISVD207 megabody proteins were incubated for 1 hour at a final concentration of 2 μg / ml in the presence of the monoclonal antibody mAb5111-human Fc. After three washes, the cells were incubated for 1 hour in the presence of 2 μg / ml anti-human IgG Fc (goat anti-human IgG Fcy fragment-specific phycoerythrin conjugate AffiniPure F(ab)2 fragment, Jackson Immuno Research), washed three times, and analyzed using flow cytometry. The inventors observed detectable fluorescence bound to yeast cells presenting the specific IL-2(K35E,C125S)_ISVD207 megabody protein and confirmed that IL-2(K35E,C125S) was well-folded in a particular IL-2(K35E,C125S) megabody protein construct (Figure 9). The epitope of mAb5111 on IL-2 is known (PDB 5UTZ), and due to the fact that in some constructs ISVD207 is inserted within or near the epitope, the epitope of mAb5111-human Fc is not present and / or inaccessible in the megabody protein. In fact, the mAb5111-human Fc antibody could not bind to any of the (AA16~31;AA70~AA86)IL-2(K35E, C125S)_ISVD207 megabody proteins in which ISVD207 was inserted near the epitope. In parallel, the expression of the same construct was tracked and confirmed by incubating clones at a final concentration of 4 μg / ml for 1 hour in the presence of mouse anti-Myc monoclonal antibody (Roche / #11 667 149 001), followed by three washes, and then incubating with anti-human IgG Fc antibody (goat anti-human IgG Fcy fragment-specific phycoerythrin conjugate AffiniPure F(ab)2 fragment, Jackson). Cells were analyzed using flow cytometry (Figure 9).

[0319] Binding of the monoclonal antibody NARA1 could only be confirmed on wild-type IL-2 because the binding epitope was disrupted in IL-2(K35E,C125S) due to the K35E mutation. Since all IL-2(K35E,C125S)_ISVD207 megabody proteins carry this mutation, no binding was observed after flow cytometry (Figure 9).

[0320] Example 3: Binding of CD25 or CD122 / CD132 to the IL-2(K35E,C125S)_ISVD207 megabody protein. Since the binding of the monoclonal antibody mAb5111-humanFc to the IL-2(K35E,C125S)_ISVD207 megabody protein was confirmed for defined clones, we investigated whether binding to the IL-2(K35E,C125S)_ISVD207 megabody protein presented on the surface of CD25 or CD122 / CD132 yeast cells was confirmed. Yeast cells expressing and presenting different IL-2(K35E,C125S)_ISVD207 megabody proteins were incubated for 1 hour at a final concentration of 4 μg / ml with a His tag (human IL-2R alpha Acrobiosystems #ILA-H52H9) in the presence of CD25 protein. In parallel experiments, yeast cells from the same batch expressing and displaying different IL-2(K35E,C125S)_ISVD207 megabody proteins were incubated for 1 hour at a final concentration of 4 μg / ml with His tags (human IL-2R beta & IL-2R gamma heterodimer protein, His tag & Twin strep tag, Acrobiosystems #ILG-H5283) in the presence of CD122 / CD132 protein. All cells were washed three times, incubated for 1 hour in the presence of mouse anti-His-PE antibody (miltenyibiotec / #130-120-718; 1 / 50 dilution), washed three times, and analyzed using flow cytometry. The inventors observed detectable fluorescence bound to yeast cells presenting a specific IL-2(K35E,C125S)_ISVD207 megabody protein and confirmed that CD25 or CD122 / CD132 can bind to certain IL-2(K35E,C125S) megabody protein constructs (Figure 9), providing evidence that the IL-2(K35E,C125S) domain within the IL-2(K35E,C125S)_ISVD207 megabody protein is well-folded. In constructs where ISVD207 was inserted near the binding site of CD122 / 132, there was little fluorescence, but binding of CD122 / 132 could be confirmed in other constructs.In fact, as an example, the inventors confirmed that CD122 / CD132 could hardly bind to the IL-2(K35E,C125S)[L132-I129]_ISVD207 megabody protein, but binding of CD122 / CD132 was observed to the IL-2(K35E,C125S)[N77-S75]_ISVD207 megabody protein. Expression of all constructs was tracked and confirmed by incubating clones at a final concentration of 4 μg / ml for 1 hour 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 (goat anti-mouse IgG Fc gamma-specific phycoerythrin conjugate AffiniPure, Jackson Immuno Research). After the three washes, cells were analyzed using flow cytometry (Figure 9).

[0321] Example 4: Binding of IL-2 megabody protein to ISVD target To further characterize the megabody proteins, similar IL-2 megabody proteins were designed using anti-HSA ISVD or anti-PD-L1 ISVD instead of anti-GFP ISVD, expressed in Pichia pastoris or CHO EBNALT85, and purified according to standard protocols. For purification purposes, the FLAG3HIS6 tag (SEQ ID NO: 35) was fused to the megabody proteins at the C-terminus.

[0322] HSA binding Human serum albumin (HSA) (Sigma-Aldrich, A8763) was biotinylated using NHS-LC-biotin (ThermoFisher, 21336) according to the manufacturer's instructions, with an average labeling level of 1. After the blockage 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 mixtures of 1 nM test compounds, each with fixed concentrations of HSA ranging from 1.13 pM to 10 μM (23 dilutions, 1 / 3 dilution ratio), were added to the plate. The mixtures were incubated at room temperature (RT) for 2 hours to allow equilibrium to be reached. After incubation with biotinylated HSA for 10 minutes, the samples were washed with 3 × 150 μL of PBS + 0.05% Tween-20. During the final detection step, 25 μL of sulfo-targeted anti-VHH antibody was added at a concentration of 2 μg / mL and incubated for 1 hour, followed by a final wash of 150 μL with 1×PBS + 0.05% Tween-20. After adding 150 μL of MSD read buffer, the plate was read with an MSD QuickPlex SQ120 reader. Data were analyzed using a 4-parameter logistic (4PL) scale fitted to a GraphPad Prism 9.

[0323] GFP binding Binding to green fluorescent protein (GFP) (Sino Biological, 13105-S07E) was investigated by surface plasmon resonance (SPR) (Cytiva, Biacore 8K+, #2626160). In short, an anti-FLAG M2 monoclonal antibody (mAb) (Sigma-Aldrich, F3165) was immobilized on a CM5 sensor (Cytiva, BR100399) using a standard amine coupling chemistry method. The test compound was injected at a flow rate of 10 μL / min for 180 seconds at various concentrations (2–60 nM). 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 enable binding to the anti-GFP ISVD / megabody protein, followed by injection of running buffer (Cytiva, BR100669, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20, pH 7.4) for 10 minutes to enable dissociation from the anti-GFP tool. The tip was regenerated using two pulses of 10 mM glycine (pH 1.5) at 45 μL / min for 30 seconds each. Binding data were collected at 25°C and analyzed according to a 1:1 binding fit model using Insight Evaluation software version 3.0.12 supplied by the manufacturer (Cytiva).

