Anti il-6 domain antibodies, pharmaceutical compositions comprising the same and use thereof

Abstract

This invention relates to compounds (e.g., ISVD, polypeptides, polypeptide derivatives) capable of binding interleukin-6 (IL-6), and their use in the treatment of inflammatory diseases such as cardiovascular diseases (CVD).

Description

Technical Field

[0001] This invention relates to compounds capable of binding interleukin-6 (IL-6) and their use in treating inflammatory diseases such as cardiovascular disease (CVD). Prior Technology

[0002] Cardiovascular disease (CVD) is a major global health burden and one of the leading causes of death worldwide. Despite advances in medical and preventative measures, the global prevalence of cardiovascular disease continues to rise, placing immense pressure on healthcare systems and the economy. Therefore, there is an urgent need to develop new therapies to more effectively prevent and treat CVD. The development of new therapies could potentially extend the lives of millions and help mitigate the social and economic impacts of cardiovascular disease.

[0003] Over the past 40 years, the most successful pharmacological approach to preventing cardiovascular disease has been actively lowering low-density lipoprotein (LDL). Lowering LDL can be achieved through lifestyle modifications such as regular exercise and following a diet rich in fruits, vegetables, whole grains, lean protein, and low in saturated and trans fats, cholesterol, and sodium, as well as / or medication. Typical LDL-lowering medications include statins, proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors, and bile acid sequestrants.

[0004] IL-6 is a multifunctional cytokine that plays a crucial role in mediating various immune responses and inflammatory processes (such as acute-phase protein upregulation or T-cell differentiation) through transient upregulation in response to infection or tissue damage. Dysregulation of IL-6 signaling is associated with the pathogenesis of a variety of diseases, including autoimmune diseases, cancer, and chronic inflammatory symptoms. IL-6 is also associated with CVD through its role in inflammation, which plays a vital role in the occurrence and progression of CVD. IL-6 can promote the production of other pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin-1β (IL-1β), which may contribute to endothelial dysfunction, plaque formation, and atherosclerosis—all hallmarks of CVD.

[0005] Targeting IL-6 has been explored as a potential therapeutic strategy for a variety of diseases, including rheumatoid arthritis, multiple myeloma, Castleman's disease, and COVID-19. One approach is to block the IL-6 receptor, which is responsible for downstream effects of IL-6 signaling. This can be achieved using monoclonal antibodies, such as tocilizumab and sarilumab, which have been approved for the treatment of rheumatoid arthritis. Another approach is to inhibit the production or activity of IL-6 through small molecule inhibitors. Although antibodies have shown therapeutic efficacy, they also have limitations, including high production costs, immunogenicity, and the need for parenteral administration. Small molecule inhibitors targeting IL-6 signaling components suffer from drawbacks such as off-target effects and limited selectivity.

[0006] Therefore, there is still a need for novel compounds that can overcome one or more limitations associated with existing treatments and allow for the effective treatment of inflammatory and / or cardiovascular diseases by targeting the IL-6 pathway. Summary of the Invention

[0007] This invention relates to highly potent anti-IL-6 compounds. As reported herein, the inventors have successfully manufactured several IL-6 antigen-binding molecules that possess properties suitable for development into therapeutic products, particularly oral therapeutic agents.

[0008] The inventors have designed anti-IL-6 compounds with robust biochemical and biophysical properties suitable for clinical-scale production (including expression, purification, and formulation). Furthermore, the compounds disclosed herein are humanizable, thus minimizing the risk of immunogenicity in human therapy.

[0009] Various aspects of the present invention are set forth below and / or in the appended claims, and other embodiments and preferred features of the invention are described below.

[0010] The object of this invention is to provide compounds (e.g., immunoglobulin single variable domain (ISVD), peptides, peptide derivatives) that can combat inflammatory diseases such as CVD, having improved preventive, therapeutic, and / or pharmacological properties, preferably possessing other advantageous properties compared to prior art (such as improved ease of preparation, good stability, improved bioavailability, improved potency, improved specificity, improved half-life, and / or reduced product cost). Specifically, this invention aims to provide ISVDs capable of binding IL-6, peptides containing them, and derivatives of such peptides.

[0011] ISVDs from different screening candidates (such as VHH) were isolated and further engineered to have a variety of advantageous characteristics, including increased bioavailability, stability, affinity and / or inhibitory activity.

[0012] In particular, the inventors have been able to develop anti-IL-6 compounds suitable for oral administration. Developing anti-IL-6 compounds suitable for oral administration is not easy; it requires developing compounds that significantly improve properties such as bioavailability, potency, half-life, and / or isoelectric point (pI), or achieving an appropriate balance in enhancing the desired function. Specifically, the inventors have been able to develop anti-IL-6 compounds suitable for oral administration due to improved bioavailability, potency, half-life, and / or isoelectric point (pI). The polypeptide derivatives described herein possess high potency, providing a sufficiently long half-life and sufficient bioavailability to allow for effective oral administration.

[0013] In the first embodiment, the present invention relates to an ISVD capable of binding IL-6. Preferably, the ISVD of the present invention is a separated ISVD. Therefore, the ISVD of the present invention may contain IL-6 binding sites. One example of an ISVD is VHH. The ISVD of the present invention may contain frame regions, such as FR1, FR2, FR3, FR4, and said frame regions may be interrupted by complementarity-determining regions (CDRs), such as CDR1, CDR2, CDR3.

[0014] In the second state, the present invention relates to a polypeptide comprising an ISVD of the first state of the present invention and further comprising an extension (such as a C-terminal extension). The length of the C-terminal extension can be from 1 to 20 amino acids, such as from 1 to 6 amino acids. The C-terminal extension may comprise one or more cysteines and / or one or more lysines. The C-terminal extension can be adapted for further derivatization. Therefore, a polypeptide of the second state according to the present invention may comprise an ISVD of the first state of the present invention, which has an extension fused at its C-terminus.

[0015] In the third state, the present invention relates to a polypeptide derivative comprising an ISVD of the first state of the present invention or a polypeptide of the second state of the present invention, and further comprising one or more substituents. The polypeptide derivative may contain two substituents. The substituent may be conjugated to a suitable amino acid moiety, such as a lysine or cysteine ​​functional group. The substituent may be attached to the C-terminal extension. Compared to the corresponding polypeptide or ISVD, the polypeptide derivative may have an extended in vivo half-life.

[0016] In the fourth state, the present invention relates to an antibody or antibody fragment or antibody derivative or polypeptide derivative, wherein the antibody or antibody fragment or antibody derivative or polypeptide derivative is capable of binding to IL-6 at an antigenic epitope containing at least mature human IL-6, such as amino acid residues 26, 30, 33, 34, 73, 74, 75, 78, 171, 175, 178, 179, 182 and 183 as shown in SEQ ID NO:89.

[0017] In the fifth state, the present invention relates to a nucleic acid molecule, preferably in a separated form, which encodes the ISVD of the first state of the present invention or the polypeptide of the second state of the present invention.

[0018] In the sixth state, the present invention relates to an expression vector comprising a nucleic acid molecule of the fifth state of the present invention.

[0019] In the seventh state, the present invention relates to a host cell carrying an expression vector of the sixth state of the present invention.

[0020] In the eighth state sample, the present invention relates to a method for producing an ISVD of the first state sample of the present invention, a polypeptide of the second state sample of the present invention, or a polypeptide derivative of the third state sample of the present invention, comprising the following steps: a. culturing host cells of the seventh state sample of the present invention under conditions that allow expression of the ISVD of the first state sample of the present invention or the polypeptide of the second state sample of the present invention; b. recovering the ISVD or polypeptide obtained in step a; and / or optionally c. attaching one or more substituents capable of prolonging the half-life of the polypeptide, optionally wherein each substituent is of chemical formula 24; and / or optionally d. purifying the ISVD or polypeptide or polypeptide derivative thus obtained.

[0021] In the ninth state, the present invention relates to a pharmaceutical composition comprising the ISVD of the first state of the present invention, or the polypeptide of the second state of the present invention, or the polypeptide derivative of the third state of the present invention.

[0022] In the tenth state sample, the present invention relates to the ISVD of the first state sample of the present invention, or the polypeptide of the second state sample of the present invention, or the polypeptide derivative of the third state sample of the present invention, or the pharmaceutical composition of the ninth state sample, for use in a pharmaceutical. In some embodiments, the present invention relates to the ISVD of the first state sample of the present invention, or the polypeptide of the second state sample of the present invention, or the polypeptide derivative of the third state sample of the present invention, or the pharmaceutical composition of the ninth state sample, for use in treating inflammatory diseases. In some embodiments, the present invention relates to the ISVD of the first state sample of the present invention, or the polypeptide of the second state sample of the present invention, or the polypeptide derivative of the third state sample of the present invention, or the pharmaceutical composition of the ninth state sample, for use in treating cardiovascular diseases.

[0023] In another state, the present invention relates to a single component (intermediate) ISVD, which is a portion of an ISVD polypeptide derivative or a VHH polypeptide derivative according to the first state, such as a specific anti-IL-6 VHH fragment or a specific anti-IL-6 VHH fragment thereof.

[0024] The polypeptide derivatives described herein are highly potent, provide a sufficiently long half-life, and / or sufficient bioavailability to allow for effective subcutaneous and oral administration.

[0025] Other forms, advantages, applications, and uses of the polypeptide and its components will become clearer from further disclosure herein. Several references are cited throughout this specification. Each reference cited herein (including all patents, patent applications, scientific literature, manufacturer's specifications, instructions, etc.), whether preceding or following, is incorporated herein in its entirety by reference. Nothing herein should be construed as an admission that the invention is not entitled to any prior invention prior to such disclosure. Simple Explanation of the Diagram

[0026] [Figures 1A-C] show non-limiting examples of anti-IL-6 peptide derivatives containing a substituent. In the embodiments shown in Figures 1A and 1B, an extension is also provided at the C-terminus or N-terminus of the ISVD, respectively. Dashed lines indicate disulfide bridges.

[0027] [Figures 1D-F] show non-limiting examples of anti-IL-6 peptide derivatives, where the substituent is an elongated portion. "E" - elongated segment; "PM" - elongated portion. Dashed lines indicate disulfide bridges.

[0028] [Figure 2] shows a non-limiting example of an anti-IL-6 peptide derivative containing a single substituent. Dashed lines represent disulfide bonds. Hydrogen atoms have been omitted for clarity.

[0029] [Figure 3A] shows a detailed illustrative description of compound 9, representing a non-limiting example of an anti-IL-6 polypeptide derivative. Dashed lines indicate disulfide bonds. Hydrogen atoms have been omitted for clarity.

[0030] [Figure 3B] shows a non-limiting illustrative description of compound 9 as shown in Figure 3A, providing details of the construct. In this embodiment, the extension is provided at the C-terminus of VHH, comprising the amino acid residue shown in SEQ ID No. 72, and is... The dashed box indicates that each of the two substituents (formula 24) is labeled as substituent 1 and substituent 2, respectively, and is marked with a dashed box. The extended portion is indicated by a dashed box. The dashed box indicates that this connection sub-element is... The dashed box indicates a disulfide bond. For clarity, hydrogen atoms have been omitted.

[0031] [Figure 4A] shows the molecular structure of the complementary site:epitope interaction between SEQ ID NO:57 and human IL-6 (hIL-6), as obtained from the X-ray crystallography experiments described in Example 2.

[0032] [Figure 4B] shows the molecular structure of the complementary site:epitope interaction portion of VHH peptides 19, 33, and 35 with human IL-6. Amino acid residues belonging to one of these VHH peptides are indicated by an asterisk "*", e.g., Trp 58 / 59. The remaining numbered amino acid residues belong to hIL-6.

[0033] [Figure 5] shows exemplary peptides containing anti-IL6 VHH and structural representations stabilized via salt bridges generated by mutations at positions 54, 56, 62, and 106. The description of each of these chemical formula representations identifies the observed anti-IL6 VHH residues. Implementation [Incorporation of Sequence List References]

[0034] This application is submitted electronically as a sequence list. The entire contents of the sequence list are hereby incorporated by reference.

[0035] There remains a need for safe and effective drugs to treat inflammatory diseases such as cardiovascular diseases. One object of this invention is to provide compounds that combine the advantageous properties of antibody drugs and small molecule drugs. For example, antibody drugs are generally able to target a broader range of antigens and receptors that are inaccessible or difficult to reach by peptides or small molecules, and often produce higher efficacy and specificity. On the other hand, small molecules can be administered orally, which is a more convenient route of administration for patients.

[0036] Therefore, one object of the present invention is to provide a highly potent compound capable of binding IL-6, having sufficient bioavailability to allow oral administration and providing a sufficiently long half-life to allow once-daily and optionally once-weekly dosing. Furthermore, alternatively, the compounds shown herein are suitable for subcutaneous injection.

[0037] This article discloses compounds capable of binding IL-6. The compounds disclosed herein exhibit high in vivo potency. For example, the in vitro potency of the compounds disclosed herein can be between 1-50 pM, such as between 5-35 pM, such as between 10-20 pM, optionally as disclosed in Example 8: Reporter Gene Assay and SPR Experiment in Section I. One example of a method for measuring potency is reporter gene assay using a recombinant cell line expressing the IL-6 and gp130 receptor, wherein IL-6 activation of the receptor leads to phosphorylation of the signal transducer and activator of transcription-3 (STAT-3) and subsequent transcription of a luciferase reporter gene with a STAT-3 response element, which can be tracked using a suitable luciferase receptor, such as a bioluminescence assay of oxidative luciferin conversion. In vitro potency can be measured as disclosed in Example 8: Reporter Gene Assay and SPR Experiment, and can be seen in the Reporter Gene Assay (RGA) section of Materials and Methods in Section I.

[0038] In addition, or alternatively, the compounds disclosed herein exhibit extended half-lives (reducing the required dosing frequency). Conveniently, compounds with extended moieties have significantly extended half-lives compared to the same compounds without the extended moiety. For example, VHH polypeptide derivatives have longer half-lives than the corresponding VHH itself. The extended half-lives are achieved by introducing substituents with extended moieties / extended components, such as fatty acid conjugation, Fc domains, FcRn-binding peptides, Fc-binding peptides, and albumin-binding peptides. The extended in vivo half-lives of substituented compounds (e.g., polypeptide derivatives or extended compounds) have been confirmed in animal models such as rats, dogs, and pigs. Unextended compounds, such as ISVDs or polypeptides, show very rapid clearance in dogs and pigs.

[0039] In addition or alternatively, the compounds disclosed herein have been engineered to be capable of oral administration and are also suitable for, for example, parenteral administration.

[0040] Conveniently, the compounds disclosed herein may have an isoelectric point between 3 and 7, such as between 3.5 and 6, such as between 4 and 5 (including the endpoints of the range). [Definitions]

[0041] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which will be clear to those skilled in the art.

[0042] Unless otherwise stated, all methods, steps, techniques, and operations not specifically described in detail can be performed and have been performed in a manner known per se, which will be clear to those skilled in the art.

[0043] Greek letters may be presented by their symbols or corresponding written names, for example: α = alpha; β = beta; ε = epsilon; γ = gamma; ω = omega; etc. In addition, the Greek letter μ may be represented by "u", for example μl = ul, or μM = uM.

[0044] The asterisk (*) in a chemical formula / structure or can be used to refer to a point of attachment.

[0045] It must be noted that, as used herein, the singular forms " [a]" and " [an]" and " [the]" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes one or more such different reagents, and reference to "the method" includes equivalent steps and methods herein known to those of ordinary skill in the art that may be modified or substituted. Unless otherwise stated, the term " [at least one]" preceding a series of elements should be understood to refer to each element in the series.

[0046] Those skilled in the art will recognize or be able to use, without more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

[0047] The term "" is used wherever it is used in this article "[and / or]" includes the meaning of "and", "or" and "all or any other combination of the elements connected by the term".

[0048] the term" "[comprise]" and its variations, such as "comprises, comprising", will be understood to imply the inclusion of the stated integers or elements or steps, or groups of integers or elements or steps, but does not exclude any other integers or elements or steps, or groups of integers or elements or steps. When used herein, the term "comprise" may be replaced by the terms "containing" or "including" or sometimes "having".

[0049] The term "" used in this article "Approximately" means roughly, roughly, or roughly. When the term "approximately" is used in conjunction with a range of values, it adjusts the range by extending the boundaries above and below the stated value. Generally, the term "approximately" can modify a value that is 20% higher or lower than the stated value (up or down), preferably 15% (up or down), more preferably 10% (up or down), and most preferably 5% (up or down).

[0050] the term" "[Affinity]" indicates the strength or stability of molecular interactions. Affinity is usually expressed as KD or the dissociation constant, with units of moles per liter (or M). Affinity can also be expressed as the binding constant KA, which is equal to 1 / K, with units of (moles per liter)⁻¹ (or M⁻¹). Here, the stability of an interaction between two molecules is primarily represented by the KD value of their interaction; those skilled in the art will understand that the relationship KA = 1 / KD can indicate the strength of the molecular interaction through its KD value, and can also be used to calculate the corresponding KA value. The thermodynamic significance of the KD value also represents the strength of the molecular interaction, as it is related to the change in binding free energy (ΔG), through the known relationship ΔG = RTIn(KD) (equivalent to ΔG = -RTln(KA)), where R is equal to the gas constant, T is equal to the absolute temperature, and In represents the natural logarithm. The KD of biological interactions considered meaningful (e.g., specific) typically ranges from 10⁻¹² M (0.001 nM) to 10⁻⁵ (10000 nM). The stronger the interaction, the lower its KD. KD can also be expressed as the ratio of the dissociation rate constant (denoted as koff) to its binding rate constant (denoted as kon) (therefore, KD = koff / kon, and KA = kon / koff). The unit of the dissociation rate constant koff is s⁻¹ (where s is the SI unit for seconds). The unit of the binding rate constant kon is M⁻¹s⁻¹. The binding rate can vary from 10² M⁻¹s⁻¹ to approximately 10⁸ M⁻¹s⁻¹, approaching the diffusion-restricted binding rate constant of bimolecular interactions.

[0051] The specific binding of antigen-binding peptides such as ISVD to an antigen or antigen determinant can be determined by any suitable method known in itself, including, for example, saturation binding assays and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assays, as well as various variations thereof known in the art; and other techniques mentioned herein.

[0052] The affinity of molecular interactions between two molecules can be measured using various techniques known per se, such as the known surface plasma resonance (SPR) biosensor technique (see, for example, Ober et al., 2001, Intern. Immunology 13:1551-1559), in which one molecule is immobilized on a biosensor chip, and another molecule flows through the immobilized molecule under flow conditions, generating kon, koff measurements, and thus KD (or KA) values. This can be performed, for example, using the known BIACORE™ instrument (Cytiva). Kinetic exclusion assays (KINEXA®) (Drake et al., 2004, Analytical Biochemistry 328:35-43) measure binding events in solution without labeling the binding ligand and are based on kinetic exclusion of the dissociation of the complex. In-solution affinity analysis can also be performed using the GYROLAB® immunoassay system, which provides automated bioanalysis and a rapid sample rotation platform (Fraley et al., 2013, Bioanalysis 5:1765-74). Those skilled in the art are aware that the measured KD corresponds to the apparent KD, and the measurement process can influence the intrinsic binding affinity of the indicator molecule in some way, such as through human factors related to the coating of a molecule on a biosensor. Furthermore, if a molecule contains more than one recognition site of another molecule, the apparent KD can be measured. In this case, the measured affinity may be affected by the strong binding of the interaction between the two molecules. In particular, accurate measurement of KD can be quite labor-intensive; therefore, apparent KD values ​​are typically measured to assess the binding strength of the two molecules. It should be noted that as long as all measurements are performed in a consistent manner (e.g., keeping the measurement conditions constant), the apparent KD measurement can be used as an approximation of the true KD; therefore, in this invention, KD and apparent KD should be of equal importance or correlation.

[0053] Amino acids are molecules containing an amino group and a carboxylic acid group, and optionally one or more additional groups commonly referred to as side chains. The term "amino acid" is used in this context. The term "[Amino Acids]" includes both quasi-amino acids (which are genetically encoded) and non-natural amino acids. Non-limiting examples of non-natural amino acids include Aib (α-aminoisobutyric acid), deaminohistidine (alternative name 3-(imidazol-4-yl)propionic acid, abbreviated as Imp (imidazolyl)), and d-isomers of quasi-amino acids. All amino acid residues within polypeptides for which optical isomers are not specified should be understood herein to refer to the l-isomer (unless otherwise indicated). Amino acid residues will be represented according to standard three-letter or one-letter amino acid codes.

[0054] The term used in this article is " "[Amino acid differences]" refers to the substitution or deletion of amino acids.

[0055] the term" "Antibody" in this document refers to a protein containing or derived from an immunoglobulin sequence that can bind to an antigen or a portion thereof. "Antibody" includes (but is not limited to) full-length antibodies containing at least four polypeptide chains: two heavy chains (HC) and two light chains (LC) linked by disulfide bonds; antibodies containing at least three polypeptide chains: two heavy chains (HC) and one light chain (LC) linked by disulfide bonds; and recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, etc.). One type of immunoglobulin is IgG. In humans, IgG types can be divided into four subtypes—IgG1, IgG2, IgG3, and IgG4—based on their heavy chain constant region sequences. Light chains can be divided into two types based on their sequence composition: κ and λ chains. An IgG molecule consists of two heavy chains (linked together by two or more disulfide bonds) and two light chains (each attached to a heavy chain by one disulfide bond). To avoid confusion, the term "antibody" also includes single-domain antibodies (sdAb).

[0056] the term" [Antigen binding site] and " The term "[antigen-binding domain]" is used interchangeably and should be understood as referring to a region of an antibody that can specifically bind to an antigen. This antigen-binding domain does not need to be in the context of the entire antibody; for example, it can be isolated (e.g., a domain-specific antibody) or present in another form, such as, as described herein, a polypeptide containing an ISVD. For example, "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments (e.g., Fab, Fab', F(ab')2, scFV, di-scFv) contain two immunoglobulin domains, particularly two variable domains, which interact to form an antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen-binding site. In this case, the complementarity-determining regions (CDRs) of the VH and VL contribute to the antigen-binding site; a total of six CDRs will participate in the formation of the antigen-binding site. An immunoglobulin single variable domain (ISVD) can specifically bind to an epitope of an antigen without pairing with additional immunoglobulin variable domains. The binding site of this immunoglobulin's single variable domain is formed by a single VH, a single VHH, or a single VL domain. Therefore, the single variable domain can be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof, as long as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0057] Immunoglobulin single variable domain (ISVD), antibody, polypeptide, or usually antigen-binding protein or antigen-binding polypeptide or fragment thereof, which can " [Combined to]” or “Can be” [Specifically binds to]”, "[Has an affinity for...]" and / or an affinity for a particular epitope, antigen, or protein (or at least a portion, fragment, or epitope thereof) "[Having specificity]" is considered to be " [Targeting] or " The epitopes, antigens, or proteins mentioned in the "[Guideline]", or those related to such epitopes, antigens, or proteins, are referred to in the "[Guideline]". [Binding] molecules, or referred to as "anti" epitopes, "anti" antigens, or "anti" proteins. For example, in the context of targeting interleukin-6 (IL-6), the IL-6-binding construct can be called "anti"-IL-6.

[0058] As used in this article, " "Conservative substitution" refers to situations where an amino acid can be substituted with another amino acid that has similar biochemical properties. For example, a basic amino acid can be substituted with another basic amino acid (e.g., lysine is substituted with arginine), an acidic amino acid can be substituted with another acidic amino acid (e.g., glutamic acid is substituted with aspartate), a neutral amino acid can be substituted with another neutral amino acid (e.g., threonine is substituted with serine), and a charged amino acid can be substituted with another charged amino acid (e.g., glutamate is substituted with aspartate). Hydrophilic amino acids can be substituted with another hydrophilic amino acid (e.g., asparagine is substituted with glutamine), hydrophobic amino acids can be substituted with another hydrophobic amino acid (e.g., alanine is substituted with valine), polar amino acids can be substituted with another polar amino acid (e.g., serine is substituted with threonine), aromatic amino acids can be substituted with another aromatic amino acid (e.g., phenylalanine is substituted with tryptophan), and aliphatic amino acids can be substituted with another aliphatic amino acid (e.g., leucine is substituted with isoleucine).

