Bivalent and multivalent albumin binders

Abstract

The present invention relates to bivalent and multivalent albumin binders. In particular, the present invention relates to novel and improved human serum albumin binders, in particular to polypeptides comprising (i) at least one immunoglobulin single variable domain (ISVD) specifically binding to serum albumin or at least one human serum albumin and (ii) at least one further moiety specifically binding to serum albumin or at least one human serum albumin.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202480060283.0, filed on September 20, 2024, entitled "Divalent and Multivalent Albumin Binding Agent". Technical Field

[0002] This technology relates to bivalent and multivalent albumin binders. In particular, this technology relates to novel and improved human serum albumin binders, especially peptides comprising (i) at least one immunoglobulin single variable domain (ISVD) specifically binding to serum albumin or at least one human serum albumin and (ii) at least one additional portion specifically binding to serum albumin or at least one human serum albumin. This technology further relates to peptides comprising an albumin binder and a human serum albumin (HSA) molecule, and to peptides comprising two HSA molecules.

[0003] This technology further relates to fusion proteins comprising polypeptides, nucleic acids encoding such molecules or portions thereof, host cells comprising such nucleic acids and / or expressing or capable of expressing such polypeptides and / or fusion proteins, compositions, and particularly pharmaceutical compositions comprising such polypeptides and / or fusion proteins, nucleic acids and / or host cells.

[0004] Furthermore, this technology relates to methods for producing such peptides and fusion proteins, and to the use of such peptides and / or fusion proteins in various applications, including but not limited to prolonging the in vivo half-life of therapeutic compounds and / or preventing and / or treating diseases and / or disorders. Background Technology

[0005] Peptides and proteins are two classes of molecules with attractive potential for therapeutic applications. However, a bottleneck for the development of peptides and proteins into clinically and commercially relevant drugs is their short half-life in the body, typically only a few minutes to a few hours.

[0006] The half-life of peptides and proteins in human serum is determined by several factors, including size, charge, proteolytic sensitivity, biological properties, and the turnover rate of the proteins they bind to. There is a relationship between the hydrodynamic radius of a peptide or protein and its serum half-life. Generally, peptides and proteins with a molecular weight less than approximately 70 kDa are primarily eliminated by renal filtration and typically have a very short serum half-life. Larger proteins may persist in circulation for several days.

[0007] Human serum albumin (HSA) and IgG are two of the most abundant soluble proteins in the blood circulation. Unlike most circulating proteins, they share a common characteristic: their serum half-life is extended by about 19 to 21 days in the human body.

[0008] HSA is the most abundant plasma protein in the blood and a carrier protein involved in many processes that maintain homeostasis (i.e., maintain osmotic pressure). Albumin is widely used as a drug delivery medium due to its high serum concentration, long half-life, non-toxicity, low immunogenicity, and its ability to be absorbed in benign tissues and bind to a variety of drugs (Mishra, V.; Heath, RJ. Structural and Biochemical Features of Human Serum Albumin Essential for Eukaryotic Cell Culture. Int. J. Mol. Sci. 2021, 22, 8411).

[0009] The prolonged half-life of serum albumin is primarily due to its protection from intracellular lysosomal degradation through binding to the neonatal Fc receptor (FcRn). FcRn is a heterodimer composed of N-glycosylated transmembrane MHC class I-like heavy chains non-covalently associated with soluble β2-microglobulin. Both IgG and albumin are ligands that bind to different epitopes of FcRn. Generally, the FcRn recycling mechanism is strictly pH-dependent, and binding to FcRn is favored at low pH levels following endosome acidification (e.g., acidic endosome pH, typically below 6.5). When albumin binds to FcRn, it escapes degradation in lysosomes. Upon returning to the cell surface, binding weakens at the extracellular physiological pH (typically around pH 7.4), leading to the release of albumin into the bloodstream (see, for example, Ward ES, Ober RJ., Targeting FcRn to Generate Antibody-Based Therapeutics. Trends Pharmacol Sci., 2018; 39(10):892-904 and Andersen et al., Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding, JBC, 2014, 289, 19: 13492-13502).

[0010] In particular, albumin is increasingly being used to improve the pharmacokinetics of short-lived small molecule drugs that can bind to albumin, as well as bioactive therapeutic peptides and proteins, through gene fusion of these molecules with the N- or C-terminus of albumin (Nilsen, J., Trabjerg, E., Grevys, A. et al., An intact C-terminal end of albumin is required for its long half-life in humans. Commun Biol, 2020, 3, 181). For example, immunoglobulin variable domain sequences (ISVDs) that can bind to serum albumin have been developed, and these immunoglobulin variable domain sequences have been described, for example, in WO 2004 / 041865, WO 2006 / 122787, WO 2012 / 175400, WO 2015 / 173325 and PCT / EP2016 / 077973, for conjugating therapeutic compounds, portions and entities to prolong serum half-life (as defined in these applications). For instance, WO 2006 / 122787 discloses a humanized serum albumin-binding nanobody known as Alb-8 (see SEQ ID NO: 5 herein) as SEQ ID NO: 62. WO 2012 / 175400 discloses a humanized serum albumin-binding nanobody known as Alb-23D as SEQ ID NO: 6. Other references disclosing ISVDs for serum albumin include WO 2003 / 035694, WO 2004 / 003019, EP 2139 918, WO 2011 / 006915 and WO 2014 / 111550.

[0011] Other albumin-binding proteins (ABPs), such as albumin-binding DARPin (designed ankyrin repeat protein) or affitin (also known as nanofitin), have also been described as scaffolds for extending the half-life of biologics (see, for example, Michot N. et al., “Albumin binding Nanofitins, a new scaffold to extend half-life of biologics - a case study with exenatide peptide”, Peptides, 2022, 152:170760 or Steiner D. et al., “Half-life extension using serumalbumin-binding DARPin® domains”, Protein Eng Des Sel, 2017, 30(9):583-591).

[0012] However, to date, no achievement has been made to extend the in vivo half-life of therapeutic peptides and small proteins to or beyond that of albumin or full-length antibodies. For example, while serum albumin has a half-life of approximately 19 days in humans, abiglutide (GLP-1-HSA fusion protein Tanzeum® (USA) or Eperzan® (EU), also known as Albugon)—a drug whose gene is fused to the N-terminus of serum albumin—has a half-life of only about 5 days. To date, other fusion partners tested clinically, such as (C-terminal peptide) CTP or (elastin-like peptide (ELP)), have not performed better, with fusion proteins having half-lives of 2.5 days and 4-5 days, respectively. For most fusion proteins, the expected optimal dosing regimen is once weekly, with some potentially requiring twice-weekly doses. While this is better than using natural peptides or proteins alone, its dosing frequency remains significantly higher than most therapeutic antibodies. Currently, almost all commercially available protein-based pharmaceutical formulations are administered intravenously or subcutaneously at high doses and frequent intervals, ultimately leading to dose-related complications and significantly reduced patient adherence.

[0013] Therefore, there is a need to prolong the serum persistence of peptide- and protein-based therapeutics, thereby achieving more uniform serum drug concentrations, lower dosages without compromising efficacy, and less frequent administration. This is likely to translate into fewer toxicities and side effects, as well as improved adherence. Summary of the Invention

[0014] Therefore, this technology provides a serum albumin binder with improved properties compared to serum albumin binders known in the art.

[0015] The inventors have identified albumin-binding peptides comprising (i) at least one immunoglobulin single variable domain (ISVD) specifically binding to serum albumin or at least one human serum albumin; and (ii) at least one additional portion specifically binding to serum albumin or at least one human serum albumin, exhibiting improved properties, particularly in increasing half-life and thus increasing therapeutic efficacy. In a preferred embodiment, the albumin-binding peptide of the present technology comprises (i) at least one immunoglobulin single variable domain (ISVD) specifically binding to serum albumin; and (ii) at least one additional portion specifically binding to serum albumin or at least one human serum albumin. In a more preferred embodiment, the albumin-binding peptide of the present technology comprises (i) at least one immunoglobulin single variable domain (ISVD) specifically binding to serum albumin; and (ii) at least one additional portion specifically binding to serum albumin (e.g., albumin-binding ISVD, albumin-binding DARPIN, albumin-binding alfaldaline, or albumin-binding domain (ABD)). Even more preferably, the albumin-binding polypeptide of the present technology comprises (i) at least one ISVD specifically bound to serum albumin; and (ii) at least one additional albumin-binding ISVD.

[0016] As described in the prior art, the albumin-binding peptides provided by this technology have the advantage of significantly increasing in vivo serum half-life compared to known extended-life peptides and proteins (including full-length immunoglobulins). For example, the in vivo serum half-life of constructs containing a single albumin-binding domain (e.g., albumin-binding ISVD) ranges from 0.5 days to 1.4 days, see, for example, Table 2 of Hoefman, S. et al., “Pre-clinical intravenous serum pharmacokinetics of albumin binding and non-half-life extended Nanobodies®”, Antibodies, 2015, 4, 141-156.

[0017] Therefore, peptides exhibiting prolonged in vivo persistence in blood circulation according to this technology can be used for a variety of applications, including but not limited to extending the in vivo half-life of (existing or future) therapeutic compounds. The benefits of extending the half-life of therapeutic molecules will be apparent to those skilled in the art. Such benefits include reduced dosage and / or reduced administration frequency, which reduces the risk of adverse events in subjects and lowers costs. Therefore, therapies with extended half-lives have significant added value in terms of pharmaceutical significance.

[0018] In a first aspect, the present technology therefore provides a polypeptide comprising (i) at least one immunoglobulin single variable domain (ISVD) specifically binding to serum albumin; and (ii) at least one additional portion specifically binding to serum albumin or at least one human serum albumin, wherein the (i) at least one ISVD comprises three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0019] a) CDR1 contains the amino acid sequence of SEQ ID NO: 1 or differs from SEQ ID NO: 1 by 3, 2 or 1 amino acids, or CDR1 contains the amino acid sequence of SEQ ID NO: 22 or differs from SEQ ID NO: 22 by 3, 2 or 1 amino acids;

[0020] b) CDR2 contains the amino acid sequence of SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 3, 2, or 1 amino acid; and

[0021] c) CDR3 contains the amino acid sequence of SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 3, 2, or 1 amino acid.

[0022] and / or

[0023] a) CDR1 contains the amino acid sequence of SEQ ID NO: 36 or differs from SEQ ID NO: 36 by 3, 2 or 1 amino acids;

[0024] b) CDR2 contains the amino acid sequence of SEQ ID NO: 37 or differs from SEQ ID NO: 37 by 3, 2, or 1 amino acid; and

[0025] c) CDR3 contains the amino acid sequence of SEQ ID NO: 38 or differs from SEQ ID NO: 38 by 3, 2, or 1 amino acid.

[0026] and / or

[0027] a) CDR1 contains the amino acid sequence of SEQ ID NO: 55 or differs from SEQ ID NO: 55 by 3, 2 or 1 amino acids;

[0028] b) CDR2 contains the amino acid sequence of SEQ ID NO: 56 or differs from SEQ ID NO: 56 by 3, 2, or 1 amino acid; and

[0029] c) CDR3 contains the amino acid sequence of SEQ ID NO: 57 or differs from SEQ ID NO: 57 by 3, 2, or 1 amino acid.

[0030] These CDR sequences are determined based on AbM numbering.

[0031] The polypeptide of this technology contains (i) at least one ISVD that is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the CDRs are as defined herein. Of course, the polypeptide and / or fusion protein of this technology may contain one or more additional ISVDs that are also substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively).

[0032] This technology also provides a fusion protein or construct comprising a polypeptide of the present technology and one or more (e.g., one, two, or more) other amino acid sequences, (binding) domains, binding units, or other portions or chemical entities. The fusion protein may preferably comprise a polypeptide of the present technology, at least one additional albumin-binding domain, and / or at least one therapeutic and / or targeting moiety. This technology also provides polypeptides, fusion proteins, and / or compositions of the present technology for use in medicine (as pharmaceutical agents).

[0033] This document also provides a composition comprising a polypeptide and / or fusion protein of the present technology, a method for producing the composition, a nucleic acid sequence encoding the composition, a vector comprising the nucleic acid sequence, and a host cell comprising the nucleic acid and / or vector of the present technology.

[0034] Finally, this technology provides a kit comprising a polypeptide, fusion protein, nucleic acid or nucleic acid sequence, vector or host cell according to this technology. Attached Figure Description

[0035] Figure 1Schematic diagrams of the constructs (fusion proteins or peptides) of Examples 1 and 2. “ALB” refers to the albumin-binding ISVD building block (ALB23002, SEQ ID NO: 4); “X,” “Y,” and “Z” represent potential future therapeutic leads. “CNB” refers to an unrelated or control ISVD building block (CNB or IRR) that is not bound to serum albumin or any other envisioned target but is simply included in the peptide construct to produce a similar-sized unrelated or control ISVD building block.

[0036] Figure 2 Following intravenous bolus administration of equimolar amounts (3.5 mg / kg for the tetravalent construct or 2.7 mg / kg for the trivalent construct in Tg32 mice), the mean (+ / - SD, n = 4 to 6) serum concentration-time curves of the fusion protein were tested.

[0037] Figure 3 A schematic diagram of the peptides and fusion proteins (constructs) used in this technique and in the examples. Detailed Implementation

[0038] definition

[0039] Unless otherwise indicated or defined, all terms used have their usual meaning in the art, which will be clear to a person skilled in the art. For example, reference standard manuals include Sambrook et al., 1989 (Molecular Cloning: A Laboratory Manual, 2nd ed., Volumes 1-3, Cold Spring Harbor Laboratory Press); Ausubel et al., 1987 (Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York); Lewin 1985 (Genes II, John Wiley & Sons, New York, NY); Old et al., 1981 (Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2nd ed., University of California Press, Berkeley, California); and Roitt et al., 2001 (Immunology, 6th ed., Mosby / Elsevier, Edinburgh). Edinburgh), Roitt et al., 2001 (Roitt's Essential Immunology, 10th edition, Blackwell Publishing, UK) and Janeway et al., 2005 (Immunobiology, 6th edition, Garland Science Publishing / Churchill Livingstone, New York) and the general background techniques cited herein.

[0040] Unless otherwise instructed, as will be clear to those skilled in the art, all methods, procedures, techniques and operations not specifically described in detail herein can be performed and have been performed in a manner known per se. For example, refer again to the standard manuals and general background techniques mentioned in this article, as well as other references cited therein; and to reviews such as Presta 2006 (Adv. Drug Deliv. Rev. [Advanced Drug Delivery Review], 58: 640), Levin and Weiss 2006 (Mol. Biosyst. [Molecular Biology Systems], 2: 49), Irving et al., 2001 (J. Immunol. Methods [Journal of Immunological Methods], 248: 31), Schmitz et al., 2000 (Placenta 21 Suppl. [Placenta 21 Supplement] A:S106), Gonzales et al., 2005 (Tumour Biol. [Tumor Biology], 26: 31), which describe techniques for protein engineering such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.

[0041] It is important to note that, as used herein, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise. Thus, for example, a reference to “a reagent” includes one or more such different reagents, and a reference to “the method” includes references to equivalent steps and methods known to those skilled in the art that can modify or replace the methods described herein.

[0042] Unless otherwise specified, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine many equivalent forms of specific embodiments of the technology described herein using only conventional experimentation. Such equivalent forms are intended to be covered by the technology.

[0043] The term “and / or” as used herein includes the meaning of “and,” “or,” and “all or any other combination of elements connected by the term.”

[0044] Throughout this specification and the claims therein, unless the context otherwise requires, the word “comprise” and its variations such as “comprises” and “comprising” should be understood to imply inclusion of an integer or group of steps of a statement, but not to exclude any other integer or group of steps. When used herein, the term “comprise” may be replaced by the terms “containing” or “including”, or sometimes by the term “having”.

[0045] As used herein, “similar” is interchangeable with similar, analogous, comparable, corresponding and identical, and is intended to have the same or common features and / or to show comparable results in a quantifiable manner, i.e., with a variability of up to 20%, 10%, more preferably 5%, or even more preferably 1%, or less.

[0046] As used in this article, the term "sequence" (e.g., in "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "V") is used in the context of "sequence" (e.g., in "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "V") HH In the context of terms such as “sequence,” “ISVD sequence,” or “protein sequence,” it should generally be understood to include both the associated amino acid sequence and the nucleic acid or nucleotide sequence encoding that amino acid sequence, unless the context requires a more restrictive interpretation. An amino acid sequence should be interpreted as an unbranched sequence of a single amino acid or two or more amino acids, depending on the context. A nucleotide sequence should be interpreted as an unbranched sequence of three or more nucleotides.

[0047] When a nucleotide or amino acid sequence is referred to as "containing" or "consistently composed of" another nucleotide or amino acid sequence, this may mean that the latter nucleotide or amino acid sequence has been incorporated into the first mentioned nucleotide or amino acid sequence, but more generally, it usually means that the first mentioned nucleotide or amino acid sequence contains a segment of nucleotide or amino acid residues within its sequence that has the same nucleotide or amino acid sequence as the latter sequence, regardless of how the first mentioned sequence was actually generated or obtained (this can be, for example, by any suitable method described herein). By way of non-limiting example, when an ISVD is referred to as containing a CDR sequence, this may mean that the CDR sequence has been incorporated into the ISVD, but more generally, it usually means that the ISVD contains a segment of amino acid residues within its sequence that has the same amino acid sequence as the CDR sequence, regardless of how the ISVD was generated or obtained. It should also be noted that when a subsequent amino acid sequence has a specific biological or structural function, it preferably has substantially the same, similar, or equivalent biological or structural function in the first mentioned amino acid sequence (in other words, the first mentioned amino acid sequence preferably enables the subsequent sequence to perform substantially the same, similar, or equivalent biological or structural function). For example, when an ISVD is referred to as containing a CDR sequence or a frame sequence, the CDR sequence and frame are preferably capable of functioning as a CDR sequence or a frame sequence, respectively, in the ISVD. Furthermore, when a nucleotide sequence is referred to as containing another nucleotide sequence, the first mentioned nucleotide sequence preferably enables the amino acid sequence encoded by the subsequent nucleotide sequence to form part of the expression product (e.g., a polypeptide) when expressed as such (in other words, the subsequent nucleotide sequence is in the same reading frame as the first mentioned larger nucleotide sequence).

[0048] Amino acids are those containing an amino group [a] (-NH4+). + 3) and carboxylic acids (-CO) - 2) Organic compounds with functional groups along with the side chains (R groups) unique to each amino acid - amino acid residues will be indicated interchangeably in this document according to the standard three-letter or one-letter amino acid codes mentioned in Table 1 below.

[0049] Table 1: Common Amino Acids

[0050]

[0051] When an amino acid residue is indicated as “X” or “Xaa”, it means that the amino acid residue is unspecified unless the context requires a more restrictive interpretation. For example, if the description provides the amino acid sequence of the CDR, where one (or more) amino acid residues are indicated by “X”, the description may further specify which amino acid residue(s) is (may) be present at that particular position in the CDR.

[0052] Amino acids are those commonly found in naturally occurring proteins and are listed in Table 1. Any amino acid sequence containing a post-translational modification can be described as an amino acid sequence with a modified position (e.g., hydroxylation or glycosylation) initially translated using the symbols shown in Table 1, but which is not explicitly shown in the amino acid sequence. This definition includes any peptide or protein that can be expressed as a sequence modification such as a linker, crosslinker, cap, non-peptide bond, etc. The terms “protein,” “peptide,” “protein / peptide,” and “polypeptide” are used interchangeably throughout this disclosure and each has the same meaning for the purposes of this disclosure. Each term refers to an organic compound consisting of a straight chain of two or more amino acids. Compounds may have ten or more amino acids; twenty-five or more amino acids; fifty or more amino acids; one hundred or more amino acids, two hundred or more amino acids, and even three hundred or more amino acids. Those skilled in the art will understand that although there is no universally accepted dividing line in the number of amino acids that distinguishes polypeptides from proteins, polypeptides generally contain fewer amino acids than proteins; those polypeptides can be prepared by chemical synthesis or recombinant methods; and those proteins are generally prepared in vitro or in vivo by recombinant methods known in the art.

[0053] By convention, the amide bonds in the primary structure of a polypeptide are written in the order of the amino acids, with the amine terminus (N-terminus) of the polypeptide always on the left and the acid terminus (C-terminus) on the right.

[0054] Any amino acid sequence containing post-translational modifications can be described as an amino acid sequence with modified sites (e.g., hydroxylation or glycosylation) that was originally translated using the symbols shown in Table 1; however, these modifications are not explicitly shown in the amino acid sequence. This definition includes any peptide or protein that can be expressed as a sequence modification such as a linker, crosslinker, cap, non-peptide bond, etc.

[0055] As used herein, the term "domain" generally refers to a globular region of an antibody chain, and particularly to a globular region of a heavy chain antibody, or to a polypeptide that is essentially composed of such a globular region. Typically, such a domain will contain, for example, a peptide ring (e.g., 3 or 4 peptide rings) stabilized as a sheet or by disulfide bonds.

[0056] The term “about” as used in the context of the parameters or parameter ranges provided herein shall have the following meaning: Unless otherwise indicated, when the term “about” is applied to a particular value or range, that value or range is interpreted as being as accurate as the method used to measure it. If no error margin is specified in the application, the last decimal place of the numerical value indicates its accuracy. In the absence of other error margins, the maximum margin is determined by applying rounding conventions to the last decimal place; for example, for a pH value of about pH 2.7, the error margin is 2.65–2.74. Any parameter indicated by the term “about” in this application is also considered to be disclosed without the term “about.” In other words, embodiments using the term “about” to refer to parameter values ​​should also describe embodiments where the numerical values ​​for said parameter are as is. For example, an embodiment specifying pH as “about pH 2.7” should also disclose an embodiment specifying pH as “pH 2.7” as is; an embodiment specifying a pH range “between about pH 2.7 and about pH 2.1” will also describe an embodiment specifying a pH range “between pH 2.7 and pH 2.1”, etc.

[0057] For the purpose of comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the corresponding nucleotides in the second nucleotide sequence] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], where each deletion, insertion, substitution, or addition of nucleotides in the second nucleotide sequence is considered a difference at a single nucleotide (position) compared to the first nucleotide sequence. Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0, with standard settings. Some other techniques, computer algorithms, and settings for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0967284, EP 1085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768. Typically, in order to determine the percentage of “sequence identity” between two nucleotide sequences according to the calculation method outlined above, the nucleotide sequence with the largest number of nucleotides is designated as the “first” nucleotide sequence, and the other nucleotide sequence is designated as the “second” nucleotide sequence.

[0058] For the purpose of comparing two or more amino acid sequences, the percentage of “sequence identity” (also referred to herein as “amino acid identity”) between a first amino acid sequence and a second amino acid sequence can be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the corresponding amino acid residues in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], where each deletion, insertion, substitution, or addition of amino acid residues in the second amino acid sequence compared to the first amino acid sequence is considered a difference at a single amino acid residue (position), i.e., an “amino acid difference” as defined herein. Alternatively, the degree of sequence identity between two amino acid sequences can be calculated again using known computer algorithms (such as those described above for determining the degree of sequence identity of nucleotide sequences) with standard settings. Typically, to determine the percentage of “sequence identity” between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is designated as the “first” amino acid sequence, and the other amino acid sequence is designated as the “second” amino acid sequence.