[0324] PD-L1 binding Binding to human PD-L1 (human PD-L1(CD274)-hFc) was investigated by surface plasmon resonance (SPR) (Cytiva, Biacore 8K+, #2743662). In short, an anti-human Fc binder was immobilized on a CM5 sensor (Cytiva, BR100399) using a standard amine coupling chemistry method. Human PD-L1-hFc was injected at a flow rate of 10 μL / min at a concentration of 1 μg / mL for 180 seconds. Subsequently, 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 injected at a flow rate of 30 μL / min for 2 minutes to enable binding to the target, followed by injection of running buffer (Cytiva, BR100669, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20, pH 7.4) for 10 minutes to enable dissociation from the target. The tip was regenerated using two 30-second pulses of 0.85% H3PO4 at 30 μL / min. Binding data were collected at 25°C and analyzed according to a 1:1 binding fit model using Insight Evaluation software version 3.0.12 supplied by the manufacturer (Cytiva).

[0325] The results are summarized in Tables 6a-6c, which show the kinetic parameters of the interaction between the IL-2 megabody protein and the multivalent protein construct (polypeptide containing the megabody protein), i.e., the megabody protein fused to ISVD. Here, ISVD targets human serum albumin (HSA) (ISVD=ALB23002; Table 6a), GFP (ISVD=ISVD207, Table 6b), and / or PD-L1 (ISVD=ISVD10F11; Table 6c). In addition, magnification differences are calculated for different compounds compared to their respective references.

[0326] [Table 10]

[0327] [Table 11]

[0328] [Table 12]

[0329] The data show that the ISVD portion retains its binding to the target when formatted in the megabody construct. For the ISVD-HSA interaction, the maximum magnification difference is 3 compared to the reference ISVD. For the ISVD-GFP interaction, the maximum magnification difference is 2, which is even lower than for the ISVD-PD-L1 interaction, further highlighting that the ISVD is fully functional when formatted in the megabody construct.

[0330] Example 5: Induction of STAT5 phosphorylation in different T cell subsets by IL-2 megabody protein To demonstrate their differential signaling profiles based on their interaction characteristics with IL-2 receptors (IL-2Rα, IL-2Rβ, IL-2Rγ), we characterized the induction of STAT5 phosphorylation in different subsets of T cells. STAT5 phosphorylation in CD8+ T cells, CD25-CD4+ T cells, and CD25+FoxP3+CD4+Treg cells was determined by flow cytometry. In short, PBMCs isolated from healthy donors within 4 hours of blood collection were removed from cryogenic storage and thawed in culture medium (RPMI 1640 supplemented with 10% thermally inactivated FBS, Sigma F7524, 1 mM sodium pyruvate, Gibco 11360-039, MEM non-essential amino acids, Gibco 11140-035, and 1× penicillin / streptomycin (Life Technologies 15140), Glutamax, 25 mM Hepes, Gibco 72400-021). The PBMCs were rested overnight in culture medium at 37°C in a 5% CO2 atmosphere at 5,000,000 cells / mL. After washing the PBMCs once with D-PBS (Gibco 14190), they were stained with ZombieNIR-fixable live / dead stain (Biolegend, 423105) in the dark for 15 minutes under RT. After washing 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 for at least 30 minutes in a 5% CO2 atmosphere. Then, an equal amount of the test compound was added, either with or without human serum albumin (HSA, final concentration 30 μM, CSL Behring 2160-679), and the cells were incubated at 37°C for 15 minutes. Next, 500 μL / well of preheated (37°C) fixation buffer I (BD Biosciences 557870) was added, and the cells were immobilized by incubation at 37°C for 15 minutes.After washing twice with FACS buffer (D-PBS supplemented with 2% thermoactivated FBS, Sigma F7524, and 0.05% sodium azide, Acros organics 19038, Gibco 14190), the cell pellet was slowly added with pre-cooled (-20°C) Perm buffer III (BD Biosciences 558050), followed by incubation on ice for 30 minutes. After two washes with FACS buffer, cells were incubated at RT for 60 minutes with a mixture of human Fc block (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 Fluor 488 (BD Bioscience 612598). After two washes with FACS buffer, cells were analyzed using a MACS Quant flow cytometer (Miltenyi Biotec). The mean fluorescence intensity (MFI) for pSTAT5-Alexa Fluor 488 staining was determined after gating with different T cell subsets. Results for IL-2(K35E,C125S) megabody proteins with HSA-targeted ISVD and control are shown in Tables 7a and 7b.

[0331] [Table 13]

[0332] [Table 14]

[0333] [Table 15]

[0334] IL-2(K35E,C125S) megabody proteins with HSA-targeted ISVD induce STAT5 (pSTAT5) phosphorylation in primary T cell subsets with different profiles. For compounds TP027 and TP028, there is at least a 100-fold difference in potency between IL-2Rβγ-(CD8+ and CD4+CD25-) expressing cells and IL-2Rαβγ-(CD4+CD25+) expressing cells. This difference is higher for wild-type IL-2 (TP027), suggesting that K35E and / or C125S influence the interaction with the IL-2 receptor, primarily IL-2Rα. For reference compound 1, engineered IL-2 with an IL-2Rβγ-bias, the ratio is below 3, indicating that IL-2Rα is not involved in downstream signaling (Klein et al. 2013:Blood 122:2278). Regarding reference compound 2, its interaction with cells that primarily express ILRβγ but not IL-2Rα is affected as expected based on the literature (Peterson et al. 2018, J Autoimmun 95:1-14).

[0335] Twelve different insertion sites were investigated in the IL-2(K35E,C125S) megabody protein containing HSA-binding ISVD, and different profiles were observed in absolute potency and the ratio of potency to IL-2Rβγ-expressing cells compared to IL-2Rαβγ-expressing cells. Compounds TP056 and TP065 showed lower potency to IL-2Rαβγ-expressing cells compared to TP027 and TP028, and their potency was similarly high in both IL-2Rβγ-expressing cells and IL-2Rαβγ-expressing cells, resulting in ratios of 2 and 4, respectively. These are similar to the profile of reference compound 1. At the opposite extreme are compounds TP063 and TP064, which mainly show lower potency to IL-2Rβγ-expressing cells compared to TP027 and TP028. They maintain good efficacy against IL-2Rαβγ-expressing cells, resulting in an increased ratio of efficacy against IL-2Rαβγ-expressing cells compared to TP028. This is similar to the profile of reference compound 2, which has an IL-2Rαβγ bias. Overall, a distinct functional profile is observed, indicating that the insertion site of ISVD into cytokines can modulate the interaction between cytokines and their receptors, resulting in differential signaling.