[0059] the term [“(cross)-block”, [(cross)-blocked], [(cross)-blocking)]”, “competitive combination”, “(cross)-competition” [(cross)-compete], [(cross)-competing] and The term "[(cross)-competition)" is used interchangeably to refer to the ability of an immunoglobulin, antibody, ISVD, peptide, or other binding agent to interfere with the binding of other immunoglobulins, antibodies, ISVDs, peptides, or binding agents to a given target. The extent to which an immunoglobulin, antibody, ISVD, peptide, or other binding agent can interfere with the binding of another to a target, and whether this constitutes cross-blocking as described in this invention, can be determined using competitive binding assays, which are common in the art, such as screening for purified ISVDs displayed on bacteriophages in a competitive ELISA. As defined herein, methods for determining whether a target-cross-blocking immunoglobulin, antibody, immunoglobulin single variable domain, peptide, or other binding agent can cross-block, competitively bind, or cross-compete are described in, for example, the methods of Xiao-Chi Jia et al. (Journal of Immunological Methods 288;91-98, 2004), Miller et al. (Journal of Immunological Methods 365;118-125, 2011), and / or those described herein.

[0060] The term used in this article is " "[Derivative]" refers to a chemically modified polypeptide having substituents covalently linked to the amino acids of that polypeptide. Generally, a polypeptide derivative refers to a polypeptide containing an ISVD, wherein the polypeptide has substituents. For example, the substituent may be covalently linked to a thiol group of cysteine. The resulting construct (i.e., the polypeptide with substituents) can be called a polypeptide derivative.

[0061] The term used in this article is " [EC]

[50] refers to the concentration required to induce 50% of the maximum potency (i.e., the usual pharmacological or therapeutic potency). The term "

[50] " as used herein refers to the concentration required to induce 50% of the maximum potency. [ED]

[50] refers to a dose that induces 50% of the pharmacological or therapeutic efficacy. Typically, EC50 / ED50 measurements are performed in pharmacological in vivo models, while IC50 measurements can be performed in vitro and in vivo.

[0062] As used in this article, the term " "Epitope" refers to a specific binding site on an antigen or antigenic structure such as an ISVD, or a polypeptide containing an ISVD, or a polypeptide derivative containing an ISVD, which has specificity and affinity. Epitopes are typically composed of surface elements of molecules (such as amino acids or sugar side chains) and usually have specific three-dimensional structural characteristics and specific charge characteristics. The difference between conformational and non-conformational epitopes is that binding to the former is lost in the presence of a denaturing agent that can destroy the structure of the protein, while binding to the latter is not. Epitopes can be linear epitopes, conformational epitopes, or hybrid epitopes. The term "epitope" can also be used to refer to structural epitopes. According to some embodiments, structural epitopes can be used to describe antigenic regions covered by antibodies (e.g., antibody footprints when binding to antigens). In some embodiments, structural epitopes can describe amino acid residues of the antigen within a specified proximity (e.g., within a specified angstrom number) of the amino acid residues of the antibody. The term "epitope" can also be used to refer to functional epitopes. According to some embodiments, functional epitopes can be used to describe the amino acid residues of an antigen that interact with the amino acid residues of an antibody in a manner that contributes to the binding energy between the antigen and the antibody. Epitopes can be determined according to different experimental techniques, also known as "epitaphing techniques." It should be understood that epitope determination can vary based on the different epitopeing techniques used, and can also vary with different experimental conditions used, such as antigen conformational changes or cleavage induced by specific experimental conditions.

[0063] As used in this article, " "[Excipients]" refers to inert substances added to pharmaceutical compositions to further facilitate the administration of the active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and starches, L-arginine, nicotinamide, SNAC, magnesium stearate, sodium stearate fumarate cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol.

[0064] As used in this article, " "[Extension]" (E) refers to a peptide or polypeptide suitable for attachment to an ISVD. The extension may be several amino acids, 1 to 10 amino acids, or longer, 10 to 30 amino acids, or very long, exceeding 30 amino acids. The extension is preferably located at the C-terminus (C-terminal extension) of the ISVD, ISVD polypeptide, ISVD polypeptide derivative, VHH, VHH polypeptide, or VHH polypeptide derivative. The extension is preferably recombinantly fused to the ISVD. In other embodiments, the extension is recombinantly expressed together with the ISVD. In other embodiments, the extension is conjugated with the ISVD in any suitable manner, such as by chemical conjugation. In other embodiments, the extension may be attached to the ISVD via other means, such as click chemistry. In some embodiments, the extension is attached to the ISVD as its C-terminal extension or its N-terminal extension. It should be understood that the resulting construct of an ISVD having an extension (e.g., a C-terminal extension) can be broadly referred to as a polypeptide, or more specifically, a polypeptide comprising an ISVD. In some embodiments, the extension is provided as a structure linked to any residue between the C-terminus and N-terminus of the ISVD, for example by yenolation. In some embodiments, the purpose of the extension is to serve as a linking site for one or more substituents (which may further comprise or be an extension portion), for example via one or more cysteines. A non-limiting example of such an extension is GQACPC (SEQ ID NO: 72). For example, in a non-limiting embodiment, the ISVD is provided as GQACPC (SEQ ID NO: 72) with a C-terminal extension amino acid residue, thus forming a polypeptide comprising the ISVD, wherein the cysteines at positions 4 and 6 of SEQ ID No. 72 may each individually serve as linking sites for substituents.

[0065] the term" "[Fatty acid]" refers to a carboxylic acid that is optionally substituted with an aliphatic chain, wherein the aliphatic chain may be saturated or unsaturated. Non-limiting examples of fatty acids are C12-C24 carboxylic acids.

[0066] " The "[frame]" or "FR" region is a variable domain region other than the highly variable residues. Therefore, the compounds disclosed in this paper include FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains from the N-terminus to the C-terminus.

[0067] As used in this article, " "[Free cysteine]" refers to a cysteine ​​residue in a polypeptide that can be reacted (e.g., chemically conjugated), and is therefore not part of a natural or engineered internal disulfide bridge. In essence, free cysteine ​​can be used for conjugation, although free cysteine ​​residues (including recombinantly introduced free cysteine) are often blocked by small thiols (such as cysteine, homocysteine, or glutathione) during the recombinant expression of the polypeptide in the host cell. The introduction of one or more free cysteine ​​residues can also be observed in the recombinant production of ISVD polypeptides. Therefore, reduction reactions can be carried out using suitable reducing agents such as disulfide reducing agents. Non-limiting examples of reducing agents are bis(p-sulfophenyl)phenylphosphine dihydrate or tris(2-carboxyethyl)phosphonate, which can be used to release and prepare free cysteine ​​for conjugation with the moiety of interest, such as, but not limited to, substituents (which may contain or be an elongated moiety).

[0068] The half-maximal inhibitory concentration (IC50) is a measure of a compound's ability to inhibit biological or biochemical functions, such as pharmacological effects. This quantitative measurement shows how much ISVD (e.g., VHH peptide) is required to inhibit half of a given biological process (or a component of a process, such as an enzyme, cell receptor, chemotaxis, degeneration, metastasis, invasion, etc.). In other words, it is the half-maximal (50%) inhibitory concentration (IC) of a substance (50% IC or IC50). For a given antagonist, such as the peptide or ISVD of the present invention (e.g., Nanobody™), its IC50 value can be calculated by determining the concentration required to inhibit half of the agonist's maximum biological response. The IC50 of a drug can be determined by establishing a dose-response curve and examining the effect of different concentrations of the antagonist (such as the ISVD of the present invention (e.g., VHH peptide)) on reversing the agonist's activity.

[0069] " The "[HPC4-tag]" refers to a 12-amino acid sequence (SEQ ID NO: 87) derived from protein C, which is a vitamin K-dependent pro-plasma zymase. Protein C is activated by proteolytic cleavage of the thrombin-thrombomodulin complex, forming antithrombin. This peptide can be expressed and detected along with the protein of interest in either an amino-terminal or carboxyl-terminal fused form.

[0070] As used in this article, " "[Host cell]" includes any type of cell system that can be engineered to generate the ISVDs disclosed herein. Host cells include (but are not limited to) cultured cells, such as mammalian cultured cells, such as CHO cells, HEK293T cells, HEK EXPI cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, fungal cells, and insect cells.

[0071] The term used in this article is " "[Water-soluble adjuvants]" refers to compounds that can increase the solubility of poorly soluble drugs in solid form. Water-soluble adjuvants can be added during the manufacturing process to improve the bioavailability of the drug after administration. These substances work by enhancing the solubility of the drug in gastric juice, thereby promoting its absorption across the intestinal wall. Non-limiting examples of water-soluble adjuvants include urea, nicotinamide, and pyridoxine hydrochloride.

[0072] As used in this article, " The term "highly variable region" refers to the amino acid residues in an antibody responsible for antigen binding. The highly variable region includes the "complementarity-determining region," also known as the "CDR."

[0073] The term "known in this art" "[Conformity]" refers to the relationship between two or more polypeptide sequences as determined by comparing sequences. In this art, "conformity" also means the degree of sequence relevance between polypeptides as determined by the number of matches between two or more amino acid residue strings. "Conformity" measures the percentage of identical matches among two or more sequences having the smaller of a gap alignment (if any) resolved by a specific mathematical model or computer program (i.e., an "algorithm"). The conformity of related polypeptides can be easily calculated using known methods.

[0074] " [IL-6] refers to interleukin-6. IL-6 may also be referred to as the "antigen" herein. The full-length amino acid sequence of hIL-6 is shown in SEQ ID NO:88. This sequence is cleaved in vivo to remove the N-terminal lead peptide, thereby producing mature IL-6. Mature hIL-6 has the amino acid sequence shown in SEQ ID NO:89. This mature sequence represents circulating IL-6 in vivo, which is a target antigen for the therapeutic and in vivo diagnostic applications described herein. Therefore, unless the context otherwise requires, IL-6 referred to herein is mature hIL-6. IL-6 may be fused with portions used for detection and / or purification and / or immobilization, such as His-tags or HPC4-tags, for example, in the assays described herein.

[0075] the term" [Immunoglobulin Single Variable Domain (ISVD)] and " [Single variable domain] or " The term "[single-domain antibody]" is used interchangeably to refer to an immunoglobulin molecule in which the antigen-binding site is located on a single immunoglobulin domain and is formed by that single immunoglobulin domain. An immunoglobulin single variable domain (ISVD) may, for example, comprise or consist of a heavy chain ISVD, such as VH, VHH, including camel VHH or humanized VHH. Preferably, it is a VHH, including camel VHH or humanized VHH. Heavy chain ISVDs may be derived from conventional four-chain antibodies or heavy chain antibodies. The term "ISVD" includes, but is not limited to, the variable domain (VHH) of camel heavy chain antibodies, domain antibodies (dAB), and shark-derived ISVDs (IgNAR domain). Similar to conventional antibodies, ISVDs can selectively bind to a specific antigen. Typically, ISVDs have a molecular weight of only 12-18 kDa, making them much smaller than conventional antibodies. For comparison, conventional antibodies typically have a molecular weight of approximately 120-160 kDa. Generally, ISVD has an amino acid sequence containing four frame regions (FR1, FR2, FR3, FR4) and three complementarity-determining regions (CDR1, CDR2, CDR3), preferably having the following formula (Formula I): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (Kabat definition).

[0076] As used in this article, the abbreviation " [V] [H] [H]" represents the variable heavy chain structural domain of the heavy chain.

[0077] Regarding CDRs, as is well known in the art, there are several conventional definitions and descriptions of CDRs for VH or VHH fragments, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the location of the structural circular region). For example, please refer to http: / / www.bioinf.org.uk / abs / .

[0078] In this paper, the CDR sequences of ISVDs (such as the VHH fragment) are determined using the Kabat definition (Kontermann and Dübel, 2010, Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, the CDRs of the variable domains are defined at positions 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3). However, it has been found that the boundaries of the CDR and FR regions may differ from the specified boundaries. However, when referring to specific amino acid residue positions (including CDRs and FRs) in the compounds described herein, consecutive numbering is used unless otherwise specified.

[0079] As used in this article, " [Isoelectric point] or " "[pI]" refers to the pH value at which the total net charge of a protein (such as an antibody) is zero. Proteins may contain many charged groups, and at the isoelectric point, the sum of all these charges is zero. Above the isoelectric point, the total net charge of the protein is negative, while below the isoelectric point, the total net charge is positive. pI can be theoretical or experimentally determined. Those skilled in the art know the methods for determining the isoelectric point of a protein. Most commonly, the isoelectric point of a protein is calculated based on its amino acid sequence. Many online tools exist for determining the isoelectric point of proteins, such as "ExPASy Compute pI / Mw," see Protein Identification and Analysis Tools on the ExPASy Server; Gasteiger E., Hoogland C., Gattiker A., ​​Duvaaud S., Wilkins MR, Appel RD, Bairoch A.; (In) John M. Walker (ed.): The Proteomics Protocols Handbook, Humana Publishing (2005), pp. 571-607. Preferably, pI is calculated using the algorithm of Skoog & Wichman, 1986, and the pKa of amino acid residues. pI can also be determined experimentally, for example, by using charge-based separation techniques such as isoelectric focusing (IEF) gel electrophoresis and capillary isoelectric focusing (cIEF) gel electrophoresis to separate charge variants.

[0080] As used in this article, the term " "[Isolated]" refers to material removed from its source or original environment (e.g., natural environment if it is naturally occurring). For example, naturally occurring polynucleotides or polypeptides present in living animals are not isolated, but the same polynucleotides or polypeptides isolated from some or all of the coexisting material in the natural system through human intervention are isolated. Such polynucleotides may be part of a carrier and / or such polynucleotides or polypeptides may be part of a composition, and are still isolated because such carriers or compositions are not part of the environment in which they are found in nature. A nucleic acid or amino acid sequence is considered "[isolated]". "[(substantially) separated (form)]" - for example, compared to the reaction medium or culture medium from which it was obtained - when it is separated from at least one other component typically associated with the said source or medium (such as another nucleic acid, another protein / peptide, another biological component or macromolecule, or at least one contaminant, impurity or minor component). In particular, a nucleic acid or amino acid sequence is considered "(substantially) separated" when it has been purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold, and up to 1000-fold or higher. "(Substantially) separated" nucleic acids or amino acids are preferably substantially homogeneous, as determined using suitable techniques, such as suitable chromatography techniques, such as polyacrylamide-gel electrophoresis.

[0081] As used in this article, the term " "[Lipophilic portion]" refers to a portion that contains a total of more than 6 and less than 30 carbon atoms, preferably more than 8 and less than 20 carbon atoms in an aliphatic hydrocarbon portion.

[0082] As used in this article, " [Oral bioavailability] or " "[Oral bioavailability]" refers to the amount of drug administered orally (estimated by the area under the plasma concentration-time curve) relative to the amount of drug administered intravenously in the systemic circulation.

[0083] The portion of an antigen-binding molecule that identifies an antigen epitope (such as immunoglobulins, conventional antibodies, immunoglobulin single variable domain (ISVD), the polypeptide of the present invention, and / or the polypeptide derivative of the present invention) is referred to as "the part of the antigen-binding molecule that identifies the antigen epitope" [Complementary position]”.

[0084] The term " "Plasma half-life" refers to the time required for half of a substance administered to a patient to be metabolized or eliminated from the patient's serum or plasma through normal biological processes.

[0085] The term used in this article is " [Extended / Extended Part]”, “ [Extension moiety] "[protracting moiety]" refers to a portion having a half-life-extending property and is used interchangeably with the term "elongator" (P). Therefore, the term "extending" refers to the extension of half-life, and thus an elongator or elongation portion is intended to extend the half-life of the ISVD and / or ISVD peptide disclosed herein. The elongator or elongation portion is optionally linked to the ISVD or peptide via a "linker" (Lp). Such a linker (Lp) may consist of one or more "linker elements." In an embodiment where the linker (Lp) consists of a single linker element, the linker (Lp) and linker element are synonymous. In an embodiment where the linker (Lp) consists of a series or at least a portion of various linker elements, the "linker element" is therefore included within the linker (Lp). Each elongator or elongation portion is preferably linked to a lysine or cysteine ​​residue exposed on the surface of one of the polypeptide backbones of the compound. The linker point is typically referred to as R1 (or R2, R3, etc., if more than one extension is being linked, where R1 ≠ R2 ≠ R3, etc.). In this regard, the elongator / extension (P) and linker (Lp) can be provided in series, for example, forming P-Lp or Lp-P, or more specifically P-Lp-Rn or Rn-Lp-P (where n is an integer, referring to the linker points R1, R2, R3, etc. mentioned above). Those skilled in the art will be able to identify other surface-exposed residues suitable for attachment. In some embodiments, an elongator / extension (P) and linker (Lp) are provided, which are linked to an amino acid of a polypeptide (such as ISVD, VHH, ISVD polypeptide, VHH polypeptide, etc.). Each of the elongator / extension (P) and linker (Lp) can be individually referred to as a "substituent element," and the combination of the elongator / extension (P) and linker (Lp) can be referred to as a "substituent." In other words, and to avoid any doubt, a substituent having a half-life extension property may be referred to as one comprising an extension moiety. It should be understood that in some non-limiting embodiments, the terms elongator or extension moiety may be used interchangeably with "substituent". This may be, for example, an embodiment in which an elongator / extension moiety (P) is linked to an amino acid of a polypeptide without a linker (Lp), such that the substituent is composed of an elongator / extension moiety (P).

[0086] For the purpose of comparing two or more nucleotide sequences, the "..." between the first nucleotide sequence and the second nucleotide sequence The percentage of sequence identity can be calculated by dividing the number of identical nucleotides at corresponding positions in the first nucleotide sequence and 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 a nucleotide in the second nucleotide sequence (compared to the first nucleotide sequence) is considered a single nucleotide (position) difference. 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. Some other techniques, computer algorithms, and settings used to determine the degree of sequence identity are described in, for example, WO 04 / 037999, EP 0967284, EP 1085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768. Typically, to determine the percentage of "sequence identity" between two nucleotide sequences according to the calculation method outlined above, the nucleotide sequence with the most nucleotides is considered the "first" nucleotide sequence, and the other nucleotide sequence is considered the "second" nucleotide sequence. For the purpose of comparing two or more amino acid sequences, the percentage of "sequence identity" between the first and second amino acid sequences is... [Sequence Consistency] percentage (also referred to here as "Sequence Consistency") [Amino acid identity] can be calculated by dividing [the number of identical amino acids at corresponding positions in the first amino acid sequence and the second amino acid sequence] by [the total number of amino acids in the first amino acid sequence] and multiplying by [100%]. Each deletion, insertion, substitution, or addition of an amino acid in the second amino acid sequence (compared to the first amino acid sequence) is considered a difference in a single amino acid residue (position), i.e., as defined herein. [Amino acid differences] Alternatively, known computer algorithms can be used to calculate the degree of sequence similarity between two amino acid sequences, such as those used above to determine the degree of sequence similarity of nucleotide sequences, similarly using standard settings. Generally, regarding the percentage of "sequence similarity" between two amino acid sequences determined according to the calculation method outlined above, the amino acid sequence with the most amino acid residues will be considered the "first" amino acid sequence, and the other amino acid sequence will be considered the "second" amino acid sequence. In this invention, similarity and consistency are determined using the Needleman program (Needleman et al., J. Mol. Biol. 1970; 48: 443-453) from the software EMBOSS-6.6.0, and 10 and 0.5 are used as parameters for gap opening and gap extensions, respectively (gap open = 10, gap extend = 0.5).

[0087] As used in this article, the term " "[Substituent]" refers to a portion of an amino acid covalently attached to a polypeptide (e.g., ISVD, VHH, ISVD polypeptide, VHH polypeptide). If a substituent is attached to a polypeptide, the polypeptide is called "substituted." When a substituent is covalently attached to a polypeptide or amino acid residue, the polypeptide or amino acid is called "carrying" the substituent. A substituent may include a series of individually defined portions; these portions may be referred to as "substituent units." Non-limiting examples of "substituent elements" are "elongators" (or "elongated portions") and "linkers."

[0088] In some embodiments, the substituent may form a non-covalent interaction with albumin, thereby promoting the circulation of the compound in the bloodstream and thus having the effect of prolonging the time the compound is present in the bloodstream. This is because the agglomerate of albumin carrying the substituent only slowly disintegrates to release the compound in its free form. Therefore, the substituent, in general, may also be referred to as an "albumin-binding moiety," and the substituent may be referred to as having a "prolonging effect." The substituent may contain a portion particularly related to albumin binding and thus prolongation, which may be referred to as an "elongator" or "prolongation moiety." The terms "elongator" and "prolongation moiety" are used interchangeably herein. An "elongator" may be a lipophilic moiety (e.g., a fatty acid). An "elongator" may be a fatty acid (e.g., a C12-C22 carboxylic acid). Non-limiting examples of "elongators" are shown in Table 7. Non-limiting examples of substituents are shown in Table 7. Other examples of suitable substituents are described, for example, WO23139187A1, WO19016300A1, WO16102562, WO21231676A1, WO22029231, and WO23031455A1.

[0089] Unless otherwise stated, "binding IL-6" means binding mature hIL-6 (SEQ ID NO:89).

[0090] The term used in this article is " "[Variant]" refers to an ISVD or polypeptide that differs from the parent ISVD or polypeptide due to the deletion, substitution, or addition of one or more amino acids, but still retains one or more specific functions or biological activities of the parent ISVD or polypeptide. [Immunoglobulin Single Variable Domain (ISVD)]

[0091] In this first embodiment, the invention relates to an ISVD capable of binding to IL-6. The ISVD disclosed herein may be a separated ISVD. Preferably, the ISVD is a VHH, such as a humanized VHH.

[0092] In some embodiments, the ISVD can specifically bind to an epitope of IL-6, wherein the epitope comprises residues 26, 30, 33, 34, 73, 74, 75, 78, 171, 172, 175, 176, 178, 179, 182, and 183 of SEQ ID NO:89.

[0093] A non-limiting example of the structure of the ISVD of the first state of the present invention may be considered to include four frame regions (FRs), such as FR1, FR2, FR3, and FR4, and said frame regions are interrupted by three CDRs, such as CDR1, CDR2, and CDR3 (as defined by Kabat). In such immunoglobulin sequences, the frame sequence can be any suitable frame sequence, and suitable examples of frame sequences will be apparent to those skilled in the art, for example, based on standard textbooks and further disclosures mentioned herein.

[0094] In some embodiments, the ISVD comprises: CDR1: EYAVG (SEQ ID NO:3), or an amino acid sequence differing from SEQ ID NO:3 by one or two amino acids; CDR2: DIGEQAENTWYAESVLG (SEQ ID NO:7), or an amino acid sequence differing from SEQ ID NO:7 by one, two, three, or four amino acids; CDR3: DKYGVGGNAQGYYDS (SEQ ID NO:17), or an amino acid sequence differing from SEQ ID NO:17 by one or two amino acids. (Kabat definition)

[0095] In some embodiments, the ISVD includes a CDR1 selected from the list consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6; a CDR2 selected from the list consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; and a CDR3 selected from the list consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20 (Kabat definition).

[0096] In some embodiments, the ISVD includes a CDR1, as shown in SEQ ID NO:3; a CDR2 selected from the list consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:12; and a CDR3 selected from the list consisting of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. (Kabat definition)

[0097] In some embodiments, the ISVD includes CDR1:NYWMY (SEQ ID NO:6); CDR2:GINTGGSTPDYADSVKG (SEQ ID NO:16); and / or CDR3:DTPRVFRLDHYSP (SEQ ID NO:20) (defined by Kabat).

[0098] In a preferred embodiment, the ISVD includes CDR1:EYAVG (SEQ ID NO:3); CDR2:DIGEQAENTWYAESVLG (SEQ ID NO:7); and / or CDR3:DKYGVGGNAQGYYDS (SEQ ID NO:17) (as defined by Kabat).

[0099] In some embodiments, the ISVD includes a CDR1 selected from Table 1, a CDR2 selected from Table 2, and a CDR3 selected from Table 3.

[0100]

[0101]

[0102]

[0103] In some embodiments, the ISVD comprises the amino acid sequence of formula II (SEQ ID No. 21): X1X2QLVESGGGX11VQPGGSLX19LSCTTSGRX28FX30X31YAVGWFRQX40PGX43EREFVAX50IGEX54AX56NTWYAX62SVX65GRFTISRDX74AKNTVYLX82MX84X85LKPEDTAVYYCAADX100YGVGGX106AQGYYDSWGQGTQVTVSS (SEQ ID NO: 21), where X1 is Q or E; X2 is L or V; X11 is L or W. X19 is K or Q; X28 is R, T, E, H or K; X30 is S, Q or D; X31 is S or E; X40 is A or G; X43 is K or Q; X50 is D or E; X54 is Q, N, T or E; X56 is E or D; X62 is H or E; X65 is K or L; X74 is E, N or D; X82 is E or Q; X84 is D or N; X85 is G or S; X100 is K or S; and X106 is N or G (sequential numbering).