[0059] Furthermore, in determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called “conserved” amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which have little or no effect on the 3D structure, function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art, for example according to WO 04 / 037999, GB 335768, WO 98 / 49185, WO 00 / 46383, and WO 01 / 09300; and the (preferred) types and / or combinations of such substitutions can be selected based on the relevant teachings from WO 04 / 037999 and WO 98 / 49185 and other references cited therein.

[0060] Such conservative substitutions are preferably substitutions in which one amino acid in the following groups (a)-(e) is replaced by another amino acid residue in the same group: (a) small aliphatic nonpolar or micropolar residues: Ala, Ser, Thr, Pro, and Gly; (b) negatively charged polar residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) positively charged polar residues: His, Arg, and Lys; (d) large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp. The particularly preferred conservative substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln, or Glu; Met is replaced by Leu, Tyr, or Ile; Phe is replaced by Met, Leu, or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp; and / or Phe is replaced by Val, Ile, or Leu.

[0061] When comparing two immunoglobulin monovariable domains, the term "amino acid difference" refers to the insertion, deletion, or substitution of a single amino acid residue at a position in the first sequence compared to the second sequence; this is understood to mean that two immunoglobulin monovariable domains may contain one, two, or more such amino acid differences.

[0062] For the purpose of comparing two or more immunoglobulin monovariable domains or other amino acid sequences, such as polypeptides of the present technology, the percentage of “sequence identity” (also referred to herein as “amino acid identity”) between a first amino acid sequence and a second amino acid sequence can be calculated or determined as described in paragraph f) on pages 49 and 50 of WO 08 / 020079 (incorporated herein by reference), such as by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the corresponding amino acid residues in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], wherein each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence compared to the first amino acid sequence is considered a difference at a single amino acid residue (position), i.e., an “amino acid difference” as defined herein; alternatively, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0, with standard settings. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2 357 768-A.

[0063] Typically, in order to determine the percentage of “sequence identity” between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is designated as the “first” amino acid sequence, and the other amino acid sequence is designated as the “second” amino acid sequence.

[0064] Furthermore, in determining the degree of sequence identity between two immunoglobulin monovariable domains, those skilled in the art may consider so-called “conserved” amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which have little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art, for example according to WO 04 / 037999, GB-A-3 357 768, WO 98 / 49185, WO 00 / 46383, and WO 01 / 09300; and the (preferred) types and / or combinations of such substitutions can be selected based on the relevant teachings from WO 04 / 037999 and WO 98 / 49185 and other references cited therein. Examples of conserved substitutions are further described below.

[0065] Any amino acid substitutions applied to the peptides described herein can also be based on analyses of amino acid variation frequencies among homologous proteins of different species developed by Schulz et al., 1978 (Principles of Protein Structure, Springer-Verlag), analyses of structure-forming potential developed by Chou and Fasman, 1975 (Biochemistry 13: 211) and 1978 (Adv. Enzymol. 47: 45-149), and analyses based on Eisenberg et al., 1984 (Proc. Natl. Acad. Sci. USA 81: 140-144), Kyte & Doolittle, 1981 (J Molec. Biol. 157: 105-132), and Goldman et al., 1986 (Ann. Rev. Biophys. Chem.). Analysis of hydrophobic patterns in proteins developed by 15: 321-353, all of which are incorporated herein by reference in their full text. Information regarding the primary, secondary, and tertiary structures of ISVD is given in the description of this paper and in the general background section cited above. Furthermore, for this purpose, V from the llama... HH The crystal structures of the domains are given, for example, by Desmyter et al., 1996 (Nature Structural Biology, 3:803), Spinelli et al., 1996 (Nature Structural Biology, 3:752-757), and Decanniere et al., 1999 (Structure, 7:361). Further details regarding the crystal structures in conventional V can be found in the prior art cited above. H V is formed in the structural domain H / V L Additional information on some amino acid residues at the interface and potential camelification substitutions at these sites.

[0066] If an immunoglobulin single variable domain and a nucleic acid sequence have 100% sequence identity along their entire length (as defined in this article), they are said to be "identical".

[0067] The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. Wild-type genes are the most frequently observed genes in a population and are therefore arbitrarily engineered to be in their "normal" or "wild-type" form. Conversely, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits sequence modifications, post-translational modifications, and / or functional characteristics (i.e., altered features) compared to a wild-type gene or gene product. Notably, naturally occurring mutants can be isolated; these are identified by the fact that they possess altered features compared to a wild-type gene or gene product. Alternatively, variants may also include synthetic molecules; for example, chemokine ligand variants may be structurally and / or functionally similar to natural chemokines but may involve artificial small molecules or synthetic peptides or synthetic proteins. Variants with different functional properties may involve hyperagonists, hyperantagonists, and other functional differences, as known to those skilled in the art.

[0068] In the context of this technology, the terms "specific," "specifically binding," or "specifically binding" refer to the number of different target molecules (such as antigens) that a particular binding unit can bind with a sufficiently high affinity (see below). "Specific," "specifically binding," or "specifically binding" may be used interchangeably herein with "selective," "selectively binding," or "selectively binding." Typically, a binding unit (such as binding ISVD) specifically binds to its designated target.

[0069] The specificity / selectivity of binding units can be determined based on affinity. Affinity represents the strength or stability of molecular interactions. Affinity is typically measured by K... D Alternatively, the dissociation constant may be given, in units of moles per liter (or M). Affinity can also be expressed as the association constant K. A It is equal to 1 / K D And the unit is (moles per liter). -1 (or M) -1 ).

[0070] Affinity is a measure of the strength of binding between a portion of a molecule and its binding site on a target molecule: K D The lower the value, the stronger the binding strength between the target molecule and the target region.

[0071] K D The value also characterizes the strength of molecular interactions in a thermodynamic sense, because it is expressed by the well-known relation DG = RT.ln(K). D (Equivalent to DG = -RT.ln(K)) A The value is related to the change in binding free energy (DG), where R is equal to the gas constant, T is equal to the absolute temperature, and ln represents the natural logarithm.

[0072] KD It can also be expressed as the dissociation rate constant of the complex (denoted as k). off ) and its association rate (denoted as k) on The ratio of (therefore K) D = k off / k on and K A = k on / k off Dissociation rate k off The unit is s -1 (Where s is the SI unit symbol for seconds). Association rate k on The unit is M -1 s -1 The association rate can reach 10. 2 M -1 s -1 To about 10 7 M -1 s -1 The variation between these values ​​approaches the diffusion-limited association rate constant of bimolecular interactions. The dissociation rate is determined by the relationship t... 1 / 2 = ln(2) / k off The half-life of the interaction with a given molecule is relevant. The dissociation rate can be as high as 10. -6 s -1 (Nearly irreversible complex, with multiple days t) 1 / 2 ) to 1 s -1 (t) 1 / 2 It varies between 0.69 s.

[0073] If the measurement process somehow affects the inherent binding affinity of the referred molecule, for example, by artifacts associated with a coating on a biosensor of a molecule, then the measured K... D It can correspond to apparent K D Furthermore, if a molecule contains more than one recognition site of another molecule or multiple molecules, the epigenetic K can be measured. D In this case, the measured affinity may be affected by the affinity of the interaction between the two molecules.

[0074] Dissociation constant (K) D () can be the actual or apparent dissociation constant that a technician clearly understands. Used to determine K D The methods will be clear to a technician, and include, for example, the techniques mentioned below. In this regard, it will also be clear that it may be impossible to measure more than 10 -4 moles per liter or 10 -3 moles per liter (e.g., 10) -2The dissociation constant (mol / L). Optionally, as will be clear to those skilled in the art, the (actual or apparent) Ki D It can be based on the (actual or apparent) association constant (K) A ), through relation (K D =1 / K A ) Calculation. K A = 1 / K D --> K A = [AB] / [A].[B].

[0075] In the context of this technology, the term "avidity" refers to the cumulative strength of multiple affinities of individual non-covalent binding interactions (such as between a protein receptor and its ligand), and may also be referred to as "functional affinity." "Affinity" differs from "affinity," which, as explained in detail above, describes the strength of a single interaction.

[0076] As used herein, the term "binding site" encompasses a region of a polypeptide responsible for selectively binding to a target antigen of interest (e.g., aChR). A binding domain contains at least one binding site. Exemplary binding domains include antibody variable domains. Antibody molecules may contain a single binding site or multiple (e.g., two, three, or four) binding sites.

[0077] The terms “variable region” and “variable domain” are used interchangeably herein and are intended to have equivalent meanings. The term “variable” refers to the fact that certain portions of the variable domains VH and VL differ widely in sequence between antibodies and are used for the binding and specificity of each particular antibody to its target antigen. However, variability is not uniformly distributed throughout the entire variable domain of the antibody. It is concentrated in three segments called “hypervariant loops” within each of the VL and VH domains, forming part of the antigen-binding site.

[0078] In the context of this technology, "acidic pH" or "acidic endogenous pH" refers to an acidic physiological pH, such as the pH inside an organ, which is typically pH < 6.8 or < 6.5, such as between 5.0 and 6.8, such as about 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, or 6.7, or such as about pH 6.4, 6.3, 6.2, 6.1, or about pH 6.0. Therefore, "acidic pH" can refer to a pH between about 6.0 and about 6.5. In the context of this technology, "neutral pH", "near-neutral pH" or "extracellular physiological pH" refers to the pH of the extracellular space, such as a pH of about 7.0 to about 7.5, such as about 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5, preferably about 7.4.

[0079] The peptides of this technology

[0080] The inventors have developed novel polypeptides comprising (i) at least one immunoglobulin single variable domain (ISVD) specifically binding to serum albumin or at least one human serum albumin; and (ii) at least one additional portion specifically binding to serum albumin or at least one human serum albumin.

[0081] In a preferred embodiment, the polypeptide of this technology comprises (i) at least one immunoglobulin single variable domain (ISVD) specifically bound to serum albumin; and (ii) at least one additional portion specifically bound to serum albumin or at least one human serum albumin.

[0082] In a more preferred embodiment, the albumin-binding polypeptide of the present technology comprises (i) at least one ISVD specifically bound to serum albumin; and (ii) at least one additional portion (e.g., albumin-binding ISVD, albumin-binding DARPIN, albumin-binding alfaldaline, or albumin-binding domain (ABD)) specifically bound to serum albumin. Even more preferably, the albumin-binding polypeptide of the present technology comprises (i) at least one ISVD specifically bound to serum albumin; and (ii) at least one additional albumin-binding ISVD.

[0083] First domain: specifically binds to serum albumin or at least one of at least one human serum albumin. ISVD

[0084] In the context of this technology, the term "immunoglobulin single variable domain" (ISVD), which can be used interchangeably with "single variable domain," defines an immunoglobulin molecule in which an antigen-binding site is located on and formed by a single immunoglobulin domain. This distinguishes ISVD from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, discFv), where two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both VH and VL contribute to the formation of the antigen-binding site; that is, a total of six CDRs will participate in the formation of the antigen-binding site.

[0085] Given the above definition, the antigen-binding domain of conventional 4-chain antibodies (such as IgG, IgM, IgA, IgD, or IgE molecules; known in the art) or Fab fragments, F(ab')2 fragments, Fv fragments such as disulfide-linked Fv or scFv fragments, or biantibodies derived from such conventional 4-chain antibodies (known in the art) are generally not considered immunoglobulin monovariable domains because in these cases, binding to the corresponding epitope of the antigen typically does not occur through a single immunoglobulin domain, but rather through a pair of related immunoglobulin domains such as light and heavy chain variable domains, i.e., through the V of the immunoglobulin domain that binds to the epitope of the corresponding antigen. H -V L Regarding what happened.

[0086] In contrast, ISVD can specifically bind to antigenic epitopes without pairing with other immunoglobulin variable domains. The binding site of an immunoglobulin single variable domain consists of a single V... H A single V HH Or a single V L Domain formation.

[0087] In the context of this technology, a single variable domain can be a sequence of light chain variable domains (e.g., V...). L Sequences) or appropriate fragments thereof; or heavy-chain variable domain sequences (e.g., V H Sequence or V HH (sequence) or a suitable fragment thereof; provided that 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).

[0088] ISVDs can be, for example, heavy chain ISVDs, such as VH V HH Including camel-derived V H Or humanized V HH Preferably, it is V. HH Including camel-derived V H Or humanized V HH Heavy chain ISVDs can be derived from conventional tetrachain antibodies or from heavy chain antibodies.

[0089] For example, an ISVD can be a single-domain antibody (or an amino acid sequence suitable for use as a single-domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb), or a Nanobody® ISVD (as defined herein, and including but not limited to V). HH ); other single variable domains, or any suitable fragment of any of them. Preferably, the ISVD is V H Humanized V H , people V H V HH Humanized V HH or camel-derived V H More preferably, ISVD is Nanobody® ISVD (such as V... HH Including humanized V HH or camel-derived V H Nanobody® or appropriate fragments thereof. Nanobody® is a registered trademark of Ablynx NV.

[0090] “V HH "Structural domain", also known as V HH V HH Antibody fragments and V HH Antibodies, initially described as "heavy chain antibodies" (i.e., "antibodies lacking the light chain"; Hamers-Casterman et al., Nature [Nature] 363: 446-448, 1993), are antigen-binding immunoglobulin variable domains. The term "V" is chosen as the preferred term. HH The term "domain" is used to combine these variable domains with the heavy chain variable domains (referred to as "V" in this paper) present in conventional 4-chain antibodies. H The structural domain) and the light chain variable structural domain (referred to in this paper as "V") present in conventional 4-chain antibodies L The structural domain is distinguished from the V. HH For further description, refer to the review article by Muyldermans et al., 2001 (Reviews in Molecular Biotechnology 74: 277-302, 2001). HHThe domains can be obtained from heavy-chain-only antibodies (HCAbs) circulating in camelids, see, for example, Muyldermans S., “A guide to: generation and design of nanobodies”, FEBS J., 2021, 288(7): 2084-2102.

[0091] Typically, immunoglobulin production involves immunizing laboratory animals, fusing immunoglobulin-producing cells to create hybridomas, and screening for the required specificity. Alternatively, immunoglobulins can be generated by screening natural or synthetic libraries (e.g., via phage display).

[0092] Immunoglobulin sequences such as Nanobody® V HH The generation of [a specific antigen] has been widely described in various publications, including, for example, WO 94 / 04678, Hamers-Casterman et al., 1993, and Muyldermans et al., 2001 (Reviews in Molecular Biotechnology 74: 277-302, 2001). In these methods, camels are immunized with a target antigen to induce an immune response against the target antigen. Further screening of the V [antigen] obtained from this immunization is then performed. HH V of the library binding target antigen HH .

[0093] In these cases, antibody production requires purified antigens for immunization and / or screening. The antigens can be purified from natural sources or during recombinant production.

[0094] Peptide fragments of such antigens can be used for screening of immunoglobulin sequences and / or immunoglobulin sequences. This technique can utilize immunoglobulin sequences from various sources, including mouse, rat, rabbit, donkey, human, and cameloid immunoglobulin sequences. This technique also includes fully human, humanized, or chimeric sequences. For example, this technique includes cameloid immunoglobulin sequences and humanized cameloid immunoglobulin sequences, or camel-derived domain antibodies, such as camel-derived dAbs as described by Ward et al. (see, for example, WO 94 / 04678 and Riechmann, Febs Lett. [European Federation of Biochemical Societies Letters], 339:285-290, 1994 and Prot. Eng. [Protein Engineering], 9:531-537, 1996). Furthermore, this technique also utilizes, for example, the formation of multivalent and / or multispecific constructs (for those containing one or more V... HHFor the preparation of multivalent and multispecific polypeptides with domains, see also Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001, and for example WO 96 / 34103 and WO 99 / 23221) fusion immunoglobulin sequences, as well as immunoglobulin sequences containing tags or other functional parts (e.g., toxins, labels, radiochemicals, etc.) that may be derived from the immunoglobulin sequences of this technique.

[0095] "Humanization V" HH "Contains V corresponding to natural occurrences" HH The amino acid sequence of the domain is the same as the amino acid sequence of the human body, but it has been "humanized," i.e., by using V, which is now present in conventional 4-chain antibodies from humans. H One or more amino acid residues at one or more corresponding positions in the domain (as shown above) replace the naturally occurring V. HH One or more amino acid residues in the amino acid sequence of a sequence (especially in a framework sequence). This can be done in a manner known per se, which will be clear to those skilled in the art, for example, based on further description herein and prior art (e.g., WO 2008 / 020079). Again, it should be noted that such humanized V HH It can be obtained in any suitable manner known in itself, and is therefore not strictly limited to the use of naturally occurring V. HH Peptides obtained by using peptides with structural domains as starting materials.

[0096] "Camel Source V" H "Contains V corresponding to natural occurrences" H The amino acid sequence of the domain is a different amino acid sequence, but it has been "camelized," i.e., by using V, which appears in heavy chain antibodies. HH One or more amino acid residues at one or more corresponding positions in the domain replace the naturally occurring V from a conventional 4-chain antibody. H One or more amino acid residues in the amino acid sequence of the domain. This can be done in a manner known per se, which will be clear to those skilled in the art, for example, based on further description herein and prior art (e.g., WO 2008 / 020079). Such "camelization" substitutions preferably insert into the domain formed and / or present in V. H -V L At the amino acid sites of the interface, and / or at so-called cameloid marker residues, as defined herein (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996), ibid.). Preferably, used for the generation or design of camel-derived V H V of the starting material or starting point HThe sequence is preferably V from mammals. H Sequence, more preferably human V H Sequences, such as V H 3. Sequence. However, it should be noted that such camel-derived V... H It can be obtained in any suitable manner known in itself, and is therefore not strictly limited to the use of naturally occurring V. H Peptides obtained by using peptides with structural domains as starting materials.

[0097] The structure of an ISVD sequence can be considered to consist of four frame regions (“FRs”), referred to in the art and herein as “Frame Region 1” (“FR1”); “Frame Region 2” (“FR2”); “Frame Region 3” (“FR3”); and “Frame Region 4” (“FR4”); these frame regions are interrupted by three complementarity determination regions (“CDRs”), referred to in the art and herein as “Complementarity Determination Region 1” (“CDR1”); “Complementarity Determination Region 2” (“CDR2”); and “Complementarity Determination Region 3” (“CDR3”).

[0098] As further described in paragraph q) on pages 58 and 59 of WO 08 / 020079, the amino acid residues of the immunoglobulin single variable domain can be determined according to the sequence of proteins of immunological interest given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH, Bethesda, Maryland, Publication No. 91). H Domains are numbered using common designations, as in the article by Riechmann and Muyldermans, 1999 (J. Immunol. Methods [Journal of Immunological Methods] 231(1-2):25-38; see, for example, this publication). Figure 2 V from camelidae animals HH Domain. It should be noted that, as in the art, V... H Domain and V HH As is well known in structural domains, the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by the Kabat number (that is, one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, generally, the numbering according to Kabat (according to Kabat annotations or Kabat numbers) may or may not correspond to the actual number of amino acid residues in the actual sequence. HDomain and V HH The total number of amino acid residues in the domain is typically in the range of 110 to 120, usually between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0099] In this application, unless otherwise indicated, the CDR sequences are determined according to the Kabat numbering with AbM CDR annotations (“according to AbM annotations” or “AbM numbering”) as described in Kontermann and Dübel (ed. 2010, Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, FR1 contains amino acid residues at positions 1-25, CDR1 contains amino acid residues at positions 26-35, FR2 contains amino acids at positions 36-49, CDR2 contains amino acid residues at positions 50-58, FR3 contains amino acid residues at positions 59-94, CDR3 contains amino acid residues at positions 95-102, and FR4 contains amino acid residues at positions 103-113.

[0100] The determination of the CDR region can also be performed using different methods. According to Kabat's CDR determination, FR1 of the immunoglobulin monovariable domain (IMDV) contains amino acid residues at positions 1-30, CDR1 of the IMDV contains amino acid residues at positions 31-35, FR2 of the IMDV contains amino acid residues at positions 36-49, CDR2 of the IMDV contains amino acid residues at positions 50-65, FR3 of the IMDV contains amino acid residues at positions 66-94, CDR3 of the IMDV contains amino acid residues at positions 95-102, and FR4 of the IMDV contains amino acid residues at positions 103-113.

[0101] In such immunoglobulin sequences, the frame sequence can be any suitable frame sequence, and examples of suitable frame sequences will be clear to those skilled in the art, for example, based on standards manuals and further disclosures and the prior art mentioned herein.

[0102] The framework sequence is preferably an immunoglobulin framework sequence or a suitable combination of framework sequences derived from immunoglobulin framework sequences (e.g., through humanization or camelification). For example, the framework sequence may be derived from a light chain variable domain (e.g., V). L Sequence) and / or heavy-chain variable structural domains (e.g., V)H Sequence or V HH A frame sequence (sequence). In a particularly preferred aspect, the frame sequence is derived from V. HH - A frame sequence of the sequence (where the frame sequence may optionally be partially or fully humanized) or a regular V sequence that has been camel-derived. H Sequences (as defined in this article).

[0103] Specifically, the framework sequence present in the ISVD sequence used in this technique may contain one or more marker residues (as defined herein), making the ISVD sequence a Nanobody® molecule, such as V HH Including humanized V HH or camel-derived V H Some preferred but non-limiting examples of such frame sequences (appropriate combinations) will become clear from further disclosure herein.

[0104] However, it should be noted that, in the context of this technology, the source of the ISVD sequence or the source of the nucleotide sequence used to express it is not limited, nor is the manner in which the ISVD sequence or nucleotide sequence is generated or obtained (or has been generated or obtained). Therefore, the ISVD sequence can be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In a particular but non-limiting respect, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to “humanized” (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, particularly partially or fully humanized V...). HH Immunoglobulin sequences, "camel-derived" (as defined herein) immunoglobulin sequences, and immunoglobulin sequences obtained by techniques such as affinity dematuration (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR transplantation, mosaicking, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques known to the technician for engineering immunoglobulin sequences; or any suitable combination of the foregoing.

[0105] Similarly, a nucleotide sequence can be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and can be, for example, a sequence isolated from a suitable naturally occurring template by PCR (e.g., DNA or RNA isolated from a cell), a nucleotide sequence isolated from a library (especially an expression library), a nucleotide sequence prepared by introducing a mutation into a naturally occurring nucleotide sequence (using any appropriate technique known per se, such as mismatch PCR), a nucleotide sequence prepared by PCR using overlapping primers, or a nucleotide sequence prepared using DNA synthesis techniques known per se.