[0336] The binding of ISVD to its target HSA may reduce its potency in a compound-dependent manner (Table 7b). In IL-2Rβγ-expressing cells, a potency reduction of up to 10-fold was observed compared to TP057. In IL-2Rαβγ-expressing cells, a potency reduction of more than 100-fold was observed compared to TP019. As expected, the potency of reference compounds without HSA-bound ISVD was not affected by the presence of HSA in the assay.

[0337] To investigate the effects of K35E and C125S mutations in IL-2, wild-type IL-2 was formatted with megabody proteins and tested for functionality (PBMC-pSTAT5). The results are summarized in Tables 8a and 8b, and Figure 10.

[0338] [Table 16]

[0339] [Table 17]

[0340] [Table 18]

[0341] Similar to IL-2(K35E,C125S) megabody proteins with HSA-targeted ISVD, IL-2 megabody proteins with HSA-targeted ISVD also induce STAT5 phosphorylation in primary T cell subsets with different profiles. The differences in EC50 values ​​and calculated fold differences compared to previous experiments may be due to differences between PBMC donors, and the overall trends and rankings remain the same. For compound TP027, there is a fold difference of 61 between the potency against IL-2Rβγ-expressing cells and the potency against IL-2Rαβγ-expressing cells. For reference compound 1, the ratio is less than 3, indicating that IL-2Rα is not involved in downstream signaling. For reference compound 2, the interaction with cells that primarily express ILRβγ but not IL-2Rα is affected as expected based on the literature.

[0342] For eight different insertion sites explored in the IL-2 megabody protein containing HSA-binding ISVD, different profiles are observed in absolute potency and the ratio of potency to IL-2Rbg-expressing cells compared to IL-2Rαβγ-expressing cells. Compounds TP115 and TP119 show lower potency to IL-2Rαβγ-expressing cells compared to TP027 and RP121, and their potency is similarly high in both IL-2Rβγ-expressing cells and IL-2Rαβγ-expressing cells, resulting in a ratio of 2. These are similar to the profile of reference compound 1. At the opposite extreme are compounds TP118 and TP072, which mainly show lower potency to IL-2Rβγ-expressing cells compared to the reference. They still exhibit good potency to IL-2Rαβγ-expressing cells, resulting in a higher ratio difference between potency to IL-2Rβγ-expressing cells and potency to IL-2Rαβγ-expressing cells. This is similar to the profile of reference compound 2, which has an IL-2Rαβγ bias. For IL-2 megabody proteins, a different functional profile is detected, indicating that the insertion site of ISVD into cytokines can modulate the interaction between cytokines and their receptors. Overall, the ranking of compounds based on the multiplier difference between potency against IL-2Rβγ-expressing cells and potency against IL-2Rαβγ-expressing cells is similar for megabody proteins containing either IL-2(K35E, C125S) (Tables 7a and 7b) or IL-2 (Tables 8a and 8b).

[0343] The binding of ISVD to its target HSA may reduce its potency, albeit in a compound-dependent manner. The greatest effect was observed with TP075, which showed a 40-fold decrease in potency against both IL-2Rβγ-expressing cells and IL-2Rαβγ-expressing cells. Apart from the megabody protein, the ISVD-cytokine construct fused from the N-terminus to the C-terminus (TP121) also showed lower potency in the presence of HSA. As expected, the potency of the reference compound without HSA-bound ISVD was not affected by the presence of HSA in the assay.

[0344] To evaluate the effect of ISVD used in megabody formats, IL-2(K35E,C125S) megabody proteins were formatted using GFP-conjugated ISVD (PSVD207) and compared to HSA-conjugated ISVD-IL-2(K35E,C125S) megabody proteins. The data are shown in Table 9.

[0345] [Table 19]

[0346] Similar to the IL-2(K35E,C125S) megabody protein with HSA-targeted ISVD, the IL-2(K35E,C125S) megabody protein with GFP-targeted ISVD (ISVD207) also induces STAT5 phosphorylation in primary T cell subsets, albeit with different profiles. For compounds TP027 and TP028, there is at least a 30-fold difference in potency between IL-2Rβγ-expressing cells and IL-2Rαβγ-expressing cells. This difference is higher in wild-type IL-2, suggesting that K35E and / or C125S influence the interaction with the IL-2 receptor, primarily IL-2Rα. For reference compound 1, the ratio is less than 3.

[0347] Three different sites in IL-2 were explored for insertion by ISVD targeting either HSA or GFP. Different profiles were observed for the three explored sites in terms of absolute potency and the ratio of potency to IL-2Rαβγ-expressing cells. Compounds TP031 and TP019 showed a lower ferricity difference between IL-2Rβγ-expressing cells and IL-2Rαβγ-expressing cells compared to TP028, while TP030 and TP018 showed a higher ferricity difference between IL-2Rβγ-expressing cells and IL-2Rαβγ-expressing cells compared to TP028.

[0348] Example 6: Activity of IL-2 megabody protein in PBMC proliferation assay The IL-2 megabody protein was characterized for its stimulation of CD4+ and CD8+ T cell proliferation in a PBMC proliferation assay using Ki67 readout. Furthermore, CD25 was added to differentiate between IL-2Rα-positive and IL-2Rα-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 supplemented with 10% thermally inactivated FBS (Sigma F9665) and 1% penicillin / streptomycin (Life Technologies, 15140), GlutaMAX® supplement, HEPES (Life Technologies-Gibco, 72400-021)). PBMCs were seeded at a rate of 300,000 cells / well in 100 μL of culture medium (CTS® OpTmizer® T cell proliferation SFM and OpTmizer® T cell proliferation supplement (Life Technologies-Gibco, A10221-01~A10484-02), 2 mM L-glutamine (Life Technologies-Gibco, A2916801), 5% CTS® immune cell SR (Life Technologies-Gibco, A25961-01), and 1% penicillin / streptomycin (Life Technologies, 15140)) in 96-well U-bottom plates (Costar, 3799). Next, equal amounts of the test compound were added, with or without human serum albumin (HSA, final concentration 30 μM, CSL Behring 2160-679), and the cells were incubated in a 5% CO2 atmosphere at 37°C for 6 days. Following incubation, Ki67 expression in CD4+ and CD8+ T cells was determined by flow cytometry using CD25 as an additional marker. The cells were transferred to V-bottom plates (Greiner, 651180), washed with D-PBS (Gibco, 14190), and then stained with ZombieAqua fixable live / dead stain (Biolegend, 423102) by RT for 15 minutes.After washing with FACS buffer (D-PBS supplemented with 2% thermally inactivated FBS and 0.05% sodium azide (Acros organics 19038)), cells were incubated with human Fc block (BD Pharmingen, 564220, 12.5 μg / mL) at RT for 15 minutes, and then stained with a mixture of anti-human CD3-APC-H7 (BD Pharmingen, 560176 / 560275), anti-human CD8-PerCP / Cy5.5 (BioLegend, 344709 / 344710), and anti-human CD25-Alexa Fluor 647 (BioLegend, 356127 / 356128) at 4°C for 30 minutes. After two washes with FACS buffer, the cells were fixed and permeabilized at room temperature for 1 hour using 1×Fix / Perm buffer from the FoxP3 / transcription factor staining buffer set (eBioscience, 00-5523-00). After two washes with 1×Perm buffer supplied in the buffer set, the cells were stained with a mixture of anti-human CD4-FITC (Biolegend, 344604) and anti-human Ki67-Brilliant Violet 421 (BioLegend, 350505 / 350506) by RT for 45 minutes. After two washes with 1×Perm buffer, the cells were resuspended in FACS buffer and analyzed using a MACS Quant flow cytometer (Miltenyi Biotec). The percentage of Ki67-positive cells was analyzed using gated CD4+ and CD8+ T cells. The results are shown in Figure 11 and Tables 10a and 10b.