[0104] In a preferred embodiment, the ISVD comprises the amino acid sequence of formula III (SEQ ID No. 22): EVQLVESGGGX11VQPGGSLX19LSCTTSGRX28FX30EYAVGWFRQX40PGX43EREFVADIGEX54AENTWYAX62SVLGRFTISRDX74AKNTVYLX82MX84X85LKPEDTAVYYCAADKYGVGGNAQGYYDSWGQGTQVTVSS (SEQ ID No. 22) NO:22), where X11 is L or W, preferably L; X19 is K or Q; X28 is R or K; X30 is S or D; X40 is A or G; X43 is K or Q; X54 is Q or N; X62 is H or E; X74 is E or D; X82 is E or Q; X84 is D or N, preferably D; and X85 is G or S (sequential numbering).

[0105] Conveniently, the ISVD has glutamic acid at amino acid position 1, and / or alanine or glycine at amino acid position 40, and / or histamine or glutamic acid at amino acid position 62, and / or glutamic acid at amino acid position 82, and / or aspartic acid at amino acid position 84, which may lead to immunosuppression. The ISVD of the present invention, having glutamic acid at amino acid position 1, and / or leucine at amino acid position 11, and / or aspartic acid at amino acid position 84, may have improved chemical stability. The pI value of the ISVD of the present invention can be conveniently reduced by having glutamic acid at amino acid position 1, and / or having glutamine at amino acid position 19, and / or having aspartic acid at amino acid position 30, and / or having glutamine at amino acid position 43, and / or having glutamic acid at amino acid position 62, and / or having aspartic acid at amino acid position 74, and / or having glutamine or glutamic acid at amino acid position 82, and / or having aspartic acid at amino acid position 84. The binding affinity of the ISVD of the present invention can be enhanced by having lysine or arginine at amino acid position 28, and / or aspartic acid or serine at amino acid position 30, and / or glutamic acid at amino acid position 31, and / or asparagine or glutamine at amino acid position 54, and / or aspartic acid or glutamic acid at amino acid position 54, and / or lysine or leucine at amino acid position 65, and / or aspartic acid or glutamic acid at amino acid position 74, and / or glycine or serine at amino acid position 85, and / or lysine at amino acid position 100, and / or asparagine at amino acid position 106.

[0106] In some embodiments, the ISVD comprises or consists of the following: SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:103.

[0107] In some embodiments, the ISVD comprises or consists of SEQ ID NO:38. In some embodiments, the ISVD comprises or consists of a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO:38.

[0108] In a preferred embodiment, the ISVD comprises or consists of the following: SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29.

[0109] In a preferred embodiment, the ISVD comprises or consists of EVQLVESGGGLVQPGGSLKLSCTTSGRRFSEYAVGWFRQAPGKEREFVADIGEQAENTWYAESVLGRFTISRDDAKNTVYLEMDGLKPEDTAVYYCAADKYGVGGNAQGYYDSWGQGTQVTVSS (SEQ ID NO:28). In some embodiments, the ISVD comprises an amino acid sequence as shown in SEQ ID NO:28.

[0110] In some embodiments, the ISVD comprises or consists of a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO:28. In some embodiments, the ISVD comprises an amino acid sequence as shown in SEQ ID NO:28. In some embodiments, the ISVD is composed of an amino acid sequence as shown in SEQ ID NO:28.

[0111] In some embodiments, the ISVD is selected from Table 4. In some embodiments, the ISVD comprises or consists of any of the sequences shown in Table 4.

[0112] In some embodiments, the ISVD contains tryptophan at position 58 or 59, which is capable of forming a cationic-π interaction with Arg179 of mature hIL-6 (SEQ ID NO: 89) and a hydrophobic interaction with Phe78 of mature hIL-6 (SEQ ID NO: 89).

[0113] In some embodiments, the ISVD can bind to IL-6 via complementary sites comprising amino acid residues 50, 53, 54, 57, 58, 59, 101, 102, 103, 104, and 105 as shown in SEQ ID NO: 89. In a preferred embodiment, the ISVD can bind to IL-6 via complementary sites comprising amino acid residues 50, 53, 54, 57, 58, 59, 60, 65, 99, 101, 102, 103, 104, 105, 106, and 110 as shown in SEQ ID NO: 28.

[0114] [Polypeptide]

[0115] In the second state, the present invention relates to a polypeptide comprising an ISVD of the first state of the present invention and an extension, optionally wherein the extension is a C-terminal extension.

[0116] In some embodiments, the extension length is 1 to 20 amino acids. In some embodiments, the extension length is 1 to 15 amino acids, such as 1 to 10 amino acids. In a preferred embodiment, the extension length is 1 to 6 amino acids.

[0117] In some embodiments, the extension segment is 4 to 6 amino acids long. In some embodiments, the extension segment is 4 amino acids long. In some embodiments, the extension segment is 5 amino acids long. In some embodiments, the extension segment is 6 amino acids long.

[0118] Those skilled in the art will understand that the extension segment can be connected to the ISVD of the first state of the present invention, as an extension segment of its C-terminus, an extension segment of its N-terminus, or an extension segment of an amino acid residue between the C-terminus and N-terminus of the ISVD. Preferably, the extension segment is connected to the ISVD as an extension of its C-terminus or its N-terminus. Even more preferably, the extension segment is connected to the ISVD of the first state of the present invention as an extension of its C-terminus.

[0119] It should also be understood that the extension can be provided in any suitable manner. For example, the extension can be reconstituted with ISVD or conjugated with ISVD in any suitable manner, such as by chemical conjugation or click chemistry.

[0120] In a preferred embodiment, the extension comprises one or more amino acid residues capable of reacting with a substituent having a half-life extension property. In a particularly preferred embodiment, the extension comprises two amino acid residues, each capable of reacting with a substituent having a half-life extension property. Non-limiting examples of such amino acid residues are lysine and cysteine. If the extension comprises two or more amino acid residues, each capable of reacting with a substituent having a half-life extension property, then each amino acid residue may be the same or different. For example, the extension may comprise, for example, lysine and cysteine, or, for example, lysine and lysine, or, for example, cysteine ​​and cysteine. Conveniently, in some embodiments, the extension comprises two cysteine ​​amino acid residues, wherein each cysteine ​​amino acid residue is each capable of reacting with a substituent having a half-life extension property. Non-limiting examples of substituents having a half-life extension property are disclosed herein.

[0121] In some embodiments, the extension segment comprises or is composed of GGGGSCPC (SEQ ID NO: 90), which may be referred to as E3. In some embodiments, the extension segment comprises or is composed of GGGGSC (SEQ ID NO: 91), which may be referred to as E4. In some embodiments, the extension segment comprises or is composed of GGGGSKPK (SEQ ID NO: 92), which may be referred to as E5. In some embodiments, the extension segment comprises or is composed of GGGGSKP (SEQ ID NO: 93), which may be referred to as E6.

[0122] In some embodiments, the extension comprises or is composed of GQACPC (SEQ ID NO: 72), which may be referred to as E2. A list of non-limiting examples of polypeptides comprising the extension of SEQ ID NO: 72 is shown in Table 6.

[0123] In some embodiments, the extension comprises or is composed of GGGGSHHHHHH (SEQ ID NO: 71), which may be referred to as E1. A list of non-limiting examples of polypeptides comprising the extension of SEQ ID NO: 71 is shown in Table 5.

[0124] In a preferred embodiment, the polypeptide is selected from the list of the following compositions: SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO:102. In a particularly preferred embodiment, the polypeptide comprises a sequence as shown in SEQ ID No. 78.

[0125] Non-limiting examples of the polypeptides of the present invention are shown in Tables 6 and 5. In these tables, each item is identified by a polypeptide ID. The components forming part of the polypeptide (e.g., ISVD and extension) can be identified from the brief description in the polypeptide column.

[0126]

[0127]

[0128] It should be understood that the above-described extension and exemplary polypeptide (comprising a VHH and an extension) are merely illustrative and not intended to limit the scope in any way. Various alternatives will be apparent to those skilled in the art. For example, it should be understood that an extension E2 (SEQ ID No. 72) may be provided at the N-terminus of the ISVD. For example, if this is VHH_1.6, a polypeptide that can be simply described as E2-[VHH_1.6] can be produced.

[0129] Those skilled in the art will also understand that a portion of any of the extensions E1-E6 can be used as an extension. In some embodiments, it may be necessary to shorten the extension, for example, shortening extension E2 (SEQ ID No. 72). For example, in one embodiment, the extension may have 4 or 5 consecutive amino acid residues of the extension E2 sequence (SEQ ID No. 72), such that the extension contains, for example, the sequence GQAC (E7; SEQ ID No. 128) or GQACP (E8; SEQ ID No. 129). In one embodiment, the extension may contain at least 5 consecutive nucleotides of SEQ ID No. 72. In another embodiment, the extension may contain or consist of the amino acid sequence GQACP (E8; SEQ ID No. 129). In another embodiment, the extension may contain at least 4 consecutive nucleotides of SEQ ID No. 72. In another embodiment, the extension may contain or consist of the amino acid sequence GQAC (E7; SEQ ID No. 128). These are merely exemplary and should not be construed as limiting in any way. [Polypeptide derivatives]

[0130] In the third state, the present invention relates to a polypeptide derivative comprising the ISVD of the first state of the present invention or the polypeptide of the second state of the present invention, and a substituent. As will be understood by those skilled in the art, the term polypeptide derivative herein refers to a product of a construct consisting of a polypeptide (i.e., a polypeptide comprising the ISVD of the first state of the present invention, or a polypeptide comprising the ISVD of the second state of the present invention) further carrying at least one substituent, as will be discussed in detail below.

[0131] As will be described in detail below, the substituent may comprise a series of individually defined portions; these portions may be referred to as "substituent elements". Non-limiting examples of "substituent elements" are "elongators / elongation portions" (P) and "linkers" (Lp). Such linkers (Lp) may consist of one or more "linker elements". For ease of understanding, a non-limiting illustrative description of an exemplary compound (compound 9) of the present invention can be seen in Figures 3A and 3B. Preferred polypeptide derivatives, as shown in Formula 31 below, are also provided.

[0132] Conveniently, the substituent can extend the half-life of the polypeptide derivative. For example, the polypeptide derivative has a longer half-life than the corresponding polypeptide.

[0133] Substituents may form non-covalent interactions with albumin, thereby promoting the circulation of the compound in the bloodstream. This prolongs the time the compound remains in the bloodstream because the agglomerate of the substituent-carrying compound and albumin slowly disintegrates to release the free form of the compound. Therefore, this substituent, as a whole, can also be called the "albumin-binding portion," and it can be said to have a "prolonging effect." Alternatively, it can be said that this substituent is the half-life-extending portion.

[0134] The substituents may contain a particularly relevant and thus elongated portion for albumin binding, which may be referred to as an "elongator" or "elongated moiety". The elongator or elongated moiety is optionally linked to the ISVD or polypeptide via a "linker" (Lp). Such a linker (Lp) may consist of one or more "linker elements". The elongator / elongated moiety (P) and the linker (Lp) may be provided in tandem, for example, forming P-Lp or Lp-P, or more specifically P-Lp-Rn or Rn-Lp-P (where Rn refers to the linking point of the ISVD or polypeptide, and n is an integer, such that each linking point can be labeled R1, R2, R3, where R1≠R2≠R3, and so on).

[0135] In other words, in some embodiments, the polypeptide derivative may have structures such as [ISVD]-E-Lp-P (see, for example, Figure 1A or 1D) or P-Lp-E-[ISVD] (see Figure 1B or 1E), where "[ISVD]" refers to the ISVD of the first state of the present invention, "E" refers to the extension segment as described above (e.g., E1, E2, E3, etc.) (such that the result of "[ISVD]-E" or "E-[ISVD]" corresponds to the polypeptide of the second state of the present invention), "Lp" refers to the linker, and "P" refers to the extension portion. It should be understood that although only one substituent is described in these examples to be attached to the extension segment E, one or more substituents may be provided, and said one or more substituents may be attached to different residues of the same extension segment E.

[0136] Furthermore, those skilled in the art should understand that the examples of the above-described polypeptide derivatives are merely exemplary and should not be construed as limiting in any way. For example, in some embodiments, the substituent may be linked to residues between the N-terminus and C-terminus of the ISVD or the polypeptide containing the ISVD, as shown in Figure 1C (or Figure 1F, if the substituent consists of an elongated portion).

[0137] The polypeptide derivatives disclosed herein conveniently have at least one, preferably two, substituents. In some embodiments, each substituent is attached to a corresponding cysteine ​​or lysine amino acid residue of the extension.

[0138] In some embodiments, the one or more substituents are linked to the polypeptide of the present invention, such as the polypeptide comprising an ISVD of the second state of the present invention. In some embodiments, the substituents are linked to the ISVD of the present invention, such as the ISVD of the first state of the present invention. [Extension sub / Extension section]

[0139] The substituent may include a portion particularly relevant to albumin binding and thus elongation, which may be referred to as an "elongator" or "elongation moiety". The terms "elongator" and "elongation moiety" are used interchangeably herein. An "elongator" may be a lipophilic moiety (e.g., a fatty acid). An "elongator" may be a fatty acid (e.g., a C16-C22 carboxylic acid). In a preferred embodiment, the elongator is a C16 or C18 carboxylic acid. Non-limiting examples of "elongators" are shown in Table 7. In the chemical structure designated as Formula 1, The term is used to describe the point where a linker or polypeptide is connected via a covalent bond.

[0140] The elongator / elongation moiety ("P") may contain a tetrazolium group. The elongator / elongation moiety may contain an aryl group. The elongator / elongation moiety may contain a sulfonic acid group. The elongator / elongation moiety may contain a phenoxy group. The elongator / elongation moiety may contain a benzoic acid group.

[0141] [Connector (L)] [p] [)]

[0142] The substituent may include a portion between the elongation portion and the linker site connecting to the amino acid residue of the polypeptide, which may be referred to as a "linker (LP)". The linker may include several "linker units". The linker element may be selected to improve the overall properties of the molecule, for example, to improve oral bioavailability, conversion half-life and / or prolongation, thereby improving the overall exposure profile after oral administration of the compound.

[0143] A series of non-limiting examples of connecting sub-elements are shown in Table 8. In chemical structures designated as chemical formulas 2-6, This is used to describe the connection point that links to an elongator or polypeptide.

[0144] [Substituents]

[0145] In some embodiments, the substituent is represented by "LP-P", wherein P comprises or is composed of a lipophilic moiety having a distal carboxylic acid, and P has an elongation property.

[0146] In some embodiments, P is chemical formula 1. In the context of the lipophilic portion, as used herein, the term "..." "[Distal carboxylic acid]" refers to a carboxylic acid that is attached to the distal (terminal) point of the lipophilic portion relative to the connection point between the lipophilic portion and the adjacent portion. For example, in the compounds described herein, the lipophilic portion having a distal carboxylic acid (e.g., Formula 1) is an extended portion, and this carboxylic acid is attached to the distal (terminal) point of the lipophilic portion relative to the connection point between the lipophilic portion and the adjacent linker element (e.g., Formula 2, Formula 3, Formula 4, Formula 5, or Formula 6), or, in the absence of a linker element, relative to the connection point between the lipophilic portion and the adjacent polypeptide. A non-limiting example of a lipophilic portion having a distal carboxylic acid is Formula 1.

[0147] In some embodiments, P is of formula 1 and Lp is a linker comprising a linker element, wherein each linker element is selected from the group consisting of formulas 2, 3, 4, 5, and 6. A series of non-limiting examples of substituents comprising lipophilic moieties are listed in Table 9. In some embodiments, the substituents are selected from Table 9.

[0148]

[0149] In some embodiments, the substituent comprises an elongator / elongation portion of Formula 1, wherein n is 14, 16, or 18; and optionally a linker, wherein the linker comprises one or more γGlu (Formula 2), and / or one or more Ado (Formula 3) and / or one or more Gly (Formula 4) and / or one or more εLys (Formula 5) and / or Formula 6. In some embodiments, the substituent is selected from Table 9. In a preferred embodiment, each substituent is Formula 24.

[0150] In a preferred embodiment, the polypeptide derivative comprises two or more substituents. These two or more substituents may be the same or different. In a particularly preferred embodiment, the polypeptide derivative comprises two substituents, each represented by chemical formula 24.

[0151] In some embodiments, the polypeptide derivative is selected from the list consisting of: compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, and compound 10. In a preferred embodiment, the polypeptide derivative is compound 9.

[0152] Non-limiting examples of this polypeptide derivative are described in the Examples section and Table 10.

[0153] 1. Cys4 and Cys6 with E2 (SEQ ID NO: 72) as substituents. 2. The polypeptide consists of VHH with an extension at the C-terminus. An exemplary structure can be seen in Figures 3A / 3B, which show compound 9.

[0154] In a preferred embodiment, the polypeptide derivative has the following structure:

[0155] In some embodiments, the polypeptide derivative has a low molecular weight. In some embodiments, the molecular weight of the polypeptide derivative is between 10 kDa and 20 kDa. In some embodiments, the molecular weight of the polypeptide derivative is about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 15.5 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, or about 20 kDa. In some embodiments, the molecular weight of the polypeptide derivative is 12-18 kDa, 13-17 kDa, or 14-16 kDa. In a preferred embodiment, the molecular weight of the polypeptide derivative is 12-18 kDa. In a more preferred embodiment, the molecular weight of the polypeptide derivative is 14-16 kDa. In a particularly preferred embodiment, the molecular weight of the polypeptide derivative is 15.5 kDa. In some embodiments, the molecular weight of the polypeptide derivative is 15 kDa.

[0156] Those skilled in the art will understand that the polypeptide derivatives of the present invention can bind to IL-6 and provide therapeutic effects, while further providing small-sized constructs, for example, with a molecular weight between 12-18 kDa. [Antibody or fragment thereof]

[0157] In the fourth state, the present invention relates to an antibody, antibody fragment, antibody derivative, or polypeptide derivative, wherein the antibody, antibody fragment, antibody derivative, or polypeptide derivative is capable of binding IL-6 at an epitope comprising at least amino acid residues 26, 30, 33, 34, 73, 74, 75, 78, 171, 175, 178, 179, 182, and 183 of SEQ ID NO: 89.

[0158] In a preferred embodiment, the antibody, antibody fragment, or antibody derivative contains monotryptophan, which is located at a position that allows the antibody, antibody fragment, or antibody derivative to form a cationic-π interaction with Arg179 and / or a hydrophobic interaction with Phe78, as shown in SEQ ID NO:89.

[0159] In a preferred embodiment, the antibody, antibody fragment, or antibody derivative is a polypeptide or polypeptide derivative containing an ISVD, wherein the ISVD contains tryptophan at position 58 or 59.

[0160] In a preferred embodiment, the antibody, antibody fragment, or antibody derivative is a polypeptide or polypeptide derivative containing an ISVD, wherein the ISVD contains monotryptophan, which is capable of forming a cationic-π interaction with Arg179 and / or a hydrophobic interaction with Phe78, as shown in SEQ ID NO:89.

[0161] In a further embodiment, the present invention relates to an antibody, antibody fragment, antibody derivative, or polypeptide derivative, wherein the antibody, antibody fragment, antibody derivative, or polypeptide derivative is capable of binding IL-6 at an epitope comprising at least amino acid residues 24, 27, 28, 31, 88, 92, 95, 99, 114, 116, 117, 118, 120, 121, 123, 124, 125, 127, 128, 138, and 139 of SEQ ID NO: 89. [Methods and applications of ISVD / peptides / peptide derivatives]

[0162] In the fifth state, the present invention relates to a nucleic acid molecule, preferably in an isolated form, encoding the ISVD of the first state of the present invention or a polypeptide of the second state of the present invention. The nucleic acid molecule may be DNA and / or RNA. Genomic DNA, cDNA, mRNA, or other RNA; synthetic sources, or any combination thereof, may encode the ISVD or polypeptide of the present invention.

[0163] In the sixth state, the present invention relates to an expression vector comprising a nucleic acid molecule of the fifth state of the present invention.

[0164] In the seventh state, the present invention relates to a host cell carrying an expression vector of the sixth state of the present invention.

[0165] In the eighth state sample, the present invention relates to a method for manufacturing an ISVD of the first state sample of the present invention, a polypeptide of the second state sample of the present invention, or a polypeptide derivative of the third state sample of the present invention, comprising the following steps: a. culturing host cells of the seventh state sample of the present invention under conditions that allow expression of the ISVD of the first state sample of the present invention or the polypeptide of the second state sample of the present invention; b. recovering the ISVD or polypeptide obtained in step a; and / or optionally c. attaching one or more substituents capable of prolonging the half-life of the polypeptide, optionally wherein each substituent is of chemical formula 24; and / or optionally d. purifying the ISVD or polypeptide or polypeptide derivative thus obtained.

[0166] In some embodiments, the ISVD of the first state sample or the polypeptide of the second state sample of the present invention is expressed in a yield of at least 0.5 g / L in the cell supernatant after initial fermentation, preferably >1 g / L. In some embodiments, the ISVD of the first state sample or the polypeptide of the second state sample of the present invention is expressed in a yield of about 0.5-5 g / L in the cell supernatant after initial fermentation. The solubility should be >5 mg / mL, preferably >20 mg / mL, wherein the ISVD or polypeptide does not significantly aggregate or degrade. [Pharmaceutical Compositions]

[0167] In the ninth state, the present invention relates to a pharmaceutical composition comprising the ISVD of the first state of the present invention, or the polypeptide of the second state of the present invention, or the polypeptide derivative of the third state of the present invention. This pharmaceutical composition is suitable for oral administration.

[0168] In a preferred embodiment, the pharmaceutical composition is a solid oral dosage form, such as tablets.

[0169] In some embodiments, the pharmaceutical composition includes an adsorption enhancer. In a preferred embodiment, the enhancer is N-(8-2-hydroxybenzoyl)amino)octanoate. In a particularly preferred embodiment, the enhancer is sodium N-(8-(2-hydroxybenzoyl)amino)octanoate (SNAC).

[0170] In some embodiments, the pharmaceutical composition includes a water-soluble adjuvant. In a preferred embodiment, the water-soluble adjuvant is nicotinamide.

[0171] In some embodiments, the composition includes a lubricant. In a preferred embodiment, the lubricant is magnesium stearate or sodium fumarate.

[0172] In some embodiments, the pharmaceutical composition comprises an ISVD of the first state of the present invention, a polypeptide of the second state of the present invention, or a polypeptide derivative of the third state of the present invention, SNAC, nicotinamide, and optionally, a lubricant, such as magnesium stearate or sodium fumarate. In a preferred embodiment, the pharmaceutical composition comprises compound 9 (Table 10) and SNAC, and optionally also comprises nicotinamide and a lubricant, such as magnesium stearate or sodium fumarate. [Medical Use]

[0173] In the tenth state sample, the present invention relates to the ISVD of the first state sample of the present invention, or the polypeptide of the second state sample of the present invention, or the polypeptide derivative of the third state sample of the present invention, or the pharmaceutical composition of the ninth state sample, for use in a pharmaceutical. In some embodiments, the present invention relates to the ISVD of the first state sample of the present invention, or the polypeptide of the second state sample of the present invention, or the polypeptide derivative of the third state sample of the present invention, or the pharmaceutical composition of the ninth state sample, for use in treating inflammatory diseases.

[0174] In some embodiments, the present invention relates to an ISVD of the first state of the present invention, or a polypeptide of the second state of the present invention, or a polypeptide derivative of the third state of the present invention, or a pharmaceutical composition of the ninth state of the present invention, for the treatment of inflammatory diseases, wherein the human individual has or is at risk of having a highly sensitive C-reactive protein (Hs-CRP) value greater than 1 mg / L, preferably greater than 1.5 mg / L, and particularly preferably greater than or equal to 2 mg / L. In some embodiments, the human individual has or is at risk of having an Hs-CRP value greater than or equal to 3 mg / L.