[0106] For a general description of ISVD, refer to the further description below and the prior art referenced herein. However, it should be noted in this regard that this specification and the prior art primarily describe the so-called "V" H Class 3 ISVD (i.e., with V) H Class 3 phylogenetic sequences such as DP-47, DP-51, or DP-29 have high sequence homology (ISVDs). However, it should be noted that this technique can generally be used with any type of ISVD in its broadest sense, and, for example, also with those belonging to the so-called "V" category. H ISVD of "Class 4" (i.e., with V) H Four types of phylogenetic sequences (such as DP-78, which have high sequence homology with ISVDs), for example, as described in WO 2007 / 118670. Preferably, the ISVD originates from the “V” category. H ISVD of "Class 3"

[0107] Typically, ISVD (especially V) HH Sequence, including (partially) humanized V HH Sequence and camel-derived V H The characteristic of an ISVD sequence may be the presence of one or more “marker residues” (as described herein) within one or more frame sequences (again, as further described herein). Therefore, generally, an ISVD can be defined as an immunoglobulin sequence having the following (general) structure:

[0108] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

[0109] FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively, with one or more marker residues as further defined herein.

[0110] Specifically, ISVD can be an immunoglobulin sequence having the following (general) structure:

[0111] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

[0112] FR1 to FR4 refer to frame regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively. The frame sequence is further defined in this paper.

[0113] More specifically, ISVD can be an immunoglobulin sequence having the following (general) structure:

[0114] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

[0115] FR1 to FR4 refer to frame regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively.

[0116] According to the Kabat number, one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 are selected from the marker residues mentioned in Table 2 below.

[0117] Table 2: Marker Residues in Nanobody® ISVD

[0118]

[0119] Therefore, Nanobody® ISVD can be defined as an amino acid sequence having the following (general) structure:

[0120] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

[0121] FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively. One or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat number are selected from the marker residues mentioned in Table 2.

[0122] Therefore, the at least one ISVD contained in the polypeptide of this technology is essentially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the CDRs are as defined herein.

[0123] In one embodiment, the ISVD contained in the polypeptide of this technology is derived from ISVD, such as from heavy chain ISVD, preferably from Nanobody® ISVD, which has been further engineered / modified to include mutations that prevent / remove binding to pre-existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO 2015 / 173325. For example, to prevent / remove the binding of a pre-existing antibody / factor, the amino acid at position 11 (according to Kabat) may be Val or Leu, preferably Val; and / or the amino acid at position 89 (according to Kabat) may preferably be Val, Thr or Leu, preferably Leu; and / or the amino acid at position 110 (according to Kabat) may preferably be Thr, Lys or Gln, preferably Thr; and / or the amino acid at position 112 (according to Kabat) may be Ser, Lys or Gln, preferably Ser; and / or the ISVD-based building block may contain a C-terminal extension of 1-5 amino acids selected from any naturally occurring amino acids.

[0124] In a preferred embodiment, the polypeptide of this technology comprises:

[0125] (i) specifically binds to at least one immunoglobulin single variable domain (ISVD) of serum albumin; and

[0126] (ii) Specifically binds to serum albumin or at least one additional portion of at least one human serum albumin.

[0127] The at least one ISVD (i) contained in the polypeptide of this technology preferably includes four frame regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3).

[0128] The CDR area is preferably the following area (according to the AbM number):

[0129] a) CDR1 contains the amino acid sequence of SEQ ID NO: 1 or differs from SEQ ID NO: 1 by 3, 2 or 1 amino acids, or CDR1 contains the amino acid sequence of SEQ ID NO: 22 or differs from SEQ ID NO: 22 by 3, 2 or 1 amino acids;

[0130] b) CDR2 contains the amino acid sequence of SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 3, 2, or 1 amino acid; and

[0131] c) CDR3 contains the amino acid sequence of SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 3, 2, or 1 amino acid.

[0132] and / or

[0133] a) CDR1 contains the amino acid sequence of SEQ ID NO: 36 or differs from SEQ ID NO: 36 by 3, 2 or 1 amino acids;

[0134] b) CDR2 contains the amino acid sequence of SEQ ID NO: 37 or differs from SEQ ID NO: 37 by 3, 2, or 1 amino acid; and

[0135] c) CDR3 contains the amino acid sequence of SEQ ID NO: 38 or differs from SEQ ID NO: 38 by 3, 2, or 1 amino acid.

[0136] and / or

[0137] a) CDR1 contains the amino acid sequence of SEQ ID NO: 55 or differs from SEQ ID NO: 55 by 3, 2 or 1 amino acids;

[0138] b) CDR2 contains the amino acid sequence of SEQ ID NO: 56 or differs from SEQ ID NO: 56 by 3, 2, or 1 amino acid; and

[0139] c) CDR3 contains the amino acid sequence of SEQ ID NO: 57 or differs from SEQ ID NO: 57 by 3, 2 or 1 amino acids.

[0140] The CDR sequence mentioned above is determined based on the AbM number.

[0141] Preferably, the polypeptide of the present invention comprises albumin-bound ISVD as a first component (i). Therefore, in a preferred embodiment, the polypeptide of the present invention comprises albumin-bound ISVD, preferably comprising albumin-bound ISVD as described herein.

[0142] In some embodiments, the at least one ISVD (i) contained in the polypeptide of the present technology comprises: CDR1 containing the amino acid sequence of SEQ ID NO: 1, CDR2 containing the amino acid sequence of SEQ ID NO: 2; and CDR3 containing the amino acid sequence of SEQ ID NO: 3, wherein these CDR sequences are determined according to AbM numbering.

[0143] In other embodiments, the at least one ISVD (i) contained in the polypeptide of this technology comprises: CDR1 containing the amino acid sequence of SEQ ID NO: 22, CDR2 containing the amino acid sequence of SEQ ID NO: 2; and CDR3 containing the amino acid sequence of SEQ ID NO: 3, wherein these CDR sequences are determined according to AbM numbering.

[0144] In other embodiments, the at least one ISVD (i) contained in the polypeptide of this technology comprises: CDR1 containing the amino acid sequence of SEQ ID NO: 36, CDR2 containing the amino acid sequence of SEQ ID NO: 37; and CDR3 containing the amino acid sequence of SEQ ID NO: 38, wherein these CDR sequences are determined according to AbM numbering.

[0145] In other embodiments, the at least one ISVD (i) contained in the polypeptide of this technology comprises: CDR1 containing the amino acid sequence of SEQ ID NO: 55, CDR2 containing the amino acid sequence of SEQ ID NO: 56; and CDR3 containing the amino acid sequence of SEQ ID NO: 57, wherein these CDR sequences are determined according to AbM numbering.

[0146] If these CRD sequences are determined according to Kabat numbering, then the at least one ISVD(i) contained in the polypeptide of this technology will preferably contain the following CDR regions:

[0147] a) CDR1 contains the amino acid sequence of SEQ ID NO: 27 or differs from SEQ ID NO: 27 by 3, 2, or 1 amino acid.

[0148] b) CDR2 contains the amino acid sequence of SEQ ID NO: 28 or differs from SEQ ID NO: 28 by 3, 2, or 1 amino acid; and

[0149] c) CDR3 contains the amino acid sequence of SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 3, 2, or 1 amino acid.

[0150] and / or

[0151] a) CDR1 contains the amino acid sequence of SEQ ID NO: 34 or differs from SEQ ID NO: 34 by 3, 2 or 1 amino acids;

[0152] b) CDR2 contains the amino acid sequence of SEQ ID NO: 35 or differs from SEQ ID NO: 35 by 3, 2, or 1 amino acid; and

[0153] c) CDR3 contains the amino acid sequence of SEQ ID NO: 38 or differs from SEQ ID NO: 38 by 3, 2, or 1 amino acid.

[0154] and / or

[0155] a) CDR1 contains the amino acid sequence of SEQ ID NO: 58 or differs from SEQ ID NO: 58 by 3, 2 or 1 amino acids;

[0156] b) CDR2 contains the amino acid sequence of SEQ ID NO: 59 or differs from SEQ ID NO: 59 by 3, 2, or 1 amino acid; and

[0157] c) CDR3 contains the amino acid sequence of SEQ ID NO: 57 or differs from SEQ ID NO: 57 by 3, 2 or 1 amino acids.

[0158] In some embodiments, the at least one ISVD (i) contained in the polypeptide of the present technology comprises: CDR1 containing the amino acid sequence of SEQ ID NO: 27, CDR2 containing the amino acid sequence of SEQ ID NO: 28; and CDR3 containing the amino acid sequence of SEQ ID NO: 3, wherein these CDR sequences are determined according to the Kabat number.

[0159] In other embodiments, the at least one ISVD (i) contained in the polypeptide of this technology comprises: CDR1 containing the amino acid sequence of SEQ ID NO: 34, CDR2 containing the amino acid sequence of SEQ ID NO: 35; and CDR3 containing the amino acid sequence of SEQ ID NO: 38, wherein these CDR sequences are determined according to Kabat numbering.

[0160] In other embodiments, the at least one ISVD (i) contained in the polypeptide of this technology comprises: CDR1 containing the amino acid sequence of SEQ ID NO: 58, CDR2 containing the amino acid sequence of SEQ ID NO: 59; and CDR3 containing the amino acid sequence of SEQ ID NO: 57, wherein these CDR sequences are determined according to Kabat numbering.

[0161] A specific example of an ISVD that specifically binds to HSA is an ISVD comprising four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3), wherein at least one ISVD that specifically binds to serum albumin has:

[0162] a) Having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity with any of the sequences as defined in SEQ ID NO: 4 to 21, 54 or 91 to 93 (wherein any C-terminal extensions that may exist and these CDRs are not considered in determining the sequence identity); and / or

[0163] b) Having no more than 7, preferably no more than 5, amino acid differences from any of the sequences as defined in SEQ ID NO: 4 to 21, 54 or 91 to 93, for example having only 3, 2 or 1 amino acid differences.

[0164] International application WO 2006 / 122787 (the contents of which are incorporated herein by reference) describes a number of ISVDs targeting (human) serum albumin. These ISVDs include those called Alb-1 (SEQ ID NO: 52 in WO 2006 / 122787) and its humanized variants (such as Alb-8 (SEQ ID NO: 62 in WO 2006 / 122787)). Similarly, these can be used to extend the half-life of biological agents according to this technology.

[0165] WO 2012 / 175400 (the contents of which are incorporated herein by reference) describes a further improved version of Alb-1, called Alb-23.

[0166] In one embodiment, the polypeptide of this technology comprises a serum albumin-binding ISVD selected from the following: Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10 (described in WO 2006 / 122787), and Alb-23. In one embodiment, the serum albumin-binding portion is Alb-8 or Alb-23 or a variant thereof, as shown on pages 7-9 of WO 2012 / 175400. In one embodiment, the serum albumin-binding portion is selected from albumin binders described in WO 2012 / 175741, WO2015 / 173325, WO 2017 / 080850, WO 2017 / 085172, WO 2018 / 104444, WO 2018 / 134235 and WO2018 / 134234, the contents of which are incorporated herein by reference. Some preferred serum albumin binders are also shown in Table 3A.

[0167] Table 3A: Serum albumin-binding ISVD / HSA sequences (“ID” refers to SEQ ID NO as used herein)

[0168]

[0169] In some embodiments, the at least one ISVD specifically bound to serum albumin contained in the polypeptide of the present technology has a sequence selected from the group consisting of SEQ ID NO: 4 to 21, 54 or 91 to 93 (Table 3A).

[0170] In some embodiments, the amino acid sequence of at least one ISVD that specifically binds to serum albumin has more than 90%, for example more than 95% or more than 99% sequence identity with Alb-23002 (SEQ ID NO: 4), Alb223 (SEQ ID NO: 18), Alb23002(E1D) (SEQ ID NO: 19), ALBX00002 (SEQ ID NO: 54), Alb82 (SEQ ID NO: 11), T023500029 (SEQ ID NO: 20), HSA006A06 (SEQ ID NO: 91) or HSA006A06-A (SEQ ID NO: 92).

[0171] In one preferred embodiment, the at least one ISVD specifically binding to serum albumin comprises the amino acid sequence of Alb-23002 (SEQ ID NO: 4) or a sequence differing from SEQ ID NO: 4 by 5, 4, 3, 2, or 1 amino acids, or is composed of such a sequence. In another preferred embodiment, the at least one ISVD specifically binding to serum albumin comprises the amino acid sequence of Alb23002 (E1D) (SEQ ID NO: 19) or a sequence differing from SEQ ID NO: 19 by 5, 4, 3, 2, or 1 amino acids, or is composed of such a sequence. In yet another preferred embodiment, the at least one ISVD specifically binding to serum albumin comprises the amino acid sequence of ALBX00002 (SEQ ID NO: 54) or a sequence differing from SEQ ID NO: 54 by 5, 4, 3, 2, or 1 amino acids, or is composed of such a sequence. In another preferred embodiment, the at least one ISVD specifically binding to serum albumin comprises the amino acid sequence T023500029 (SEQ ID NO: 20) or a sequence differing from SEQ ID NO: 20 by 5, 4, 3, 2, or 1 amino acids, or is composed of such sequence. In another preferred embodiment, the at least one ISVD specifically binding to serum albumin comprises the amino acid sequence Alb 82 (SEQ ID NO: 11), or a sequence having at least 99% identity with SEQ ID NO: 11, or a sequence differing from SEQ ID NO: 11 by 5, 4, 3, 2, or 1 amino acids, or is composed of such sequence. In another preferred embodiment, the at least one ISVD specifically binding to serum albumin comprises the amino acid sequence Alb223 (SEQ ID NO: 18) or a sequence differing from SEQ ID NO: 18 by 5, 4, 3, 2, or 1 amino acids, or is composed of such sequence. In another preferred embodiment, the at least one ISVD that specifically binds to serum albumin comprises the amino acid sequence HSA006A06 (SEQ ID NO: 91) or a sequence differing from SEQ ID NO: 91 by 5, 4, 3, 2, or 1 amino acids, or is composed of such sequence. In another preferred embodiment, the at least one ISVD that specifically binds to serum albumin comprises the amino acid sequence HSA006A06-A (SEQ ID NO: 92) or a sequence differing from SEQ ID NO: 92 by 5, 4, 3, 2, or 1 amino acid, or is composed of such sequence.In another embodiment, the at least one ISVD that specifically binds to serum albumin comprises an amino acid sequence of ALB1 (SEQ ID NO: 93) or a sequence that differs from SEQ ID NO: 93 by 5, 4, 3, 2 or 1 amino acids, or is composed of such sequence.

[0172] In other embodiments, the amino acid sequence of at least one ISVD specifically binding to serum albumin has more than 90%, for example, more than 95%, or more than 99% sequence identity with T023500029 (SEQ ID NO: 20). In other embodiments, the amino acid sequence of at least one ISVD specifically binding to serum albumin has more than 90%, for example, more than 95%, or more than 99% sequence identity with Alb-23002 (SEQ ID NO: 4). In other embodiments, the amino acid sequence of at least one ISVD specifically binding to serum albumin has more than 90%, for example, more than 95%, or more than 99% sequence identity with Alb23002(E1D) (SEQ ID NO: 19). In other embodiments, the amino acid sequence of at least one ISVD specifically binding to serum albumin has more than 90%, for example, more than 95%, or more than 99% sequence identity with ALBX00002 (SEQ ID NO: 54). In other embodiments, the amino acid sequence of at least one ISVD specifically binding to serum albumin has more than 90%, for example, more than 95%, or more than 99% sequence identity with Alb 82 (SEQ ID NO: 11). In other embodiments, the amino acid sequence of at least one ISVD specifically binding to serum albumin has more than 90%, for example, more than 95%, or more than 99% sequence identity with Alb223 (SEQ ID NO: 18). In other embodiments, the amino acid sequence of at least one ISVD specifically binding to serum albumin has more than 90%, for example, more than 95%, or more than 99% sequence identity with HSA006A06 (SEQ ID NO: 91). In other embodiments, the amino acid sequence of at least one ISVD specifically binding to serum albumin has more than 90%, for example, more than 95%, or more than 99% sequence identity with HSA006A06-A (SEQ ID NO: 92). In other embodiments, the amino acid sequence of at least one ISVD that specifically binds to serum albumin has more than 90%, for example more than 95% or more than 99% sequence identity with ALB1 (SEQ ID NO: 93).

[0173] When an ISVD bound to human serum albumin differs from the corresponding reference CDR sequence (such as the sequences defined above according to AbM numbers as SEQ ID NO: 1 to 3) in at least one CDR by 2 or 1 amino acid, the ISVD preferably has at least half the binding affinity to human serum albumin compared to the construct ALB23002 (SEQ ID NO: 4), and more preferably has at least the same binding affinity to human serum albumin, wherein the binding affinity is measured using the same method (e.g., surface plasmon resonance). When the amino acid sequence of the ISVD that binds to human serum albumin differs from the amino acid sequence of the corresponding ISVD by 5, 4, 3, 2, or 1 amino acid, the ISVD preferably has at least half the binding affinity to human serum albumin compared to the construct ALB23002 (SEQ ID NO: 4), and more preferably has at least the same binding affinity to human serum albumin, wherein the binding affinity is measured using the same method (e.g., surface plasmon resonance).

[0174] In a more preferred embodiment, the at least one ISVD specifically bound to serum albumin contained in the polypeptide of this technology comprises or is composed of the amino acid sequence of Alb-23002 (SEQ ID NO: 4).

[0175] When such an ISVD bound to human serum albumin has a C-terminal position in the polypeptide of this technology, it may exhibit a C-terminal alanine (A) or C-terminal glycine (G) extension, and is preferably selected from SEQ ID NO: 7, 8, 10, 12, 13, 14, 15, 16, 17, 92 and 18 (see Table 3A). In a preferred embodiment, the ISVD bound to human serum albumin has a position other than the C-terminal position (i.e., not the C-terminal ISVD of the polypeptide of this technology) and is selected from SEQ ID NO: 15 to 17 (see Table 3A).

[0176] In certain embodiments, a polypeptide comprising an ISVD having one or more CDRs having a difference of one, two, three, or four amino acids, as defined herein, binds to serum albumin with substantially the same affinity as a polypeptide comprising an amino acid sequence or polypeptide comprising a CDR having no difference of four, three, two, or one amino acid, as defined herein, compared to a polypeptide comprising an amino acid sequence or CDR having no difference of one, two, three, or four amino acids, as defined herein, the affinity being measured by surface plasmon resonance.

[0177] Compared with the sequence of SEQ ID NO: 4, the at least one serum albumin-binding ISVD contained in the polypeptide of this technology preferably further contains (at least): one or more humanized substitutions;

[0178] and / or

[0179] One or more mutations that reduce the binding of a pre-existing antibody (i.e., amino acid substitution, deletion, or addition, and especially substitution); and optionally contain one or more additional mutations as described herein (e.g., to improve the chemical stability of the peptide).

[0180] For suitable humanization substitutions (and suitable combinations thereof), refer, for example, to WO 09 / 138519 (or to the prior art cited in WO 09 / 138519) and WO 08 / 020079 (or to the prior art cited in WO 08 / 020079), and Tables A-3 to A-8 from WO 08 / 020079 (which are lists showing possible humanization substitutions). Some preferred but non-limiting examples of such humanization substitutions are Q108L and A14P or suitable combinations thereof. Such humanization substitutions may also be suitably combined with one or more other mutations as described herein, such as one or more mutations that reduce the binding of a pre-existing antibody.

[0181] For appropriate mutations (and appropriate combinations of such mutations) that can reduce the binding of pre-existing antibodies, see, for example, WO 2012 / 175741 and WO 2015 / 173325, and also, for example, WO 2013 / 024059 and WO 2016 / 118733.

[0182] Generally, if an amino acid is substituted in one or more or all of the CDRs, the subsequently obtained "substituted" sequence is preferably at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, even more particularly preferably 80%, or even more than 90% identical to the "original" CDR sequence. This means that it depends on the length of the CDR to what extent it is identical to the "substituted" sequence. For example, a CDR having 5 amino acids is preferably 80% identical to its substituted sequence, such that at least one amino acid is substituted. Thus, the CDR of an amino acid sequence, ISVD, or polypeptide can have different degrees of identity with its substituted sequence; for example, CDR1 may have 80%, while CDR3 may have 90%.

[0183] Preferred amino acid substitutions are conservative substitutions. Such conservative substitutions are preferably substitutions in which one amino acid in the following groups (a)-(e) is replaced by another amino acid residue in the same group: (a) small aliphatic nonpolar or micropolar residues: Ala, Ser, Thr, Pro, and Gly; (b) negatively charged polar residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) positively charged polar residues: His, Arg, and Lys; (d) large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp. Other preferred conservative substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln, or Glu; Met is replaced by Leu, Tyr, or Ile; Phe is replaced by Met, Leu, or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp; and / or Phe is replaced by Val, Ile, or Leu. However, any substitution (including non-conservative substitution) is contemplated, provided that the polypeptide retains its ability to specifically bind to epitopes on albumin as described herein, and / or its CDR has at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, and more particularly preferably 80% identity with the “original” CDR sequence.

[0184] In some embodiments, ISVD is a (single) domain antibody, V HH Humanized V HH or camel-derived V H Nanobody® is preferred.

[0185] Preferably, the first domain (i) contained in the polypeptide of this technology is at least one ISVD that specifically binds to serum albumin or contains at least one ISVD that specifically binds to serum albumin, as described herein.

[0186] In another embodiment, the first domain (i) included in the polypeptide of the present invention may be or may include at least one human serum albumin. Serum albumin may be human serum albumin (HSA, for example, AAA98797 as defined in SEQ ID NO: 39 or P02768-1 as defined in SEQ ID NO: 40) or its derivatives, variants, or fragments, as described in detail below. In one embodiment, the first domain (i) included in the polypeptide of the present invention is at least one HSA protein as defined in SEQ ID NO: 40, or an HSA protein having an amino acid sequence having at least 85%, for example at least 90%, or at least 95%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 40, or containing such an HSA protein.

[0187] Second domain: specifically binds to serum albumin or at least one other human serum albumin. outside part

[0188] In certain embodiments, the polypeptide disclosed herein comprises (i) at least one ISVD as defined herein and (ii) at least one portion comprising serum albumin.

[0189] In a particular embodiment, serum albumin is human serum albumin (such as AAA98797 as defined in SEQ ID NO: 39 or P02768-1 as defined in SEQ ID NO: 40) or its polymorphic variants or isotypes. Preferably, serum albumin is human serum albumin, which is or contains the protein sequence as defined in SEQ ID NO: 40, or has an amino acid sequence having at least 85%, for example at least 90%, or at least 95%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 40.

[0190] In the context of this technology, the term "serum albumin protein" refers to serum albumin, such as human serum albumin (e.g., as defined in SEQ ID NO: 39 and 40) or its derivatives, variants or fragments.

[0191] The size of albumin derivatives, their variants, or fragments can vary depending on the size of the fragment, the number of domains, the size of the non-albumin portion of the polypeptide, etc. However, preferably, the size of albumin derivatives, variants, or fragments is in the range of 40-80 kDa, more preferably in the range of 50-70 kDa, more preferably in the range of 55-65 kDa, and most preferably around 60 kDa.

[0192] Human serum albumin is a preferred serum albumin according to the present invention, and is a protein composed of about 585 amino acid residues and having a molecular weight of about 67 kDa (e.g., SEQ ID NO: 39 or SEQ ID NO: 40, preferably SEQ ID NO: 40). Those skilled in the art will understand that natural alleles may exist that have substantially the same properties as human serum albumin, but have one or more amino acid variations compared to SEQ ID NO: 39 or SEQ ID NO: 40, and the inventors also envision using such natural alleles as serum albumin according to the present invention.