[0349] [Table 20]

[0350] [Table 21]

[0351] [Table 22]

[0352] As expected based on pSTAT5 data (Table 7), IL-2(K35E,C125S) megabody proteins with HSA-targeted ISVD can promote the proliferation of different immune cell populations (CD8+CD25-, CD4+CD25-, and CD4+CD25+). For compound TP027, there is a 160-fold difference in potency between IL-2Rβγ-(CD8+CD25- and CD4+CD25-) expressing cells and IL-2Rαβγ-(CD4+CD25+) expressing cells. For reference compound 1, the ratio is less than 3. Although a complete dose-response curve was not obtained for reference compound 2 against CD8+CD25- and CD4+CD25- T cells, this compound maintained its potency against CD4+CD25+ T cells compared to wild-type human IL-2 (TP027).

[0353] Different insertion sites explored in the IL-2(K35E,C125S) megabody protein containing HSA-binding ISVD show different profiles in absolute potency and the ratio of potency to IL-2Rαβγ-expressing cells. Compounds TP056 and TP065 exhibit lower potency to IL-2Rαβγ-expressing cells compared to TP027, with very similar potency in both IL-2Rβγ-expressing and IL-2Rαβγ-expressing cells, resulting in ratios of 0.2 and 0.3, respectively. This is consistent with pSTAT5 data (Table 7). At the opposite extreme is compound TP064, which exhibits lower potency to IL-2Rβγ-expressing cells, mainly compared to TP027. However, this protein still exhibits good potency to IL-2Rαβγ-expressing cells. This is consistent with the pSTAT5 data (Table 7) and similar to the profile of reference compound 2, which has an IL-2Rαβγ bias. Overall, a distinct functional profile is detected, indicating that the insertion site of ISVD into cytokines can modulate the interaction between cytokines and their receptors.

[0354] The compound's bias toward IL-2Rβγ or IL-2Rαβγ did not change upon binding of ISVD to its target. In contrast to observations made in the pSTAT5 assay, no effect of HSA binding was observed in the growth assay. This is presumably due to the nature of the characterization assay, where the growth assay is performed over 6 days, in contrast to the 15-minute incubation in the pSTAT5 assay, which allows for equilibrium conditions.

[0355] To investigate the effects of K35E and C125S mutations in IL-2 on PBMC proliferation assays, wild-type IL-2 was formatted with megabody proteins. The results are summarized in Tables 11a and 11b, and Figure 11.

[0356] [Table 23]

[0357] [Table 24]

[0358] [Table 25]

[0359] Similar to IL-2(K35E,C125S) megabody proteins with HSA-targeted ISVDs, IL-2 megabody proteins with HSA-targeted ISVDs can also promote the proliferation of different immune cell populations (CD8+CD25-, CD4+CD25-, and CD4+CD25+). For compound TP027, there is a 50-fold difference in potency between IL-2Rβγ-(CD8+CD25- and CD4+CD25-) expressing cells and IL-2Rαβγ-(CD4+CD25+) expressing cells.

[0360] For eight different insertion sites explored in the IL-2 megabody protein containing HSA-binding ISVD, different profiles were observed in absolute potency and the ratio of potency to IL-2Rαβγ-expressing cells. Compounds TP115 and TP119 showed lower potency to IL-2Rαβγ-expressing cells compared to TP027, and their potency was very similar in both IL-2Rβγ-expressing 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. AlphaFold models for TP115 and TP119 (Figures 12 (SA17669) and 13 (SA17658)) show that the ISVD is positioned toward the IL-2Rα protein, thereby interfering with the IL-2-IL-2Rα interaction. At the opposite end of the spectrum are compounds TP118 and TP072, which primarily exhibit lower potency towards IL-2Rβγ-expressing cells compared to reference TP027. They still possess good potency towards IL-2Rαβγ-expressing cells, resulting in a higher factorial difference in potency between IL-2Rβγ-expressing cells and IL-2Rαβγ-expressing cells. This is consistent with pSTAT5 data (Table 8) and similar to the profile of reference compound 2. The AlphaFold model for TP118 (Figure 14 (SA17678)) shows that ISVD is positioned toward the IL-2Rγ protein, thereby interfering with the IL-2-IL-2Rγ interaction while maintaining binding to IL-2Rα. The AlphaFold model for TP116 (Figure 15 (SA17659)) shows that ISVD is positioned toward the IL-2Rβ protein, thereby interfering with the IL-2-IL-2Rβ interaction. Generally, for IL-2 megabody proteins, different functional profiles are detected, indicating that the insertion site of ISVD into cytokines can modulate the interaction between cytokines and their receptors.Furthermore, the ranking of compounds based on the ratio difference between their efficacy against IL-2Rβγ-expressing cells and their efficacy against IL-2Rαβγ-expressing cells is similar for megabody proteins containing either IL-2(K35E, C125S) (Tables 10a and 10b) or IL-2 (Tables 11a and 11b).

[0361] The compound's bias toward the IL-2Rβγ or IL-2Rαβγ receptor complex did not change upon binding of ISVD to its target. In contrast to observations made in the pSTAT5 assay, no effect of HSA binding was observed in the PBMC proliferation assay. This is presumably due to the nature of the characterization assay, where the PBMC proliferation assay is performed over 6 days, in contrast to the 15-minute incubation in the pSTAT5 assay, which allows for equilibrium conditions.

[0362] Example 7: Binding of IL-2 megabody protein to the IL-2 receptor IL-2Rα binding Binding to human IL-2Rα (ACROBiosystems, ILA-H5251) was investigated by surface plasmon resonance (SPR) (Cytiva, Biacore 8K+, #2743662). In short, anti-human Fc binders were immobilized on a CM5 sensor (Cytiva, 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 for 2 minutes at a flow rate of 30 μL / min as a 7-step dilution series (dilution ratio 2.5) starting at 500 or 100 nM, to enable binding to the target, followed by dissociation from the target with a 10-minute running buffer (Cytiva, BR100669, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20, pH 7.4). The tip was regenerated using two 30-second pulses of 0.85% H3PO4 at 30 μL / min. Binding data were collected at 25°C and analyzed according to a 1:1 binding fit model using Insight Evaluation software version 3.0.12 supplied by the manufacturer (Cytiva).