[0175] In some embodiments, the present invention relates to an ISVD of the first state of the present invention, or a polypeptide of the second state of the present invention, or a polypeptide derivative of the third state of the present invention, or a pharmaceutical composition of the ninth state of the present invention, for the treatment of inflammatory diseases, wherein the human individual has or is at risk of having a highly sensitive C-reactive protein (hsCRP) level greater than or equal to 2 mg / L (hsCRP). 2 mg / L). In some embodiments, the human individual has or is at risk of having a high-sensitivity C-reactive protein (hsCRP) level greater than or equal to 3 mg / L (hsCRP). 3 mg / L).

[0176] In some embodiments, the present invention relates to an ISVD of the first state of the present invention, or a polypeptide of the second state of the present invention, or a polypeptide derivative of the third state of the present invention, or a pharmaceutical composition of the ninth state of the present invention, for the treatment of cardiovascular diseases.

[0177] In some embodiments, the present invention relates to an ISVD of the first state of the present invention, or a polypeptide of the second state of the present invention, or a polypeptide derivative of the third state of the present invention, or a pharmaceutical composition of the ninth state of the present invention, for the treatment of atherosclerotic cardiovascular disease (ASCVD).

[0178] In some embodiments, the present invention relates to an ISVD of the first state of the present invention, or a polypeptide of the second state of the present invention, or a polypeptide derivative of the third state of the present invention, or a pharmaceutical composition of the ninth state of the present invention, for the treatment of atherosclerotic cardiovascular disease (ASCVD), which is defined as stroke and / or myocardial infarction (MI) and / or peripheral artery disease (PAD).

[0179] In some embodiments, the present invention relates to an ISVD of the first state of the present invention, or a polypeptide of the second state of the present invention, or a polypeptide derivative of the third state of the present invention, or a pharmaceutical composition of the ninth state of the present invention, for the treatment of stroke and / or myocardial infarction (MI) and / or peripheral artery disease (PAD).

[0180] In some embodiments, the present invention relates to an ISVD of the first state of the present invention, or a polypeptide of the second state of the present invention, or a polypeptide derivative of the third state of the present invention, or a pharmaceutical composition of the ninth state of the present invention, for the manufacture of a medicament for treating atherosclerotic cardiovascular disease (ASCVD).

[0181] In some embodiments, the present invention relates to a method for treating an inflammatory disease, the method comprising administering to an individual an effective amount of an ISVD of the first state of the present invention, or a polypeptide of the second state of the present invention, or a polypeptide derivative of the third state of the present invention, or a pharmaceutical composition of the ninth state of the present invention.

[0182] In some embodiments, the present invention relates to a method for treating cardiovascular disease, the method comprising administering to an individual an effective amount of an ISVD of the first state of the present invention, or a polypeptide of the second state of the present invention, or a polypeptide derivative of the third state of the present invention, or a pharmaceutical composition of the ninth state of the present invention.

[0183] In some embodiments, the present invention relates to a method for treating cardiovascular disease, the method comprising administering to an individual suffering from chronic kidney disease (CKD) an effective amount of an ISVD of the first state of the present invention, or a polypeptide of the second state of the present invention, or a polypeptide derivative of the third state of the present invention, or a pharmaceutical composition of the ninth state of the present invention.

[0184] In some embodiments, the present invention relates to a method for treating cardiovascular disease, the method comprising administering to an individual suffering from stroke and / or myocardial infarction (MI) and / or peripheral artery disease (PAD) an effective amount of the first state form of the present invention, or the second state form of the present invention, or the third state form of the present invention, or the ninth state form of the present invention, a pharmaceutical composition thereof.

[0185] In some embodiments, the present invention relates to a method for treating atherosclerotic cardiovascular disease (ASCVD), the method comprising administering to an individual suffering from stroke and / or myocardial infarction (MI) and / or peripheral artery disease (PAD) an effective amount of the first state form of the present invention, or the second state form of the present invention, or the third state form of the present invention, or the ninth state form of the present invention, a pharmaceutical composition.

[0186] As used herein, the term "treatment" means any medical intervention for a human subject in need. Treatment can be preventative, preventative, palliative, symptomatic, and / or curative. The timing and purpose of treatment may vary depending on the subject's health condition. [List of Examples]

[0187] The present invention is further described by way of the following non-limiting embodiments. [Immunoglobulin Single Variable Domain (ISVD)]

[0188] 1. An immunoglobulin single variable domain (ISVD) having the following complementarity-determining region (CDR) sequences: CDR1: EYAVG (SEQ ID NO:3), or an amino acid sequence differing from SEQ ID NO:3 by one or two amino acids; CDR2: DIGEQAENTWYAESVLG (SEQ ID NO:7), or an amino acid sequence differing from SEQ ID NO:7 by one, two, three, or four amino acids; CDR3: DKYGVGGNAQGYYDS (SEQ ID NO:17), or an amino acid sequence differing from SEQ ID NO:17 by one or two amino acids. (Kabat definition)

[0189] 2. An immunoglobulin single variable domain (ISVD) comprising: a CDR1 selected from the list consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6; a CDR2 selected from the list consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; and a CDR3 selected from the list consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. (Kabat definition)

[0190] 3. An ISVD comprising or consisting of the following: X1X2QLVESGGGX11VQPGGSLX19LSCTTSGRX28FX30X31YAVGWFRQX40PGX43EREFVAX50IGEX54AX56NTWYAX62SVX65GRFTISRDX74AKNTVYLX82MX84X85LKPEDTAVYYCAADX100YGVGGX106AQGYYDSWGQGTQVTVSS (SEQ ID NO:21), where X1 is Q or E; X2 is L or V; X11 is L or W; X19 is K or Q; X28 is R, T, E, H, or K; X30 is S, Q, or D; X31 is S or E; X40 is A or G; X43 is K or Q; X50 is D or E; X54 is Q, N, T, or E; X56 is E or D; X62 is H or E; X65 is K or L; X74 is E, N, or D; X82 is E or Q; X84 is D or N; X85 is G or S; X100 is K or S; and X106 is N or G. (Numbered consecutively)

[0191] 4. The ISVD as described in Example 3, where X1 is Q.

[0192] 5. The ISVD as described in Example 3, where X1 is E.

[0193] 6. The ISVD as described in any of Examples 3 to 5, wherein X11 is L.

[0194] 7. The ISVD as described in any of Examples 3 to 5, wherein X11 is W.

[0195] 8. The ISVD as described in any of Examples 3 to 7, wherein X19 is K.

[0196] 9. The ISVD as described in any of Examples 3 to 7, wherein X19 is Q.

[0197] 10. The ISVD as described in any of Examples 3 to 9, wherein X28 is R.

[0198] 11. The ISVD as described in any of Examples 3 to 9, wherein X28 is T.

[0199] 12. The ISVD as described in any of Examples 3 to 9, wherein X28 is E.

[0200] 13. The ISVD as described in any of Examples 3 to 9, wherein X28 is H.

[0201] 14. The ISVD as described in any of Examples 3 to 9, wherein X28 is K.

[0202] 15. The ISVD as described in any of Examples 3 to 14, wherein X30 is S.

[0203] 16. The ISVD as described in any of Examples 3 to 14, wherein X30 is Q.

[0204] 17. An ISVD as described in any of Examples 3 to 14, wherein X30 is D.

[0205] 18. IS`VD as described in any of Examples 3 to 17, wherein X31 is S.

[0206] 19. An ISVD as described in any of Examples 3 to 17, wherein X31 is E.

[0207] 20. An ISVD as described in any of Examples 3 to 17, wherein X40 is A.

[0208] 21. The ISVD as described in any of Examples 3 to 17, wherein X40 is G.

[0209] 22. The ISVD as described in any of Examples 3 to 21, wherein X43 is K.

[0210] 23. The ISVD as described in any of Examples 3 to 21, wherein X43 is Q.

[0211] 24. An ISVD as described in any of Examples 3 to 23, wherein X50 is D.

[0212] 25. An ISVD as described in any of Examples 3 to 23, wherein X50 is E.

[0213] 26. The ISVD as described in any of Examples 3 to 25, wherein X54 is Q.

[0214] 27. An ISVD as described in any of Examples 3 to 25, wherein X54 is N.

[0215] 28. An ISVD as described in any of Examples 3 to 25, wherein X54 is T.

[0216] 29. An ISVD as described in any of Examples 3 to 25, wherein X54 is E.

[0217] 30. An ISVD as described in any of Examples 3 to 29, wherein X56 is E.

[0218] 31. The ISVD as described in any of Examples 3 to 29, wherein X56 is D.

[0219] 32. The ISVD as described in any of Examples 3 to 31, wherein X62 is H.

[0220] 33. The ISVD as described in any of Examples 3 to 31, wherein X62 is E.

[0221] 34. The ISVD as described in any of Examples 3 to 33, wherein X65 is K.

[0222] 35. The ISVD as described in any of Examples 3 to 33, wherein X65 is L.

[0223] 36. The ISVD as described in any of Examples 3 to 35, wherein X74 is E.

[0224] 37. The ISVD as described in any of Examples 3 to 35, wherein X74 is N.

[0225] 38. The ISVD as described in any of Examples 3 to 35, wherein X74 is D.

[0226] 39. An ISVD as described in any of Examples 3 to 38, wherein X82 is E.

[0227] 40. The ISVD as described in any of Examples 3 to 38, wherein X82 is Q.

[0228] 41. The ISVD as described in any of Examples 3 to 40, wherein X84 is D.

[0229] 42. The ISVD as described in any of Examples 3 to 40, wherein X84 is N.

[0230] 43. The ISVD as described in any of Examples 3 to 42, wherein X85 is G.

[0231] 44. The ISVD as described in any of Examples 3 to 42, wherein X85 is S.

[0232] 45. An ISVD as described in any of Examples 3 to 44, wherein X100 is K.

[0233] 46. ​​An ISVD as described in any of Examples 3 to 44, wherein X100 is S.

[0234] 47. An ISVD as described in any of Examples 3 to 46, wherein X106 is N.

[0235] 48. The ISVD as described in any of Examples 3 to 46, wherein X106 is G.

[0236] 49. An ISVD comprising or consisting of the following: SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:103.

[0237] 50. An ISVD comprising or consisting of SEQ ID NO:38.

[0238] 51. An ISVD comprising or consisting of EVQLVESGGGLVQPGGSLKLSCTTSGRRFSEYAVGWFRQAPGKEREFVADIGEQAENTWYAESVLGRFTISRDDAKNTVYLEMDGLKPEDTAVYYCAADKYGVGGNAQGYYDSWGQGTQVTVSS (SEQ ID NO:28).

[0239] 52. An immunoglobulin single variable domain (ISVD) comprising the following amino acid sequence: EVQLVESGGGLVQPGGSLKLSCTTSGRRFSEYAVGWFRQAPGKEREFVADIGEQAENTWYAESVLGRFTISRDDAKNTVYLEMDGLKPEDTAVYYCAADKYGVGGNAQGYYDSWGQGTQVTVSS (SEQ ID NO:28).

[0240] 53. An immunoglobulin single variable domain (ISVD) consisting of the following amino acid sequence: EVQLVESGGGLVQPGGSLKLSCTTSGRRFSEYAVGWFRQAPGKEREFVADIGEQAENTWYAESVLGRFTISRDDAKNTVYLEMDGLKPEDTAVYYCAADKYGVGGNAQGYYDSWGQGTQVTVSS (SEQ ID NO:28).

[0241] 54. An ISVD comprising or consisting of the following sequence: a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO:38 or SEQ ID NO:28.

[0242] 55. The ISVD as described in Example 2, wherein the ISVD has the following complementary determination region (CDR) sequences: CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGEQAENTWYAESVLG (SEQ ID NO: 7); and / or CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17). (Kabat definition)

[0243] 56. The ISVD as described in Example 2, wherein the ISVD has the following complementarity-determining region (CDR) sequences: CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGEQAENTWYAESVLG (SEQ ID NO: 7); and CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17). (Kabat definition)

[0244] 57. The ISVD as described in Example 2, wherein the ISVD has the following complementary determination region (CDR) sequences: CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGENAENTWYAESVLG (SEQ ID NO: 8); and / or CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17). (Kabat definition)

[0245] 58. The ISVD as described in Example 2, wherein the ISVD has the following complementary determination region (CDR) sequences: CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGENAENTWYAHSVLG (SEQ ID NO: 9); and / or CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17). (Kabat definition)

[0246] 59. The ISVD as described in Example 2, wherein the ISVD has the following complementary determination region (CDR) sequences: CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGEQAENTWYAHSVLG (SEQ ID NO: 12); and / or CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17). (Kabat definition)

[0247] 60. The ISVD as described in Example 2, wherein the ISVD has the following complementary determination region (CDR) sequences: CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGEQAENTWYAESVLG (SEQ ID NO: 7); and / or CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17). (Kabat definition)

[0248] 61. The ISVD as described in Example 2, wherein the ISVD has the following complementarity-determining region (CDR) sequences: CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGEQAENTWYAHSVLG (SEQ ID NO: 12); and / or CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17). (Kabat definition)

[0249] 62. The ISVD as described in Example 2, wherein the ISVD has the following complementary determination region (CDR) sequences: CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGENAENTWYAESVLG (SEQ ID NO: 8); and / or CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17). (Kabat definition)

[0250] 63. The ISVD as described in Example 2, wherein the ISVD has the following complementarity-determining region (CDR) sequences: CDR1: EYAMA (SEQ ID NO: 5); CDR2: DIGESGGTWYADSVKG (SEQ ID NO: 14); and / or CDR3: DSYGVGGGAERYYDS (SEQ ID NO: 19). (Kabat definition)

[0251] 64. The ISVD as described in Example 2, wherein the ISVD has the following complementarity-determining region (CDR) sequences: CDR1: SYAVG (SEQ ID NO: 4); CDR2: DIGENADNTWYAHSVKG (SEQ ID NO: 15); and / or CDR3: DSYGVGGGAQGYYDS (SEQ ID NO: 18). (Kabat definition)

[0252] 65. The ISVD as described in Example 2, wherein the ISVD has the following complementary determinant region (CDR) sequences: CDR1: NYWMY (SEQ ID NO: 6); CDR2: GINTGGSTPDYADSVKG (SEQ ID NO: 16); and / or CDR3: DTPRVFRLDHYSP (SEQ ID NO: 20). (Kabat definition)

[0253] 66. An ISVD as described in any of Examples 1 to 65, wherein the ISVD comprises one or more substituents.

[0254] 67. The ISVD as described in any of Examples 1 to 66, wherein the substituent is a conservative substituent.

[0255] 68. The ISVD as described in any of Examples 1 to 67, wherein the ISVD is a separated ISVD.

[0256] 69. An ISVD as described in any of Examples 1 to 68, wherein the ISVD is a VHH, such as a humanized VHH.

[0257] 70. The ISVD as described in any of Examples 1 to 69, wherein the ISVD is capable of binding IL-6, preferably hIL-6 (SEQ ID NO: 89).

[0258] 71. An ISVD as described in any of Examples 1 to 70, wherein the ISVD has a complementary site comprising amino acid residues D50, E53, N54, N57, T58, W59, Y60, K65, D99, Y101, G102, V103, G104, G105, G106 and Y110 (numbered consecutively), or wherein the ISVD has a complementary site comprising amino acid residues D50, E53, Q54, N57, T58, W59, Y60, L65, D99, Y101, G102, V103, G104, G105, N106 and Y110 (numbered consecutively).

[0259] 72. An ISVD as described in any of Examples 1 to 71, wherein the ISVD has a pI value ranging from 3 to 6.

[0260] 73. An ISVD as described in any of Examples 1 to 71, wherein the ISVD has a pI value ranging from 3.5 to 5.5.

[0261] 74. An ISVD as described in any of Examples 1 to 71, wherein the ISVD has a pI value ranging from 4.0 to 5.0.

[0262] 75. An ISVD as described in any of Examples 1 to 71, wherein the ISVD has a pI value ranging from 4.5 to 5.0.

[0263] 76. An ISVD as described in any of Examples 1 to 71, wherein the ISVD has a pI value of about 3.5, about 3.6, about 3.7, about 3.8, 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, or about 5.5. [Polypeptide]

[0264] 77. A polypeptide comprising an ISVD as described in any one of Examples 1 to 76 and an extension segment.

[0265] 78. The polypeptide as described in Example 77, wherein the extension is a C-terminal extension.

[0266] 79. The polypeptide as described in Example 77, wherein the extension is an N-terminal extension.

[0267] 80. The polypeptide as described in Example 77, wherein the extension is located between the C-terminus and N-terminus of the ISVD.

[0268] 81. The polypeptide as described in any of Examples 77 to 80, wherein the length of the extension is 1 to 6 amino acid residues.

[0269] 82. The polypeptide as described in any of Examples 77 to 80, wherein the length of the extension is 1 to 10 amino acid residues.

[0270] 83. The polypeptide as described in any of Examples 77 to 80, wherein the length of the extension is 1 to 20 amino acid residues.

[0271] 84. The polypeptide as described in any of Examples 77 to 80, wherein the length of the extension is 1 to 13 amino acid residues.

[0272] 85. The polypeptide as described in any of Examples 77 to 84, wherein the length of the extension is 4 to 6 amino acid residues.

[0273] 86. The polypeptide as described in any of Examples 77 to 85, wherein the length of the extended segment is 4 amino acid residues.

[0274] 87. The polypeptide as described in any of Examples 77 to 85, wherein the length of the extension is 5 amino acid residues.

[0275] 88. The polypeptide as described in any of Examples 77 to 85, wherein the length of the extension is 6 amino acid residues.

[0276] 89. The polypeptide as described in any of Examples 77 to 88, wherein the extension comprises a cysteine ​​amino acid residue.

[0277] 90. The polypeptide as described in any of Examples 77 to 88, wherein the extension comprises two cysteine ​​amino acid residues.

[0278] 91. The polypeptide as described in any of Examples 77 to 88, wherein the extension comprises two or more cysteine ​​amino acid residues.

[0279] 92. The polypeptide as described in any of Examples 77 to 88, wherein the extension comprises an amino acid residue capable of reacting with the half-life extension portion.

[0280] 93. The polypeptide as described in any of Examples 77 to 88, wherein the extension comprises two amino acid residues capable of reacting with the half-life extension portion.

[0281] 94. The polypeptide as described in Example 93, wherein each of the two amino acid residues is selected from Lys or Cys.

[0282] 95. The polypeptide as described in Example 93, wherein each of the two amino acid residues is Cys.

[0283] 96. The polypeptide as described in any of Examples 77 to 91, wherein the extension comprises at least one amino acid residue capable of reacting with a substituent.

[0284] 97. The polypeptide as described in Example 96, wherein the at least one amino acid residue capable of reacting with a substituent is at least two amino acid residues capable of reacting with a substituent.

[0285] 98. The polypeptide as described in any of Examples 96 to 97, wherein each of the at least one amino acid residue is selected from Lys and Cys.

[0286] 99. The polypeptide as described in any of Examples 96 to 97, wherein each of the at least one amino acid residue is Cys.

[0287] 100. The polypeptide as described in any of Examples 77 to 84 and 91 to 94, wherein the extension comprises or is composed of an amino acid sequence as shown in any of SEQ ID No. 72, 71, 90, 91, 92, 93.

[0288] 101. The polypeptide as described in Example 100, wherein the extension comprises or is composed of an amino acid sequence as shown in any one of SEQ ID No. 72, 90, 91, 92, 93.

[0289] 102. The polypeptide as described in Example 100, wherein the extension comprises an amino acid sequence as shown in SEQ ID No. 72.

[0290] 103. The polypeptide as described in Example 100, wherein the extension consists of an amino acid sequence as shown in SEQ ID No. 72.

[0291] 104. The polypeptide as described in any of Examples 77 to 84 and 91 to 94, wherein the extension comprises four consecutive amino acid residues of the sequence shown in SEQ ID No. 72.

[0292] 105. The polypeptide as described in Example 104, wherein the extension comprises or is composed of an amino acid sequence as shown in SEQ ID No. 128.

[0293] 106. The polypeptide as described in any of Examples 77 to 84 and 91 to 94, wherein the extension comprises five consecutive amino acid residues of the sequence shown in SEQ ID No. 72.

[0294] 107. The polypeptide as described in Example 106, wherein the extension comprises or is composed of an amino acid sequence as shown in SEQ ID No. 129.

[0295] 108. The polypeptide as described in Examples 69 or 70, wherein the extension comprises or is composed of the amino acid residue GQACPC (SEQ ID NO: 72).

[0296] 109. The polypeptide as described in any of Examples 69 to 83, wherein the polypeptide is capable of binding IL-6, preferably hIL-6 (SEQ ID NO: 89). [Polypeptide derivatives]

[0297] 110. A polypeptide derivative comprising the polypeptide as described in any one of Examples 77 to 109, wherein the polypeptide derivative comprises a substituent.

[0298] 111. The polypeptide derivative as described in Example 110, wherein the substituent is capable of prolonging the half-life and / or binding albumin.

[0299] 112. A polypeptide derivative comprising an ISVD as described in any of Examples 1 to 76, or a polypeptide as described in any of Examples 77 to 109, wherein the polypeptide derivative comprises two substituents.

[0300] 113. The polypeptide derivative as described in Example 112, wherein at least one of the two substituents is capable of prolonging the half-life and / or binding albumin.

[0301] 114. The polypeptide derivative as described in Example 112, wherein each of the two substituents is capable of prolonging the half-life and / or binding albumin.

[0302] 115. The polypeptide derivative as described in Example 112, wherein each of the two substituents is capable of extending the half-life compared to an unsubstituented ISVD or polypeptide.

[0303] 116. The polypeptide derivative as described in Example 112, wherein each of the two substituents is capable of binding albumin.

[0304] 117. The polypeptide derivative as described in Example 112, wherein each / substituent is a half-life extension portion.

[0305] 118. The polypeptide derivative as described in any of Examples 110 to 117, wherein each / the substituent comprises chemical formula 1.

[0306] 119. The polypeptide derivative as described in any of Examples 110 to 117, wherein the substituent comprises

[0307] Where n = 12, 14, 16, 18 or 20.

[0308] 120. The polypeptide derivative as described in any of Examples 112 to 117, wherein each of the two substituents comprises

[0309] Where n = 12, 14, 16, 18 or 20.

[0310] 121. The polypeptide derivative as described in any of Examples 110 to 120, wherein each / the substituent further comprises one or more of the following chemical formulas: chemical formula 2, chemical formula 3, chemical formula 4, chemical formula 5 or chemical formula 6.

[0311] 122. The polypeptide derivative as described in any of Examples 110 to 120, wherein each / the substituent further comprises one or more of the following chemical formulas:

[0312] 123. The polypeptide derivative as described in any of Examples 110 to 122, wherein each / the substituent comprises or substantially consists of an elongated portion and at least one linker.

[0313] 124. The polypeptide derivative as described in any of Examples 110 to 122, wherein each / the substituent comprises an elongation portion and at least one linker.

[0314] 125. The polypeptide derivative as described in any of Examples 110 to 122, wherein each / the substituent comprises an elongation portion and a linker.

[0315] 126. The polypeptide derivative described in any of Examples 123 to 125, wherein the extended portion is of Formula 1; HOOC-Benzene-O-(CH2)x-CO-*; or HO-S(=O)2-(CH2)x-CO-*; and wherein x is an integer in the range of 8 to 18.

[0316] 127. The polypeptide derivative as described in any of Examples 123 to 125, wherein the extended portion comprises , where n = 12, 14, 16, 18 or 20; HOOC-benzene-O-(CH2)x-CO-*, where x is an integer in the range of 8 to 18; or HO-S(=O)2-(CH2)x-CO-*, where x is an integer in the range of 8 to 18.

[0317] 128. The polypeptide derivative as described in any of Examples 123 to 127, wherein the extended portion comprises , where n = 12, 14, 16, 18 or 20.

[0318] 129. The polypeptide derivative as described in any of Examples 123 to 127, wherein the extended portion comprises , where n=14.

[0319] 130. The polypeptide derivative as described in Example 121 or Example 123, wherein the linker comprises chemical formula 2, chemical formula 3, chemical formula 4, chemical formula 5 or chemical formula 6.

[0320] 131. The polypeptide derivative as described in any of Examples 123 to 129, wherein the linker comprises One or more.

[0321] 132. The polypeptide derivative as described in any of Examples 123 to 131, wherein the at least one linker is composed of one or more linker elements.

[0322] 133. The polypeptide derivative as described in Example 132, wherein the linker element is selected from...