[0193] According to this technology, the term "(serum) albumin derivative" refers to a non-natural engineered molecule that contains or is composed of one or more portions of one or more domains of the specified serum albumin.

[0194] The term “(serum) albumin variant” includes albumin or albumin derivatives wherein the albumin or albumin derivative is altered by chemical means, such as post-translational derivatization or peptide modification, such as PEGylation and / or conjugation of a desired portion (e.g., a therapeutic portion) to a thiol group (e.g., provided by unpaired cysteine). The terms “derivative” and “variant” may or may not be used interchangeably.

[0195] The term "(serum) albumin fragment" refers to a polypeptide that has one or more amino acids deleted from the amino and / or carboxyl termini of serum albumin and / or retains the ability to bind to FcRn within an internal region of serum albumin. Fragments may comprise or consist of a single, uninterrupted sequence derived from human serum albumin, or these fragments may comprise or consist of two or more sequences derived from human serum albumin.

[0196] In a particular embodiment, the at least one additional portion comprising serum albumin is a portion, fragment, derivative, or variant of serum albumin.

[0197] In a particular embodiment, the at least one portion comprising serum albumin is human serum albumin or a portion, fragment, derivative, or variant of human serum albumin.

[0198] In other specific embodiments, the polypeptide disclosed herein comprises (ii) at least one additional portion of serum albumin (e.g., specifically bound to human serum albumin (such as AAA98797 as defined in SEQ ID NO: 39 or P02768-1 as defined in SEQ ID NO: 40) or a (polymorphic) variant or isotype thereof.

[0199] Preferably, the polypeptide of this technology comprises the following as a second component: (ii) at least one additional portion of serum albumin (e.g., specifically bound to human serum albumin (such as AAA98797 as defined in SEQ ID NO: 39 or P02768-1 as defined in SEQ ID NO: 40) or a (polymorphic) variant or isotype thereof.

[0200] In a further specific embodiment, the polypeptide of this technology contains (ii) at least one additional portion specifically bound to serum albumin, and amino acid residues on serum albumin that do not participate in the binding of serum albumin to FcRn.

[0201] In a further specific embodiment, the polypeptide of the present technology contains (ii) a portion that specifically binds to at least one additional portion of serum albumin that specifically binds to domain II of human serum albumin.

[0202] In a particular embodiment, the polypeptide of the present technology contains (ii) at least one additional portion that specifically binds to serum albumin, which is a peptide or protein containing between 5 and 500 amino acids.

[0203] In a further specific embodiment, the polypeptide of this technology contains (ii) that specifically binds to at least one portion of serum albumin selected from the group consisting of: Affibody®, scFv, Fab, designed ankylosing repeat protein (DARPin®), albumin-binding domain (ABD), Nanofitin® (also known as affitin), and immunoglobulin variable domain sequence (ISVD).

[0204] For example, the albumin-binding domain (ABD) is described in Hopp J. et al., “The effects of affinity and valency of analbumin-binding domain (ABD) on the half-life of a single-chain diabody-ABD fusion protein”, Protein Eng Des Sel., 2010, 23(11):827-34. For example, peptides of this technology may contain an ABD comprising or composed of SEQ ID NO: 90 (LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA).

[0205] The specific binding to at least one additional portion of serum albumin (ii) may preferably be an albumin-binding ISVD as described in the context of component (i) of the polypeptide of this technology. For example, the specific binding to at least one additional portion of serum albumin (ii) may be an ISVD comprising a CDR1 containing the amino acid sequence of SEQ ID NO: 1, a CDR2 containing the amino acid sequence of SEQ ID NO: 2, and a CDR3 containing the amino acid sequence of SEQ ID NO: 3, wherein these CDR sequences are determined according to an AbM number. For example, the specific binding to at least one additional portion of serum albumin (ii) may be an ISVD comprising a CDR1 containing the amino acid sequence of SEQ ID NO: 22, a CDR2 containing the amino acid sequence of SEQ ID NO: 2, and a CDR3 containing the amino acid sequence of SEQ ID NO: 3, wherein these CDR sequences are determined according to an AbM number. For example, (ii) specifically binding to at least one additional portion of serum albumin may be an ISVD comprising CDR1 containing the amino acid sequence of SEQ ID NO: 36, CDR2 containing the amino acid sequence of SEQ ID NO: 37, and CDR3 containing the amino acid sequence of SEQ ID NO: 38, wherein these CDR sequences are determined according to the AbM number. For example, (ii) specifically binding to at least one additional portion of serum albumin may be an ISVD comprising CDR1 containing the amino acid sequence of SEQ ID NO: 55, CDR2 containing the amino acid sequence of SEQ ID NO: 56, and CDR3 containing the amino acid sequence of SEQ ID NO: 57, wherein these CDR sequences are determined according to the AbM number. For example, (ii) specifically binding to at least one additional portion of serum albumin may be an ISVD comprising or composed of the following sequences: as defined in any one of SEQ ID NO: 4 to 21, 54 or 91 to 93, or a sequence having at least 90%, for example at least 95% or at least 99% identity with the amino acid sequence of any one of SEQ ID NO: 4 to 21, 54 or 91 to 93.

[0206] In a further specific embodiment, the portion of the polypeptide of the present technology that specifically binds to at least one portion of serum albumin is at least one ISVD that specifically binds to domain II of serum albumin (e.g., domain II of human serum albumin).

[0207] In other embodiments, the portion (ii) of the polypeptide of this technology that specifically binds to at least one serum albumin is at least one DARPin that specifically binds to serum albumin. For example, the at least one DARPin may comprise or consist of the following: SEQ ID NO: 88, or a polypeptide having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 88.

[0208] In other embodiments, the portion (ii) of the polypeptide of this technology that specifically binds to at least one portion of serum albumin is at least one afetin that specifically binds to serum albumin. For example, the at least one afetin may comprise or consist of the following: SEQ ID NO: 89, or a polypeptide having at least 90%, for example at least 95%, or at least 97% or at least 99% sequence identity with SEQ ID NO: 89.

[0209] In other embodiments, the portion (ii) of the polypeptide of this technology that specifically binds to at least one serum albumin is at least one albumin-binding domain (ABD) that specifically binds to serum albumin. For example, the at least one ABD may comprise or consist of the following: SEQ ID NO: 90, or a polypeptide having at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 90.

[0210] Table 3B. Serum albumin-binding protein sequences (“ID” refers to SEQ ID NO as used herein)

[0211]

[0212] The peptides of this technology

[0213] Therefore, as described above, the polypeptide of this technology comprises (i) at least one immunoglobulin single variable domain (ISVD) specifically bound to serum albumin or at least one human serum albumin and (ii) at least one additional portion specifically bound to serum albumin or at least one human serum albumin.

[0214] Components (i) and (ii) may be directly connected to each other (as described, for example, in WO 99 / 23221) and / or may be connected to each other by one or more suitable spacers or joints or any combination thereof.

[0215] Those skilled in the art will recognize suitable spacers or linkers for peptides, and these spacers or linkers can generally be any linker or spacer used in the art for linking amino acid sequences. Preferably, the linker or spacer is suitable for constructing proteins or peptides intended for pharmaceutical use.

[0216] For example, the linker can be a suitable amino acid sequence, and particularly an amino acid sequence having between 1 and 50, preferably 1 and 30, for example 1 and 10 amino acid residues. Some preferred examples of such amino acid sequences include, for example, (gly) as described in WO 99 / 42077. x ser y ) z The adapters include gly-ser type adapters (e.g., (gly4ser)3 or (gly3ser2)3) and the 30GS, 15GS, 9GS, and 7GS adapters described in the Ablynx applications mentioned herein (see, for example, WO06 / 040153 and WO 06 / 122825), as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (e.g., described in WO 94 / 04678). Preferred adapters are illustrated in Table 4, particularly 9GS, 20GS, and 35GS, SEQ ID NO: 45, 49, or 52, more preferably 9GS and 35GS, and most preferably the 35GS adapter.

[0217] Table 4: Connector Sequence (“ID” refers to SEQ ID NO as used herein)

[0218]

[0219] Some other particularly preferred connectors are polyalanine (e.g., AAA) as well as connectors GS30 (SEQ ID NO: 85 in WO 06 / 122825) and GS9 (SEQ ID NO: 84 in WO 06 / 122825).

[0220] The length, flexibility, and / or other properties of the linker used (though not critical, as they are typically specific to linkers used in ScFv fragments) may have some influence on the properties of the final peptide of this technique, including, but not limited to, affinity, specificity, or affinity for albumin or one or more other antigens. Based on the disclosure herein, those skilled in the art will optionally be able to determine the optimal linker for a particular peptide used in this technique after a limited number of routine experiments.

[0221] Other suitable linkers typically contain organic compounds or polymers, particularly those suitable for use with proteins in pharmaceutical applications. For example, poly(ethylene glycol) moieties have been used to link antibody domains, see, for example, WO2004 / 081026.

[0222] In one embodiment, the polypeptide of this technology comprises two ISVDs as components (i) and (ii), wherein each ISVD independently includes three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0223] a) CDR1 contains the amino acid sequence of SEQ ID NO: 1 or differs from SEQ ID NO: 1 by 3, 2 or 1 amino acids, or CDR1 contains the amino acid sequence of SEQ ID NO: 22 or differs from SEQ ID NO: 22 by 3, 2 or 1 amino acids;

[0224] b) CDR2 contains the amino acid sequence of SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 3, 2, or 1 amino acid; and

[0225] c) CDR3 contains the amino acid sequence of SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 3, 2, or 1 amino acid.

[0226] and / or

[0227] a) CDR1 contains the amino acid sequence of SEQ ID NO: 36 or differs from SEQ ID NO: 36 by 3, 2 or 1 amino acids;

[0228] b) CDR2 contains the amino acid sequence of SEQ ID NO: 37 or differs from SEQ ID NO: 37 by 3, 2, or 1 amino acid; and

[0229] c) CDR3 contains the amino acid sequence of SEQ ID NO: 38 or differs from SEQ ID NO: 38 by 3, 2, or 1 amino acid.

[0230] and / or

[0231] a) CDR1 contains the amino acid sequence of SEQ ID NO: 55 or differs from SEQ ID NO: 55 by 3, 2 or 1 amino acids;

[0232] b) CDR2 contains the amino acid sequence of SEQ ID NO: 56 or differs from SEQ ID NO: 56 by 3, 2, or 1 amino acid; and

[0233] c) CDR3 contains the amino acid sequence of SEQ ID NO: 57 or differs from SEQ ID NO: 57 by 3, 2 or 1 amino acids.

[0234] These CDR sequences are determined based on AbM numbering.

[0235] Therefore, in one embodiment, the polypeptide of this technology comprises:

[0236] -(i) At least one immunoglobulin single variable domain (ISVD) specifically binds to serum albumin, wherein (i) at least one ISVD comprises three complementarity-determining regions (CDR1 to CDR3, respectively):

[0237] a) CDR1 contains the amino acid sequence of SEQ ID NO: 1 or differs from SEQ ID NO: 1 by 3, 2 or 1 amino acids, or CDR1 contains the amino acid sequence of SEQ ID NO: 22 or differs from SEQ ID NO: 22 by 3, 2 or 1 amino acids;

[0238] b) CDR2 contains the amino acid sequence of SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 3, 2, or 1 amino acid; and

[0239] c) CDR3 contains the amino acid sequence of SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 3, 2, or 1 amino acid.

[0240] and / or

[0241] a) CDR1 contains the amino acid sequence of SEQ ID NO: 36 or differs from SEQ ID NO: 36 by 3, 2 or 1 amino acids;

[0242] b) CDR2 contains the amino acid sequence of SEQ ID NO: 37 or differs from SEQ ID NO: 37 by 3, 2, or 1 amino acid; and

[0243] c) CDR3 contains the amino acid sequence of SEQ ID NO: 38 or differs from SEQ ID NO: 38 by 3, 2, or 1 amino acid.

[0244] and / or

[0245] a) CDR1 contains the amino acid sequence of SEQ ID NO: 55 or differs from SEQ ID NO: 55 by 3, 2 or 1 amino acids;

[0246] b) CDR2 contains the amino acid sequence of SEQ ID NO: 56 or differs from SEQ ID NO: 56 by 3, 2, or 1 amino acid; and

[0247] c) CDR3 contains the amino acid sequence of SEQ ID NO: 57 or differs from SEQ ID NO: 57 by 3, 2, or 1 amino acid.

[0248] These CDR sequences are determined based on AbM numbering; and

[0249] -(ii) Specifically binds to at least one additional portion of serum albumin, wherein the at least one additional portion specifically bound to serum albumin is an ISVD, wherein the (ii) ISVD comprises three complementarity-determining regions (CDR1 to CDR3, respectively):

[0250] a) CDR1 contains the amino acid sequence of SEQ ID NO: 1 or differs from SEQ ID NO: 1 by 3, 2 or 1 amino acids, or CDR1 contains the amino acid sequence of SEQ ID NO: 22 or differs from SEQ ID NO: 22 by 3, 2 or 1 amino acids;

[0251] b) CDR2 contains the amino acid sequence of SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 3, 2, or 1 amino acid; and

[0252] c) CDR3 contains the amino acid sequence of SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 3, 2, or 1 amino acid.

[0253] and / or

[0254] a) CDR1 contains the amino acid sequence of SEQ ID NO: 36 or differs from SEQ ID NO: 36 by 3, 2 or 1 amino acids;

[0255] b) CDR2 contains the amino acid sequence of SEQ ID NO: 37 or differs from SEQ ID NO: 37 by 3, 2, or 1 amino acid; and

[0256] c) CDR3 contains the amino acid sequence of SEQ ID NO: 38 or differs from SEQ ID NO: 38 by 3, 2, or 1 amino acid.

[0257] and / or

[0258] a) CDR1 contains the amino acid sequence of SEQ ID NO: 55 or differs from SEQ ID NO: 55 by 3, 2 or 1 amino acids;

[0259] b) CDR2 contains the amino acid sequence of SEQ ID NO: 56 or differs from SEQ ID NO: 56 by 3, 2, or 1 amino acid; and

[0260] c) CDR3 contains the amino acid sequence of SEQ ID NO: 57 or differs from SEQ ID NO: 57 by 3, 2, or 1 amino acid.

[0261] These CDR sequences are determined based on AbM numbering.

[0262] The two ISVDs included as components (i) and (ii) may be the same or different, and each of the two ISVDs preferably includes the three CDRs as defined above. Therefore, in one embodiment, the two ISVDs included as components (i) and (ii) in the polypeptide of this technology are ISVDs containing or composed of the following sequences: sequences selected from SEQ ID NO: 4 to 21, 54, or 91 to 93, or sequences containing or composed of sequences having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with sequences selected from SEQ ID NO: 4 to 21, 54, or 91 to 93. Examples of polypeptides containing two ISVDs as components (i) and (ii) are illustrated in Table 5 as SEQ ID NO: 74-78, 147-149, and 152-154. Therefore, in one embodiment, the polypeptide of this technology may contain two ISVDs, which may be the same or different, directly connected or connected through a peptide linker, preferably connected through a peptide linker, such as the peptide linkers shown in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)).

[0263] For example, component (i) may be selected from an ISVD that comprises or is composed of: SEQ ID NO: 11, 12, 54, 18, 4, 20, 91 or 92, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 11, 12, 54, 18, 4, 20, 91 or 92, or a protein composed of such a sequence. For example, component (ii) may be selected from an ISVD that comprises or is composed of: SEQ ID NO: 11, 12, 54, 18, 4, 20, 91 or 92, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 11, 12, 54, 18, 4, 20, 91 or 92, or a protein composed of such a sequence. Components (i) and (ii) may be directly connected or connected via peptide connectors, preferably via peptide connectors, such as those shown in Table 4 (e.g., 9GS connector (SEQ ID NO: 45) or 35GS connector (SEQ ID NO: 52)).

[0264] In one embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or consists of SEQ ID NO: 91, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 91, and the polypeptide of the present invention comprises the following ISVD as component (ii), which comprises or consists of SEQ ID NO: 92, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 92. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)). For example, a polypeptide may comprise or consist of the following: SEQ ID NO: 74, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 74, or a polypeptide consisting of that sequence.

[0265] In one embodiment, the polypeptide of this technology comprises two ISVDs as components (i) and (ii), which comprise or consist of the following: SEQ ID NO: 91, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 91, or a protein composed of such sequence. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)).

[0266] In one embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or consists of SEQ ID NO: 91, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 91, and the polypeptide of the present invention comprises the following ISVD as component (ii), which comprises or consists of SEQ ID NO: 18, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)). For example, a polypeptide may comprise or consist of the following: SEQ ID NO: 76, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 76, or a polypeptide consisting of that sequence.

[0267] In one embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or consists of: SEQ ID NO: 91, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 90, and the polypeptide of the present invention comprises the following ISVD as component (ii), which comprises or consists of: SEQ ID NO: 4, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)).

[0268] In one embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or consists of SEQ ID NO: 11, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 11, and the polypeptide of the present invention comprises the following ISVD as component (ii), which comprises or consists of SEQ ID NO: 12, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 12. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)).

[0269] In one embodiment, the polypeptide of this technology comprises two ISVDs as components (i) and (ii), which comprise or consist of the following: SEQ ID NO: 11, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 11, or a protein composed of such sequence. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)).

[0270] In one embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or consists of SEQ ID NO: 54, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 54, and the polypeptide of the present invention comprises the following ISVD as component (ii), which comprises or consists of SEQ ID NO: 18, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)).

[0271] In one embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or consists of SEQ ID NO: 54, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 54, and the polypeptide of the present invention comprises the following ISVD as component (ii), which comprises or consists of SEQ ID NO: 4, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)).

[0272] In one embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or consists of SEQ ID NO: 54, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 54, and the polypeptide of the present invention comprises the following ISVD as component (ii), which comprises or consists of SEQ ID NO: 18, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)). For example, a polypeptide may comprise or consist of the following: SEQ ID NO: 77, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 77, or a polypeptide consisting of the sequence.

[0273] In one embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or consists of SEQ ID NO: 20, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 20, and the polypeptide of the present invention comprises the following ISVD as component (ii), which comprises or consists of SEQ ID NO: 18, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)). For example, a polypeptide may comprise or consist of the following: SEQ ID NO: 78, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 78, or a polypeptide consisting of the sequence.

[0274] In one embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or consists of SEQ ID NO: 20, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 20, and the polypeptide of the present invention comprises the following ISVD as component (ii), which comprises or consists of SEQ ID NO: 4, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)).

[0275] In one embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or consists of SEQ ID NO: 88, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 88, and the polypeptide of the present invention comprises the following ISVD as component (ii), which comprises or consists of SEQ ID NO: 18, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)). For example, a polypeptide may comprise or consist of the following: SEQ ID NO: 79, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 79, or a polypeptide consisting of such sequence.

[0276] In one embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or consists of SEQ ID NO: 88, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 88, and the polypeptide of the present invention comprises the following ISVD as component (ii), which comprises or consists of SEQ ID NO: 4, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)).

[0277] In one embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or consists of SEQ ID NO: 90, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 90, and the polypeptide of the present invention comprises the following ISVD as component (ii), which comprises or consists of SEQ ID NO: 18, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)). For example, a polypeptide may comprise or consist of the following: SEQ ID NO: 81, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 81, or a polypeptide consisting of such sequence.

[0278] In one embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or consists of SEQ ID NO: 90, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 90, and the polypeptide of the present invention comprises the following ISVD as component (ii), which comprises or consists of SEQ ID NO: 4, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 9GS linker (SEQ ID NO: 45) or 35GS linker (SEQ ID NO: 52)).

[0279] In one embodiment, the polypeptide of the present invention comprises two ISVDs, wherein each ISVD comprises or is composed of: SEQ ID NO: 4, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4. Therefore, in one embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 4, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or is composed of a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or is composed of a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or is composed of a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4. Components (i) and (ii) can be directly connected or connected via peptide connectors, preferably via peptide connectors, such as those shown in Table 4 (e.g., 35GS connector (SEQ ID NO: 52) or 9GS connector (SEQ ID NO: 45)).

[0280] Preferably, if the ISVD is located at the C-terminal portion of the polypeptide of the present invention, it contains a C-terminal A. For example, if the ISVD is ALB23002 (SEQ ID NO: 4) and it is located at the C-terminus of the polypeptide, then the ISVD contains a C-terminal A (i.e., the C-terminal ISVD would then be Alb223, SEQ ID NO: 18). Therefore, in one embodiment, the polypeptide of the present invention contains the following ISVD as component (i), which contains or is composed of: SEQ ID NO: 4, or contains a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, and the polypeptide of the present invention contains the following ISVD as component (ii), which contains or is composed of: SEQ ID NO: 18, or contains a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18, or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18. Components (i) and (ii) can be directly connected or connected via peptide connectors, preferably via peptide connectors, such as those shown in Table 4 (e.g., 35GS connector (SEQ ID NO: 52) or 9GS connector (SEQ ID NO: 45)).

[0281] In other embodiments, the polypeptide of this technology comprises two HSA proteins as components (i) and (ii). These two HSA proteins may be the same or different. In one embodiment, the HSA protein comprises or is composed of: SEQ ID NO: 39 or 40, or a protein comprising a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 39 or 40, preferably SEQ ID NO: 40, or a protein comprising a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 40, or a protein comprising a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 40. An example of a polypeptide comprising two HSAs as components (i) and (ii) is illustrated in Table 5 as SEQ ID NO: 87.

[0282] The polypeptide of this technology may also include an ISVD as defined herein and an HSA protein as defined herein.

[0283] In one embodiment, the polypeptide of this technology comprises at least one immunoglobulin single variable domain (ISVD) specifically binding to serum albumin as component (i), wherein the at least one ISVD includes three complementarity-determining regions (CDR1 to CDR3, respectively):

[0284] a) CDR1 contains the amino acid sequence of SEQ ID NO: 1 or differs from SEQ ID NO: 1 by 3, 2 or 1 amino acids, or CDR1 contains the amino acid sequence of SEQ ID NO: 22 or differs from SEQ ID NO: 22 by 3, 2 or 1 amino acids;

[0285] b) CDR2 contains the amino acid sequence of SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 3, 2, or 1 amino acid; and

[0286] c) CDR3 contains the amino acid sequence of SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 3, 2, or 1 amino acid.

[0287] and / or

[0288] a) CDR1 contains the amino acid sequence of SEQ ID NO: 36 or differs from SEQ ID NO: 36 by 3, 2 or 1 amino acids;

[0289] b) CDR2 contains the amino acid sequence of SEQ ID NO: 37 or differs from SEQ ID NO: 37 by 3, 2, or 1 amino acid; and

[0290] c) CDR3 contains the amino acid sequence of SEQ ID NO: 38 or differs from SEQ ID NO: 38 by 3, 2, or 1 amino acid.