[0363] IL-2Rβ binding The binding of human IL-2Rβ (ACROBiosystems, ILB-H5253) was investigated by surface plasmon resonance (SPR) (Cytiva, Biacore 8K+, #2743662). Briefly, an anti-human Fc binder was immobilized on a CM5 sensor (Cytiva, BR100399) using a standard amine coupling chemistry method. 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 for 2 minutes at a flow rate of 30 μL / min as a 7-step dilution series (dilution ratio 2.5) starting at 500 or 250 nM, to enable binding to the target, followed by dissociation from the target with a 10-minute running buffer (Cytiva, BR100669, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20, pH 7.4). The tip was regenerated using two 30-second pulses of 0.85% H3PO4 at 30 μL / min. Binding data were collected at 25°C and analyzed according to a 1:1 binding fit model using Insight Evaluation software version 3.0.12 supplied by the manufacturer (Cytiva).

[0364] IL-2Rβ / γ binding The binding of IL-2Rβ / γ (human IL-2RB(ECD)(T191C)-THR-zipper) to an in-house manufactured IL-2Rβ / γ was explored using surface plasmon resonance (SPR) (Cytiva, Biacore 8K+, #2626160). In short, Strep-Tactin(registered trademark)XT(Iba Life) The Sciences Twin-Strep-Tag Capture Kit (2-4370-000) was immobilized onto a CM5 sensor (Cytiva, BR100399) as described in the kit protocol. Human IL-2Rβ / γ was injected at a flow rate of 10 μL / min for 180 seconds at a concentration of 0.75 μg / mL. Subsequently, the test compound was injected at a flow rate of 30 μL / min for 2 minutes as a seven-step dilution series (dilution ratio 2.5) starting at 250 or 150 nM, to enable binding to the target, followed by dissociation from the target with a 10-minute running buffer (Cytiva, BR100669, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20, pH 7.4). The tip was subjected to a 3M run at 30 μL / min for 70 seconds. Regeneration was performed using three pulses of GuHCl. Binding data were collected at 25°C and analyzed according to a 1:1 binding fit model using Insight Evaluation software version 3.0.12 supplied by the manufacturer (Cytiva).

[0365] The results are summarized in Tables 12a, 12b, and 12c.

[0366] [Table 26]

[0367] [Table 27]

[0368] [Table 28]

[0369] As expected from pSTAT5 and proliferation assays, IL-2 megabody proteins exhibit differential binding profiles to cytokine receptors. Compounds TP115, TP116, and TP119 show a significant decrease in affinity for IL-2Rα. Compound TP116 also has a decreased affinity for IL-2Rβ, but can still bind to the IL-2Rβ / γ heterodimer. A similar trend is observed for compound TP118. Differential binding to the IL-2Rβ / γ heterodimer is not very pronounced in the current assay. Overall, these different binding profiles suggest that the insertion site of ISVD into cytokines can modulate the interaction between cytokines and their receptors.

[0370] Example 8: Activity of IL-2 megabody protein in tetanus toxoid (TT) recall assay The multivalent construct, i.e., the megabody protein fused to ISVD, was tested for primary T cell activation (monitored via IFNγ production) in an autologous tetanus toxoid recall assay. Briefly, PBMCs isolated from healthy donors were removed from cryogenic storage and thawed in culture medium (RPMI 1640 medium supplemented with 10% thermally inactivated FBS (Sigma F9665) and 1% penicillin / streptomycin (Life Technologies, 15140), GlutaMAX® supplement, and 25 mM HEPES (Life Technologies-Gibco, 72400-021). Monocytes were isolated by negative magnetic separation according to the supplier's instructions for the EasySep Human Monocyte Isolation Kit (Stemcell Technologies, 19359) and human Mo-DC differentiation medium (Miltenyi Monocytes were cultured for 7 days in Biotec (130-094-812) (with the same amount of fresh medium added on day 3). After 7 days, monocytes had differentiated into immature dendritic cells (iDCs). The iDCs were harvested and frozen in liquid nitrogen for later use. PBMCs from the same donor were cultured in assay medium (RPMI 1640 medium supplemented with 10% heat-inactivated human AB serum (BioIVT, SM-612-HSI) and 1% penicillin / streptomycin, GlutaMAX® supplement, 25 mM HEPES (Life Cells were thawed in assay medium (Technologies-Gibco, 72400-021) and cultured in assay medium supplemented with 0.5 μg / mL tetanus toxoid (TT) (Calbiochem, 582231) at 37°C for 7 days under a 5% CO2 atmosphere. After 7 days, TT-specific T cells were enriched. Cells were harvested and frozen in liquid nitrogen for later use. For the characterization assay, iDCs were thawed in assay medium and seeded at 5000 cells / well in assay medium supplemented with 0.5 μg / mL TT in a 96-well U-bottom plate (Corning, 3799), and incubated at 37°C under a 5% CO2 atmosphere for 4 hours. Autologous TT-enriched T cells were thawed in assay medium and 100,000 cells / well were added to iDCs, and serially diluted polyvalent constructs were added. After 3 days of co-culture, the cell supernatant was collected and the IFNγ concentration was determined by ELISA.The results are shown in Figure 16 and Table 13.

[0371] [Table 29]

[0372] All tested compounds, whether containing IL-2 alone, anti-PD-L1 ISVD alone, or a combination of both in different formats, induced dose-dependent IFNγ production in the TT recall assay. The best efficacy was observed for the IL-2-only construct. Combining IL-2 with anti-PD-L1 ISVD resulted in decreased efficacy but increased effectiveness, with higher levels of IFNγ production detected, demonstrating the added effect of combining IL-2 with anti-PD-L1 ISVD on primary T cell functionality. This was observed in combination treatments, N-terminal to C-terminal ISVD-cytokine fusion, and fusions of megabody proteins and ISVD megabodies (multivalent constructs), indicating that both IL-2 and anti-PD-L1 ISVD are functional in different formats. The ALB-IL-2 megabody protein (TP021) exhibited better efficacy compared to the anti-PD-L1-IL-2 megabody protein (TP048), but behaved similarly to IL-2 alone, exhibiting lower efficacy.