[0323] 134. The polypeptide derivative as described in any of Examples 110 to 133, wherein each / the substituent is selected from Formula 7, Formula 8, Formula 9, Formula 10, Formula 11, Formula 12, Formula 13, Formula 14, Formula 15, Formula 16, Formula 17, Formula 18, Formula 19, Formula 20, Formula 21, Formula 22, Formula 23 and Formula 24.

[0324] 135. The polypeptide derivative as described in any of Examples 110 to 133, wherein each / the substituent is selected from... Formula 13) as well as

[0325] 136. The polypeptide derivative as described in Example 135, wherein each / the substituent is selected from... as well as

[0326] 137. The polypeptide derivative as described in Example 135, wherein each / the substituent is

[0327] 138. The polypeptide derivative as described in any of Examples 110 to 137, having the chemical formula 31:

[0328] 139. A polypeptide derivative comprising an ISVD as shown in SEQ ID NO:28, wherein the ISVD is fused to a C-terminal extension consisting of SEQ ID NO:72, and wherein each hemisacyl group of the C-terminal extension carries a substituent as shown in Formula 24.

[0329] 140. A polypeptide derivative comprising an immunoglobulin single variable domain (ISVD) as shown in SEQ ID NO:28, wherein the ISVD is fused to a C-terminal extension consisting of SEQ ID NO:72, and wherein each hacystine nucleotide of the C-terminal extension carries a substituent comprising the following structure:

[0330] 141. A polypeptide derivative capable of binding IL-6, comprising an immunoglobulin single variable domain (ISVD), an extension, and two substituents, wherein the ISVD comprises the following complementarity-determining regions (CDRs): CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGEQAENTWYAESVLG (SEQ ID NO: 7); and CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17) (as defined by Kabat), wherein the extension is attached to the C-terminus of the ISVD and comprises an amino acid sequence as shown in SEQ ID No. 72, and wherein each of the substituents is attached to a cysteine ​​in the extension, and each of the substituents comprises the following structure

[0331] 142. A polypeptide derivative capable of binding IL-6, comprising an immunoglobulin single variable domain (ISVD), an extension, and two substituents, wherein the ISVD comprises the following complementarity-determining regions (CDRs): CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGEQAENTWYAESVLG (SEQ ID NO: 7); and CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17) (as defined by Kabat), wherein the extension is attached to the C-terminus of the ISVD and comprises an amino acid sequence as shown in SEQ ID No. 72, and wherein a first substituent is attached to a cysteine ​​at position 4 of the extension, and a second substituent is attached to a cysteine ​​at position 6 of the extension, and each of the substituents comprises the following structure

[0332] 143. A polypeptide derivative capable of binding IL-6, comprising an immunoglobulin single variable domain (ISVD), an extension, a first substituent, and a second substituent, wherein the ISVD comprises the following complementarity-determining regions (CDRs): CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGEQAENTWYAESVLG (SEQ ID NO: 7); and CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17) (as defined by Kabat), wherein the extension comprises an amino acid sequence as shown in SEQ ID No. 72, having cysteine ​​at positions 4 and 6, and the extension is attached to the C-terminus of the ISVD, wherein the first substituent is attached to the cysteine ​​at position 4 of the extension, and the second substituent is attached to the cysteine ​​at position 6 of the extension, and wherein each of the first and second substituents comprises the following structure

[0333] 144. A polypeptide derivative capable of binding IL-6, comprising an immunoglobulin single variable domain (ISVD), an extension, a first substituent, and a second substituent, wherein the ISVD comprises the following complementarity-determining regions (CDRs): CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGEQAENTWYAESVLG (SEQ ID NO: 7); and CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17) (as defined by Kabat), wherein the extension comprises an amino acid sequence as shown in SEQ ID No. 72, wherein cysteine ​​is present at positions 4 and 6 of SEQ ID No. 72, and the extension is connected to the C-terminus of the ISVD, wherein the first substituent is connected to the cysteine ​​at position 4 of SEQ ID No. 72, and the second substituent is connected to the cysteine ​​at position 6 of SEQ ID No. 72, and wherein each of the first and second substituents comprises the following structure

[0334] 145. A polypeptide derivative capable of binding IL-6, comprising a VHH, an extension, a first substituent, and a second substituent, wherein the VHH comprises the following complementarity-determining regions (CDRs): CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGEQAENTWYAESVLG (SEQ ID NO: 7); and CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17) (as defined by Kabat), wherein the extension is attached to the C-terminus of the VHH and comprises an amino acid sequence as shown in SEQ ID No. 72, wherein the first substituent is attached to cysteine ​​at position 4 of SEQ ID No. 72, and the second substituent is attached to cysteine ​​at position 6 of SEQ ID No. 72, and wherein each of the first and second substituents comprises the following structure

[0335] 146. A polypeptide derivative capable of binding IL-6, comprising an immunoglobulin single variable domain (ISVD), an extension, and two substituents, wherein the ISVD comprises the following complementarity-determining regions (CDRs): CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGEQAENTWYAESVLG (SEQ ID NO: 7); and CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17) (as defined by Kabat), wherein the extension is attached to the C-terminus of the ISVD and consists of an amino acid sequence as shown in SEQ ID No. 72, and wherein each of the substituents is attached to a cysteine ​​and comprises the following structure

[0336] 147. A polypeptide derivative capable of binding IL-6, comprising an immunoglobulin single variable domain (ISVD), an extension, and two substituents, wherein the ISVD comprises an amino acid sequence as shown in SEQ ID No. 28, wherein the extension is attached to the C-terminus of the ISVD and comprises an amino acid sequence as shown in SEQ ID No. 72, and wherein each of the substituents is attached to a cysteine ​​in the extension and comprises the following structure

[0337] 148. A polypeptide derivative capable of binding IL-6, comprising an immunoglobulin single variable domain (ISVD), an extension, and two substituents, wherein the ISVD comprises an amino acid sequence as shown in SEQ ID No. 28, wherein the extension is attached to the C-terminus of the ISVD and comprises an amino acid sequence as shown in SEQ ID No. 72, and wherein a first substituent is attached to a cysteine ​​at position 4 of the extension, and a second substituent is attached to a cysteine ​​at position 6 of the extension, and each of the substituents comprises the following structure

[0338] 149. A polypeptide derivative capable of binding IL-6, having the structure of chemical formula 31:

[0339] 150. The polypeptide derivative as described in any of Examples 110 to 149, wherein the ISVD is VHH, preferably a humanized VHH.

[0340] 151. The polypeptide derivative as described in any of Examples 110 to 150, wherein the polypeptide derivative has an in vitro potency of about 1 to 50 pM.

[0341] 152. The polypeptide derivative as described in any of Examples 110 to 150, wherein the polypeptide derivative has an in vitro potency of about 5 to 35 pM.

[0342] 153. The polypeptide derivative as described in any of Examples 110 to 150, wherein the polypeptide derivative has an in vitro potency of about 10 to 20 pM.

[0343] 154. The polypeptide derivative as described in any of Examples 110 to 150, wherein the polypeptide derivative has an in vitro potency of about 12 to 19 pM.

[0344] 155. The polypeptide derivative as described in any of Examples 151 to 154, wherein the in vitro potency is measured as described in Example 8.1: in vitro activity against IL-6 VHH in the stat-3-luc reporter gene assay.

[0345] 156. The polypeptide derivative as described in any of Examples 151 to 154, wherein the in vitro potency is measured by stat-3-luc reporter gene assay.

[0346] 157. A polypeptide derivative, as shown in Figure 3A.

[0347] 158. A polypeptide derivative, as shown in Figure 3A or 3B.

[0348] 159. A polypeptide derivative, as shown in Figure 3B.

[0349] 160. A polypeptide derivative, as shown in chemical formula 31:

[0350] 161. A polypeptide derivative comprising the polypeptide as described in any one of Examples 77 to 109, and further comprising a half-life extended portion.

[0351] 162. The polypeptide derivative as described in Example 160, wherein the extended half-life portion is an albumin binder, a fatty acid, an Fc domain, an FcRn binder, or an Fc-binding peptide.

[0352] 163. The polypeptide derivative as described in any of Examples 110 to 161, wherein the polypeptide derivative is capable of binding IL-6, preferably hIL-6 (SEQ ID NO: 89).

[0353] 164. The polypeptide derivative as described in any of Examples 110 to 162, wherein the molecular weight of the polypeptide derivative is 10-20 kDa, such as 10-20 kDa, 12-18 kDa, 13-17 kDa and 14-16 kDa.

[0354] 165. The polypeptide derivative as described in any of Examples 110 to 162, wherein the molecular weight of the polypeptide derivative is 12-18 kDa.

[0355] 166. The polypeptide derivative as described in any of Examples 110 to 162, wherein the molecular weight of the polypeptide derivative is 14-16 kDa.

[0356] 167. The polypeptide derivative as described in any of Examples 110 to 162, wherein the molecular weight of the polypeptide derivative is about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, or about 20 kDa.

[0357] 168. The polypeptide derivative as described in any of Examples 110 to 162, wherein the molecular weight of the polypeptide derivative is 14 kDa.

[0358] 169. The polypeptide derivative as described in any of Examples 110 to 162, wherein the molecular weight of the polypeptide derivative is 15 kDa.

[0359] 170. The polypeptide derivative as described in any of Examples 110 to 162, wherein the molecular weight of the polypeptide derivative is 15.5 kDa.

[0360] 171. The polypeptide derivative as described in any of Examples 110 to 162, wherein the molecular weight of the polypeptide derivative is 16 kDa.

[0361] 172. The polypeptide derivative as described in any of Examples 110 to 170, wherein the polypeptide derivative has a pI value in the range of 3.5-5.5, such as 4.0-5.0.

[0362] 173. The polypeptide derivative as described in any of Examples 110 to 170, wherein the polypeptide derivative has a pI value of about 3.5, about 3.6, about 3.7, about 3.8, 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, or about 5.5. [Further Examples]

[0363] 174. A nucleic acid molecule, preferably in an isolated form, encoding an ISVD as described in any one of Examples 1 to 76 or a polypeptide as described in any one of Examples 77 to 109.

[0364] 175. An expression vector comprising the nucleic acid molecule as described in Example 173.

[0365] 176. A host cell carrying an expression vector as described in Example 174.

[0366] 177. A method for producing an ISVD as described in any one of Examples 1 to 76, or a polypeptide as described in any one of Examples 77 to 109, or a polypeptide derivative as described in any one of Examples 110 to 172, comprising the steps of: a. culturing host cells as described in Example 175 under conditions that allow the expression of the ISVD as described in any one of Examples 1 to 76 or the polypeptide as described in any one of Examples 77 to 109; b. recovering the ISVD or the polypeptide; c. optionally attaching one or more substituents capable of prolonging the half-life of the polypeptide, optionally wherein each substituent is of chemical formula 24; and d. optionally purifying the ISVD or polypeptide or polypeptide derivative thus obtained.

[0367] 178. An isolated ISVD used as an intermediate in the manufacture of an anti-IL6 polypeptide derivative, comprising the following complementarity-determining region (CDR) sequences: CDR1: EYAVG (SEQ ID NO:3), or an amino acid sequence differing from SEQ ID NO:3 by one or two amino acids; CDR2: DIGEQAENTWYAESVLG (SEQ ID NO:7), or an amino acid sequence differing from SEQ ID NO:7 by one, two, or three amino acids; CDR3: DKYGVGGNAQGYYDS (SEQ ID NO:17), or an amino acid sequence differing from SEQ ID NO:17 by one or two amino acids. (Kabat definition).

[0368] 179. An isolated immunoglobulin single variable domain (ISVD) used as an intermediate in the manufacture of an anti-IL6 polypeptide derivative, comprising the following complementarity-determining region (CDR) sequences: CDR1: EYAVG (SEQ ID NO:3); CDR2: DIGEQAENTWYAESVLG (SEQ ID NO:7); and CDR3: DKYGVGGNAQGYYDS (SEQ ID NO:17). (Kabat definition). [Pharmaceutical Compositions]

[0369] 180. A pharmaceutical composition comprising an ISVD as described in any one of Examples 1 to 76, or a polypeptide as described in any one of Examples 77 to 109, or a polypeptide derivative as described in any one of Examples 110 to 172.

[0370] 181. An oral pharmaceutical composition comprising an ISVD as described in any one of Examples 1 to 76, or a polypeptide as described in any one of Examples 77 to 109, or a polypeptide derivative as described in any one of Examples 110 to 172.

[0371] 182. The pharmaceutical composition as described in Example 180, wherein the composition comprises an adsorption enhancer.

[0372] 183. A pharmaceutical composition as described in Example 180 or Example 181, wherein the composition comprises N-(8-2-hydroxybenzoyl)amino)octanoate.

[0373] 184. A pharmaceutical composition as described in Example 180 or Example 181, wherein the composition comprises sodium N-(8-(2-hydroxybenzoyl)amino)octanoate.

[0374] 185. The pharmaceutical composition as described in any of Examples 180 to 183 further comprises nicotinamide.

[0375] 186. The pharmaceutical composition as described in any of Examples 180 to 184, wherein the composition is a solid composition. [Medical Use]

[0376] 187. The ISVD as described in any of Examples 1 to 76, or the polypeptide as described in any of Examples 77 to 109, or the polypeptide derivative as described in any of Examples 110 to 172, is used in a pharmaceutical.

[0377] 188. The polypeptide derivatives described in any of Examples 110 to 172 are used in pharmaceuticals.

[0378] 189. The ISVD as described in any of Examples 1 to 76, or the polypeptide as described in any of Examples 77 to 109, or the polypeptide derivative as described in any of Examples 110 to 172, is used to treat inflammatory diseases.

[0379] 190. An ISVD as described in any of Examples 1 to 76, or a polypeptide as described in any of Examples 77 to 109, or a polypeptide derivative as described in any of Examples 110 to 172, is intended for the treatment of inflammatory diseases, wherein the human individual has or is at risk of having a high-sensitivity C-reactive protein (Hs-CRP) value greater than 1 mg / L, preferably greater than 1.5 mg / L.

[0380] 191. An ISVD as described in any of Examples 1 to 76, or a polypeptide as described in any of Examples 77 to 109, or a polypeptide derivative as described in any of Examples 110 to 172, is intended for the treatment of inflammatory diseases in which the human individual has or is at risk of having a high-sensitivity C-reactive protein (hs-CRP) level greater than or equal to 2 mg / L (hsCRP). 2 mg / L).

[0381] 192. The ISVD as described in any of Examples 1 to 76, or the polypeptide as described in any of Examples 77 to 109, or the polypeptide derivative as described in any of Examples 110 to 172, is used to treat cardiovascular diseases.

[0382] 193. The ISVD as described in any of Examples 1 to 76, or the polypeptide as described in any of Examples 77 to 109, or the polypeptide derivative as described in any of Examples 110 to 172, is used to treat atherosclerotic cardiovascular disease.

[0383] 194. The ISVD as described in any of Examples 1 to 76, or the polypeptide as described in any of Examples 77 to 109, or the polypeptide derivative as described in any of Examples 110 to 172, is for the treatment of atherosclerotic cardiovascular disease (ASCVD), defined as stroke and / or myocardial infarction (MI) and / or peripheral artery disease (PAD).

[0384] 195. An ISVD as described in any of Examples 1 to 76, or a polypeptide as described in any of Examples 77 to 109, or a polypeptide derivative as described in any of Examples 110 to 172, for the treatment of stroke and / or myocardial infarction (MI) and / or peripheral artery disease (PAD).

[0385] 196. An ISVD as described in any of Examples 1 to 76, or a polypeptide as described in any of Examples 77 to 109, or a polypeptide derivative as described in any of Examples 110 to 172, for use in the manufacture of a medicament for the treatment of cardiovascular diseases such as atherosclerotic cardiovascular diseases.

[0386] 197. An ISVD as described in any of Examples 1 to 76, or a polypeptide as described in any of Examples 77 to 109, or a polypeptide derivative as described in any of Examples 110 to 172, for the manufacture of a medicament for the treatment of cardiovascular diseases, such as for the treatment of stroke and / or myocardial infarction (MI) and / or peripheral artery disease (PAD).

[0387] 198. An ISVD as described in any of Examples 1 to 76, or a polypeptide as described in any of Examples 77 to 109, or a polypeptide derivative as described in any of Examples 110 to 172, for use in the manufacture of a medicament for treating inflammatory diseases.

[0388] 199. A method for treating an inflammatory disease, the method comprising administering to an individual an effective amount of an ISVD as described in any one of Examples 1 to 76, or a polypeptide as described in any one of Examples 77 to 109, or a polypeptide derivative as described in any one of Examples 110 to 172.

[0389] 200. A method for treating an inflammatory disease, the method comprising administering to an individual having or at risk of having a high-sensitivity C-reactive protein (Hs-CRP) level greater than 1 mg / L, preferably greater than 1.5 mg / L, an effective amount of an ISVD as described in any of Examples 1 to 76, or a polypeptide as described in any of Examples 77 to 109, or a polypeptide derivative as described in any of Examples 110 to 172.

[0390] 201. A method for treating an inflammatory disease, the method comprising administering to an individual having or at risk of having a high-sensitivity C-reactive protein (Hs-CRP) value greater than or equal to 2 mg / L an effective amount of an ISVD as described in any of Examples 1 to 76, or a polypeptide as described in any of Examples 77 to 109, or a polypeptide derivative as described in any of Examples 110 to 172.

[0391] 202. A method for treating cardiovascular disease, the method comprising administering to an individual an effective amount of an ISVD as described in any one of Examples 1 to 76, or a polypeptide as described in any one of Examples 77 to 109, or a polypeptide derivative as described in any one of Examples 110 to 172.

[0392] 203. A method for treating cardiovascular disease, the method comprising administering to an individual suffering from stroke and / or myocardial infarction (MI) and / or peripheral artery disease (PAD) an effective amount of ISVD as described in any one of Examples 1 to 76, or a polypeptide as described in any one of Examples 77 to 109, or a polypeptide derivative as described in any one of Examples 110 to 172.

[0393] 204. A method for treating cardiovascular disease, the method comprising administering to an individual suffering from chronic kidney disease (CKD) an effective amount of ISVD as described in any one of Examples 1 to 76, or a polypeptide as described in any one of Examples 77 to 109, or a polypeptide derivative as described in any one of Examples 110 to 172.

[0394] 205. A method for treating atherosclerotic cardiovascular disease, the method comprising administering to an individual an effective amount of ISVD as described in any one of Examples 1 to 76, or a polypeptide as described in any one of Examples 77 to 109, or a polypeptide derivative as described in any one of Examples 110 to 172.

[0395] 206. A method for treating atherosclerotic cardiovascular disease, the method comprising administering to an individual suffering from stroke and / or myocardial infarction (MI) and / or peripheral artery disease (PAD) an effective amount of ISVD as described in any one of Examples 1 to 76, or a polypeptide as described in any one of Examples 77 to 109, or a polypeptide derivative as described in any one of Examples 110 to 172. [tablet]

[0396] 207. An ISVD, polypeptide, or polypeptide derivative capable of binding to IL-6 at an epitope comprising at least amino acid residues 26, 30, 33, 34, 74, 75, 78, 171, 175, 178, 179, 182, and 183 of SEQ ID NO: 89. (sequentially numbered)

[0397] 208. An ISVD, polypeptide, or polypeptide derivative capable of binding to IL-6 at an episite comprising at least amino acid residues 26, 30, 33, 34, 73, 74, 75, 78, 171, 175, 178, 179, 182, and 183 of SEQ ID NO: 89. (sequentially numbered)

[0398] 209. An ISVD, polypeptide, or polypeptide derivative as described in any of Examples 1 to 172, comprising a tryptophan residue capable of forming a cationic-π interaction with Arg179 and / or a hydrophobic interaction with Phe78, as shown in SEQ ID NO: 89. (Serial numbered)

[0399] 210. An ISVD, polypeptide, or polypeptide derivative capable of binding to IL-6 at an episite comprising at least amino acid residues 24, 27, 28, 31, 88, 92, 95, 99, 114, 116, 117, 118, 120, 121, 132, 124, 125, 127, 128, and 139 of SEQ ID NO: 89. (sequentially numbered)

[0400] 211. An ISVD, polypeptide, or polypeptide derivative capable of binding to IL-6 at an episite comprising at least amino acid residues 24, 27, 28, 31, 88, 92, 95, 99, 114, 116, 117, 118, 120, 121, 132, 124, 125, 127, 128, 138, and 139 of SEQ ID NO: 89. (sequentially numbered)

[0401] 212. An ISVD, polypeptide, or polypeptide derivative of hIL-6 (SEQ ID NO:89) having a KD of 10-60 pM, such as 11-40 pM, such as 12-30 pM.

[0402] 213. An ISVD, polypeptide, or polypeptide derivative as described in any of Examples 1 to 172, which is capable of binding hIL-6 (SEQ ID NO: 89) and has a KD of 10-60 pM, such as 11-40 pM, such as 12-30 pM.

[0403] Equivalents: Those skilled in the art will recognize or be able to use numerous equivalents of specific embodiments of the invention, without exceeding conventional experimental verification. These equivalents are intended to fall within the scope of protection of the appended claims. [Example]

[0404] [List of Abbreviations]

[0405] CDR: Complementarity Determinant Region

[0406] EC50 half-maximum effective concentration

[0407] ED50 (half maximum effective dose)

[0408] FACS: Fluorescence-Activated Cell Sorter

[0409] FBS / FCS: Fetal bovine / calf serum

[0410] FR: Framework Area

[0411] HSA: Hepes buffered saline solution

[0412] HSA: Human serum albumin

[0413] IC50 half-maximum inhibition concentration

[0414] ISVD: Immunoglobulin Single Variable Domain

[0415] IV: Intravenous

[0416] MSX: L-methionine sulfadiazine

[0417] NAM: Nicotinamide

[0418] MRTHL: Mean Residence Time Terminal Half-Life

[0419] OEG: 8-Amino-3,6-dioxa-octanoic acid

[0420] PBMC: Peripheral blood mononuclear cells

[0421] PBS: Phosphate-buffered saline

[0422] PCR: Polymerase chain reaction

[0423] PK: Pharmacokinetics

[0424] PO: via oral

[0425] RGA: Reporter Gene Assay

[0426] RT-PCR: Reverse Transcription Polymerase Chain Reaction

[0427] SAA1: Serum amyloid A1

[0428] SIA: Sequence-identical analogues

[0429] SC: Subcutaneous

[0430] SNAC: Sodium N-[8-(2-hydroxybenzoyl)amino]octanoate

[0431] SPR: Surface Plasma Resonance

[0432] STAT: signal transducers and transcription activators

[0433] T½: Half-life

[0434] TMB: 3,3',5,5'-Tetramethylbenzidine [Materials and Methods] Preparation of reference anti-IL6 Mab and reference anti-IL6 Fab

[0435] Reference anti-IL6 Mab (SEQ ID No. 130, having heavy chain: SEQ ID NO: 97 & light chain: SEQ ID NO: 101) (Table 35 Item 1) and its Fab derivative, which may be referred to as reference anti-IL6 Fab (VH-CH1 of heavy chain: SEQ ID NO: 100; light chain: SEQ ID NO: 101), may be prepared according to the procedure described in WO 2008 / 065378.

[0436]

[0437] [ General Molecular Biology ]

[0438] For general molecular biology techniques, see Molecular Cloning: A Laboratory Manual (4th edition, 2014, edited by Sambrook, Fritsch and Maniatis, CSHL Publishing, Cold Spring Harbor, NY USA). [V] [H] [General characteristics of H]

[0439] [V in HEK293 cells] [H] [General characteristics of H]

[0440] Plastids used for transient expression in HEK293 cells were purchased from Twist Biosciences or ThermoFisher Scientific. The plastid from Twist Biosciences was based on the pTT vector, described in Durocher, Y. et al., (2002) Nucleic Acid Res, 30:E9, while the plastid from ThermoFisher Scientific was based on the pcDNA34-Topo vector (ThermoFisher Scientific). HEK293 suspension cells (293Expi, Invitrogen) were transiently transfected with the plastids basically according to the manufacturer's instructions. 293Expi cells were typically passaged every 3–4 days in Expi293F expression medium (Invitrogen, catalog number A1435104) supplemented with 1% P / S (GIBCO catalog number 15140-122). Expi293F cells were transfected at a cell density of 2.5–3 milligrams / mL using Expifectamine. For each liter of Expi293F cells, transfection was performed by diluting a total of 1 mg of plasmid DNA in 50 mL of Optimem (GIBCO, catalog number 51985-026, dilution A) and 2.7 mL of Expifectamine in 50 mL of Optimem (dilution B). For co-transfection (i.e., Fab and mAb), plasmids were used at a 1:1 ratio. Diluents A and B were mixed and incubated at room temperature for 10–20 minutes. Subsequently, the transfection mixture was added to Expi293F cells, and the cells were incubated at 37°C in a humidified incubator equipped with an orbital rotor (85–140 rpm). One day after transfection, the transfected cells were supplemented with 5 mL of ExpiFectamine 293 Transfection Enhancer 1 and 50 mL of ExpiFectamine 293 Transfection Enhancer 2. Typically, 4–5 days after transfection, the cell culture supernatant was collected by centrifugation followed by filtration.