[0291] and / or

[0292] a) CDR1 contains the amino acid sequence of SEQ ID NO: 55 or differs from SEQ ID NO: 55 by 3, 2 or 1 amino acids;

[0293] b) CDR2 contains the amino acid sequence of SEQ ID NO: 56 or differs from SEQ ID NO: 56 by 3, 2, or 1 amino acid; and

[0294] c) CDR3 contains the amino acid sequence of SEQ ID NO: 57 or differs from SEQ ID NO: 57 by 3, 2, or 1 amino acid.

[0295] These CDR sequences are determined based on AbM numbering.

[0296] And includes as component (ii) at least one additional portion specifically bound to serum albumin, wherein the at least one additional portion specifically bound to serum albumin is DARPin, preferably comprising or consisting of DARPin of SEQ ID NO: 88, or comprising or consisting of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 88.

[0297] For example, in one embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 18, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18, and the polypeptide of the present technology comprises the following DARPin as component (ii), which comprises or is composed of: SEQ ID NO: 88, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 88. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 35GS linker (SEQ ID NO: 52) or 9GS linker (SEQ ID NO: 45)).

[0298] In another embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or is composed of SEQ ID NO: 4, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, and the polypeptide of the present invention comprises the following DARPin as component (ii), which comprises or is composed of SEQ ID NO: 88, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 88. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 35GS linker (SEQ ID NO: 52) or 9GS linker (SEQ ID NO: 45)).

[0299] Examples of polypeptides containing ISVD as component (i) and DARPin as component (ii) are illustrated in Table 5 as SEQ ID NO: 79 or 150.

[0300] In another embodiment, the polypeptide of this technology comprises at least one immunoglobulin single variable domain (ISVD) specifically binding to serum albumin as component (i), wherein the at least one ISVD includes three complementarity-determining regions (CDR1 to CDR3, respectively):

[0301] a) CDR1 contains the amino acid sequence of SEQ ID NO: 1 or differs from SEQ ID NO: 1 by 3, 2 or 1 amino acids, or CDR1 contains the amino acid sequence of SEQ ID NO: 22 or differs from SEQ ID NO: 22 by 3, 2 or 1 amino acids;

[0302] b) CDR2 contains the amino acid sequence of SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 3, 2, or 1 amino acid; and

[0303] c) CDR3 contains the amino acid sequence of SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 3, 2, or 1 amino acid.

[0304] and / or

[0305] a) CDR1 contains the amino acid sequence of SEQ ID NO: 36 or differs from SEQ ID NO: 36 by 3, 2 or 1 amino acids;

[0306] b) CDR2 contains the amino acid sequence of SEQ ID NO: 37 or differs from SEQ ID NO: 37 by 3, 2, or 1 amino acid; and

[0307] c) CDR3 contains the amino acid sequence of SEQ ID NO: 38 or differs from SEQ ID NO: 38 by 3, 2, or 1 amino acid.

[0308] and / or

[0309] a) CDR1 contains the amino acid sequence of SEQ ID NO: 55 or differs from SEQ ID NO: 55 by 3, 2 or 1 amino acids;

[0310] b) CDR2 contains the amino acid sequence of SEQ ID NO: 56 or differs from SEQ ID NO: 56 by 3, 2, or 1 amino acid; and

[0311] c) CDR3 contains the amino acid sequence of SEQ ID NO: 57 or differs from SEQ ID NO: 57 by 3, 2, or 1 amino acid.

[0312] These CDR sequences are determined based on AbM numbering.

[0313] And includes as component (ii) at least one additional portion specifically bound to serum albumin, wherein the at least one additional portion specifically bound to serum albumin is afetin, preferably comprising or consisting of afetin consisting of: SEQ ID NO: 89, or comprising or consisting of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 89.

[0314] For example, in one embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 18, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18, and the polypeptide of the present technology comprises the following affixine as component (ii), which comprises or is composed of: SEQ ID NO: 89, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 89. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 35GS linker (SEQ ID NO: 52) or 9GS linker (SEQ ID NO: 45)).

[0315] In another embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 4, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, and the polypeptide of the present invention comprises the following affitine as component (ii), which comprises or is composed of: SEQ ID NO: 89, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 89. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 35GS linker (SEQ ID NO: 52) or 9GS linker (SEQ ID NO: 45)).

[0316] Examples of polypeptides containing ISVD as component (i) and affitin as component (ii) are illustrated in Table 5 as SEQ ID NO: 80.

[0317] In another embodiment, the polypeptide of this technology comprises at least one immunoglobulin single variable domain (ISVD) specifically binding to serum albumin as component (i), wherein the at least one ISVD includes three complementarity-determining regions (CDR1 to CDR3, respectively):

[0318] a) CDR1 contains the amino acid sequence of SEQ ID NO: 1 or differs from SEQ ID NO: 1 by 3, 2 or 1 amino acids, or CDR1 contains the amino acid sequence of SEQ ID NO: 22 or differs from SEQ ID NO: 22 by 3, 2 or 1 amino acids;

[0319] b) CDR2 contains the amino acid sequence of SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 3, 2, or 1 amino acid; and

[0320] c) CDR3 contains the amino acid sequence of SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 3, 2, or 1 amino acid.

[0321] and / or

[0322] a) CDR1 contains the amino acid sequence of SEQ ID NO: 36 or differs from SEQ ID NO: 36 by 3, 2 or 1 amino acids;

[0323] b) CDR2 contains the amino acid sequence of SEQ ID NO: 37 or differs from SEQ ID NO: 37 by 3, 2, or 1 amino acid; and

[0324] c) CDR3 contains the amino acid sequence of SEQ ID NO: 38 or differs from SEQ ID NO: 38 by 3, 2, or 1 amino acid.

[0325] and / or

[0326] a) CDR1 contains the amino acid sequence of SEQ ID NO: 55 or differs from SEQ ID NO: 55 by 3, 2 or 1 amino acids;

[0327] b) CDR2 contains the amino acid sequence of SEQ ID NO: 56 or differs from SEQ ID NO: 56 by 3, 2, or 1 amino acid; and

[0328] c) CDR3 contains the amino acid sequence of SEQ ID NO: 57 or differs from SEQ ID NO: 57 by 3, 2, or 1 amino acid.

[0329] These CDR sequences are determined based on AbM numbering.

[0330] And includes as component (ii) at least one additional portion specifically bound to serum albumin, wherein the at least one additional portion specifically bound to serum albumin is an albumin-binding domain (ABD), preferably comprising or consisting of the following ABD: SEQ ID NO: 90, or comprising a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 90, or consisting of such sequence, or a 9GS adapter (SEQ ID NO: 45).

[0331] For example, in one embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of SEQ ID NO: 18, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18, and the polypeptide of the present technology comprises the following albumin-binding domain (ABD) as component (ii), which comprises or is composed of SEQ ID NO: 90, or comprises a sequence or protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 90. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 35GS linker (SEQ ID NO: 52) or 9GS linker (SEQ ID NO: 45)).

[0332] In another embodiment, the polypeptide of the present invention comprises the following ISVD as component (i), which comprises or is composed of SEQ ID NO: 4, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, and the polypeptide of the present invention comprises the following albumin-binding domain (ABD) as component (ii), which comprises or is composed of SEQ ID NO: 90, or comprises a sequence or a protein having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 90. Components (i) and (ii) may be directly linked or linked via peptide linkers, preferably via peptide linkers, such as peptide linkers illustrated in Table 4 (e.g., 35GS linker (SEQ ID NO: 52) or 9GS linker (SEQ ID NO: 45)).

[0333] Examples of polypeptides containing ISVD as component (i) and ABD as component (ii) are illustrated in Table 5 as SEQ ID NO: 81 or 151.

[0334] Therefore, in a preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 11, or comprises a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 11, or comprises a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following ISVD as component (ii), which comprises or is composed of: SEQ ID NO: 11, or comprises a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 11, or comprises a polypeptide composed of such sequence.

[0335] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 54, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 54, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following ISVD as component (ii), which comprises or is composed of: SEQ ID NO: 18, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18, or a polypeptide composed of such sequence.

[0336] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 54, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 54, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following ISVD as component (ii), which comprises or is composed of: SEQ ID NO: 4, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or a polypeptide composed of such sequence.

[0337] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 20, or comprises a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 20, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following ISVD as component (ii), which comprises or is composed of: SEQ ID NO: 18, or comprises a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18, or a polypeptide composed of such sequence.

[0338] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 20, or comprises a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 20, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following ISVD as component (ii), which comprises or is composed of: SEQ ID NO: 4, or comprises a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or a polypeptide composed of such sequence.

[0339] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 91, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 91, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following ISVD as component (ii), which comprises or is composed of: SEQ ID NO: 92, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 92, or a polypeptide composed of such sequence.

[0340] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 91, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 91, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following ISVD as component (ii), which comprises or is composed of: SEQ ID NO: 18, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18, or a polypeptide composed of such sequence.

[0341] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 91, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 91, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following ISVD as component (ii), which comprises or is composed of: SEQ ID NO: 4, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or a polypeptide composed of such sequence.

[0342] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 4, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following ISVD as component (ii), which comprises or is composed of: SEQ ID NO: 18, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18, or a polypeptide composed of such sequence.

[0343] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 4, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following ISVD as component (ii), which comprises or is composed of: SEQ ID NO: 4, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or a polypeptide composed of such sequence.

[0344] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 18, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following DARPin as component (ii), which comprises or is composed of: SEQ ID NO: 88, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 88, or a polypeptide composed of such sequence.

[0345] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 4, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following DARPin as component (ii), which comprises or is composed of: SEQ ID NO: 88, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 88, or a polypeptide composed of such sequence.

[0346] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 18, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following affitin as component (ii), which comprises or is composed of: SEQ ID NO: 89, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 89, or a polypeptide composed of such sequence.

[0347] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 4, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following affitin as component (ii), which comprises or is composed of: SEQ ID NO: 89, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 89, or a polypeptide composed of such sequence.

[0348] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 18, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following albumin-binding domain (ABD) as component (ii), which comprises or is composed of: SEQ ID NO: 90, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 90, or a polypeptide composed of such sequence.

[0349] In another preferred embodiment, the polypeptide of the present technology comprises the following ISVD as component (i), which comprises or is composed of: SEQ ID NO: 4, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following albumin-binding domain (ABD) as component (ii), which comprises or is composed of: SEQ ID NO: 90, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 90, or a polypeptide composed of such sequence.

[0350] In another embodiment, the polypeptide of the present technology comprises the following HSA as component (i), which comprises or is composed of: SEQ ID NO: 40, or comprises a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 40, or a polypeptide composed of such sequence, and the polypeptide of the present technology comprises the following HSA as component (ii), which comprises or is composed of: SEQ ID NO: 40, or comprises a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 40, or a polypeptide composed of such sequence.

[0351] As described above, preferably, the components (i) and (ii) of the polypeptide of this technology are linked by peptide connectors, such as the peptide connectors shown in Table 4, preferably 35GS connectors (SEQ ID NO: 52) or 9GS connectors (SEQ ID NO: 45).

[0352] Table 5. Examples of peptides of this technology (“ID” refers to SEQ ID NO as used herein)

[0353]

[0354] In one embodiment, the polypeptide of this technology comprises:

[0355] (i) Specifically binds to at least one immunoglobulin single variable domain (ISVD) of serum albumin as defined herein, wherein the ISVD preferably does not contain SEQ ID NO: 4 or SEQ ID NO: 18 or an ISVD consisting of SEQ ID NO: 4 or SEQ ID NO: 18; and

[0356] (ii) At least one human serum albumin.

[0357] Therefore, this technology provides a polypeptide comprising or composed of the following sequences: sequences selected from SEQ ID NO: 74-81, 87 and 147-152, or sequences comprising or composed of sequences having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with sequences selected from SEQ ID NO: 74-81, 87 and 147-154.

[0358] Fusion protein

[0359] This technology further provides a fusion protein or construct comprising a polypeptide of this technology. In addition to the polypeptide, the fusion protein of this technology may also contain additional groups, residues, parts, or binding units. As will be apparent to those skilled in the art from the further disclosure herein, these additional groups, residues, parts, binding units, or amino acid sequences may or may not provide further functionality to the polypeptide of this technology, and may or may not alter the properties of the polypeptide of this technology.

[0360] One or more additional groups, residues, portions or binding units contained in the fusion protein of this technology may be directly linked to the polypeptide of this technology (as described, for example, in WO 99 / 23221) and / or linked through one or more suitable spacers or linkers or any combination thereof.

[0361] Suitable spacers or linkers for use in the fusion protein of this technology will be apparent to those skilled in the art, and are generally any linker or spacer used in the art for linking amino acid sequences. Preferably, the linker or spacer is suitable for constructing proteins or peptides intended for pharmaceutical use. The linker in the fusion protein of this technology may be the same as the linker for components (i) and (ii) of the peptide of this technology described above. Preferred linkers are illustrated in Table 4, particularly 9GS, 20GS, and 35GS, SEQ ID NO: 45, 49, or 52, more preferably 9GS and 35GS, and even more preferably 35GS.

[0362] It should be understood that the order of the polypeptide and other groups, residues, portions, or binding units (if present) in the fusion protein of this technology can be selected according to the needs of those skilled in the art and may depend on the relative affinity of the polypeptide and other groups, residues, portions, or binding units (if present) at their positions in the fusion protein. Whether the fusion protein includes one or more linkers to allow the polypeptide and optionally other groups, residues, portions, or binding units to interconnect is a matter of design choice. However, some orientations, with or without linkers, may provide preferred binding characteristics compared to other orientations. This technology covers all different possible orientations.

[0363] When two or more linkers are used in the peptide of this technique, these linkers may be the same or different. Similarly, based on the disclosure herein, those skilled in the art will optionally be able to determine the optimal linker for a particular fusion protein used in this technique after a limited number of routine experiments.

[0364] Typically, for ease of expression and production, the fusion protein of this technique will be a linear protein. However, the technique is not limited to this in the broadest sense. For example, when the fusion protein of this technique contains three or more domains (e.g., at least two domains are contained in the polypeptide as components (i) and (ii) and at least one additional domain is used to form the fusion protein), they can be linked together using a connector with three or more "arms," ​​each "arm" connecting to one domain, thereby providing a "star" construct. Circular constructs can also be used, but this is generally a subpreferable option.

[0365] The additional groups, residues, portions, or binding units included in the fusion protein of this technology may be one or more additional immunoglobulins, thereby forming a (fusion) protein (the fusion protein of this technology). In a preferred but non-limiting aspect, the one or more additional groups, residues, portions, or binding units are ISVDs. Even more preferably, the one or more additional groups, residues, portions, or binding units are selected from the group consisting of: domain antibodies, ISVDs suitable for use as domain antibodies, single-domain antibodies, ISVDs suitable for use as single-domain antibodies, "dAb", ISVDs suitable for use as dAbs, V HH Humanized V HH Camel-based V H Or Nanobody® V HH Preferably, ISVD is derived from V. H or V HHMore preferably, the ISVD is a single-domain antibody (dAb), and even more preferably, a Nanobody® ISVD. For example, in the context of this technology, additional groups, residues, portions, or binding units contained in the fusion protein of this technology, together with the polypeptide of this technology, constitute one or more ISVDs, as described in this application in the context of the polypeptide of this technology.

[0366] The at least one additional group contained in the fusion protein of this technology may be any albumin binding agent described in the “First Domain (i)” and “Second Domain (ii)” sections of this specification.

[0367] Therefore, in one embodiment, the fusion protein of this technology comprises a polypeptide of this technology and at least one additional group, residue, portion, or binding unit, wherein the at least one additional group, residue, portion, or binding unit is an ISVD, the ISVD comprising three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0368] a) CDR1 contains the amino acid sequence of SEQ ID NO: 1 or differs from SEQ ID NO: 1 by 3, 2 or 1 amino acids, or CDR1 contains the amino acid sequence of SEQ ID NO: 22 or differs from SEQ ID NO: 22 by 3, 2 or 1 amino acids;

[0369] b) CDR2 contains the amino acid sequence of SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 3, 2, or 1 amino acid; and

[0370] c) CDR3 contains the amino acid sequence of SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 3, 2, or 1 amino acid.

[0371] and / or

[0372] a) CDR1 contains the amino acid sequence of SEQ ID NO: 36 or differs from SEQ ID NO: 36 by 3, 2 or 1 amino acids;

[0373] b) CDR2 contains the amino acid sequence of SEQ ID NO: 37 or differs from SEQ ID NO: 37 by 3, 2, or 1 amino acid; and

[0374] c) CDR3 contains the amino acid sequence of SEQ ID NO: 38 or differs from SEQ ID NO: 38 by 3, 2, or 1 amino acid.

[0375] and / or

[0376] a) CDR1 contains the amino acid sequence of SEQ ID NO: 55 or differs from SEQ ID NO: 55 by 3, 2 or 1 amino acids;

[0377] b) CDR2 contains the amino acid sequence of SEQ ID NO: 56 or differs from SEQ ID NO: 56 by 3, 2, or 1 amino acid; and

[0378] c) CDR3 contains the amino acid sequence of SEQ ID NO: 57 or differs from SEQ ID NO: 57 by 3, 2, or 1 amino acid.

[0379] These CDR sequences are determined based on AbM numbering.

[0380] In one embodiment, the additional group, residue, portion, or binding unit contained in the fusion protein of this technology is an ISVD, which contains or is composed of the following sequences: sequences selected from SEQ ID NO: 4 to 21, 54, or 91 to 93, or sequences containing or composed of sequences having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with sequences selected from SEQ ID NO: 4 to 21, 54, or 91 to 93.

[0381] In another embodiment, the additional group, residue, portion, or binding unit included in the fusion protein of this technology is an ISVD, which comprises or consists of the following sequences: sequences selected from SEQ ID NO: 4 and 18, or sequences that have at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with sequences selected from SEQ ID NO: 4 and 18, or sequences composed of such sequences. If the additional group, residue, portion, or binding unit included in the fusion protein of this technology is located at the C-terminus of the fusion protein, then the additional group, residue, portion, or binding unit included in the fusion protein of this technology may have a C-terminal alanine residue. For example, if the additional group, residue, portion, or binding unit included in the fusion protein of this technology is an ISVD comprising or composed of SEQ ID NO: 4, then it may contain a C-terminal alanine residue (SEQ ID NO: 18).

[0382] In one embodiment, the additional group, residue, portion or binding unit contained in the fusion protein of the present technology is an ISVD, which comprises or consists of the following: SEQ ID NO: 20, or a sequence containing or consisting of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 20.

[0383] In another embodiment, the additional group, residue, portion or binding unit contained in the fusion protein of this technology is an ISVD, which contains or is composed of the following: SEQ ID NO: 54, or a sequence containing or composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 54.

[0384] In another embodiment, the additional group, residue, portion or binding unit contained in the fusion protein of this technology is an ISVD, which contains or is composed of the following: SEQ ID NO: 91, or a sequence containing or composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 91.

[0385] In another embodiment, the additional group, residue, portion or binding unit contained in the fusion protein of this technology is an ISVD, which contains or is composed of the following: SEQ ID NO: 92, or a sequence containing or composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 92.

[0386] The additional groups, residues, portions or binding units contained in the fusion protein of this technology may also be human serum albumin, such as the human serum albumin illustrated in SEQ ID NO: 39 or 40, preferably SEQ ID NO: 40, or an HSA protein containing or composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 39 or 40, preferably SEQ ID NO: 40.

[0387] In one embodiment, the additional groups, residues, portions, or binding units included in the fusion protein of this technology are DARPin, afetin, or albumin-binding domains (ABD), as described above. In this case, the additional groups, residues, portions, or binding units included in the fusion protein of this technology may be proteins comprising or composed of the following sequences: sequences selected from SEQ ID NO: 88-90, or sequences comprising or composed of sequences having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 88-90.

[0388] Therefore, in one embodiment, the additional group, residue, portion or binding unit contained in the fusion protein of this technology is DARPin, which contains or is composed of the following: SEQ ID NO: 88, or a sequence containing or composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 88.

[0389] In another embodiment, the additional group, residue, portion or binding unit contained in the fusion protein of this technology is afetin, which comprises or consists of: SEQ ID NO: 89, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 89, or a sequence consisting of such sequence.

[0390] In another embodiment, the additional group, residue, portion or binding unit contained in the fusion protein of this technology is an ABD, which comprises or consists of the following: SEQ ID NO: 90, or a sequence containing or consisting of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 90.

[0391] In one embodiment, the fusion protein of this technology comprises three ISVDs, wherein each ISVD comprises or consists of the following: SEQ ID NO: 4, or comprises or consists of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4. Preferably, the three ISVDs are directly linked or linked via peptide linkers, preferably linked via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 35GS linker (SEQ ID NO: 52) or 9GS linker (SEQ ID NO: 45)). Therefore, in a preferred embodiment, the fusion protein comprises: three ISVDs comprising or consisting of SEQ ID NO: 4; and two peptide linkers (preferably as illustrated in SEQ ID NO: 52 or SEQ ID NO: 45, and even more preferably as illustrated in SEQ ID NO: 52) that link the ISVDs to each other. Preferably, the ISVD located at the C-terminal portion of the fusion protein comprises a C-terminal A. If the C-terminal ISVD is Alb2003 (SEQ ID NO: 4), then since it contains the C-terminal A, the ISVD will contain SEQ ID NO: 18 or consist of SEQ ID NO: 18.

[0392] In another embodiment, the fusion protein of this technology comprises a polypeptide containing two ISVDs, which contain or consist of the following: SEQ ID NO: 4, or a sequence having at least 90%, for example, at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or a sequence composed of such a sequence. The fusion protein of this technology also includes an additional portion, which is an ISVD containing or consist of the following: SEQ ID NO: 18, or a sequence having at least 90%, for example, at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18, or a sequence composed of such a sequence. Preferably, the three ISVDs are directly linked or linked via peptide linkers, preferably via peptide linkers, such as those shown in Table 4 (e.g., 35GS linker (SEQ ID NO: 52) or 9GS linker (SEQ ID NO: 45)). Therefore, in a preferred embodiment, the fusion protein comprises or consists of: three ISVDs, two of which contain or consist of SEQ ID NO: 4, and one of which contains or consists of SEQ ID NO: 18; and two peptide linkers (preferably as illustrated in SEQ ID NO: 52 or SEQ ID NO: 45) that link the ISVDs together. Preferably, the ISVD located at the C-terminal portion of the fusion protein contains a C-terminal A (SEQ ID NO: 18). For example, in this embodiment, the fusion protein may comprise or consist of: SEQ ID NO: 82, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 82, or a protein comprising or consisting of that sequence. For example, in this embodiment, the fusion protein may comprise or consist of the following: SEQ ID NO: 85, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 85, or a protein composed of such sequence.