[0373] Example 9: Design of a cyclically substituted variant of interferon alpha-2a (IFNA2a). To design the IFNA2a_NbALB23 megabody protein, a fully folded, cyclically substituted version of interferon alpha-2a (IFNA2a) is required. For this purpose, we examined the structure of IFNA2a (PBD:1ITF,3S9D), designed a theoretical construct by opening one site shown in Figure 17, and introducing a peptide linker to connect the C-terminus of IFNA2a to its N-terminus. Finally, we constructed two constructs and cloned them into a yeast display vector containing all the previously described auxiliary proteins so that the cyclically substituted variant could be presented on the surface of EBY100 yeast cells. In one construct, the last five amino acids of IFNA2a were deleted, and a 3-amino acid peptide linker (SEQ ID NO: 57) was introduced to generate a cyclically substituted variant of IFNA2a called IFNA2a[D77-W76]V2 (SEQ ID NO: 58), by connecting the C-terminus (residue 160) of IFNA2a to its N-terminus. 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 connect the C-terminus (residue 163) of IFNA2a to its N-terminus. This cyclically substituted variant of IFNA2a is called IFNA2[D77-W76]V4 (SEQ ID NO: 59). In parallel, wild-type IFNA2a (SEQ ID NO: 56) was cloned and presented on the surface of yeast cells.

[0374] Example 10: Binding of specific anti-IFNA2a monoclonal to interferon alpha-2a and its cyclically substituted variant. A circulatingly replaced version of IFNA2a was expressed and presented 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 to IFNA2a (SEQ ID NO: 56), yeast cells expressing different constructs were incubated for 1 hour at a final concentration of 2.5 μg / ml in the presence of the monoclonal antibody mAb93452 (human IFN-alpha-2 / IFNA2a antibody, R&D systems: MAB93452). Simultaneously, EBY100 yeast cells that did not express any construct were incubated for 1 hour in the presence of the monoclonal antibody mAb93452 (human IFN-alpha-2 / IFNA2a antibody, R&D systems: MAB93452) and served as a negative control. After three washes, all cells were incubated for 1 hour in the presence of 2 μg / ml anti-mouse IgG Fc (goat anti-mouse IgG Fc gamma-specific phycoerythrin conjugate AffiniPure, Jackson Immuno Research), washed three times, and analyzed using flow cytometry. More fluorescence was observed in yeast cells bound to either IFNA2a (SEQ ID NO: 56), IFNA2[D77-W76]V2 (SEQ ID NO: 58), or IFNA2[D77-W76]V4 (SEQ ID NO: 59) after incubation with monoclonal mAb93452 (Figure 18), but no clear shift in fluorescence was observed in EBY100 yeast cells that did not express any construct. Expression of all constructs was tracked and confirmed by incubating clones at a final concentration of 4 μg / ml for 1 hour 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 (goat anti-mouse IgG Fc gamma-specific phycoerythrin conjugate AffiniPure, Jackson Immuno Research). After the three washes, cells were analyzed using flow cytometry (Figure 18).

[0375] Example 11: Binding of IFNAR2 to interferon alpha-2a and its cyclically substituted variant. A circulatingly replaced version of IFNA2a was expressed and presented 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 to IFNA2a (SEQ ID NO: 56), yeast cells expressing different constructs were incubated for 1 hour at a final concentration of 4 μg / ml in the presence of his-tagged IFNAR2 (human IFN-alpha / beta R2 protein, His-tagged, Acrobiosystems). Simultaneously, EBY100 yeast cells expressing no constructs were incubated for 1 hour in the presence of IFNAR2 (human IFN-alpha / beta R2 protein, His-tagged, Acrobiosystems) to serve as a negative control. After three washes, all cells were incubated for 1 hour in the presence of mouse anti-His antibody-PE (miltenyibiotec / #130-120-718; 1 / 50 dilution), washed three times, and analyzed using flow cytometry. More fluorescence was observed in yeast cells bound to IFNA2a (SEQ ID NO: 56), IFNA2a[D77-W76]V2 (SEQ ID NO: 58), or IFNA2[D77-W76]V4 (SEQ ID NO: 59) after incubation with IFNAR2 (Figure 19), while EBY100 yeast cells not expressing any construct showed a smaller shift in fluorescence. Expression of all constructs was tracked and confirmed by incubating clones at a final concentration of 4 μg / ml for 1 hour in the presence of mouse anti-Myc monoclonal antibody (Roche / #11 667 149 001), followed by three washes, and then incubation in the presence of anti-mouse IgG-Fc-PE (goat anti-mouse IgG Fc gamma-specific phycoerythrin conjugate AffiniPure, Jackson Immuno Research). After the three washes, cells were analyzed using flow cytometry (Figure 19).

[0376] Example 12: Design of a 31 kDa antigen-binding chimeric protein constructed from a circulatingly substituted variant of interferon alpha-2a inserted into the first β-turn connecting β-chains A and B of anti-HSA ISVD. Based on the successful design of the IL-2(K35E,C125S)_ISVD207 megabody protein, and understanding that the inventors can create cyclically substituted variants of IFNA2a, the inventors also designed an ISVD molecule to be grafted onto interferon alpha-2a (IFNA2a).

[0377] The topology of interferon alpha-2a (IFNA2a PDB 1ITF, 3S9D, Figure 17) was investigated, and ISVD ALB23002 was grafted onto different sites. Twenty-three different versions of the IFNA2_ALB23 megabody protein were created. The 31 kDa megabody proteins described herein are chimeric polypeptides in which a portion of a single-domain immunoglobulin is linked to a portion of a scaffold protein linked according to Figure 1. The immunoglobulin domain used here is anti-HSA ISVD, as shown in SEQ ID NO: 55. The scaffold protein is IFNA2a (SEQ ID NO: 56). All parts were linked together by 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 in SEQ ID NO: 55), short peptide linker (SEQ ID NO: 5), C-terminal portion of IFNA2a (amino acid positions X2 to 165 in SEQ ID NO: 56), peptide linker (amino acid positions 1 to X1 in SEQ ID NO: 56) to connect the C-terminal portion of IFNA2a to its N-terminus (SEQ ID NO: 57), N-terminal portion of IFNA2, short peptide linker (SEQ ID NO: 5), followed by β-chains B-G of anti-HSA ISVD (residues 16-126 in SEQ ID NO: 55). Alpha-fold models of several (non-limiting) examples of IFNA2a_ALB23002 megabody proteins are shown in Figures 20-23 (SEQ ID NOs: 60-63).

[0378] Example 13: Binding of IFNA2a megabody protein to IFNα receptor IFNAR2 and ISVD-targeted human serum albumin (HSA) IFNAR2 binding Binding to human IFNAR2 (Sino Biological, 10359-H02H) was investigated by surface plasmon resonance (SPR) (Cytiva, Biacore 8K+, #2626160). In short, anti-human Fc binders were immobilized on a CM5 sensor (Cytiva, BR100399) using standard amine coupling chemistry. Human IFNAR2 was injected at 1 or 20 μg / mL at a flow rate of 10 μL / min for 180 seconds. Next, the test compound was injected at a flow rate of 30 μL / min for 2 minutes as a seven-step dilution series starting from 500, 100, 50, or 25 nM (dilution ratio 2.5) to enable binding to the target, followed by dissociation from the target with a 10-minute running buffer (Cytiva, BR100669, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20, pH 7.4). The tip was regenerated using two 30-second pulses of 0.85% H3PO4 at 30 μL / min. Binding data were collected at 25°C and analyzed according to a 1:1 binding fit model using Insight Evaluation software version 3.0.12 supplied by the manufacturer (Cytiva).