[0441] [V] [H] [General characteristics of H in Saccharomyces cerevisiae]

[0442] The plasmid for expressing the VHH polypeptide in *Saccharomyces cerevisiae* was constructed by transfecting a synthetic DNA fragment encoding the VHH polypeptide, obtained from Geneart AG (Regensburg, Germany), into a yeast multicopy vector derived from cPOT-type expressor plasmids (previously described in EP0171142). The resulting plasmids were transformed into yFI3104 (a proprietary *Saccharomyces cerevisiae* strain) using the freeze-EZ yeast transformation kit (Zymo Research, CA), according to the manufacturer's instructions or other standard yeast transformation methods. Yeast transformers were screened using glucose as a carbon source on agar plates containing 1% yeast extract, 2% peptone, and 2% glucose. As described by Verduyn et al. (Verduyn, C., Postma, E., Scheffers, WA, Van Dijken, JP (1992) Yeast 8, 501-517), yeast strains containing plastids encoding the VHH polypeptide were cultured in minimal medium with the addition of 7 g / L yeast extract and 210 g / L glucose. The yeast supernatant was harvested by centrifugation. [V] [H] [General purification and analysis of H]

[0443] VHH peptides with or without a hexahistine tag were purified by immobilized metal affinity chromatography (IMAC) on Ni-Excel (Cytiva) resin with imidazole or acidic eluent, followed by a desalting step (PD column with Sephadex G25 resin, Cytiva), and, if necessary, gel filtration chromatography in PBS or HBS (Superdex 200 or Superdex 75 column, Cytiva). VHH peptides without a hexahistine tag were purified by multimodal resin (Cytiva), eluented with acidic eluent, followed by a desalting step (e.g., PD column with Sephadex G25 resin, Cytiva), and, if necessary, gel filtration chromatography in PBS or HBS (Superdex 200 or Superdex 75 column, Cytiva). Protein integrity was analyzed using high performance size exclusion liquid chromatography (SE-HPLC) on an Agilent LC 1100 / 1200 system with a BIOSEP-SEC-3000 300×7.8 mm column (Phenomenex, catalog number OOH-2146-K0) and a run buffer consisting of 200 mM sodium phosphate (pH 6.9), 300 mM NaCl, and 10% isopropanol. The molecular weight of the purified VHH peptide batch was analyzed using electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) on a 6280 Agilent system (Agilent Technologies) with a MassPREP desalting (Waters) column, in a staged elution process at 0.4 mL / min in buffer A consisting of MQ-H2O / 0.1% formic acid and buffer B consisting of acetonitrile / 0.1% formic acid. The final protein concentration was measured using a NanoDrop™ spectrometer (Thermo Scientific) and theoretically calculated extinction coefficients. [ Anti-IL-6 V ] [ H ] [ Differential scanning fluorescence (Nano DSF) of H ]analyze

[0444] The pyrolysis folding phenomenon was measured using a Prometheus NT.48 instrument from Nanotemper Technologies. A linear thermal gradient (1–1.5 °C / min, from 20 °C to 95 °C) was used and fluorescence at 350 and 330 nm was measured. The first derivative of the fluorescence ratio (F350 / F330) was determined from the midpoint (Tm) of the first pyrolysis folding reaction.

[0445] Nano DSF addresses the thermal stability issues of various variants of the VHH peptide. The final compound concentration is approximately 1 mg / ml, and the buffer concentration is 20 mM HEPES, pH 7.4, containing 150 mM NaCl. [ Reporter gene sequencing (RGA) ]

[0446] In detail, the reporter gene assay (RGA) tests the ability of anti-IL-6 VHH to inhibit IL-6-induced STAT3 phosphorylation, thereby inhibiting the transcription of luciferase reporter genes in reporter cells expressing IL-6R and gp130.

[0447] To this end, an IL-6 reporter cell line was developed by stably transfecting HEK293 cells (ATCC CRL-1573) with reporter plasmids encoding a STAT3 response element coupled to luciferase. A single cell line was isolated as the reporter cell line. Upon exposure to IL-6, the transcription factor STAT3 was phosphorylated, activating the transcription of the downstream luciferase gene of this response element. IL-6 activity could be directly measured by detecting bioluminescence during the conversion of luciferin, the luciferase receptor, to oxidized luciferin.

[0448] For this assay, HEK293-STAT3-Luc cells were cultured in DMEM GlutaMAX (Gibco 31966-021), 10% fetal bovine serum (FBS, Gibco 10091-148), 1% penicillin / streptomycin (P / S, Lonza DE17-602E), and 1 μg / ml puromycin (Gibco #A11138-03). On the day of the experiment, the cells were resuspended in the assay medium (DMEM w / o phenol red (Gibco #31053), 10% FBS 1% P / S), and 12,500 cells / well were seeded at 22.5 μl / well in a 384-well plate (Greiner #781086).

[0449] Anti-IL-6 VHH was serially diluted and pre-incubated for 30 minutes in assay medium containing 1 nM hIL-6 (internal). 2.5 μl / well was added to the seeded cells, with duplicate or triple replicates to obtain a final concentration of 100 pM IL-6. After incubation overnight at 37°C, 25 μl / well of 5% CO2 detection reagent (Steady-GLO, Promega E2510) was added, and the dish was incubated at room temperature for 15 minutes. luminescence was then detected using an EnVision multi-mode disk reader (PerkinElmer). To determine the IC50 for each assay, the raw data were fitted with 4-parameters using GraphPad Prism software (GraphPad Prism version 9.0.1 for Windows, GraphPad Software, San Diego, California USA). [ISVD, peptides and peptide derivatives / their constructs]

[0450] The following list of ISVDs, peptides, and / or peptide derivatives is a non-exhaustive list of exemplary structures, which may be discussed in the Examples section and should be understood as presented for ease of reference. These lists and tables are merely illustrative and should not be construed as limiting in any way. [ Immunoglobulin Single Variable Domain (ISVD) ]

[0451] The following are examples of immunoglobulin single variable domains (ISVDs) containing a VHH. Non-restrictive VHH examples provided in Table 11 are further identified by their amino acid sequences and SEQ ID Nos in their respective columns. The following are merely illustrative and should not be construed as limiting in any way.

[0452] [ polypeptides & polypeptide derivatives / compounds

[0453] The following are examples of peptides and / or peptide derivatives. Peptides will be assigned a designation of type "peptide_n", where n is an integer used for identification (e.g., peptide_1, peptide_2, peptide_3, and so on). Similarly, peptide derivatives will be assigned a designation with the prefix "compound", such that the resulting designation (ID) is of type "compound_n", where n is an integer used for identification (e.g., compound 1, compound 2, compound 3, and so on). In the table below, it should be understood that any reference to a given position of a substituent in an extension refers to the relative position from the N-terminus to the C-terminus of the full length of the amino acid sequence of the extension. For example, if the position of a substituent is referred to as "Cys at position 4 of extension", it should be understood to refer to the cysteine ​​residue found at position 4 of the full length of the extension sequence, as defined in the corresponding extension column for a given peptide derivative, starting from residue number 1 closest to the N-terminus. Furthermore, it should be understood that when referring to an extension, the position column refers to the location of the extension relative to VHH. For example, if the item status in the location column of the extension is "C-terminus", it should be understood that it means that the extension is found at the C-terminus of the VHH of the polypeptide, which can also be used for polypeptides found in the section on polypeptide derivatives.

[0454] Chemical formula 24 refers to the substituents as indicated in Table 9. The extension / tag is identified by its SEQ ID No. If it is an extension that is a combination of sub-elements (e.g., an extension with a his-tag), then the ID will identify the name of the sub-element, and the SEQ ID No. refers to the complete sequence of the extension.

[0455] The following are merely illustrative and should not be construed as limiting in any way. The peptide derivative ID "Compound 13" is intentionally omitted.

[0456] [Example 1: V] [H] [Immunization and Construction of the H Library]

[0457] The purpose of this example is to construct an anti-IL-6 VHH library for the development of suitable anti-IL-6 VHH peptides for further development into drug candidates for oral and / or subcutaneous delivery.

[0458] First, an anti-IL-6 VHH library was generated. A group of VHHs with the highest potential in terms of appropriate binding affinity to KD, IC50 value, and biophysical stability were selected for further optimization, including reduced pI values, humanization, and the addition of elongators, to develop, for example, an optimized oral anti-IL-6 drug candidate. [ 1.1 Immunization ]

[0459] Single-domain antibodies were obtained from llamas immunized with the N-terminal His-tagged human IL-6 (SEQ ID NO: 82; hIL-6-His) protein antigen. Animals were immunized six times weekly via subcutaneous injection (100 μg antigen for the first two injections, and 50 μg antigen for the remaining injections). One week after the final booster, serum was collected to determine antibody titers against biotinylated hIL-6-His (SEQ ID NO: 82; bio-hIL-6-His) and biotinylated cynomolgus IL-6 (SEQ ID NO: 83; bio-cyIL-6-His) with an N-terminal His-tagged antibody, which were measured by ELISA. In this ELISA, 96-well discs (Maxisorp; Nunc) were coated with 5 μg / ml of neutral avidin (Thermo Scientific) diluted in PBS (pH 7.4) and incubated overnight at 4°C. The next day, the dish was washed with 0.05% PBS-Tween (pH 7.4) and blocked with 1% casein (Sigma) at room temperature for 2 hours. After blocking, 1 nM of bio-hIL-6-His and bio-cyIL-6-His antigens (diluted in 0.1% casein / PBS (pH 7.4)) were captured, followed by the addition of serum samples (pH 7.4) sequentially diluted in 0.1% casein / PBS. The presence of anti-hIL-6 antibody was confirmed using mouse anti-lamb IgG antibody, followed by donkey anti-mouse immunoglobulin peroxidase conjugate antibody (Jackson ImmunoResearch). The dish was incubated at room temperature for 1 hour. Between each incubation step, the dish was washed with 0.05% PBS-Tween (pH 7.4) and a final wash with PBS (pH 7.4) was performed before color development. The substrate solution (TMB solution) and H2SO4 were added sequentially to the dish for ELISA signal development. The ELISA signal was obtained at 450nm using a disk reader.

[0460] [ 1.2 Gene Bank Construction ]

[0461] RNA was extracted from the PBMCs of two immunized llamas. The extracted RNA was used for cDNA synthesis using random primers. The cDNA was used for primary PCR amplification using untagged primers that adhered to the leader sequence and the CH1 region of the hinge, followed by a secondary PCR amplification to introduce restriction endonuclease sites for selecting the VHH gene in a phage vector. The gene library was electroporated into TG1 *E. coli* cells, and the bacterial glycerol stock solution of the immunobank was stored at -80°C. [ 1.3 V ] [ H ] [ H separation ]

[0462] A phage system created from llama VHH library aggregates was used for three consecutive rounds of phage display screening, employing biotinylated human IL-6 Avi (bio-hIL-6-Avi-His, SEQ ID NO:96) and bio-cyIL-6-His proteins (SEQ ID NO:83) with N-terminal His tags, at pH 7.4 The concentration of PBS buffer was decreased (50 nM, 5 nM, and 0.5 nM for bio-hIL-6-Avi-His and bio-cyIL-6-His in the first round, respectively (pre-captured on microbeads); 50 nM, 5 nM, and 0.5 nM for the second round; and 50 nM, 5 nM, 0.5 nM, and 0.05 nM for the last round) to wash off nonspecific phages. Then, the phage extract was prepared with trypsin-containing buffer (total extract), and trypsin activity was inhibited with 4-(2-aminoethyl)-benzenesulfonylfluorine. Specific extraction was performed using anti-IL-6 monoclonal antibody (see "anti-IL6 Mab"; SEQ ID No. 130, with heavy chain: SEQ ID NO: 97 & light chain: SEQ ID NO: 101). The phage extracted from the counter-cyclic phage was subjected to a series of dilutions and used to infect exponentially growing TG1. The infected TG1 was plated in LB+Carb100+Glu2% plates, and enrichment values ​​were calculated against the background (without screening antigen).

[0463] Selected strains from the second and third rounds of screening were placed into 96-well master discs, and the binding of periplasmic extract (PE) to bio-hIL-6-Avi-His (SEQ ID NO: 96) and bio-hIL-6-Avi-His (SEQ ID NO: 83) was tested via avidin capture and measured using a binding ELISA. For the PE binding ELISA, MaxiSorp™ high protein binding capacity 96-well ELISA discs were coated with 5 μg / ml avidin (Thermo Scientific), diluted in PBS (pH 7.4), and incubated overnight at 4°C. The next day, the discs were washed with 0.05% PBS-Tween (pH 7.4) and blocked with 1% casein (Sigma) at room temperature for 2 hours. Following blocking, 1 nM of bio-hIL-6-Avi-His (SEQ ID NO: 96) and bio-cyIL-6-His (SEQ ID NO: 83) antigens were captured and diluted in 0.1% casein / PBS (pH 7.4). Periplasmic extract containing soluble VHH was then added to the dish and incubated at a 1:5 dilution in 0.1% casein / PBS (pH 7.4). Detection of PE sample binding was performed using mouse anti-c-Myc antibody (Roche), followed by a secondary antibody anti-mouse immunoglobulin peroxidase conjugate (Jackson ImmunoResearch). The dish was incubated at room temperature for 1 hour, washed with 0.05% PBS-Tween (pH 7.4) between incubation steps, and finally washed with PBS (pH 7.4) before color development. The acceptor solution (TMB solution) and H2SO4 were added sequentially to the dish for ELISA signal development. The ELISA signal was obtained at 450nm using a disk reader.

[0464] The VHH gene of positive binders was sequenced. Variants were classified into selected strains based on different HCDR3 sequences. Based on sequence similarity and on / off rate kinetics, 182 VHH sequences were selected for further identification using a Fortebio instrument (Octet Fortebio Red384 from Sartorius) according to the vendor description.

[0465] Specifically, 22 sequences were selected for comprehensive identification, including affinity measurements, in vitro assays, and biophysical assays. Table 14 provides an overview of the selected VHH sequences, and Table 15 further lists the characteristics of the VHH peptides. As will be understood by those skilled in the art, the VHH peptide sequences listed below comprise the VHH sequence itself and the C-terminal His tag GGGGSHHHHHH (extension E1, SEQ ID NO. 71), which may alternatively be referred to as the C-terminal extension of VHH.

[0466]

[0467]

[0468] Table 15 shows that peptides 19 (SEQ ID No. 57), 35 (SEQ ID No. 86), 33 (SEQ ID No. 84), and 34 (SEQ ID No. 85) not only exhibit sequence diversity, but each VHH sequence also demonstrates optimized stability, expressibility, binding affinity, and potency. Therefore, these four peptides were selected for further optimization and development. [Example 2: Tablet Puzzle]

[0469] The purpose of this example is to resolve the protein crystal structures of selected anti-IL-6 VHH peptides (SEQ ID No: 84, 86, and 57) that are complexed with human IL-6, in order to determine the complementary site and epitope interactions between the selected anti-IL-6 VHH peptides and human IL-6. The selected anti-IL-6 VHH peptides are peptide_33 (SEQ ID No. 84), peptide_35 (SEQ ID No. 86), and peptide_19 (SEQ ID No. 57), detailed in Table 14.

[0470] This result enhances our understanding of the detailed molecular mechanisms of action and guides further optimization of VHH peptides, particularly by introducing mutations in the CDR ring and other complementary residues to further improve potency. [ 2.1 Preparation of Crystal Structure ]

[0471] A mixture of IL-6 (1 equivalent) and VHH peptide (1.2 equivalent) dissolved in HBS buffer was incubated at 4°C for 60 minutes and purified by size exclusion chromatography on a Superdex 75 16 / 60 column, using a solution of 10 mM Tris pH 7.5 and 150 mM sodium chloride. The SEC extract peaks of the purified complex were collected, aliquoted, combined, and concentrated to 5–20 mg / mL, and used for a 96-well sitting drop crystallization assay using a commercially available crystallization sieve.

[0472] [2.1.1 Crystallization and structural determination of SEQ ID NO:84 (peptide 33) complexed with IL-6.]

[0473] The crystals were obtained under the following storage conditions: 2M ammonium sulfate, 0.1M Bis-Tris, pH 6.5. The crystals were cryoprotected in glycerol and then frozen in liquid nitrogen. X-ray diffraction data were collected on a Rigaku FR-X rotating anode with a Rigaku Dectris Pilatus3R 1M detector and processed in XDS / XSCALE to a resolution of 2.4 Å in space group 5 (C2). The structure was determined using molecular substitution, run in Phoenix, using the following PDB items as search models: IL-6 (PDBid: 1alu) and VHH template (PDBid: 5ivo). The solution from the molecular substitution was in space group 5 (C2) and contained two IL-6 / SEQ ID NO: 84 complexes in the asymmetric unit. The structure was refined to a resolution of 2.4 Å in Phoenix.refine. The two complexes in the asymmetric unit showed high structural similarity, with an RMSD of 0.286 Å calculated using "Align" in PyMOL. Both IL-6 molecules in the asymmetric unit also showed high structural similarity to the IL-6 PDB project 1alu: the RMSDs of both IL-6 molecules calculated using "Align" in PyMOL were below 0.48 Å.

[0474] [2.1.2 Crystallization and structural determination of SEQ ID NO:86 (peptide 35) complexed with IL-6.]

[0475] The crystals were obtained under the following storage conditions: 0.2 M magnesium chloride hexahydrate, 0.1 M sodium HEPES, pH 7.5, and 25% w / v PEG 3350. The crystals were cryoprotected in glycerol and then frozen in liquid nitrogen. Synchrotron X-ray diffraction data were collected at the Swiss Light Source and processed in XDS / XSCALE to a resolution of 3.0 Å in space group 90 (P4212). The structure was determined using molecular substitution, run in Phoenix, using the following PDB items as search models: IL-6 (PDBid: 1alu) and VHH template (PDBid: 5ivo). The solution from the molecular substitution was in space group 90 (P4212) and contained two IL-6 / SEQ ID NO: 86 complexes in the asymmetric unit. The structure was refined to a resolution of 3.0 in Phoenix.refine. The two complexes in the asymmetric unit showed high structural similarity, with an RMSD of 0.337 Å calculated using "Align" in PyMOL. Both IL-6 molecules in the asymmetric unit also showed high structural similarity to the IL-6 PDB project 1alu: the RMSDs of both IL-6 molecules calculated using "Align" in PyMOL were below 0.58 Å.

[0476] [2.1.3 Crystallization and structural determination of SEQ ID NO:57 (peptide 19) complexed with IL-6.]

[0477] The crystals were obtained under the following storage conditions: 2.0 M ammonium sulfate, 0.1 M sodium acetate 4.6. The crystals were cryoprotected in glycerol and then frozen in liquid nitrogen. Synchrotron X-ray diffraction data were collected at the Swiss Light Source and processed in XDS / XSCALE to a resolution of 2.4 Å in space group 20 (C2221). The structure was determined using molecular substitution, run in Phoenix, using IL-6 / SEQ ID NO:33 as the search model. The solution from the molecular substitution was in space group 20 (C2221) and contained an IL-6 / SEQ ID NO:57 complex in the asymmetric unit. The structure was refined to a resolution of 2.4 Å in Phoenix.refine. Both IL-6 molecules in the asymmetric unit showed a high structural similarity to the IL-6 pdb item 1alu: RMSD = 0.554 Å, calculated in PyMOL using "Align". [ 2.2 Localization of antigenic epitopes and complementary sites ]

[0478] The epitopes of IL-6 and the complementary bits of VHH are described using two different methods.

[0479] [Method 1]

[0480] The difference in surface contact area between the complex and the free component was calculated using PyMOL InterfaceResidues with a cutoff value of 1.0, thereby defining the interface residues.

[0481] [Method 2]

[0482] Method 2 is based on the distance between the amino acid residues of IL-6 and the amino acid residues of VHH, using a distance cutoff value of <4.5 Å for manual inspection.

[0483] The crystal structures of SEQ ID NO:84 and SEQ ID NO:86, which are complexed with IL-6, each contain two complexes (see Tables 16 and 17. SEQ ID NO:84 and SEQ ID NO:86 are denoted as "Comp1" and "Comp2").

[0484] The crystal structure of the complex of SEQ ID NO:57 and IL-6 contains only one complex in the asymmetric unit, and is called SEQ ID NO:57 complex 1.

[0485] Epitopes and complementary residues of the IL-6 / peptide complex are listed in Tables 16 and 17, respectively.

[0486]

[0487]

[0488] As shown in Figure 4A, aromatic π-cation interactions between the aromatic side chain of Trp59 (peptide_19; SEQ ID NO:57) and the basic side chain of Arg179 (human IL-6) can be observed. This is important for the very strong affinity and potency of VHH and its variants observed in vitro and in vivo.

[0489] Based on crystallographic experiments, molecular structures were obtained at resolutions of 2.4 and 3.0 Å, revealing detailed epitope-complementary site interactions between human IL-6 and the tested anti-IL-6 VHH peptide. These data were used as input for optimizing the VHH peptide, as described below, as part of the design cycle optimization. [ 2.3 Computer Models for Structural Prediction ]

[0490] AlphaFold-Multimer version 2.2.0 is used to generate the complex structure of VHH and hIL-6 using a sequence-based approach. AlphaFold-Multimer version 2.2.0 generates multiple structures for each model with default values ​​and selects the top model based on the ranking_confidence score, choosing the highest-ranked structure. Ranking_confidence is a linear combination of the interface score ipTM (interface prediction template simulation score) and the overall structure score pTM: 0.8ipTM + 0.2pTM. The analysis method for interface residues is the same as in Method 1.

[0491]

[0492]

[0493] Tables 18 and 19 show a comparison between experimentally determined complex structures and computer-generated structural predictions and high-resolution resolved complementary-epitaxe information. We observed structural differences within angstroms in the protein backbone alignment between the two methods, and high accuracy in side chain prediction was also observed when the backbone prediction was accurate. The results indicate that the two methods yield nearly consistent results for complementary-epitaxe determination. This further rationalizes the prediction of epitopes / complementary sites in structurally unresolved complexes.

[0494] [Example 3: Design, Cycling, and Generation of Peptides] [ 3.1 Designing a Loop ]

[0495] To optimize the anti-IL-6 peptide, a series of design cycles were performed. These design cycles were performed to optimize one or more of the following: ˙ achieving complementarity-epitope interaction by introducing mutations that enhance affinity and functional potency; ˙ enhancing oral bioavailability by reducing the pI value of the anti-IL-6 VHH peptide; ˙ reducing the risk of immunogenicity from human exposure by humanizing the llama-derived VHH sequence; ˙ introducing a fatty acid elongation moiety (substituent) by conjugating the elongation moiety to a cysteine ​​residue in the C-terminal extension of the VHH peptide; and ˙ stabilizing volatile residues, such as mutating the N-terminal glutamine to glutamic acid to inhibit the formation of pyroglutamic acid. [ 3.2 Production of polypeptides ]

[0496] [V generated as part of the design cycle] [H] [Examples of H-peptides.]

[0497] As described above, as part of the design cycle optimization, Table 21 below shows peptides optimized through mutation to increase potency / affinity, through mutation to increase oral bioavailability, through mutation to reduce immunogenicity risk, and / or through mutation to stabilize volatile residues. This design cycle is based on the immunochemical and construction of the peptide_19 (SEQ ID No. 57):VHH library originally shown in Example 1. For easy reference, Table 21 provides the basic VHH for each peptide, and Table 12 can also be consulted for detailed composition of the example peptides.

[0498] For example, starting from polypeptide_19, SEQ ID No. 57 (or more specifically, VHH-component polypeptide_19), polypeptide_29 is generated and contains the mutations shown below: ˙ Mutations that increase potency / affinity: T28R, S31E, N54Q, D56E, H62E, K65L, N74D, S85G, S100K, G106N; ˙ Mutations that increase oral bioavailability: Q1E, S31E, H62E, K65L, N74D, Q82E, N84D; ˙ Mutations that reduce the risk of immunogenicity: Q1E, L2V, W11L, H62E, Q82E, N84D; and ˙ Mutations that stabilize volatile residues: Q1E, W11L.