[0393] In another embodiment, the fusion protein of the present invention comprises a polypeptide comprising two ISVDs. One of the two ISVDs comprises or consists of SEQ ID NO: 54, or comprises a sequence or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 54, and the other of the two ISVDs comprises or consists of SEQ ID NO: 20, or comprises a sequence or consists of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 20, and the fusion protein of the present invention comprises an additional portion, which is an ISVD comprising or consists of SEQ ID NO: 18, or comprises a sequence or consists of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18. Preferably, the three ISVDs are directly linked or linked via peptide linkers, preferably via peptide linkers, such as those illustrated in Table 4 (e.g., 35GS linker (SEQ ID NO: 52) or 9GS linker (SEQ ID NO: 45)). Therefore, in a preferred embodiment, the fusion protein comprises: three ISVDs, one of which contains or is composed of SEQ ID NO: 54, another containing or is composed of SEQ ID NO: 20, and another containing or is composed of SEQ ID NO: 18; and two peptide linkers (preferably as illustrated in SEQ ID NO: 52 or SEQ ID NO: 45) linking the ISVDs to each other. Preferably, the ISVD located at the C-terminal portion of the fusion protein contains a C-terminal A (SEQ ID NO: 18). For example, in this embodiment, the fusion protein may comprise or consist of the following: SEQ ID NO: 83, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 83, or a protein composed of such sequence.

[0394] In another embodiment, the fusion protein of this technology comprises: a polypeptide comprising an ISVD comprising or consisting of: SEQ ID NO: 4, or comprising or consisting of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4; and a DARPin comprising or consisting of: SEQ ID NO: 88, or comprising or consisting of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 88; and an additional portion, the additional portion being an ISVD comprising or consisting of: SEQ ID NO: 18, or comprising or consisting of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18. Preferably, the two ISVDs and DARPin are directly linked or linked via peptide linkers, preferably via peptide linkers, such as those illustrated in Table 4 (e.g., 35GS linker (SEQ ID NO: 52) or 9GS linker (SEQ ID NO: 45)). Thus, in one embodiment, the fusion protein comprises: two ISVDs comprising or consisting of SEQ ID NO: 4; and DARPin comprising or consisting of SEQ ID NO: 88; and two peptide linkers (preferably as illustrated in SEQ ID NO: 52 or SEQ ID NO: 45) that link the ISVDs to each other and link the ISVDs to DARPin. Preferably, the ISVD located at the C-terminal portion of the fusion protein comprises a C-terminal A (SEQ ID NO: 18). Therefore, in a preferred embodiment, the fusion protein comprises: two ISVDs, one of which comprises or is composed of SEQ ID NO: 4, and the other ISVD comprises or is composed of SEQ ID NO: 18; a DARPin comprising or is composed of SEQ ID NO: 88; and two peptide linkers (preferably as illustrated in SEQ ID NO: 52 or SEQ ID NO: 45) that connect the ISVDs to each other and link the ISVDs to the DARPin. The ISVD located at the C-terminal portion of the fusion protein preferably comprises a C-terminal A (SEQ ID NO: 18).For example, in this embodiment, the fusion protein may comprise or consist of the following: SEQ ID NO: 84, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 84, or a protein composed of such sequence.

[0395] In another embodiment, the fusion protein of the present invention comprises a polypeptide comprising two ISVDs, which comprise or consist of: SEQ ID NO: 4, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or a sequence composed of such a sequence; and the fusion protein of the present invention comprises two additional portions, which are two ISVDs, one of which comprises or consists of: SEQ ID NO: 4, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, or a sequence composed of such a sequence; and the other ISVD comprises or consists of: SEQ ID NO: 18, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18, or a sequence composed of such a sequence. Preferably, the four ISVDs are directly linked or linked via peptide linkers, preferably via peptide linkers, such as those illustrated in Table 4 (e.g., 35GS linker (SEQ ID NO: 52) or 9GS linker (SEQ ID NO: 45)). In one embodiment, the fusion protein comprises the four ISVDs as described above and two peptide linkers (preferably illustrated in SEQ ID NO: 52 or SEQ ID NO: 45) linking the ISVDs to each other. Preferably, the ISVD located at the C-terminal portion of the fusion protein comprises a C-terminal A (SEQ ID NO: 18). Thus, in a preferred embodiment, the fusion protein comprises: four ISVDs, three of which comprise or consist of SEQ ID NO: 4, and one ISVD comprises or consists of SEQ ID NO: 18; and two peptide linkers (preferably illustrated in SEQ ID NO: 52 or SEQ ID NO: 45) linking the ISVDs to each other. The ISVD located at the C-terminal portion of the fusion protein preferably includes a C-terminal A (SEQ ID NO: 18). For example, in this embodiment, the fusion protein may include or consist of the following: SEQ ID NO: 85, or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 85, or a protein consisting of such a sequence.

[0396] In another embodiment, the fusion protein of this technology comprises a polypeptide comprising two ISVDs. One of the ISVDs comprises or consists of SEQ ID NO: 54, or comprises a sequence or a sequence having at least 90%, for example, at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 54, and the other ISVD comprises or consists of SEQ ID NO: 20, or comprises a sequence or consists of a sequence having at least 90%, for example, at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 20, and the fusion protein of this technology comprises two additional portions, which are two ISVDs. One of the ISVDs comprises or consists of SEQ ID NO: 4, or comprises a sequence or consists of a sequence having at least 90%, for example, at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, and the other ISVD comprises or consists of SEQ ID NO: 4. 18. Or a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18, or a sequence composed of such a sequence. Preferably, the four ISVDs are directly linked or linked via peptide linkers, preferably linked via peptide linkers, such as peptide linkers as illustrated in Table 4 (e.g., 35GS linker (SEQ ID NO: 52)). In one embodiment, the fusion protein comprises: four ISVDs comprising or composed of SEQ ID NO: 54, 20, 4, and 18; and two peptide linkers (preferably as illustrated in SEQ ID NO: 52) that link the ISVDs to each other. Preferably, the ISVD located at the C-terminal portion of the fusion protein comprises a C-terminal A (SEQ ID NO: 18).

[0397] In a preferred embodiment, the fusion protein of this technology is selected from: SEQ ID NO: 82-86, or a sequence containing at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 82-86, or a sequence composed of such a sequence.

[0398] Table 6. Examples of fusion proteins of this technology (“ID” refers to SEQ ID NO as used herein)

[0399]

[0400] In some embodiments, the fusion protein of this technology comprises or is composed of the following:

[0401] - Two target building blocks, preferably two target ISVDs, are directly connected to each other or connected via a connector, preferably connected via a connector selected from Table 4 (e.g., 9SG connector), see, for example, SEQ ID NO: 45;

[0402] - Two albumin-binding ISVDs, preferably wherein each ISVD comprises or is composed of: SEQ ID NO:4, or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO:4, wherein if one of the albumin-binding ISVDs is located at the C-terminus of the fusion protein, the C-terminal ISVD comprises a C-terminal alanine (i.e., SEQ ID NO: 18 or comprises or is composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18), wherein the two albumin-binding ISVDs are directly connected to each other or connected by a adapter, preferably connected by an adapter selected from Table 4 (e.g., a 9SG adapter), see, for example, SEQ ID NO: 45, and wherein one of these target building blocks is directly or connected by a adapter to one of the albumin-binding ISVDs, preferably connected by an adapter selected from Table 4 (e.g., a 9SG adapter), see, for example, SEQ ID NO: 45.

[0403] In other embodiments, the fusion protein of this technology comprises or is composed of the following:

[0404] - Two target building blocks, preferably two target ISVDs, are directly connected to each other or connected via a connector, preferably connected via a connector selected from Table 4 (e.g., 9SG connector), see, for example, SEQ ID NO: 45;

[0405] - Two albumin-binding ISVDs, preferably wherein each ISVD comprises or is composed of: SEQ ID NO:4, or comprises or consists of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO:4, wherein if one of the albumin-binding ISVDs is located at the C-terminus of the fusion protein, the C-terminal ISVD comprises a C-terminal alanine (i.e., SEQ ID NO: 18 or comprises or consists of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18), wherein the two albumin-binding ISVDs are directly connected to each other or connected by a adapter, preferably connected by an adapter selected from Table 4 (e.g., a 35SG adapter), see, for example, SEQ ID NO: 52, and wherein one of these target building blocks is directly or connected by an adapter to one of the albumin-binding ISVDs, preferably connected by an adapter selected from Table 4 (e.g., a 9SG adapter), see, for example, SEQ ID NO: 45.

[0406] In other embodiments, the fusion protein of this technology comprises or is composed of the following:

[0407] - Two target building blocks, preferably two target ISVDs, are directly connected to each other or connected via a connector, preferably connected via a connector selected from Table 4 (e.g., 9SG connector), see, for example, SEQ ID NO: 45;

[0408] - Two albumin-binding ISVDs, preferably wherein each ISVD comprises or is composed of: SEQ ID NO:4, or comprises or consists of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO:4, wherein if one of the albumin-binding ISVDs is located at the C-terminus of the fusion protein, the C-terminal ISVD comprises a C-terminal alanine (i.e., SEQ ID NO: 18 or comprises or consists of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18), wherein the two albumin-binding ISVDs are directly connected to each other or connected by a adapter, preferably connected by an adapter selected from Table 4 (e.g., a 20SG adapter), see, for example, SEQ ID NO: 49, and wherein one of these target building blocks is directly or connected by an adapter to one of the albumin-binding ISVDs, preferably connected by an adapter selected from Table 4 (e.g., a 9SG adapter), see, for example, SEQ ID NO: 45.

[0409] In other embodiments, the fusion protein of this technology comprises or is composed of the following:

[0410] - Three target building blocks, preferably three target ISVDs, are directly connected to each other or connected by connectors, preferably connected by connectors selected from Table 4 (e.g., 9SG connectors), see, for example, SEQ ID NO: 45;

[0411] An albumin-binding ISVD, preferably wherein the ISVD comprises or is composed of: SEQ ID NO: 4, or comprises or consists of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4, wherein if the albumin-binding ISVD is located at the C-terminus of the fusion protein, the C-terminal ISVD comprises a C-terminal alanine (i.e., SEQ ID NO: 18 or comprises or consists of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18), and wherein one of these target building blocks is directly or via a linker to the albumin-binding ISVD, preferably via a linker selected from Table 4 (e.g., the 9SG linker), see, for example, SEQ ID NO: 45.

[0412] In one embodiment, the fusion protein of this technology comprises or is composed of the following sequences: sequences selected from SEQ ID NO: 82-86, or sequences comprising or composed of sequences having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with sequences selected from SEQ ID NO: 82-86.

[0413] Preferred fusion proteins and peptides of this technology comprise or consist of the following: fusion proteins or peptides having at least 90%, for example at least 95% or at least 99% identity with the amino acid sequence of any one of SEQ ID NO: 76, 82, 85 or 150.

[0414] The additional groups, residues, portions, or binding units included in the fusion protein of this technology may be, for example, chemical groups, residues, or portions, which may or may not have biological and / or pharmacological activity. For example, but not limited to, as further described herein, this group may be linked to a peptide of this technology. The fusion protein of this technology may also include additional groups having certain functional groups, such as labels, toxins, one or more linkers, binding sequences, etc. These additional functional groups include both amino acid-based groups and non-amino acid-based groups.

[0415] The fusion protein of this technology may additionally include (in addition to the polypeptide of this technology) one or more targeting moieties. As defined herein, a “targeting moiety” is any group, residue, portion, or binding unit that can be directed by its binding to a target.

[0416] Furthermore, the fusion protein of this technology may additionally include one or more therapeutic moieties. As defined herein, a "therapeutic moiety" is any group, residue, portion, or binding unit capable of exerting therapeutic activity in animals and / or humans. The therapeutic moieties may also be in the form of a precursor, which is then activated to exert its therapeutic activity.

[0417] Typically, proteins or peptides containing a single albumin-binding unit will be referred to in this document as “monovalent peptides,” “monovalent fusion proteins,” or “monovalent constructs.”

[0418] A polypeptide comprising two or more binding units (such as the polypeptide or fusion protein of the present invention) will be referred to herein as a “multivalent polypeptide,” “multivalent fusion protein,” or “multivalent construct.” A polypeptide or fusion protein of the present application comprising two albumin-binding moieties will be referred herein as a “bivalent polypeptide,” “bivalent protein,” or “bivalent construct.” In another embodiment, the polypeptide or fusion protein is at least “trivalent,” meaning it comprises at least three binding moieties (e.g., three binding moieties) or is composed of at least three binding moieties (e.g., three binding moieties). In another embodiment, the polypeptide or fusion protein is at least “tetravalent,” meaning it comprises at least four binding moieties (e.g., four moieties) or is composed of at least four binding moieties (e.g., four moieties). Therefore, the polypeptide or fusion protein of the present invention can be “trivalent,” “quadrivalent,” “pentavalent,” “hexavalent,” “heptavalent,” “octavalent,” “nonavalent,” etc., meaning the polypeptide or fusion protein comprises three, four, five, six, seven, eight, nine, etc. binding moieties or is composed of three, four, five, six, seven, eight, nine, etc. binding moieties.

[0419] As described herein, the multivalent polypeptides or proteins of this technology may be, for example, but not limited to, multispecific (e.g., bispecific or trispecific) or multicomplementary (e.g., bicomplementary) constructs (or constructs that are both multicomplementary and multispecific), and may be, for example, polypeptides or proteins containing at least two binding domains or binding units each targeting different epitopes on the same subunit, polypeptides or proteins containing at least two binding domains or binding units each having different biological functions (e.g., a binding domain that can block or inhibit receptor-ligand interactions, and a binding domain that does not block or inhibit receptor-ligand interactions), or polypeptides or proteins containing at least two binding domains or binding units each targeting different targets.

[0420] The term "multispecific" refers to binding to multiple different target molecules. Therefore, multivalent polypeptides or proteins can be "bispecific," "trispecific," "quadrispecific," etc., meaning they can bind to two, three, four, or other different target molecules respectively. For a general description of multivalent and multispecific polypeptides containing one or more ISVDs and their preparation, see also Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology, 74 (2001), 277-302; and, for example, WO 96 / 34103, WO 99 / 23221, WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO 2009 / 068627.

[0421] It should be understood that the sequence of portions in the peptides and / or fusion proteins of this technology can be selected according to the needs of those skilled in the art and may depend on the relative affinity of these binding domains at their positions within the peptide. Whether the peptide or fusion protein includes one or more linkers to interconnect the binding domains and optional additional groups, residues, or portions is a matter of design choice, as described in detail in this application. However, some orientations, with or without linkers, may provide preferred binding characteristics compared to other orientations. This technology covers all different possible orientations.

[0422] When the polypeptide or fusion protein of this technology has an ISVD at its C-terminus, the C-terminal ISVD (and therefore, the entire compound of this technology by extension) preferably has a C-terminal extension at its C-terminus. This C-terminal extension will be directly linked to the last C-terminal amino acid residue of the C-terminal ISVD, which, according to Kabat, is typically the amino acid residue at position 113 (unless the ISVD contains one or more amino acid deletions such that the sequence of the ISVD ends before position 113). Therefore, typically, the C-terminal extension will be directly linked to the C-terminal VTVSS sequence (SEQ ID NO: 94) of the C-terminal ISVD or the C-terminal sequence of the C-terminal ISVD corresponding to the C-terminal ISVD sequence (e.g., where the C-terminal sequence of the C-terminal ISVD contains one or more substitutions or deletions compared to the usual VTVSS sequence (e.g., T110K, T110Q, S112K, or S112K).

[0423] Generally, any C-terminal extension used herein (i.e., at the C-terminus of the polypeptide or fusion protein of this technique) is typically as described in WO 2012 / 174741 or WO 2015 / 173325 (see also, for example, WO 2103 / 024059 and WO2016 / 118733). In particular, the C-terminal extension may have formula (X). n , wherein n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4 or 5 (and preferably 1 or 2, for example 1); and each X is independently selected from naturally occurring amino acid residues (preferably naturally occurring) amino acid residues (although according to a preferred aspect, it does not contain any cysteine ​​residues), and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[0424] Based on this type of C-end extension X (n) Some preferred but non-limiting aspects of X and n may be as follows:

[0425] a) n = 1 and X = Ala;

[0426] b) n = 2 and each X = Ala;

[0427] c) n = 3 and each X = Ala;

[0428] d) n = 2 and at least one X = Ala (the remaining one or more amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile).

[0429] e)n = 3 and at least one X = Ala (the remaining one or more amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile).

[0430] f)n = 3 and at least two X = Ala (the remaining one or more amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile).

[0431] g)n = 1 and X = Gly;

[0432] h)n = 2 and each X = Gly;

[0433] i)n = 3 and each X = Gly;

[0434] j)n = 2 and at least one X = Gly (the remaining one or more amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile).

[0435] k)n = 3 and at least one X = Gly (the remaining one or more amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile).

[0436] l)n = 3 and at least two X = Gly (the remaining one or more amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile).

[0437] m)n = 2 and each X = Ala or Gly;

[0438] n)n = 3 and each X = Ala or Gly;

[0439] o)n = 3 and at least one X = Ala or Gly (the remaining one or more amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile); or

[0440] p)n = 3 and at least two X = Ala or Gly (the remaining one or more amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile).

[0441] Of particular preference are aspects (a), (b), (c), (g), (h), (i), (m), and (n), especially aspects where n = 1 or 2, and particularly aspects where n = 1.

[0442] It should also be noted that, preferably, any C-terminal extensions present in the polypeptides and / or fusion proteins of this technology do not contain (free) cysteine ​​residues (unless said cysteine ​​residues are used or intended for further functionalization, such as polyethylene glycol conversion).

[0443] Some particular, but non-limiting, examples of useful C-terminal extensions are the following amino acid sequences: A, AA, AAA, G, GG, GGG, AG, GA, AAG, AGG, AGA, GGA, GAA, or GAG, preferably A. Also preferably, when the polypeptide and / or fusion protein of the present technology has an ISVD at its C-terminus, then (at least) the C-terminal ISVD preferably comprises (even more preferably, in addition to the C-terminal extension as described herein) one or more mutations that reduce the binding of a pre-existing antibody (i.e., as described herein with respect to serum albumin binders of the present technology, and more generally described in WO 2012 / 175741 and WO 2015 / 173325, and also, for example, more generally described in WO 2013 / 024059 and WO 2016 / 118733).

[0444] More generally, according to a particular aspect of the present technology, when the polypeptide and / or fusion protein of the present technology contains two or more ISVDs, preferably all of these ISVDs (again, if the polypeptide and / or fusion protein of the present technology has an ISVD at its C-terminus, preferably in addition to the C-terminal extension linked to the C-terminal ISVD) contain mutations that reduce binding to pre-existing antibodies.

[0445] When the polypeptide and / or fusion protein of this technology has an ISVD at its N-terminus, the N-terminal ISVD (and therefore, by extension, the entire polypeptide and / or fusion protein of this technology) preferably contains a D at position 1.

[0446] Specificity

[0447] The specific binding of the binding units of the peptides and / or fusion proteins of this technology (e.g., ISVD, HSA, DARPin, afetin, or ABD as described herein) to their designated targets can be determined by any suitable means known per se (including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assay) and various variants thereof known in the art; as well as other techniques mentioned herein. The dissociation constant can be the actual or apparent dissociation constant that will be clear to a person skilled in the art. Methods for determining the dissociation constant will be clear to a person skilled in the art and include, for example, the techniques mentioned below. In this regard, it is also clear that it may be impossible to measure more than 10 -4 moles per liter or 10 -3 moles per liter (e.g., 10) -2 The dissociation constant (mol / L). Optionally, as will be clear to those skilled in the art, the (actual or apparent) dissociation constant can be based on the (actual or apparent) association constant (K). A ) through relation [KD = 1 / K A The affinity of molecular interactions between two molecules can be measured using various techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, for example, Ober et al., 2001, Intern. Immunology [International Immunology] 13: 30 1551-1559). As used herein, the term “surface plasmon resonance” (SPR) refers to an optical phenomenon that allows the analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and another molecule flows through the immobilized molecule under flowing conditions, thereby generating k on k off Measured values, and thus K. D (or K) A This can be achieved, for example, using well-known BIAcore® systems (BIAcore International AB, GE Healthcare company, Uppsala, Sweden and Piscatave, New Jersey) or ProteOn. TM(Bio-Rad Laboratories, Inc.) systems are used. For detailed descriptions, see Jonsson et al. (1993, Ann. Biol. Clin. [Annals of Clinical Biology] 51: 19-26), Jonsson et al. (1991, Biotechniques [Biotechnology] 11: 620-627), Johnson et al. (1995, J. Mol. Recognit. [Journal of Molecular Recognition] 8: 125-131), and Johnson et al. (1991, Anal. Biochem. [Analytical Biochemistry] 198: 268-277). Another well-known biosensor technique for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, for example, Abdiche et al., 2008, Anal. Biochem. [Analytical Biochemistry] 377: 209-217). As used herein, the term “biolayer interferometry” or “BLI” refers to a label-free optical technique for analyzing the interference patterns of light reflected from two surfaces: an internal reference layer (reference beam) and an immobilized protein layer on the biosensor tip (signal beam). Changes in the number of molecules bound to the biosensor tip cause a shift in the interference pattern, which is reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the surface of the biosensor tip. Because interactions can be measured in real time, association and dissociation rates, as well as affinity, can be determined. BLI can be performed, for example, using the well-known Octet® system (ForteBio, a division of Pall Life Sciences, Menlo Park, USA). Alternatively, affinity can be measured using the KinExA® platform (Sapidyne Instruments Inc., Boise, USA) in kinetic size exclusion assays (KinExA) (see, for example, Drake et al. 2004, Anal. Biochem. [Analytical Biochemistry], 328: 35-43). As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrium solution of an antibody / antigen complex is passed through a chromatographic column containing beads pre-coated with an antigen (or antibody), thereby binding the free antibody (or antigen) to the coated molecule. Detection of the thus captured antibody (or antigen) is accomplished using a fluorescently labeled protein that binds the antibody (or antigen). The GYROLAB® immunoassay system provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al., 2013, Bioanalysis 5: 1765-74).

[0448] Typically, the binding units of this technology (e.g., ISVD, HSA, DARPin, afetin, or ABD as described herein) or peptides or fusion proteins will dissociate with the following dissociation constant (K). D ) Combined with its target: 10 -5 Up to 10 -12 mol / L or less, and preferably 10 -7 Up to 10 -12 mol / L or less, and more preferably 10 -8 Up to 10 -12 moles per liter (i.e., association constant (K)) A ) is 10 5 Up to 10 12 Liters per mole or greater, and preferably 10. 7 Up to 10 12 liters per mole or greater, and more preferably 10. 8 Up to 10 12 (liters / moles). Any value greater than 10 -4 moles / liter of K D Value (or anything below 10) 4 K in liters / moles A The K value is generally considered to indicate nonspecific binding. K is considered to indicate specific biological interactions (e.g., the binding of an immunoglobulin sequence to an antigen). D Usually in 10 -5 mol / L (10000 nM or 10 µM) to 10 -12 The range is mol / L (0.001 nM or 1 pM) or lower. Therefore, specific / selective binding could mean: using the same measurement method, such as SPR, the binding unit (or peptide containing the binding unit) is in the range of 10... -5 Up to 10 -12 K moles per liter or less D The value is bound to its target and is greater than 10. -4 moles / liter of K D The value is combined with the relevant target.

[0449] In certain embodiments, the binding unit of this technology (e.g., ISVD, HSA, DARPin, afetin, or ABD as described herein) or peptide or fusion protein comprises at least one component with a content between 10 5 M -1 With 10 11 M -1 Between, for example, between 10 6 M -1 With 10 11 M -1 Affinity (K) betweenA It specifically binds to the domains of serum albumin (e.g., HSA).