[0379] HSA binding The binding of the compound to human serum albumin (HSA) (Sigma Aldrich, A8763) was investigated using surface plasmon resonance (SPR) (Cytiva, Biacore 8K+, #2626160). In short, HSA was immobilized on a C1 sensor (Cytiva, BR100535) using a standard amine coupling chemistry method. Subsequently, the test compound was injected at a flow rate of 30 μL / min for 2 minutes as a nine-step dilution series starting at 2500 nM (dilution ratio 2.5) to enable binding to the target, followed by dissociation from the target with a 10-minute running buffer (Cytiva, BR100669, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20, pH 7.4). The tips were regenerated using two 30-second pulses of 10 mM glycine (pH 1.5) at a flow rate of 30 μL / min. Binding data were collected at 25°C and analyzed according to a 1:1 binding fit model using Insight Evaluation software version 3.0.12 supplied by the manufacturer (Cytiva).

[0380] The objective of the megabody construct design was to modulate the interaction between IFNA2a and its high-affinity receptor IFNAR2 while maintaining the interaction between ISVD and its target. Scanning the IFNA2a cytokine yields a range of affinities (KD) to IFNAR2, with either higher affinity (Cluster C) or lower affinity (Cluster B) compared to the reference compound TP088. Compounds with similar affinity to TP088 are grouped into Cluster A. Compounds with the same ISVD insertion site in the same region of the IFNA2a cytokine exhibit similar affinities. The data are shown in Table 14. The AlphaFold models for TP093 (Cluster B, Figure 24) and TP109 (Cluster C, Figure 25) show that ISVD is positioned toward the low-affinity receptor IFNAR1 and away from the high-affinity receptor IFNAR2. The binding kinetics actually show a limited effect on affinity to IFNAR2, with a 5-fold decrease and a 10-fold increase for TP093 and TP109, respectively. On the other hand, the AlphaFold models for TP095 (cluster B, Figure 26) and TP098 (cluster B, Figure 27) indicate that ISVD is directed toward anti-affinity IFNAR2. For TP095, a 70-fold decrease in affinity to IFNAR2 was observed, while for TP098, the binding was too low to determine affinity. Furthermore, different megabody proteins may have similar functional consequences (i.e., the same clustering) through different mechanisms. In the case of compounds in which ISVD is directed toward IFNAR1 (e.g., TP093), the regulation of cytokine activity may result from inhibiting heterodimerization of the receptor complex. In the case of other compounds in which ISVD is directed toward IFNAR2 (e.g., TP095), the effect may result from a direct influencing interaction with IFNAR2.

[0381] [Table 30]

[0382] [Table 31]

[0383] A KD of 1–10 nM is expected for the ISVD-HSA interaction. Binding of ISVD to its target HSA is not hindered by formatting to the IFNA2a megabody protein. For all megabody proteins, the binding affinity to HSA is within 3.5 times the difference compared to the reference compound TP113.

[0384] Example 14: Activity of IFNA2a megabody protein in STAT1 phosphate assay Megabody proteins were characterized for their induction of STAT1 signaling in A549 cells (human lung cancer, ATCC CCL-185), and their differential signaling profiles were demonstrated. STAT1 phosphorylation was determined by flow cytometry. Briefly, A549 cells were cultured in culture medium (Ham's F-12K (Kaighn's) medium supplemented with 10% thermally inactivated FBS; Life Technologies-Gibco 21127 and Sigma F9665, respectively) at 37°C in a 5% CO2 atmosphere. On the day of the assay, A549 cells were collected, washed with D-PBS (Gibco 14190), and then stained with ZombieNIR-fixable live / dead stain (Biolegend, 423105) in the dark at RT for 15 minutes. After washing with culture medium, 150,000 cells were seeded in 75 μl of culture medium in each well of a 96-well U-bottom plate (Costar 3799) and incubated at 37°C for at least 30 minutes in a 5% CO2 atmosphere. Then, an equal volume of megabody protein or human serum albumin (HSA, final concentration 30 μM, CSL Behring 2160-679) was added, and the cells were incubated at 37°C for 15 minutes. Next, the cells were immobilized by adding 150 μl / well of preheated (37°C) fixation buffer I (BD Biosciences 557870) and incubating at 37°C for 15 minutes. After two washes with FACS buffer (D-PBS supplemented with 2% thermoactivated FBS, Sigma F7524, and 0.05% sodium azide, Acros organics 19038, Gibco 14190), pre-cooled (-20°C) Perm buffer III (BD Biosciences 558050) was slowly added to the cell pellet, followed by incubation on ice for 30 minutes. After two washes with FACS buffer, the cells were incubated with human Fc block (BD Pharmingen 564220, 12.5 μg / mL) at 4°C for 15 minutes, followed by the addition of PE-conjugated anti-human STAT1 (pY701) (BD Bioscience 562069), and incubation in the dark at RT for 60 minutes.After two washes with FACS buffer, cells were analyzed using a MACS Quant flow cytometer (Miltenyi Biotec). The median fluorescence intensity (MFI) of pSTAT1-PE staining was determined after gating in live cells. The results are shown in Figure 28 and Table 15.

[0385] [Table 32]

[0386] [Table 33]

[0387] Scanning the IFNA2a cytokine yields a range of potencies, with either better (Cluster C) or worse (Cluster B) potencies compared to the reference compound. Compounds exhibiting similar potency to TP088 are grouped into Cluster A. Compounds with the same ISVD insertion site in the same region of the IFNA2a cytokine exhibit similar functionality. Functionality correlates with binding data (Table 14). HSAs can further modulate functionality, and the effect is compound-dependent.