[0499]

[0500]

[0501] As observed in Table 22, the peptides shown exhibit reduced pI values ​​(pI calculated as described herein), obtained through mutations of specific surface-exposed residues, as shown in Table 21. As can be observed, these peptides show a clear reduction in pI values ​​compared to the reference peptides. The tables also show Tm measurements of the peptides shown, which were measured according to the General Materials and Methods: Differential Scanning Fluorescence (Nano DSF) Analysis for Anti-IL-6 VHH. All of the above peptides exhibited relatively high melting temperatures, between 48–60 °C, with most between 50–60 °C, which is an acceptable melting temperature for pharmaceutical compounds suitable for human administration, where the normal physiological temperature is 37 °C.

[0502] Specifically, compared to reference peptide 19, peptide 29 exhibited a reduced pI value of 5.8 while maintaining a suitable melting temperature of 59°C. Therefore, this peptide, or more specifically, the underlying VHH therein, provides the desired pharmaceutical properties, such as pI value, melting temperature, and potency (see further examples below). Further details will be provided in the examples below. [Example 4: In vitro activity of anti-IL-6 VHH variant in STAT-3-LUC reporter gene assay] [Antifraction of mutated IL-6 interacting residues in VHH_1.6] [ Purpose ]

[0503] The purpose of this example is to test the ability of anti-IL-6 VHH VHH_1.6 anti-CDR / FR residue variants to inhibit IL-6-dependent STAT3 signaling in vitro, thereby suppressing IL-6 activity. Therefore, this provides insight into the effects of individual CDR / FR residues on the interaction between anti-IL-6 VHH_1.6 and human IL-6. Data are listed in Tables 23 and 24. [ method ]

[0504] [Computer-induced mutagenesis sampling for positive and favorable mutations to improve binding]

[0505] X-ray crystal structure of the VHH-IL-6 protein complex was obtained. The structure was cleaned using PyMOL (Schrödinger, LLC) to remove any missing residues or atoms. Hydrogen atoms were added, and the structure was minimized using RosettaSuite to mitigate any spatial conflicts. Residues within a 5 Å range on the IL-6 surface were identified as potential mutation sites. For each identified residue, computer-generated site saturation mutagenesis (SSM) was performed, mutating the residue to all 19 possible amino acid substitutions by computation. The effect on binding energy was assessed using the Rosetta scoring function (specifically, the scoring function named ref2015). Substituents from the reference polypeptide 29 (SEQ ID No. 67) containing VHH_1.6 are summarized in Table 23. To better simulate backbone movement upon introduction of mutations, a backbone perturbation of 0.2 Å was allowed.

[0506] Molecular dynamics (MD) simulations were performed on the highest-ranking mutations in the SSM to further assess binding affinity and generalize binding kinetics. AMBER99SB force fields were applied to the proteins, and simulations were performed using the GROMACS software suite. Each system was minimized, equilibrated, and then subjected to a 100 ns production run at 300 K and 1 atm pressure. Selected trajectories were extracted using the Rosetta Cartesian ddg application to calculate binding energies.

[0507] [Report Genetic Assay (RGA)]

[0508] The reporter gene assay was performed as described in the Materials and Methods section. Reported values ​​are the average of two technical replicates (Table 24). [ result ]

[0509] As described above, the design cycle for peptide_29 is indicated as the starting point. Each mutation explored as part of the design cycle, compared to peptide_19 (SEQ ID No. 57, originally indicated in Example 1: Immunochemicals and Construction of the VHH Library), was defolded to better understand the individual role of peptide_19. Table 23 below provides an overview of the peptides in this example, where the peptide column provides information for each item, the modification column indicates residue modifications compared to peptide_29, and the VHH column indicates the underlying VHH for each of these peptides. Detailed composition of the example peptides can also be found in Table 12. Table 24 shows the potency (IC50) of the anti-IL6 VHH peptides for each item and their relative potency compared to the reference peptide_29. Tables 23 and 24 further indicate the reference peptide_19 for illustrative purposes.

[0510]

[0511]

[0512] As shown in Table 24, peptide 29 exhibits a potency of 0.034 nM. Mutant peptides generally result in lower potency. For example, compared to peptide 29, peptides 54, 55, and 56 showed potency reductions of 4.4-fold, 8.1-fold, and 4.9-fold, respectively. These mutant peptides incorporated mutations at positions 54, 56, and 62, highlighting the importance of these residues. Furthermore, a 2.3-fold decrease in potency was also observed in mutant peptide 57 with a mutation at position 65, while a 3.8-fold decrease in potency was observed in mutant peptide 62 with a mutation at position 106.

[0513] This data indicates that the mutations identified in the design cycle of peptide_29 played a role in achieving higher potency, which is desirable. In some cases, antifold data showed that altering some of these residues resulted in a 2-fold or more fold loss in potency. The data showed that residues 54Q, 56E, 62E, and 106N, found in peptide_29 (or more specifically in basal VHH_1.6), play a role in increasing the salt bridge between anti-IL6 VHH and IL-6 (54Q and 56E) or generating new salt bridges (62E & 106N). Figure 5 shows a structural analysis of the salt bridge stability at positions 54, 56, 62, and 106, confirming better binding to IL-6, thereby improving the potency of peptide_29. [Example 5: In vitro activity of anti-IL-6 VHH_1.6 and comparative agents in STAT-3-LUC reporter gene assay] [ Purpose ]

[0514] The purpose of this example is to compare the efficacy of VHH_1.6 with other IL-6 inhibitory antibodies in inhibiting IL-6-dependent STAT3 signaling and thus inhibiting IL-6 activity in vitro. [ method ]

[0515] The reported gene assay was performed as described in the Materials and Methods section. The reported values ​​are the average of two technical replicates.

[0516] As described above, peptide 29 (containing VHH 1.6) and reference anti-IL6 Fab were prepared.

[0517] Following the general descriptions in "General Performance of VHH" and "General Purification and Analysis of VHH" in the Materials and Methods section above, two comparative constructs were recombined for face-to-face comparison of IC50 measurements and calculation of relative potency. A sequence equivalent (SIA) of the anti-IL-6R domain (SEQ ID No. 163) of the bispecific domain antibody vobarilizumab (CAS Registry No.: 1628814-88-9) was prepared as the first comparative. Furthermore, a sequence equivalent (SIA) (SEQ ID No. 165) of the anti-IL-6 VHH construct 7G05 in the multispecific ISVD construct described in WO2022 / 129572 was prepared as the second comparative. [ result ]

[0518]

[0519] As shown above, the reference anti-IL6 Fab and peptide_29 exhibited comparable potency at 39 and 44 pM, respectively. Furthermore, the sequence equivalents (SIAs) of the anti-IL-6R domain and the anti-IL-6 VHH construct block 7G05 of the bispecific antibody vobarilizumab were observed to have higher IC50 values, both approximately 2.6 times higher than peptide_29. In other words, this data indicates that the comparative drug was 2.6 times less potent than peptide_29. [ in conclusion ]

[0520] As can be clearly seen from the above, peptide_29 exhibits significantly higher potency compared to the comparative anti-IL6 construct. Peptide_29, or more specifically, the base VHH_1.6, has been shown to provide potency similar to that presented by the reference anti-IL6 Fab. It is understood that achieving high potency is a highly desirable characteristic. [Example 6: Generation and Identification of Peptide Derivatives]

[0521] To improve pharmacokinetic properties, a polypeptide system containing two free cysteine ​​residues in its C-terminal extension is conjugated with two substituents, each substituent comprising a fatty acid extension and a linker element. The resulting construct forms a polypeptide derivative, as further described below.

[0522] To attach substituents to peptides, intermediate reagents in the form of modified substituents (e.g., chemical formula 28, see Table 26) are used.

[0523] Intermediate reagents containing the extended portion are prepared as described in WO 2016 / 102562. Non-limiting examples of intermediate reagents suitable for other suitable substituents (e.g., the substituents shown in Table 9) are shown in Table 26 below: [ Conjugation, purification and analysis ]

[0524] In a solution of purified VHH peptides prepared with water, PBS, or HBS containing conjugated cysteine ​​residues, 5 equivalents of BSPP (bis(p-sulfophenyl)phenylphosphine dihydrate dipotassium salt) or 1.1 equivalents of TCEP (tris(2-carboxyethyl)phosphine hydrochloride), which is cysteine-terminated (i.e., cysteine ​​is blocked in recombinant expression in host cells, with small thiols such as cysteine, homocysteine, glutathione, etc.), are added. After stirring for 1 to 2 hours, the pH is adjusted to 8.5 with aqueous NaOH solution, and 5 equivalents of 0.1 M NaHCO3 aqueous solution of intermediate reagent (Formula 28) are added, with each cysteine ​​in each VHH peptide in its free form. The mixture is gently stirred in the dark for 1.5–16 hours. The reaction mixture is diluted with water and then purified using an Äkta system via anion exchange chromatography (AIEX). The VHH peptides with conjugated side chains are purified using AIEX chromatography. Therefore, AIEX Source 30Q resin packed in a suitable column is used in conjunction with a sodium chloride or ammonium acetate gradient program mounted on an Äkta Avant chromatography system. The buffer system used consists of an equilibration buffer of 20 mM Tris (pH 8.5) and an extraction buffer of 20 mM Tris and 1 M NaCl (pH 8.5) or 20 mM Tris and 1 M ammonium acetate (pH 8.5). The reaction mixture is adjusted to pH 8.5 and diluted with MilliQ-H2O or equilibration buffer to a conductivity below 4 mS / cm. The sample is applied to the column, and the column is washed with 5 to 10 column volumes of equilibration buffer after application. Separation chromatography is then performed using a shallow gradient of 30 to 50 column volumes. The gradient used ranges from 0% to 50%, depending on the pI of the purified VHH polypeptide derivative. Generally, unconjugated raw VHH peptides are extracted early in the gradient flush, monoconjugated VHH peptides are extracted midway through the gradient flush, and multiconjugated VHH peptides (meaning each VHH peptide molecule has more than one conjugated side chain) are extracted late in the gradient flush. The main peaks are aliquoted and pooled to obtain 90% to 99% high-purity biconjugated VHH peptide formulations. Purity analysis is performed using reverse-phase high-performance liquid chromatography (RP-UPLC) on a Waters Acquity UPLC system equipped with UV and FLD sensors, using a 1000 Å, 2.7 μm, 150 x 2.1 mm HALO diphenyl column (Scantec Nordic USDPF001316) and a run buffer consisting of A) 0.1% v / v TFA aqueous solution and B) 0.09% v / v TFA acetonitrile solution. The column temperature is set at 60 °C.The gradient program is as follows: 1) 0.0-8.0 min: 20-50% B, 2) 8.0-8.1 min: 50-80% B, 3) 8.1-9.0 min: 80% B, 4) 9.0-9.1 min: 80-20% B, and 5) 9.1-11.0 min: 20% B. Unconjugated pristine VHH peptides are flushed at 4.6 to 4.8 min. Monoconjugated VHH peptide derivatives are flushed at 5.1 to 5.6 min. Biconjugated VHH peptide derivatives are flushed at 5.7 to 5.8 min, followed by multiconjugated VHH peptides. The integrity of the conjugated VHH peptide was analyzed using a SE-HPLC method established on an Agilent LC 1100 / 1200 system and a BIOSEP-SEC-3000 300×7.8 mm column (Phenomenex, catalog number OOH-2146-K0) with a run buffer consisting of 200 mM sodium phosphate (pH 6.9), 300 mM NaCl, and 10% isopropanol. The molecular weight of the conjugated VHH peptide was analyzed using ESI-TOF-MS on a 6280 Agilent system (Agilent Technologies) with a MassPREP desalting (Waters) column at a flow rate of 0.4 mL / min in buffer A consisting of MilliQ-H2O / 0.1% formic acid and buffer B consisting of acetonitrile / 0.1% formic acid. ESI-TOF-MS was also used to evaluate, in conjunction with reverse-phase UPLC purification, the quality of intact protein collected from the extraction peak indicated by the RP-UPLC run, and the quality of trypsin-digested peptides. The protein concentration of batch-prepared conjugated VHH peptide derivatives was measured using a NanoDrop™ spectrometer (Thermo Scientific) with theoretically calculated extinction coefficients. [ Examples of polypeptide derivatives ]

[0525] For ease of reference, an overview of the synthesized peptide derivatives is shown in Table 27. Further overviews of the peptide derivatives can be found in Table 12. Identification data for the peptide derivatives are shown in Table 28. It should be understood that these are merely illustrative and should not be construed as limiting in any way.

[0526] 1. Two substituents. One substituent is attached to Cys at position 4 of SEQ ID NO:72, and the other substituent is attached to Cys at position 6. See Figure 3A / 3B for a schematic diagram of compound 9.

[0527] 1. For detailed information on the method, please refer to the RP-UPLC method using HALO DiPhenyl columns provided in the section on conjugation, purification and analysis.

[0528] [ Characteristics of the selected polypeptide derivative ]

[0529]

[0530] As observed in Tables 27, 28, and 29, the specified compound constructs exhibit high purity and correctly measured molecular weights, as well as reduced pI values ​​(as calculated herein) obtained via mutations of specific surface-exposed residues (as previously noted in Table 21) and via diacid substituent conjugates. Therefore, these compounds again demonstrate a significant reduction in pI values ​​compared to reference peptide 19 and / or the corresponding peptide construct without substituents, as discussed above with reference to Tables 21 and 22. Table 29 also shows Tm measurements of the indicated compounds / peptide derivatives, measured according to the section on materials and methods: differential scanning fluorescence (Nano DSF) analysis of anti-IL-6 VHH. All compounds exhibited relatively high melting temperatures, ranging from 48.7 to 56.1 °C, with most above 51 °C, which is an acceptable melting temperature for pharmaceutical compounds suitable for human administration, where the normal physiological temperature is 37 °C.

[0531] Using the transient mammalian expression system Expi293F, the expression levels of the precursor peptides that produced these peptide conjugates ranged from 74 to 143 mg / L, which is an acceptable initial expression level for further scale-up development and manufacturing efforts.

[0532] Specifically, compound 9 exhibits a pI value of 4.61 and a melting temperature of 56.1 °C, which are ideal for drug constructs. Further details will be provided in the examples below. [ in conclusion ]

[0533] The generation of peptides and peptide derivatives as described above provides for the development of constructs exhibiting desired pharmaceutical properties. These peptides and / or peptide derivatives exhibit low pI values ​​while maintaining suitable melting temperatures and good expressibility. For example, compound 9 exhibits a low pI value, a high melting temperature, and good expressibility. [Example 7: In vitro activity of anti-IL-6 VHH variant in STAT-3-LUC reporter gene assay, effect of fatty acid substituents] [ Purpose ]

[0534] The purpose of this example is to test the effect of the length of the C-terminal fatty acid elongator on the efficacy of variants in inhibiting IL-6-dependent STAT3 signaling, thereby inhibiting IL-6 activity in vitro. This elucidates the effect of serum albumin binding on the efficacy of anti-IL-6 VHH peptide derivatives in binding to human IL-6. [ method ]

[0535] The reported gene assay was performed as described in the Materials and Methods section. Reported values ​​are the average of two technical replicates or two experimental replicates (n=2). [ result ]

[0536] Table 30 shows the polypeptide derivatives tested as part of this experiment, labeled "compounds," and provides the detailed composition of each of these polypeptide derivatives. The detailed composition of these polypeptide derivatives can also be found in Table 12. It can be seen that compounds 14 to 19 are polypeptide derivatives containing VHH_1.6, which include a C-terminal extension with one or two cysteines. These cysteines are examples of having one or more substituents conjugated with different fatty acids.

[0537] Table 33 shows the mean and relative IC50 values ​​for the non-elongated polypeptide (polypeptide_29) containing the same base VHH (VHH_1.6). The polypeptide derivatives labeled as compounds have one or more substituents linked to the polypeptide, comprising a fatty acid elongation. The effects of conjugated 1 or 2 substituents were observed, while the effects of different types of elongated portions were further explored using C16, C18, and C20 fatty acids. Polypeptide derivatives labeled as compounds 14 and 17 contain 1x or 2x C16 fatty acids (Formula 24). Polypeptide derivatives labeled as compounds 15 and 18 contain 1x or 2x C18 fatty acids (Formula 26). Polypeptide derivatives labeled as compounds 16 and 19 contain 1x or 2x C20 fatty acids (Formula 27). For comparative purposes, Table 33 also provides the efficacy of polypeptide_29 containing the same base VHH (VHH_1.6) and an elongation without any substituents.

[0538]

[0539] [ in conclusion ]

[0540] Peptide derivatives containing fatty acid substituents exhibited IC50 values ​​comparable to the non-elongated reference peptide 29. The data indicate that the potency remained stable regardless of the presence of a single or double substituent on the peptide, or whether the substituent contained formulas 24, 25, or 26. Compound 17, with two substituents (one attached to a cysteine ​​residue at position 4 of the C-terminal elongation and the other to a cysteine ​​residue at position 6 of the C-terminal elongation), resulted in minimal variation in the potency of this peptide derivative. Example 8: Reporter gene sequencing and SPR experiment ]

[0541] In vitro identification was performed using reporter gene assays and assays based on direct SPR binding as part of VHH optimization for the peptide and peptide derivative forms. [ 8.1: Anti-IL-6 V in stat-3-luc reporter gene assay ] [ H ] [ In vitro activity of H ]

[0542] [Purpose]

[0543] The purpose of this study is to test the ability of anti-IL-6 VHH to inhibit IL-6-dependent STAT3 signaling in vitro, thereby inhibiting IL-6 activity.

[0544] This assay is used to detect additional modifications, such as pI mutations, deimmunization mutations, and fatty acid conjugation, that do not lead to an undesirable reduction in the potency of VHH peptides and / or peptide derivatives, including in the presence of HSA.

[0545] [method]

[0546] The reported gene assays were performed as described in the Materials and Methods section. However, it is worth noting that when testing for acetylated VHH, FBS was replaced with 1% human serum albumin (HSA, (Sigma #A9511)). The reported values ​​are averages from all experiments.

[0547] The sequence equivalent (SIA) of the bispecific domain antibody vobarilizumab (CAS Registry No.: 1628814-88-9, SEQ ID No. 164) and its anti-IL-6R domain (SEQ ID No. 163, discussed in Table 26 above) are shown for comparative purposes. The bispecific domain antibody vobarilizumab and its anti-IL-6R domain are recombinantly generated as generally described in the Materials and Methods section above, under "General Characterization of VHH" and "General Purification and Analysis of VHH".

[0548] It also shows the reference anti-IL6 Fab described in detail above.

[0549] [result]

[0550] Table 32 below shows the IC50 values ​​of selected anti-IL-6 VHH peptides (labeled by peptide ID) or peptide derivatives (labeled by compound ID) in media containing 1% human serum albumin (HSA) or without HSA. For ease of reference, the baseline VHH for each of these peptides / peptide derivatives is shown. Detailed compositions of exemplary peptides / peptide derivatives can also be found in Table 12. IC50 was determined by fitting a 4-parameter model to 12 consecutive 1:3 dilutions.

[0551]

[0552] [in conclusion]

[0553] Anti-IL-6-VHH peptides / peptide derivatives showed IC50 values ​​ranging from 10 to 117 pM for inhibiting IL-6 signaling (Table 32).

[0554] As can be seen, the selected initial VHH polypeptide_19 (SEQ ID NO:57) exhibits an IC50 potency of approximately 117 pM. Introducing selected mutations at Cys4 and Cys6 in the C-terminal extension E2 of SEQ ID No.72 and conjugating two C16 diacid fatty acids (Formula 24) at the C-terminus further demonstrates a suitable IC50 potency of approximately 60 pM or lower in the presence of HSA. For example, in the presence of HSA, compound 9 exhibits an IC50 potency of 14 pM.

[0555] The data also show that the potency of these peptides (or more specifically, the basic VHH) is not affected by the fatty acid substituents attached to them, as discussed in Example 7: In vitro activity of the anti-IL-6 VHH variant in STAT-3-LUC reporter gene assays, the effect of fatty acid substituents. Conversely, the sequence equivalent of vobarilizumab (CAS Registry No.: 1628814-88-9, SEQ ID No. 164) results in a complete loss of potency when conjugated with its anti-HSA domain. The data indicate that while the sequence equivalent of vobarilizumab (SEQ ID No. 163) with the anti-IL-6R domain exhibits some low-potency IL-6 signaling inhibition (as discussed in Table 25 above), the inclusion of the anti-HSA domain in vobarilizumab results in a loss of the IL-6 inhibitory properties of the construct.

[0556] Furthermore, the anti-IL-6-VHH with fatty acid conjugation, referred to in this paper as polypeptide derivatives (labeled as compounds 1-10 in Table 32), did not show a decrease in activity when HSA was present in the assay medium (0% vs. 1% HSA).

[0557] Introducing potency-enhancing mutations, deimmunization mutations, and volatile-stabilizing mutations (exemplary mutations are shown in Table 21) results in VHH peptide derivatives exhibiting high IC50 potency ranging from 12 to 25 pM (Table 32, items 16A-23B). These results further demonstrate that the IC50 potency provided by the peptide derivatives described herein is, if not better, comparable to that provided by the reference anti-IL6 Fab. This high potency is promising for the development of oral drug candidates, particularly antibody-backbone-based oral drug candidates, as oral bioavailability is typically lower compared to other administration routes.

[0558] These high potency effects are combined with the previously discussed low pI, high melting temperature, and good expressibility. For example, the examples above show that compound 9 provides a low pI of 4.61 and a high melting temperature of 56.1 °C, while Table 32 above further demonstrates that the same peptide derivatives provide a high IC50 potency of 14–22 pM (items 21A, 21B), which is maintained or even enhanced in the presence of HSA. This combination of characteristics is ideal and demonstrates the potential applicability of such peptide derivatives for therapeutic products. [ 8.2: SPR analysis using IL-6 with the HPC4-tagged label as the analyte. ]

[0559] [Purpose]

[0560] The purpose of this example is to estimate the binding constant of the selected anti-IL-6 VHH to human IL-6. Complementary sites: Epitope interactions can be optimized by introducing specific mutations targeting, for example, those that reduce pI, deimmunize, or improve function. Measuring the binding constant of the modified peptide ensures that these modifications do not negatively affect the binding affinity to human IL-6.

[0561] [SPR analysis of IL-6 with HPC4 tag]

[0562] The binding of purified anti-IL-6 VHH to recombinant human HPC4-tagged IL-6 (SEQ ID NO:98) was detected by surface plasma resonance (SPR).

[0563] In short, the anti-HPC4 antibody (as previously described (Rezaie et al. (1992) Expression and Purification of a Soluble Tissue Factor Fusion Protein with an Epitope for an Unusual Calcium-Dependent Antibody. Protein Expression and Purification 3:453-460; Stearns et al. (1988) The Interaction of a Ca2+-dependent Monoclonal Antibody with the Protein C Activation Peptide Region. JBC 263:826-832)) was immobilized on a CM4 sensing chip (Cytiva) or Xantec HLC200M (Xantec) at pH 5 using standard amine coupling chemistry. Immobilization at 10 μl / min for 420 seconds yielded a fixed level of 5500-6500 RU. IL-6 was captured at a flow rate of 10 μl / min for 30 seconds.

[0564]

[0565]

[0566] Subsequently, VHH peptides of the concentration series shown in Table 33 were injected for 200 seconds at a flow rate of 50 μL / min to allow binding with the anti-IL-6 VHH peptide. This was followed by injection of running buffer (10 mM HEPES, 150 mM sodium chloride, 5 mM CaCl2, 0.05% (v / v) surfactant P20, 1 mg / mL bovine serum albumin, pH 7.4) for 600 seconds to allow dissociation from IL-6. The running buffer was also used to dilute both the anti-IL-6 VHH and IL-6. Regeneration of the wafer was achieved using a regeneration buffer consisting of running buffer (CaCl2-free) containing 50 mM EDTA, with a contact time of 30 seconds, applied twice to the surface at a flow rate of 30 μL / min. Binding data were collected at 25 °C using a Biacore instrument (Cytiva AB, Uppsala) or a Bruker SPR32 (Bruker Daltonics). Analyze the data in a 1:1 model using BiaEvaluation, Biacore Insight Evaluation (Cytiva AB, Uppsala), or the Bruker Analyzer (Bruker Daltonics).