[0450] In certain embodiments, the polypeptide and / or fusion protein comprises at least one component with a content between 10 -6 M and 8.5 10 -11 M or lower, for example, between 10 -6 M and 10 -11 The dissociation constant (K) between M and lower D It specifically binds to the structural domains of serum albumin (e.g., ISVD, HSA, DARPin, afetin, or ABD as described herein). Preferably, K D It is determined by Kinexa, BLI, or SPR, for example, by SPR.

[0451] In certain embodiments, the binding units of this technology (e.g., ISVD, HSA, DARPin, afetin, or ABD as described herein) or peptides or fusion proteins are generally preferably such that they are at a concentration of 10 -5 Up to 10 -12 mol / L or less, and preferably 10 -7 Up to 10 -12 mol / L or less, and more preferably 10 -8 Up to 10 -12 dissociation constant (K) per mole / liter D ) and / or at least 10 7 M -1 Preferably at least 10 8 M -1 More preferably at least 10 9 M -1 For example, at least 10 12 M -1 The binding affinity of the antigen to human serum albumin is as determined using ProteOn. Preferably, the serum albumin binder of this technology (e.g., ISVD, HSA, DARPin, afetin, or ABD as described herein) will bind to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, for example less than 500 pM, as determined using ProteOn.

[0452] In certain embodiments, the peptides and / or fusion proteins of this technology comprise at least one association rate constant (k) selected from the group consisting of... on ) specifically binds to the domains of serum albumin (e.g., ISVD, HSA, DARPin, afetin, or ABD as described herein): at least about 10 2M -1 s -1 At least about 10 3 M -1 s -1 At least about 10 4 M -1 s -1 At least about 10 5 M -1 s -1 At least about 10 6 M -1 s -1 At least about 10 7 M -1 s -1 and at least about 10 8 M -1 s -1 Preferably, it is measured by surface plasmon resonance or BLI.

[0453] In certain embodiments, the peptides and / or fusion proteins of this technology comprise at least one dissociation rate constant (kdissociation rate constant) selected from the group consisting of... off Specifically binds to the domains of serum albumin (e.g., ISVD, HSA, DARPin, afetin, or ABD as described herein): up to approximately 10 -1 s -1 At most about 10 -2 s -1 At most about 10 -3 s -1 At most about 10 -4 s -1 At most about 10 -5 s -1 and at most about 10 -6 s -1 Preferably, it is measured by surface plasmon resonance or BLI.

[0454] According to different aspects of this technology, binding units (e.g., ISVD, HSA, DARPin, afetin, or ABD as described herein), peptides, and / or fusion proteins are generally also preferably made to have cross-reactivity between human serum albumin and serum albumin from at least one, preferably at least two, more preferably at least three, up to substantially all of the following mammalian species: rats, mice, rabbits, guinea pigs, pigs, sheep, cows, and cynomolgus monkeys. In particular, serum albumin binders (binding units (e.g., ISVD, HSA, DARPin, afetin, or ABD as described herein), peptides, and / or fusion proteins) according to different aspects of this technology may make them (at least) cross-reactive between human serum albumin and at least one, preferably at least two, more preferably all three, of human serum albumin and rat serum albumin, mouse serum albumin, and cynomolgus monkey serum albumin. In this respect, compared with serum albumin binders that have the same CDR (according to AbM number) as Alb-11 and / or Alb23002 (E1D), the serum albumin binders of this technology may have improved cross-reactivity (particularly between human serum albumin on one side and rat and / or mouse serum albumin on the other side), see, for example, SEQ ID NO: 9 (Alb-11) or SEQ ID NO: 19 (Alb23002 (E1D)).

[0455] When a binding unit (e.g., ISVD, HAS, DARPin, affixine, or ABD as described herein) or peptide or fusion protein of this technology is described as exhibiting “improved cross-reactivity with human and non-human primate serum albumin” compared to another binding unit (e.g., ISVD, HSA, DARPin, affixine, or ABD as described herein) or peptide or fusion protein, this means that for the binding unit (e.g., ISVD, HSA, DARPin, affixine, or ABD as described herein) or peptide or fusion protein, the ratio of its binding activity to human serum albumin and non-human primate serum albumin (e.g., expressed in the term K) is... D or k off The ratio (indicated by the assay) is lower than the same ratio calculated for other binding units (e.g., ISVD, HSA, DARPin, afetin, or ABD) or peptides or fusion proteins as described herein. The good cross-reactivity with human and non-human primate serum albumin allows for the assessment of the toxicity of serum albumin-binding peptides according to this technique in preclinical studies in non-human primates.

[0456] In a particular embodiment, the at least one serum albumin-binding domain contained in the polypeptide of the present invention makes it (at least) cross-reactive between human serum albumin and cynomolgus monkey serum albumin, and preferably also cross-reactive between human serum albumin and / or cynomolgus monkey serum albumin on one hand and at least one, preferably both, of rat serum albumin and porcine serum albumin on the other hand. For convenience, amino acid segments considered to be part of the putative epitopes of the polypeptide of the present invention have been highlighted in the sequence of serum albumin. Without limitation to any particular mechanism or hypothesis, it is assumed that the polypeptide and fusion protein of the present invention are (substantially) capable of binding to the corresponding segment (one or more amino acid residues) of the amino acid residues present in the amino acid sequence of those mammalian serum albumins that cross-react with the polypeptide and fusion protein of the present invention.

[0457] Generally, when a polypeptide of the present technology containing at least one serum albumin-binding portion can bind to human serum albumin with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, and can also bind to serum albumin from those of the aforementioned species with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, the polypeptide can be considered to have cross-reactivity between human serum albumin and serum albumin from one of the aforementioned other species, and both of these affinities are determined as using SPR.

[0458] Preferably, when the polypeptide and / or fusion protein of the present invention comprises or is composed of two or more target building blocks that specifically bind to serum albumin, it is preferred that each of these building blocks binds to different albumin molecules at least at physiological albumin concentrations. Therefore, at least at physiological albumin concentrations, it is preferred that at least two albumin-binding building blocks (preferably all albumin-binding building blocks) contained in the polypeptide or fusion protein of the present invention bind to at least two albumin molecules (i.e., each albumin-binding building block binds to a different albumin molecule). Therefore, when the polypeptide and / or fusion protein of the present invention comprises or is composed of two or more albumin-binding building blocks, it is preferred that the two or more albumin-binding building blocks of the polypeptide and / or fusion protein of the present invention do not bind to the same serum albumin molecule. For example, in the polypeptide and / or fusion protein of the present invention comprising or composed of two albumin-binding building blocks, it is preferred that the polypeptide and / or fusion protein of the present invention binds to two serum albumin molecules simultaneously. For example, in the polypeptides and / or fusion proteins of the present invention that contain or consist of three albumin-binding building blocks, it is preferred that the polypeptides and / or fusion proteins of the present invention simultaneously bind three serum albumin molecules.

[0459] In embodiments of the present invention, the polypeptide and / or fusion protein comprises or consists of: (i) at least one ISVD specifically binding to serum albumin; and (ii) at least one additional portion specifically binding to serum albumin, each of (i) and (ii) not binding to the same or overlapping epitopes on serum albumin. In other embodiments, if the present invention's polypeptide and / or fusion protein comprises or consists of: (i) at least one ISVD specifically binding to serum albumin; and (ii) at least one additional portion specifically binding to serum albumin, each of (i) and (ii) (and each of other albumin-binding building blocks (if present)) binds to the same or overlapping epitopes on serum albumin, but each of the albumin-binding building blocks contained in the polypeptide and / or fusion protein binds to a different albumin molecule at least at physiological albumin concentrations.

[0460] half life

[0461] As used herein, the term "half-life" is generally defined as described in paragraph o) on page 57 of WO 2008 / 020079, and as mentioned therein, refers to the time taken for the serum concentration of a compound or polypeptide to decrease by 50% in vivo, for example, due to degradation of the sequence or compound and / or clearance or chelation of the sequence or compound by natural mechanisms. The in vivo half-life of polypeptides and / or fusion proteins of this technology can be determined in any manner known per se (e.g., by pharmacokinetic analysis). Suitable techniques will be apparent to those skilled in the art and can be, for example, generally described as in paragraph o) on page 57 of WO 2008 / 020079. As also mentioned in paragraph o) on page 57 of WO 2008 / 020079, half-life can be measured using, for example, t 1 / 2 -α、t 1 / 2 The half-life is expressed using parameters such as -β and the area under the curve (AUC). In this regard, it should be noted that, as used herein, the term "half-life" specifically refers to t... 1 / 2 -β or terminal half-life (where t) 1 / 2-α and / or AUC or both may be disregarded). See, for example, standard manuals such as Kenneth, A et al.: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). Also see “Pharmacokinetics”, M Gibaldi & D Perron, by Marcel Dekker, 2nd revised edition (1982). Similarly, the terms “increased half-life” or “increased half-life” are also as defined in paragraph o) on page 57 of WO 2008 / 020079, and in the presence or absence of t 1 / 2 In the case of an increase in -α and / or AUC or both, specifically referring to t 1 / 2 -β increases.

[0462] The half-life in a mammalian species (e.g., mouse) will depend primarily on, among a range of factors, the binding properties (e.g., affinity) of the polypeptide and / or fusion protein of the present invention to serum albumin of said mammalian species and the half-life of naïve serum albumin in said species. According to a preferred embodiment of the present invention, when the polypeptide and / or fusion protein of the present invention exhibits cross-reactivity (as defined herein) between human serum albumin and serum albumin from another mammalian species (e.g., mouse), the half-life of the polypeptide and / or fusion protein of the present invention determined in said species is preferably at least 5%, for example at least 10%, more preferably at least 25%, for example about 50%, and possibly up to 500%, for example 100%, 150%, or 200% of the half-life of naïve serum albumin in said species. Preferably, the half-life of the polypeptide and / or fusion protein of the present invention determined in said species is preferably at least 100%, for example at least 150%, more preferably at least 200%, or greater than the half-life of naïve serum albumin in said species. Therefore, the half-life of the polypeptides and / or fusion proteins of this technology identified in the said species is preferably at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.7 times, or at least 1.8 times, preferably at least 2 times, preferably at least 2.3 times, or at least 2.4 times, or at least 2.5 times, or at least 3 times, at least 4 times, for example at least 5 times, for example at least 10 times, or more than 20 times, greater than the half-life of pure serum albumin in the said species. In particular, the serum half-life of the polypeptides and / or fusion proteins in humans is at least 5%, for example at least 10%, at least 25%, at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% of the half-life of serum albumin in humans. Therefore, the half-life of the polypeptides and / or fusion proteins of the present technology identified in the species is preferably at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.7 times, preferably at least 2 times, preferably at least 2.1 times, or at least 2.2 times, or at least 2.3 times, or at least 2.4 times, or at least 3 times, or at least 4 times, for example at least 5 times, for example at least 10 times, or more than 20 times, greater than the half-life of serum albumin (HSA) in humans.

[0463] Furthermore, if the polypeptide according to this technology is fused with another part (e.g., one or more therapeutic parts), it will have an increased half-life compared to the other part itself (e.g., another one or more therapeutic parts itself).

[0464] The polypeptides and / or fusion proteins described herein preferably have a half-life that is at least 2 times, for example at least 5 times, preferably at least 10 times or more than 20 times, for example more than 50 times, more than 100 times, more than 500 times, and preferably more than 1,000 times greater than the corresponding other part itself (e.g., the therapeutic part itself) (as measured in humans or suitable animals such as mice or cynomolgus monkeys).

[0465] Furthermore, compared to peptides comprising a single albumin-binding moiety itself (e.g., a single albumin-binding ISVD, or a single other albumin-binding moiety (ABD, Darpin, afetin) itself), the peptides and / or fusion proteins of this technology will have an increased half-life. Specifically, compared to constructs disclosed in the prior art that comprise a therapeutic portion and a single known half-life-extending portion, the peptides according to this technology will have an increased half-life.

[0466] The peptides and / or fusion proteins described herein preferably have a half-life that is at least 1.1, 1.2, 1.3, 1.4, 1.5, preferably at least 2, preferably at least 2.4, at least 3, at least 4, for example at least 5, for example at least 10, or more than 20 times greater than the half-life of corresponding constructs disclosed in the prior art that contain a single known extended half-life (as measured in humans or suitable animals such as mice or cynomolgus monkeys). In particular, the peptides and / or fusion proteins described herein preferably have a half-life that is at least 1.5, or at least 1.7, or at least 1.8, preferably at least 2, for example at least 2.1, at least 2.2, at least 2.3, at least 2.4, or at least 2.5 times greater than the half-life of corresponding constructs disclosed in the prior art that contain a single known extended half-life (as measured in humans or suitable animals such as mice or cynomolgus monkeys).

[0467] The peptides and / or fusion proteins described herein preferably have a half-life that is at least 1.1, 1.2, 1.3, 1.4, 1.5, preferably at least 2, preferably at least 2.3, or at least 2.4, or at least 2.5, at least 3, at least 4, for example at least 5, for example at least 10, or more than 20 times greater than that of therapeutic constructs disclosed in the prior art that include a therapeutic portion and a single known extended half-life portion (as measured in humans or suitable animals such as mice or cynomolgus monkeys). In particular, the peptides and / or fusion proteins described herein preferably have a half-life that is at least 1.5 times, or at least 1.7 times, or at least 1.8 times, preferably at least 2 times, for example at least 2.1 times, at least 2.2 times, at least 2.3 times, or at least 2.4 times, or at least 2.5 times greater than that of therapeutic constructs disclosed in the prior art that include a therapeutic portion and a single known extended half-life portion (as measured in humans or suitable animals such as mice or cynomolgus monkeys).

[0468] As described above, in one aspect, the polypeptide according to the present technology can be used to increase one or more immunoglobulin single variable domains (ISVDs) (e.g., domain antibodies, single domain antibodies, "dAb", V). HH Or Nanobody® V HH (e.g., V) HH Humanized V HH or camel-derived V H For example, camel-derived human V H The half-life of ).

[0469] Methods for preparing peptides and / or fusion proteins of this technology

[0470] Another embodiment of this technology relates to a method for producing polypeptides and / or fusion proteins of this technology.

[0471] As described in detail above, the polypeptides and / or fusion proteins according to the present technology comprise (i) at least one immunoglobulin single variable domain (ISVD) specifically bound to serum albumin or at least one human serum albumin and (ii) at least one additional portion specifically bound to serum albumin or at least one human serum albumin.

[0472] Therefore, the peptides of this technology can generally be prepared by a method comprising at least one step of appropriately linking one or more components (i) and (ii) (including a linker (if present)) to each other, for example linking an ISVD, a peptide linker (e.g., 35GS, 9GS or 20GS) and a domain containing serum albumin or a serum albumin-binding domain to each other.

[0473] The fusion protein of this technology can be prepared by a method similar to that used to prepare the peptides of this technology.

[0474] The peptides and fusion proteins of this technology can also be prepared by methods that typically include at least the following steps: providing a nucleic acid encoding the peptide or fusion protein of this technology, expressing the nucleic acid in an appropriate manner, and recovering the expressed peptide of this technology. Such methods can be carried out in ways known per se, which will be apparent to those skilled in the art, for example, based on the methods and techniques further described herein. The process of designing / selecting and / or preparing the peptide or fusion protein of this technology, starting from a peptide or fusion protein containing at least one component (e.g., component (i) or (ii)), is also referred to herein as “formatting” the peptide or fusion protein of this technology. Based on the disclosure herein, those skilled in the art will be aware of examples of ways in which peptides or fusion proteins of this technology can be formatted, and examples of such formats.

[0475] Those skilled in the art are familiar with methods for linking two polypeptides to prepare the polypeptides and / or fusion proteins of this technology. For example, the method may include the following steps:

[0476] a) As described above, provide at least one ISVD or HSA (i) and an additional part or HSA (ii);

[0477] c) Optionally provide an additional group, residue, part or binding unit;

[0478] b) As described herein, all peptides are linked together directly or via a linker (see, for example, Table 4).

[0479] For example, the method may include the following steps:

[0480] a) As described above, select at least one ISVD or HSA (i) and another part or HSA (ii), and optionally select at least one other group, residue, part or binding unit;

[0481] b) Design a gene construct encoding a protein sequence containing the polypeptide described in a); and

[0482] c) Introduce the gene construct into an expression system to obtain a polypeptide or fusion protein of the present technology as described above in this specification.

[0483] In the context of this technology, the location of each of the components ((i) and (ii)) within the polypeptide of this technology is not limited. For example, the first component (i) may be located in the N-terminal portion of the polypeptide, while the at least one additional portion (ii) specifically bound to serum albumin or at least one human serum albumin may be located in the C-terminal portion of the polypeptide. Furthermore, the first component (i) may be located in the C-terminal portion of the polypeptide, while the at least one additional portion (ii) specifically bound to serum albumin or at least one human serum albumin may be located in the N-terminal portion of the polypeptide. This also applies to the fusion proteins of this technology; the polypeptide of this technology may be located in the C-terminal portion of the fusion protein, while the at least one additional group, residue, portion, or binding unit may be located in the N-terminal portion of the fusion protein, and vice versa (the polypeptide of this technology may be located in the N-terminal portion of the fusion protein, while the at least one additional group, residue, portion, or binding unit may be located in the C-terminal portion of the fusion protein).

[0484] Nucleic acid molecules

[0485] This technology also provides nucleic acid molecules encoding polypeptides or fusion proteins of this technology.

[0486] A “nucleic acid molecule” (used interchangeably with “nucleic acid”) is a chain of nucleotide monomers linked together by a phosphate backbone to form a nucleotide sequence. Nucleic acids can be used to transform / transfect host cells or host organisms, for example, for the expression and / or production of polypeptides or fusion proteins. A suitable (non-human) host or host cell for production purposes will be clear to a person skilled in the art and can be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line or any suitable fungus, prokaryotic or eukaryotic organism. Hosts or host cells containing nucleic acids encoding protein-based vector building blocks and / or molecules (or parts thereof) of this technology are also covered in this technology.

[0487] Nucleic acids can be, for example, DNA, RNA, or hybrids thereof, and may also contain (e.g., chemically modified) nucleotides, such as PNA. They can be single-stranded or double-stranded. In one embodiment, it is in the form of double-stranded DNA. For example, the nucleotide sequence of this technology can be genomic DNA or cDNA.

[0488] The nucleic acids of this technique can be prepared or obtained in a manner known per se, and / or isolated from suitable natural sources. Nucleotide sequences encoding naturally occurring (poly)peptides can, for example, be subjected to site-directed mutagenesis to provide nucleic acid molecules encoding polypeptides with sequence variations. Furthermore, it will be apparent to those skilled in the art that, in order to prepare nucleic acids, several nucleotide sequences (e.g., at least one nucleotide sequence encoding a target moiety) and nucleic acids, for example, encoding one or more adapters, can be linked together in a suitable manner.

[0489] The techniques used to generate nucleic acids will be clear to a technician and may include, for example, but not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more portions thereof); introducing mutations that result in the expression of a truncated expression product; and introducing one or more restriction sites (e.g., to form boxes and / or regions that can be easily digested and / or ligated using appropriate restriction enzymes) and / or introducing mutations through PCR reactions using one or more “mismatched” primers.

[0490] carrier

[0491] A vector comprising a nucleic acid molecule encoding a polypeptide or fusion protein of this technology is also provided.

[0492] The vectors used in this article are suitable agents for carrying genetic material into cells. Vectors include naked nucleic acids (such as plasmids or mRNA) or nucleic acids embedded in larger structures (such as liposomes or viral vectors).

[0493] In some embodiments, the vector comprises at least one nucleic acid optionally linked to one or more regulatory elements, such as one or more suitable promoters, enhancers, terminators, etc. In one embodiment, the vector is an expression vector, i.e., a vector suitable for expressing a encoded polypeptide or construct under appropriate conditions, such as when the vector is introduced into a cell (e.g., human). DNA-based vectors include the presence of elements for transcription (e.g., promoters and polyA signals) and translation (e.g., Kozak sequences).

[0494] In one embodiment, within the vector, the at least one nucleic acid and the regulatory element are "operably linked" to each other, which generally means that they are in a functional relationship. For example, a promoter is considered "operably linked" to a coding sequence (where the coding sequence should be understood as being "under the control" of the promoter) if it is capable of initiating or otherwise controlling / regulating transcription and / or expression of the coding sequence. Typically, when two nucleotide sequences are operably linked, the two nucleotide sequences will be in the same orientation and usually in the same reading frame. The two nucleotide sequences are also usually substantially contiguous, but this may not be necessary.

[0495] In one embodiment, any regulatory elements of the vector enable it to provide its intended biological function in the intended host cell or host organism.

[0496] For example, a promoter, enhancer, or terminator should be "operable" in the intended host cell or host organism, meaning, for example, that the promoter should be able to initiate or otherwise control / regulate the transcription and / or expression of a nucleotide sequence (e.g., a coding sequence) to which it is operably linked.

[0497] The host cell of this technology

[0498] The nucleic acids and / or gene constructs of this technology (the nucleic acids of this technology) can be used to transform host cells or host organisms, i.e., to express and / or produce the polypeptides and / or fusion proteins of this technology. The preferred host is a non-human host. A suitable host or host cell will be clear to a technician and may be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, prokaryotic or eukaryotic organism, such as: bacterial strains, including but not limited to: Gram-negative strains, such as *Escherichia coli* strains; *Proteus* strains, such as *Proteus mirabilis* strains; *Pseudomonas* strains, such as *Pseudomonas fluorescens* strains; and Gram-positive strains, such as *Bacillus* strains, such as *Bacillus subtilis* strains or *Bacillus brevis* strains; *Streptomyces* strains, such as *Streptomyces lividans* strains; *Staphylococcus* strains, such as *Staphylococcus carinatum* strains; and *Lactococcus* strains, such as *Lactococcus lactis* strains; fungal cells, including but not limited to: cells from *Trichoderma* species, such as cells from *Trichoderma reesei*; and cells from *Neurospora*, such as cells from *Neurospora crassa*. Cells of *Crassula crassa*; cells of scabies, such as those from *Sordaria macrospora*; cells of *Aspergillus*, such as those from *Aspergillus niger* or *Aspergillus sojae*; or cells from other filamentous fungi; yeast cells, including but not limited to cells from yeast species, such as *Saccharomyces cerevisiae*; cells of fissile yeasts, such as those from *Schizosaccharomyces pombe*; cells of *Pichia pastoris*, such as those from *Pichia methanolica*; cells of *Hansenula polymorpha*, such as those from *Hansenula polymorpha*; cells of *Kluyveromyces lactis*, such as those from *Kluyveromyces lactis*; cells of *Arxella*, such as those from *Arxella adenine-degrading*. Cells of *Adeninivorans*; cells of *Yarrowia*, such as cells of *Yarrowia lipolytica*; amphibian cells or cell lines, such as oocytes of *Xenopus*.Insect-derived cells or cell lines, such as those derived from Lepidoptera, including but not limited to Spodoptera SF9 and Sf21 cells or Drosophila-derived cells, such as Schneider and Kc cells; plant or plant cells, such as tobacco plants; and / or mammalian cells or cell lines, such as those derived from humans, mammalian cells or cell lines, including but not limited to CHO cells, BHK cells (e.g., BHK-21 cells), and human cells or cell lines, such as HeLa, COS (e.g., COS-7), and PER.C6 cells; and all other host or host cells known per se for expressing and producing antibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv fragments), which will be clear to a person skilled in the art. Also refer to the general background techniques cited above, and to references such as WO 94 / 29457; WO 96 / 34103; WO 99 / 42077; Frenken et al. 1998 (Res. Immunol. [Immunological Research] 149: 589-99); Riechmann and Muyldermans 1999 (J. Immunol. Met. [Journal of Immunology and Metabolism] 231: 25-38); van der Linden 2000 (J. Biotechnol. [Journal of Biotechnology] 80: 261-70); Joosten et al. 2003 (Microb. Cell Fact. [Microbial Cell Factory] 2: 1); Joosten et al. 2005 (Appl. Microbiol. Biotechnol. [Applied Microbiology and Biotechnology] 66: 384-92); and other references cited herein.