[0388] Example 15: Antiproliferative activity of IFNA2a megabody protein against RPMI 8226 cells and NCI-H929 cells We characterized the antiproliferative effects of megabody proteins on RPMI 8226 (a human B lymphocyte cell line derived from plasmacytoma, ATCC CCL-155) and NCI-H929 cells (a human myeloma cell line, DSMZ ACC 163). Cells were cultured in RPMI 1640, Glutamax, 25mM Hepes, Gibco 72400-021) supplemented with RPMI 8226 cell-specific culture medium (10% thermo-inactivated FBS, Sigma F9665, 1mM sodium pyruvate, Gibco 11360-039, and 1% penicillin / streptomycin, Gibco 15140-122) and NCI-H929 cell-specific culture medium (10% thermo-inactivated FBS, Sigma F9665, 1mM sodium pyruvate, Gibco 11360-039, 50μM beta-mercaptoethanol, Gibco 21985-023, and 1% penicillin / streptomycin, Gibco 15140-122). Cells were grown in Hepes (Gibco 72400-021). Cells were harvested on the day of the assay and seeded at 5,000 cells / well in 40 μl of culture medium in a 384-well flat, clear-bottomed white TC-treated plate (Corning, 3765). Equivalent volumes of the compound, or the compound combined with human serum albumin (HSA, final concentration 30 μM, CSL Behring 2160-679), were then added, and the cells were incubated at 37°C for 3 days in a 5% CO2 atmosphere. Next, 40 μl of the supernatant was removed and replaced with 40 μl of CellTiter-Glo reagent (Promega, G7570), followed by resuspension, shaking, and incubation for 10 minutes. Finally, luminescence was measured using an EnVision instrument (PerkinElmer). The results are shown in Figure 29 and Table 16.

[0389] [Table 34]

[0390] [Table 35]

[0391] Scanning the IFNA2a cytokine yields a range of potencies, with some exhibiting better (Cluster C) or worse (Cluster B) potencies compared to the reference compound TP088. Compounds exhibiting similar potency to TP088 are grouped into Cluster A. Compounds with the same ISVD insertion site in the same region of the IFNA2a cytokine exhibit similar functionality. Functionality correlates with binding data and pSTAT1 data (Tables 14 and 15). HSA can further modulate functionality, and the effect is compound-dependent.

[0392] Example 16: Design of a cyclically substituted variant of interleukin-18. To design the IL18_ISVD207 megabody protein fusion, a fully folded, cyclically substituted version of interleukin-18 (IL18) is required. For this purpose, the structure of IL18 (PBD:1J0S, 3WO4, 3F62) was examined, and a theoretical construct was designed in which one site is opened and a peptide linker is introduced to connect the C-terminus of IL18 to its N-terminus, as shown in Figure 30. Ultimately, three constructs were made and cloned into a yeast display vector using all the auxiliary proteins as previously described, so that the cyclically substituted IL18 variant could be presented on the surface of yeast cells. In one construct, the first three amino acids of IL18 were deleted, and an 11-amino acid peptide linker (SEQ ID NO: 65) was introduced to connect the C-terminus of IL18 to its cleaved N-terminus, generating a cyclically substituted variant of IL18 called IL18[K70-E69]V1b (SEQ ID NO: 66). In the second construct, the C-terminus of IL18 was linked to the N-terminus by a 12-amino acid peptide linker (SEQ ID NO: 67), and this variant was called IL18[K70-E69]V5b (SEQ ID NO: 68). In the third construct, the first five amino acids of IL18 (SEQ ID NO: 64) were deleted, and a 5-amino acid peptide linker (SEQ ID NO: 69) was introduced to link the C-terminus of IL18 to the cleaved N-terminus, generating a cyclically substituted variant of IL18 called IL18[K70-E69]V7 (SEQ ID NO: 70).

[0393] Expression of all constructs was tracked and confirmed by a clear shift in fluorescence, indicated by incubation of clones at a final concentration of 4 μg / ml for 1 hour 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 (goat anti-mouse IgG Fc gamma-specific phycoerythrin conjugate AffiniPure, Jackson Immuno Research). After the three washes, cells were analyzed using flow cytometry (Figure 31). All IL18 wild-type and three cyclically substituted IL18 variants can be expressed on the surface of yeast cells.

[0394] Example 17: Binding of specific anti-IL18 monoclonal to a cyclically substituted variant of interleukin-18. A circulatingly substituted version of IL18 was expressed and presented on the surface of EBY100 yeast cells. To analyze the folding of IL18[K70-E69]V1b (SEQ ID NO: 66), IL18[K70-E69]V5b (SEQ ID NO: ...

Claims

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

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

3. The chimeric protein according to claim 1 or 2, wherein the ISVD and the cytokine are fused via 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 in the cytokine, or between one β chain and one α helix.

5. The ISVD mentioned above is V Ｈ or V ＨＨ Preferably, the ISVD is V ＨＨ More preferably humanized V ＨＨ Or Camelization V Ｈ The chimeric protein according to any one of claims 1 to 4.

6. The chimeric protein according to any one of claims 1 to 5, wherein the cytokine is an interleukin or an interferon, preferably the interleukin is interleukin-2 (IL-2) or interleukin-18 (IL-18), and / or the interferon is interferon (IFN) alpha 2a (IFNA2A).

7. According to the IMGT classification, the cytokines undergo the following turns of the ISVD: a. In the first β-turn connecting β-chains A and B of the ISVD; or b. In the β-turns connecting the β-chain C and C' of the ISVD; or c. In the β-turns connecting the β-chains C'' and D of the ISVD; or d. In the β-turns connecting the β-chains D and E of the ISVD; or e. During the β-turn connecting β-chains E and F of the ISVD A chimeric protein according to any one of claims 1 to 6, wherein it is fused to the ISVD at an internal fusion site located in one of the ISVDs.

8. The chimeric protein according to any one of claims 1 to 7, wherein the cytokine is an interleukin, and 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 includes an ISVD containing a sequence defined by 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 is a sequence defined by SEQ ID NOs: 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 SEQ ID NOs: 4, 58, 59, 64, 66 A chimeric protein according to any one of claims 1 to 8, comprising a cytokine containing a sequence having at least 80% identity with 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.

11. The chimeric protein is a sequence defined by SEQ ID NOs: 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 SEQ ID NOs: 7-25, 36-54, 60 A chimeric protein according to any one of claims 1 to 10, comprising or consisting of a sequence having at least 80% identity with ~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.

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

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

14. A method for altering and / or modifying cytokine signaling by fusing a cytokine to an ISVD, and / or influencing, altering and / or modifying receptor oligomerization upon binding of the cytokine to at least one of its receptors or receptor subunits, wherein the internal fusion site of the ISVD is used for inserting the cytokine or a circulatingly substituted cytokine, and in the ISVD, the internal fusion site is located within a loop or turn between two secondary structural elements.

15. A method for regulating cytokine signaling, - A step of providing a chimeric protein as defined in any one of claims 1 to 11 or a polypeptide as defined in claim 12, - A step of screening chimeric proteins or polypeptides, wherein the cytokine contained therein exhibits modified cytokine signaling compared to the cytokine not fused to the ISVD, Methods that include...

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

17. A chimeric protein as defined in any one of claims 1 to 11 or a polypeptide as defined in claim 12, for use in pharmaceuticals.

18. A chimeric protein as defined in any one of claims 1 to 11 or a polypeptide as defined in claim 12 for use in the treatment of said cancer and / or inflammatory diseases, preferably the cancer being a solid tumor and / or a humoral tumor.

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