[0567] [result]

[0568] The table below lists the binding constants of peptides and peptide derivatives calculated based on kinetic data obtained on HPC4-human IL6. For ease of reference, the base VHH for each of these peptides / peptide derivatives is shown. Detailed compositions of exemplary peptides / peptide derivatives can also be found in Table 12.

[0569] The calculation method for a is KD = koff / kon.

[0570] [in conclusion]

[0571] It can be observed that low pM KD affinity is achieved throughout the design cycle, and only a minor effect on binding affinity is observed throughout the optimization of the design cycle. In other words, an affinity for achieving and maintaining a suitable pM range for this peptide derivative can be observed. For example, VHH peptide derivative compound 1 shows an affinity of approximately 45-63 pM, while other peptide derivatives such as compounds 4-10 show an affinity between 14 and 34 pM.

[0572] The combination of pM-level affinity with high efficiency, low pI, and high melting temperature is ideal. Among them, compound 9, in addition to having high efficiency, low pI, and high melting temperature, also exhibits an ideal KD of 18-22 pM. [In vivo experiments]

[0573] A series of in vivo experiments were conducted on polypeptide derivatives of compounds 1, 2, 3, 7, and 9.

[0574] Example 9: In vivo intravenous pharmacokinetic study in rats, the role of fatty acid substituents; Example 10: Dose-response study in an acute mouse model of human IL-6; Example 11: Intravenous PK in miniature pigs; and Example 13: Oral and intravenous pharmacokinetic study in dogs: VHH polypeptide derivatives formulated with SNAC and NAM, as described below, showing in vivo data. [Example 9: In vivo intravenous pharmacokinetics study in rats, the effect of fatty acid substituents] [ Purpose ]

[0575] The purpose of this study is to test the effect of the type and number (1 and 2) of fatty acid substituents in the anti-IL-6 VHH compound on pharmacokinetic (PK) parameters after intravenous administration to healthy Sprague Dawley rats. [ method ]

[0576] A liquid formulation of the anti-IL-6 VHH polypeptide derivative (referred to as the compound in the Examples section) was administered intravenously (IV) (IV formulation: 20 mM Hepes, 150 mM NaCl, pH 7.4). The formulated compound was administered intravenously to Sprague Dawley rats in parallel groups, who had been acclimatized indoors and maintained in their cages with free access to standard food and water for at least one week prior to the study. On the day of administration, all rats were acclimatized in the operating room for 30 minutes.

[0577] Different anti-IL6 VHH variants were administered at time 0, and blood samples were collected 5 minutes later, followed by blood samples at 4, 24, 48, 96, 144, 168, and 240 hours post-administration. Blood samples were taken from the hypoglossal plexus of the tongue (150 μl) in EDTA-coated vials (Microvette 200K3E, reference number 20.1288.100, Sarstedt). Plasma was separated (4°C, 8000 RPM, 5 min) and transferred to a Mirconic tube to measure plasma exposure. Plasma concentrations of the VHH peptide conjugate compound were determined using a His-tagged immunoassay, employing an anti-VHH antibody (Novo Nordisk, Denmark) against the test compound and an anti-His-tagged antibody. Here, 96-well MaxiSorp discs (Nunc, 439454) were coated with 2 μg / ml His-tagged antibody (R&D systems, MAB050) and washed and blocked using PBS; 0.05% Tween 20; 1% BSA; pH 7.4. After the washing step, compound-specific calibrators (0, 2.7, 8.2, 24.7, 74, 222, 667, 2000 pM) in 1% rat EDTA plasma and rat EDTA plasma samples at a minimum dilution of 100 times were placed on the discs to capture VHH conjugated compounds via their His tags. After an additional washing step, a self-biotinylated VHH-specific antibody (0.5 nM) was added to the discs to form a sandwich ELISA. After the final washing step, horseradish peroxidase (HRP)-streptavidin was added to the discs as the detection reagent. After adding a chromogenic acceptor (e.g., TMB (3,3',5,5'-tetramethylbenzidine)), the amount of biotin-labeled antibody bound to the VHH peptide derivative is detected. Optical density is measured using a spectrometer (e.g., a SpectraMax® M2 spectrometer (Molecular Devices)). The reaction is proportional to the concentration of peroxidase, which in turn is proportional to the concentration of the VHH peptide-derived compound. [ result ]

[0578] The peptide derivatives tested as part of this experiment are labeled "compounds" and correspond to those described in detail in Example 7: In vitro activity of the anti-IL-6 VHH variant in the STAT-3-LUC reporter gene assay, and the effect of fatty acid substituents. Table 30 shows the peptide derivatives tested and provides the detailed composition of each peptide derivative. The detailed composition of these peptide derivatives can also be found in Table 12.

[0579] It can be seen that compounds 14 to 19 are polypeptide derivatives containing VHH_1.6 and a C-terminal extension with one or two cysteines. These cysteines are examples of having one or more substituents conjugated with different fatty acids.

[0580] The results are provided in Table 34 below, showing the terminal half-life (T½) of the polypeptide derivatives (labeled as compounds), and an unsubstituted (i.e., non-elongated) polypeptide (polypeptide_29) containing the same base VHH (VHH_1.6) as a reference. Results for another unsubstituted polypeptide (polypeptide_63) are also provided. The polypeptide derivatives labeled as compounds have one or more substituents linked to the polypeptide, which contain a fatty acid elongation. For ease of reference, these are noted in the substituent notes column.

[0581]

[0582] Data show that unelongated peptides (also known as unsubstituted peptides), peptides 29 and 63, were rapidly eliminated in rats, with half-lives just over half an hour. All peptide derivatives with fatty acid diacid conjugations showed an increase in half-life of at least one order of magnitude. More specifically, compounds 15–19 showed approximately 40–60 times greater half-lives compared to the unsubstituted VHH peptides (peptides 29 and 63). [ in conclusion ]

[0583] The provision of substituents in fatty diacids indicates an increased half-life.

[0584] Given the observation that these polypeptide derivatives with fatty diacid substituents exhibit conserved potency compared to unsubstituted polypeptides (peptide_29) containing the same VHH (see above), this improved pharmacokinetic outcome of these polypeptide derivatives is promising, especially when this PK property is combined with other desired drug properties. For example, compound 17, consistent with the polypeptide derivative identified herein as compound 9 (see Table 12, VHH_1.6 with two 24-substituents), exhibits a high half-life of 29.6 hours, while also demonstrating high potency in other examples. [Example 10: Dose-response study in an acute mouse model of human IL-6]

[0585] The aim of this study was to investigate the ability of anti-IL-6 VHH to inhibit the response of the biomarker serum amyloid A1 (SAA1) in an acute human IL-6 mouse model. SAA1 was chosen as an in vivo readout biomarker because it is an established pro-atherosclerotic effector molecule regulated by IL-6 levels and has been used in mouse models of atherosclerosis (Thompson JC et al. (2015)). Transient increases in serum amyloid A are sufficient to increase atherosclerosis (J Lipid Res. 56(2):286-93). SAA1 levels are associated with cardiovascular disease, synergistically induce IL-6 levels, and promote single nucleotide diversity in the disease (Hagihara K (2004) IL-6 plays a critical role in the synergistic induction of human serum amyloid A (SAA) gene when stimulated with proinflammatory cytokines as analyzed with an SAA isoform real-time quantitative RT-PCR assay system. Biochem Biophys Res Commun. 314(2):363-9; Carty CL et al. (2009) Association of genetic variation in serum amyloid-A with cardiovascular disease and interactions with IL-6, IL1RN, IL1beta and TNF genes in the Cardiovascular Health Study. J Atheroscler Thromb. 16(4):419-30). Therefore, based on the use of SAA1 in human clinical trials and its sensitivity in acute mouse models, SAA1 was selected as the primary readout.

[0586] In this in vivo mouse model, human IL-6 (hIL-6, SEQ ID NO:82) was administered to healthy mice to elicit an acute-phase reactant response (measured as SAA1). Due to the limited cross-reactivity of the tested anti-IL-6 VHH peptide derivative (see above) with mouse IL-6, the response was not satisfactory. [ method ]

[0587] Healthy male BALB / c mice (Charles River, Germany, n=7-8 per group) were intravenously injected (iv) with an IL-6 antagonist. Thirty minutes later, hIL-6 (in-house manufactured (SEQ ID NO:82)) was injected intravenously via the tail vein at a dose of 3.6 nmol / kg. Five (5) hours after hIL-6 injection, blood samples were collected to determine SAA1 concentrations using a commercially available ELISA kit (Mouse SAA ELISA Kit, R&D Systems, Bio-Techne brand, MSAA00). Compound concentrations were determined via luminescent oxygen channel immunoassay (LOCI).

[0588] Data were normalized based on a window between levels in healthy control animals (set as 100%) and levels after SAA1 upregulation via hIL-6 alone (set as 0%). The half-maximum effective dose (ED50) was provided as the dose of the antagonist that reduced SAA1 by 50% compared to the hIL-6-upregulated group. Based on plasma concentrations of the compound (exposure), if available, the half-maximum effective concentration (EC50) was reported as the plasma concentration of the antagonist that reduced SAA1 by 50%, compared to the hIL-6-upregulated group. [ result ]

[0589] The results are shown in Table 35. Detailed compositions of these polypeptide derivatives can also be found in Table 12.

[0590] [ in conclusion ]

[0591] Data showed that SAA1 decreased in a dose-dependent manner in mice treated with anti-IL-6 VHH peptide derivatives. The most potent peptide derivatives tested were compounds 7 and 9, with EC50 efficacies of approximately 36 and 25 nM, respectively, comparable to those observed in in vitro reporter gene assays (see above). [Example 11:] Mini Pigs' Veins Comparison [ Target ]

[0592] The aim of this study was to determine the pharmacokinetic parameters following intravenous administration of anti-IL-6 VHH peptide derivatives. All peptide derivatives tested contained a C-terminal VHH extension with two substituents, including a C16 diacid fatty acid elongator moiety to prolong the half-life. Detailed compositions of the peptide derivatives described below can also be found in Table 12. VHH peptides (i.e., non-extended VHH constructs) showed short half-lives (less than 15 hours in miniature pigs; data not shown). [ method ]

[0593] The study subjects were female Ellegaard Minipigs A / S (Dalmose, Denmark), weighing approximately 25 kg. The dose was 5 nmol / kg, administered intravenously via a permanent catheter, with n=3 per group. The vehicle used for the single-domain antibody was 20 mM HEPES, 150 mM NaCl, pH 7.4, with a target concentration of 100 nmol / ml.

[0594] Blood samples were collected over a period of up to 912 hours (39 days) via an indwelling permanent venous catheter and collected in EDTA tubes (Sarstedt, Germany). Plasma was separated and compound concentrations were analyzed using a luminescent oxygen channel immunoassay (LOCI).

[0595] Non-compartmental (NCA) pharmacokinetic analysis was performed using Phoenix® WinNonlin® v.8.1 (Certara LP Princeton, NJ, USA). AUC was calculated using the Linear Up / Log Down method. The terminal elimination phase was fitted using uniformly weighted linear regression. Nominal sampling time and actual dose were used for NCA.

[0596] The results are shown in Table 36. Detailed compositions of these polypeptide derivatives can also be found in Table 12.

[0597] nd = Not determined. 1. Sampling time was too short to accurately estimate pharmacokinetic parameters. No clinical observations related to the test compound were observed during the study. [ in conclusion ]

[0598] The observed half-lives of the tested peptide derivatives (labeled as compounds in the table above) ranged from 165 to 236 hours, which is more than 10 times longer than that of the VHH peptide (i.e., the non-elongated construct). Since the reported half-life of porcine albumin is approximately 197 hours, the observed half-lives of the peptide derivatives are close to the theoretical value. In other words, the observed half-lives are close to the theoretically achievable upper limit of the albumin-based elongated portion of candidate drug molecules (such as VHH peptide derivatives) (Dich J et al. (1963) Metabolism and Distribution of I-labeled Albumin in the Pig. Can J Comp Med Vet Sci. 27(11):269-73). [Example 12: Anti-IL-6 V with SNAC and nicotinamide] [H] [Formulations of H-peptide derivatives suitable for oral administration]

[0599] For oral administration testing, tablets containing sodium octanoate (SNAC), nicotinamide (NAM), and magnesium stearate (MgSt) or sodium stearate fumarate (SSF) as excipients for oral administration were prepared following the procedure below.

[0600] [Method 1: Dry granulation and mixing]

[0601] The mixture was first manually geometrically mixed with SNAC and nicotinic acid amide, then mixed on a turbo mixer at 25 rpm for 7 minutes. In a second mixing step, a lubricant (MgSt or SSF) was added manually geometrically, followed by mixing on a turbo mixer at 25 rpm for 2 minutes. The mixture was then used for further processing.

[0602] [Method 2: Dry Granulation]

[0603] Dry granulation was simulated using a flat punch measuring 10mm (width) and 20mm (length) on a STYL'One Evo compaction simulator, via roller pressing. The setup was to simulate the Gerteis Mini-Pactor at a machine speed of 3 rpm.

[0604] After dry granulation, the ribs are manually sieved into granules using an 800μm conical sieve on a Gerteis hand mill. These granules are then used for further compression processing.

[0605] [Method 3: Direct Compression Mixing]

[0606] If the formulation does not include a dry granulation step, pre-formulated intermediate particles containing SNAC, nicotinamide, and SSF are used, mixed with the spray-dried anti-IL-6 VHH conjugate. The intermediate particles are then manually geometrically mixed with the anti-IL-6 VHH, followed by mixing at 25 rpm for 7 minutes on a turbo mixer. This product is intended for further compression processing.

[0607] [Method 4: Compression of Tablets]

[0608] The ingots were pressed into oval shapes using a set of punches measuring 8mm x 14mm or 8.1mm x 14.3mm on the STYL'One Evo compaction simulator. The setup simulated a Fette 102i rotary press at a machine speed of 20 rpm. The filling depth was adjusted to obtain ingots with a target weight based on the composition. The compression force ranged from 10kN to 20kN to obtain ingots with an apparent density of approximately 1.18-1.22 g / m³.

[0609] [Method 5: Tablet Analysis]

[0610] To analyze the content of anti-IL-6 VHH conjugates, SNAC, and NAM, the tablets were weighed before extraction. The tablets were dissolved in a relevant amount of 50 mM phosphate buffer (pH 7.4) containing 20% ​​acetonitrile for 2 hours. The centrifuged sample was transferred to an appropriate HPLC vial. Standards for anti-IL-6 VHH conjugates, SNAC, and NAM were prepared using the same diluent as the sample. The contents of anti-IL-6 VHH conjugates, SNAC, and NAM were determined using a UPLC system.

[0611] [Preparation of oral tablet formulations]

[0612] The formulations were prepared according to Table 37. The components of the formulations were prepared by combining the methods defined above. Formulations 1-4 and 8 were manufactured using the dry granulation mixing, dry granulation, and tablet pressing methods (methods 1, 2, and 4). Formulations 5-7 were manufactured using the direct compression mixing and tablet pressing methods (methods 3 and 4).

[0613] All tablet formulations were analyzed using Method 5. Detailed compositions of the polypeptide derivatives used can also be found in Table 12 (i.e., the compound components listed below).

[0614] [Example 13: Oral and intravenous pharmacokinetics studies in dogs: V formulated with SNAC and NAM] [H] [H-peptide derivative] [ Purpose ]

[0615] The aim of this study was to evaluate the effect of anti-IL-6 VHH peptide derivatives containing a fatty acid extension moiety as part of a substituent on prolonging half-life, and to assess whether the decrease in pI value leads to an increase in oral bioavailability. [ method ]

[0616] Appropriate formulations of the anti-IL-6 VHH peptide derivatives were administered orally to a beagle group via intravenous injection (IV formulations based on HBS buffer) or as tablets, as described in Example 12: Formulations of anti-IL-6 VHH peptide derivatives with SNAC and nicotinamide suitable for oral administration. Dogs were administered the medication in the morning after an overnight fast, and remained fasted for 4 hours after the single dose. In a subset of the oral studies (§), approximately 3 nmol / kg of glucagon was administered subcutaneously to dogs 10 minutes prior to oral administration. Blood samples were collected immediately before administration, and subsequently at various time points. Plasma concentrations of VHH peptides with fatty acid substituents were measured using a bead-based immunoassay called alphaELISA, in which recombinant human IL-6 receptor protein (Novo Nordisk, Denmark) and an anti-γ-Glu linker antibody conjugated to the test molecule's side chain motif were bound to the VHH peptide. First, compound-specific calibrators (0, 6, 13, 25, 50, 100, 200, 400, 800, 1600, 3200, 6400 pM) from canine EDTA plasma and canine EDTA plasma samples were added at a minimum dilution of 30-fold to 384-well plates (Revivity AlphaPlate-384, 6005359), along with biotinylated human IL-6 receptor protein and an anti-γ-Glu linker antibody conjugated to alphaELISA receptor beads, to capture the VHH peptide compound. After standing overnight (16-24 hours), donor beads coated with α-streptavidin were added to a 384-well dish. When the analyte appeared in the sample, the donor and acceptor beads aggregated. Upon excitation, the photosensitizer within the donor beads converted ambient oxygen into an excited singlet state. This singlet oxygen diffused to 200 nm, triggering a chemiluminescence reaction in the acceptor beads, producing light emission. The luminescence readings were measured using a spectrometer (e.g., Enspire (Perkin Elmer)). This reaction is proportional to the concentration of the VHH peptide compound.

[0617] In a separate, additional experiment, plasma samples were prepared to determine and analyze plasma concentrations of compounds 1–3, 7, and 9, as well as an example VHH peptide (i.e., without fatty acid substituents), provided by LC-MS analysis. Calibrators were prepared by adding the analytes to blank canine plasma to achieve a final concentration typically in the range of 1–500 nM. The calibrator, plasma blank, or study sample was mixed 1:1 with 8 M guanidine hydrochloride (GndHCl) to a final concentration of 4 M GndHCl and incubated at 37 °C for 30 min to dissociate non-covalent protein interactions. The supernatant was then diluted with two volumes of Milli-Q filtered water (hereinafter referred to as Milli-Q water, containing 1% formic acid) and injected into the LC-MS system. The system used was a Transcend II TLX-2 system equipped with a Thermo Scientific Ultimate 3000 RSLC pump and a TriPlus RSI autosampler, coupled to a Thermo Scientific Q-Exactive Plus mass spectrometer. LC was performed at room temperature using a Cyclone column (CH-953288, Thermo Scientific) as the first-dimensional capture column, and analyzed at 60 °C using an XBridge Protein BEH C4 column (2.1 x 50 mm, Waters). Mobile phase A consisted of Milli-Q water, 1% formic acid, and 5% methanol / acetonitrile (50 / 50), while mobile phase B consisted of methanol / acetonitrile (50 / 50), 1% formic acid, and 5% water. Gradient extraction was performed using a ramp gradient from 0% mobile phase B to 30% mobile phase B in 0.25 min, then from 30% mobile phase B to 45% mobile phase B in 1.17 min, and then from 45% mobile phase B to 100% mobile phase B in 1.17 min. A Q-Exactive Plus mass spectrometer (ThermoFisher Scientific) was used as the detector in single-ion monitoring mode (m / z 1624.8–1628.8). The concentrations of the test compounds in plasma samples were calculated using linear calibration curves (weighted 1 / x2). Quality control samples of the analyte were also included. The deviation between the nominal and calculated concentrations in the calibrators and quality control samples was less than 15%, and the deviation for LLOQ samples was less than 20%.

[0618] Based on these exposure data, non-compartmental pharmacokinetic parameters were calculated using Phoenix WinNonlin or the open-source statistical analysis software R (software suite "NonCompart"). [ result ]

[0619] Tables 39 and 38 below present the results for five VHH polypeptide derivatives (labeled as compounds), and show a) the effect of fatty acid elongation on half-life, and b) that reducing pI improves oral bioavailability.

[0620] For detailed composition of these polypeptide derivatives, please refer to Table 12.

[0621] More specifically, Table 38 below shows the results for the first group, in which the IV half-lives of compounds 1–3, 7, and 9, as well as the reference VHH peptide (unsubstituted) peptide_63, were collected. For each of these items, n was 2, and a dose of 0.05 mg / kg was administered to each animal.

[0622]

[0623] As seen above, compounds 1-3, 7 and 9 all provide half-lives that are orders of magnitude higher than those provided by the reference VHH peptide (peptide_63).

[0624] Table 39 below provides a second set of results for the oral bioavailability data of compounds 1-3, 7, and 9. Although the results below show the compound IDs, it should be understood that the IV formulation is based on HBS buffer and is an oral tablet, as described in Example 12: Formulations of Anti-IL-6 VHH Peptide Derivatives with SNAC and Nicotinamide Suitable for Oral Dosage Formation. Compound IDs are used for ease of reference.

[0625]

[0626] For example, regarding oral bioavailability observed in dogs, peptide derivatives formulated with NAM / SNAC showed significant effects, enabling oral bioavailability to reach or exceed clinically relevant levels. The achieved levels were particularly relevant to other desired drug properties achievable with these same compounds, such as potency and half-life. [ in conclusion ]

[0627] Excellent half-life and oral bioavailability were observed. Comparison of the VHH peptide derivative (i.e., the VHH peptide with a fatty diacid substituent) with the unsubstituted reference VHH peptide (peptide_63) confirmed a two-order-of-magnitude increase in half-life. Furthermore, the bioavailability compared to IV was observed in the range of approximately 1–2%.

[0628] Taking into account efficacy, half-life, and oral bioavailability, the VHH peptide derivatives revealed in this paper are expected to become first-in-class anti-inflammatory oral drugs and antibody-analysts targeting IL-6.

[0629] For example, compound 9 has shown an ideal balance of high efficacy, long half-life and high bioavailability, highlighting its suitability as a therapeutic product.

[0630] Although certain features of the invention have been set forth and described herein, many modifications, substitutions, variations, and equivalents will be contemplated by those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and alterations falling within the true spirit of the invention.

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Claims

1. A polypeptide derivative capable of binding IL-6, comprising a VHH, an extension, a first substituent, and a second substituent, wherein the VHH comprises the following complementarity-determining regions (CDRs): CDR1: EYAVG (SEQ ID NO: 3); CDR2: DIGEQAENTWYAESVLG (SEQ ID NO: 7); and CDR3: DKYGVGGNAQGYYDS (SEQ ID NO: 17) (as defined by Kabat), wherein the extension is attached to the C-terminus of the VHH and comprises an amino acid sequence as shown in SEQ ID No. 72, wherein the first substituent is attached to cysteine ​​at position 4 of SEQ ID No. 72, and the second substituent is attached to cysteine ​​at position 6 of SEQ ID No. 72, and wherein each of the first and second substituents comprises the following structure (Chemical Formula 24).

2. The polypeptide derivative as claimed in claim 1, wherein the molecular weight of the polypeptide derivative is between 12 and 18 kDa.

3. The polypeptide derivative as described in claim 1, which is: (SEQ ID NO: 78).

4. A pharmaceutical composition comprising a polypeptide derivative as described in claim 1 and a pharmaceutically acceptable carrier, diluent, or excipient.

5. The pharmaceutical composition as claimed in claim 4, wherein the composition further comprises sodium N-(8-(2-hydroxybenzoyl)amino)octanoate (SNAC).

6. The composition as described in claim 5, wherein the composition further comprises nicotinamide.

7. A pharmaceutical composition comprising a polypeptide derivative as described in claim 3 and a pharmaceutically acceptable carrier, diluent, or excipient.

8. The pharmaceutical composition as claimed in claim 7, wherein the composition further comprises sodium N-(8-(2-hydroxybenzoyl)amino)octanoate (SNAC).

9. The composition as described in claim 8, wherein the composition further comprises nicotinamide.

10. Use of a polypeptide derivative as described in claim 1 for the preparation of a medicament for treating inflammatory diseases.

11. As requested in item 10, wherein the inflammatory disease is a cardiovascular disease.

12. As requested in claim 11, wherein the cardiovascular disease is atherosclerotic cardiovascular disease (ASCVD).

13. Use of a polypeptide derivative as described in claim 3 for the preparation of a medicament for treating inflammatory diseases.

14. As requested in claim 13, wherein the inflammatory disease is a cardiovascular disease.

15. As requested in claim 14, wherein the cardiovascular disease is atherosclerotic cardiovascular disease (ASCVD).

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