[0499] To express peptides and / or fusion proteins in cells, they can also be expressed as so-called “intrabody”, as described, for example, in WO 94 / 02610, WO 95 / 22618 and US 7004940; WO 03 / 014960; Cattaneo and Biocca 1997 (Intracellular Antibodies: Development and Applications, Landes and Springer-Verlag); and Kontermann 2004 (Methods 34: 163-170).

[0500] According to a preferred but non-limiting embodiment of the present technology, the polypeptides and / or fusion proteins of the present technology are produced in bacterial cells (particularly bacterial cells suitable for large-scale drug production, such as cells of the strains described above).

[0501] According to another preferred but non-limiting embodiment of the present technology, the polypeptides and / or fusion proteins of the present technology are produced in yeast cells (especially yeast cells suitable for large-scale drug production, such as cells of the species described above).

[0502] According to another preferred but non-limiting embodiment of the present technology, the polypeptides and / or fusion proteins of the present technology are produced in mammalian cells (particularly in human cells or human cell lines, and more particularly in human cells or human cell lines suitable for large-scale drug production, such as the cell lines mentioned above).

[0503] The appropriate techniques for transforming the host or host cells used in this technology will be clear to those skilled in the art and may depend on the intended host cell / host organism and the gene construct to be used. See again the aforementioned manual and patent application.

[0504] Following transformation, steps can be performed to detect and select those host cells or host organisms that have been successfully transformed using the nucleotide sequence / gene construct of this technology. This could be, for example, a selection step based on selectable markers present in the gene construct of this technology, or a step involving, for example, detecting peptides of this technology using specific antibodies.

[0505] Transformed host cells (which may be in the form of stable cell lines) or host organisms (which may be in the form of stable mutant systems or strains) form another aspect of this technology.

[0506] Preferably, these host cells or host organisms are such that they express or (at least) are able to express (e.g., under suitable conditions) the polypeptides and / or fusion proteins of this technology (and in the case of a host organism: expressed in at least one of its cells, parts, tissues, or organs). This technology also includes, for example, further generations, progeny, and / or offspring of the host cells or host organisms of this technology obtained through cell division or through sexual or asexual reproduction.

[0507] Therefore, in another aspect, this technology relates to expressing (or being able to express, where appropriate) the polypeptides and / or fusion proteins of this technology; and / or a host or host cell containing nucleic acids encoding such polypeptides and / or fusion proteins. Some preferred but non-limiting examples of such hosts or host cells may be generally as described in WO 04 / 041867, WO 04 / 041865, or WO 09 / 068627. For example, the polypeptides and / or fusion proteins of this technology may advantageously be expressed, produced, or manufactured in mammalian cells such as Chinese hamster ovary cells (CHO cells) or in suitable yeast strains such as Pichia pastoris. Reference is also made to WO 04 / 25591, WO 10 / 125187, WO 11 / 003622, and WO 12 / 056000, which also describe the expression / production of immunoglobulin monovariable domains and polypeptides containing such immunoglobulin monovariable domains in Pichia pastoris and other host / host cells.

[0508] To generate / obtain the expression of the polypeptides and / or fusion proteins of this technology, the transformed host cells or transformed host organisms are typically maintained, sustained, and / or cultured under conditions that enable the expression / production of the (desired) polypeptides and / or fusion proteins of this technology. Appropriate conditions will be clear to those skilled in the art and generally depend on the host cells / host organisms used and the regulatory elements controlling the expression of the (relevant) nucleotide sequences of this technology. Again, refer to the manuals and patent applications mentioned above in the paragraph concerning the gene constructs of this technology.

[0509] Typically, suitable conditions may include the use of a suitable culture medium, the presence of a suitable food source and / or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducing factor or compound (e.g., when the nucleotide sequence of this technology is under the control of an inducible promoter); all of these can be selected by a person skilled in the art. Similarly, under such conditions, the polypeptides and / or fusion proteins of this technology may be expressed constitutively, transiently, or only upon appropriate induction.

[0510] Those skilled in the art will also understand that the peptides and / or fusion proteins of this technology can (firstly) be produced in an immature form (as described above), and then undergo post-translational modifications, depending on the host cell / host organism used. Furthermore, the peptides and / or fusion proteins of this technology can be glycosylated, again depending on the host cell / host organism used.

[0511] The polypeptides and / or fusion proteins of this technology can then be isolated from the host cells / host organism and / or from the culture medium in which the host cells or host organism are cultured, using protein isolation and / or purification techniques known per se, such as (preparative) chromatography and / or electrophoresis, differential precipitation, affinity techniques (e.g., using a specific cleavable amino acid sequence fused with the polypeptide or construct of this technology) and / or preparative immunological techniques (i.e., using antibodies against the amino acid sequence to be isolated).

[0512] For example, a polypeptide or protein is considered "substantially separated" when it is separated from at least one other component (e.g., another protein / peptide, another biological component or macromolecule, or at least one contaminant, impurity, or minor component) that is normally associated with it in the source or culture medium, compared to the natural biological source of the polypeptide or protein and / or the reaction or culture medium in which it is obtained. In particular, a polypeptide or protein 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 more. A polypeptide or protein "substantially separated" is preferably substantially homogeneous, as determined using suitable techniques (e.g., suitable chromatographic techniques, such as polyacrylamide gel electrophoresis).

[0513] Compositions, vaccines, and treatments and / or preventive methods

[0514] This technology also relates to compositions comprising peptides and / or fusion proteins incorporating this technology. Such compositions may be pharmaceutical compositions. The compositions may further comprise at least one pharmaceutically acceptable carrier, diluent, or excipient and / or adjuvant, and optionally comprise one or more additional pharmaceutically active peptides and / or compounds.

[0515] In the methods described above, the peptides and / or fusion proteins can be administered in any suitable manner, depending on the specific pharmaceutical formulation or composition to be used. Therefore, the peptides and / or fusion proteins and / or compositions comprising them of this technology can be administered, for example, orally, intraperitoneally, intravenously, subcutaneously, intramuscularly, or via any other route of administration bypassing the gastrointestinal tract, intranasally, transdermally, topically, via suppositories, or by inhalation, again depending on the specific pharmaceutical formulation or composition to be used. They can also be administered via mucosal delivery, such as oral delivery or intranasal administration. Clinicians will be able to select the appropriate route of administration and the appropriate pharmaceutical formulation or composition to be used for such administration based on the disease or disorder to be prevented or treated and other factors known to the clinician.

[0516] An effective amount of peptides and / or fusion proteins, or compositions containing them, can be administered to a subject to provide the desired therapeutic outcome.

[0517] As used herein, the term "therapeutic agent" means any agent that can be used for the prevention, treatment, and / or management of a disease or disorder or one or more symptoms thereof. In some embodiments, the term "therapeutic agent" refers to peptides and / or fusion proteins of the present technology and / or compositions comprising them. Preferably, a therapeutic agent is an agent known to be used, or already used, or currently used for the treatment, prevention, and / or management of a disease or disorder or one or more symptoms thereof.

[0518] As used herein, in the context of this technology, "therapeutic effective amount" refers to a single treatment or a combination of treatments that provides therapeutic benefit in the treatment and / or management of a disease and / or disorder. In one aspect, therapeutic effective amount means a treatment amount sufficient to cure, alter, stabilize, or control a disease and / or disorder or one or more symptoms thereof. In another aspect, therapeutic effective amount means a treatment amount sufficient to alleviate the symptoms of a disease and / or disorder. In yet another aspect, therapeutic effective amount means a treatment amount sufficient to delay or minimize the spread of a disease and / or disorder.

[0519] As used herein, the term "therapeutic" means any treatment regimen, method, and / or agent that can be used to treat, prevent, and / or manage a disease and / or disorder or its symptoms. In some embodiments, the terms "therapies" and "therapy" refer to biological, supportive, and / or other therapies that can be used to treat, prevent, and / or manage a disease and / or disorder or one or more symptoms of it that are known to those skilled in the art (such as medical personnel).

[0520] As used herein, in the context of administering one or more therapies to a subject, the terms “treat,” “treatment,” and “treating” refer to a reduction or improvement in the progression, severity, and / or duration of a disease or disorder, and / or an improvement in one or more symptoms resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more preventative or therapeutic agents). In the context of this technology, the terms “treat,” “treatment,” and “treating” may refer to therapeutic and / or prophylactic / preventive treatment. The term “preventive treatment” refers to a therapy that reduces susceptibility to a clinical condition. Therefore, the terms “treat,” “treatment,” and “treating,” and their equivalents, refer to achieving the desired pharmacological or physiological effect, encompassing any treatment of a pathological condition, disease, or disorder in mammals, including humans. The effect may be preventive in relation to the complete or partial prevention of a pathological condition, disease, or disorder or its symptoms, and / or therapeutic in relation to the partial or complete cure of a pathological condition, disease, or disorder and / or adverse reactions attributable to such pathological condition, disease, or disorder. That is, “treatment” includes (1) preventing the occurrence or recurrence of a pathological condition, disease, or disorder in a subject, (2) suppressing a pathological condition, disease, or disorder, such as preventing its development, (3) stopping or terminating a pathological condition, disease, or disorder or at least stopping or terminating the symptoms associated with it, so that the host is no longer troubled by such pathological condition, disease, or disorder or its symptoms, such as causing the pathological condition, disease, or disorder or its symptoms to subside, or (4) alleviating, reducing, or improving a pathological condition, disease, or disorder or its associated symptoms, wherein improvement is broadly used to refer to a reduction in the order of magnitude of at least one parameter (e.g., inflammation, pain, or immunodeficiency).

[0521] The polypeptides or fusion proteins of this technology and / or compositions comprising them shall be administered according to a treatment regimen suitable for the prevention and / or treatment of the disease and / or disorder to be prevented or treated. Clinicians will generally be able to determine the appropriate treatment regimen based on factors such as: the stage of the disease and / or disorder to be treated, the severity of the disease and / or disorder to be treated and / or the severity of its symptoms, the specific polypeptide, fusion protein, or composition of this technology to be used, the specific route of administration and the pharmaceutical formulation or composition to be used, the patient's age, sex, weight, diet, general condition, and similar factors known to the clinician.

[0522] Typically, the treatment regimen will involve administering one or more peptides or fusion proteins of this technology, or one or more compositions comprising them, in one or more pharmaceutically effective amounts or doses. The specific amount or dose to be administered can be determined by the clinician based on the factors described above.

[0523] Typically, the methods described above utilize a single polypeptide or fusion protein of this technology. However, the combination of two or more polypeptides and / or fusion proteins of this technology is also within the scope of this technology.

[0524] The peptides or fusion proteins of this technology can also be used in combination with one or more other pharmaceutically active compounds or principal components, i.e., as a combination therapy, which may or may not result in a synergistic effect. Similarly, based on the above factors and expert judgment, clinicians will be able to select such additional compounds or principal components, as well as appropriate combination therapy regimens.

[0525] In particular, the peptides or fusion proteins of this technology can be used in combination with other pharmaceutically active compounds or principal components used for or potentially used to prevent and / or treat the diseases and / or disorders cited herein, with or without the possibility of a synergistic effect. Examples of such compounds and principal components, as well as the routes, methods, and pharmaceutical formulations or compositions for their administration, will be clear to clinicians.

[0526] When two or more substances or principal components are used as part of a combination therapy, they may be administered substantially at the same time or at different times via the same route of administration or via different routes of administration (e.g., substantially simultaneously, consecutively, or according to an alternating regimen). When substances or principal components are to be administered simultaneously via the same route of administration, they may be administered as different pharmaceutical formulations or compositions, or as part of a combination of pharmaceutical formulations or compositions, as will be clear to a person skilled in the art.

[0527] In one aspect, this disclosure provides methods for administering polypeptides, fusion proteins, and / or compositions comprising the present technology. In some embodiments, the polypeptides, fusion proteins, and compositions comprising the present technology are administered as pharmaceutical compositions. In addition to the polypeptides, fusion proteins, and compositions comprising the present technology, the pharmaceutical composition may also include a pharmaceutically acceptable carrier.

[0528] Because the polypeptides, fusion proteins, and compositions comprising them of this technology have extended half-lives, they are preferably administered into circulation. Therefore, they can be administered in any suitable manner that allows the compounds or polypeptides of this technology to enter circulation, such as intravenous, intramuscular, intradermal, intranasal, and pulmonary administration. Similarly, suitable methods and routes of administration will be apparent to those skilled in the art, for example, through the teachings of published patent applications of Ablynx NV, such as WO 04 / 041862, WO2006 / 122786, WO 2008 / 020079, WO 2008 / 142164, or WO 2009 / 068627.

[0529] The phrase “pharmaceutically acceptable” as used herein refers to compounds, materials, compositions, and / or dosage forms that, to the extent of reasonable medical judgment, are suitable for use in contact with tissues of humans and animals without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, and have a proportionate and reasonable benefit / risk ratio.

[0530] As used herein, the phrase “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, which participates in the transport or translocation of the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense that it is compatible with other components of the formulation and is harmless to the patient.

[0531] Methods for preparing these formulations or compositions include the steps of associating an immunoglobulin monovariable domain or polypeptide construct with a carrier and optionally one or more auxiliary components. Generally, formulations are prepared by uniformly and tightly associating an immunoglobulin monovariable domain or polypeptide construct with a liquid carrier or a finely fragmented solid carrier, or both, and then shaping the product if necessary.

[0532] This technology further provides peptides and / or fusion proteins and / or pharmaceutical compositions of the present technology as vaccines or immunogenic compositions. Therefore, this technology further provides a vaccine comprising peptides, fusion proteins and / or pharmaceutical compositions of the present technology. As described above, the vaccine can be administered to subjects in need. For example, the vaccine can be administered via mucosal application, such as orally or intranasally.

[0533] As used herein, the term "vaccine" refers to a substance or composition that establishes or improves immunity against a specific disease by inducing an adaptive immune response, including immune memory; that is, a substance or composition that, when administered to a subject in an effective amount, stimulates the production of protective antibodies or a protective T-cell response. Vaccines can be prophylactic or therapeutic. In one aspect, the vaccine of this technology is a prophylactic vaccine.

[0534] Vaccines based on this technology may contain adjuvants. As used herein, the term "adjuvant" refers to a substance that, when added to an immunogenic agent, nonspecifically enhances or strengthens the recipient host's immune response to the agent upon exposure to the mixture.

[0535] The accompanying drawings, sequence listings, and experimental sections / examples are for further illustration of the technology only and should not be construed or interpreted as limiting the scope of the technology and / or the appended claims in any way, unless otherwise expressly indicated herein.

[0536] The embodiments shown and discussed in this specification are intended only to teach the best known methods of making and using the technology to those skilled in the art. As will be understood by those skilled in the art based on the foregoing teachings, modifications and variations may be made to the above embodiments of the technology without departing from the technology. Therefore, it should be understood that the technology may be practiced in ways other than those specifically described within the scope of the claims and their equivalents.

[0537] The present technology will now be further described with reference to the following non-limiting preferred aspects, examples and figures.

[0538] All references cited throughout this application (including bibliographic references, granted patents, published patent applications and co-pending patent applications), especially the teachings cited above, are hereby expressly incorporated herein by reference.

[0539] Other sequences

[0540] Table A. Sequence of CDRs and frames based on AbM numbers (“ID” refers to a given SEQ ID NO)

[0541]

[0542] Example

[0543] Example 1: Generation, protein expression, and purification of Nanobody® ISVD expression constructs (fusion proteins)

[0544] Multivalent Nanobody® (ISVD) proteins (peptides, fusion proteins, or constructs containing ISVD, as defined herein) were expressed in *Pichia pastoris*. The yeast expression vector contained encoding information for the AOX1 promoter and terminator, the bleomycin (Zeocin) resistance gene, and the *Saccharomyces cerevisiae* α-mating factor signal peptide. ISVDs were bound to GS adapters (SEQ ID NO: 45, 49, or 52) and cloned into expression vectors using the Golden Gate cloning technique (Engler C, Marillonnet S. Golden Gate cloning. [Golden Gate Cloning] Methods Mol Biol. [Molecular Biology Methods] 2014; 1116:119-31). The expression vectors contained two BpiI restriction sites, a monovalent ISVD for cloning PCR amplification, and a GS adapter contained in one or more vectors. All these elements were flanked by BpiI sites. Unique nucleotide overhangs at each position in the cloning cassette allowed for seamless ligation in a predefined order. Following Sanger sequence confirmation, plasmid DNA derived from *E. coli* TOP10 was linearized and transformed via electroporation into an internally prepared highly active *Pichia pastoris* strain NRRL Y-11430 (ATCC 76273). Multivalent constructs containing at least one ALB23002 ISVD (SEQ ID NO: 4) were purified on Amsphere A3 (JSR) or MabCapture A (Poros) resin, followed by a desalting step (using a PD column from GE Healthcare with Sephadex G25 resin). Concentrations were determined by OD280 / OD340 measurements. Quality control was performed by SDS-PAGE and mass spectrometry analysis.

[0545] Example 2: Albumin binding study of Nanobody® ISVD expression construct (fusion protein)

[0546] A set of ISVD expression constructs were generated, typically consisting of: (i) one or two albumin-binding ISVDs (ALB23002, SEQ ID NO: 4); and (ii) two or three target ISVDs (X, Y, Z) to represent potential future therapeutic clues; or three control ISVD constructs (“CNBs”, also known as “IRRs”) that do not bind to serum albumin or any other envisioned target, but are simply included in the peptide construct to produce a similar size. See also Figure 1 Different ISVDs were fused with GS adapters (as detailed herein, see SEQ ID NO: 45, 49 and 52) and C-terminal alanine was added.

[0547] We first evaluated the feasibility of simultaneous HSA binding in SPR at pH 7.4 and whether this was affected by the linker length between the two albumin-binding constructs. For this evaluation, target X was immobilized on a CM5 (Cytiva, S series, BR100399) sensor via amine coupling using a Biacore 8k+ device. These formats were captured on this target via the first construct of the construct, followed by injection of HSA at progressively increasing concentrations over 120 seconds, ranging from 10 nM to 30 µM, in MCK assays. As a control, formats with only one albumin-binding construct were included. The binding levels of HSA on formats containing two ALB constructs were significantly higher than the control. We estimated the amount of HSA molecules bound to constructs containing one or more ISVDs (either peptides or constructs containing ISVDs) based on the capture-to-binding ratio and the molecular weight of both the construct containing the ISVD and the HSA. In determining the theoretical number of HSA molecules bound, the number of bound HSA molecules increased with increasing HSA concentration until saturation was reached. Compared to a maximum of 0.93 for a construct containing ISVD with a single albumin-binding building block, we obtained a theoretical number of HSA molecules in the range of 1.45 to 1.69 at saturation (reaching 30 µM HSA) if two albumin-binding building blocks were present. This indicates that simultaneous HSA binding is possible if two albumin-binding building blocks are present, and this applies to all tested linker lengths (9GS, 20GS, or 35GS, SEQ ID NO: 45, 49, or 52, respectively) (see Table 7).

[0548] Table 7. SPR constructs containing ISVD (fusion proteins) simultaneously with HSA binding

[0549]

[0550] Constructs containing 9GS and 35GS linkers between albumin-binding building blocks were selected for further characterization. The affinity of the purified ISVD expression constructs for human and mouse serum albumin (HSA and MSA, respectively) at pH 6.0 and pH 7.4 was determined using a Biacore 8K+ instrument. HSA or MSA (HSA: Sigma-Aldrich-Sigma, catalog number A8763; MSA: Albumin Bioscience, catalog number 2601) was immobilized on the S-series sensor chip C1. The fusion proteins described in Table 7 above were injected at nine different concentrations (between 0.6 and 2000 nM) and associated at a rate of 30 µL / min for 120 seconds, followed by dissociation for 600 seconds. Sensor plot evaluation was based on fitting of the bivalent analytes. The affinity of HSA and MSA is shown in Table 8.

[0551] Table 8. HSA and MSA binding of Nanobody® ISVD constructs (fusion proteins) at pH 6.0 and pH 7.4

[0552]

[0553] Italics and underlines: Indicative value

[0554] Example 3: Development and Optimization of Serum PK Assay

[0555] A specific and sensitive ligand binding assay was developed to measure the concentrations of IRR0064, F027301978, T026301360, and T026301365 in mouse serum. The assay was performed at room temperature using Superblock T20. TMMSD GOLD 96-well SMALLSPOT® plates (Meso Scale Discovery) coated with streptavidin were blocked for 30 minutes. The plates were then washed and incubated at 600 rpm for 1 hour at room temperature with a 1.0 µg / mL biotinylated universal mAb for the different ISVD builders. Calibrators and QCs were prepared in pooled mouse serum. After washing the plates, the calibrators, QCs, and samples were applied to the plates in 0.1% casein PBS at an MRD of 20 and incubated at 600 rpm for 1 hour at room temperature. After washing, the plates were incubated at 600 rpm for 1 hour at room temperature with a 2.0 µg / mL sulfonic acid-labeled mAb for the specific ISVD builder, depending on the construct being evaluated. After washing the plate, 2x MSD read buffer (MSD) was added, and the plate was read on a Quickplex SQ 120 fan-shaped imager (MSD).

[0556] The concentrations of F027301978 and T026301360 in cynomolgus monkeys were measured using the same settings, but with the following changes: calibrators and QC were prepared in the combined cynomolgus monkey serum, and the MRD was applied to 100.

[0557] Example 4: Contains one or two serum albumin-binding ISVDs (ALB23002) and additional target-binding ISVD building blocks (X, Y). Pharmacokinetics of fusion protein in mice

[0558] Pharmacokinetic studies were performed in TG32 (B6.Cg-Fcgrttm1Dcr-Tg(FCGRT) 32Dcr / Dcr) mice to evaluate the half-life of a fusion protein (a multivalent construct containing ISVDs) consisting of two target ISVD building blocks (X, Y) fused to a single albumin-binding ISVD Alb23002 SEQ ID NO: 4 (trivalent construct) or 2Alb23002 ISVD SEQ ID NO: 4 (tetravalent construct). A construct containing three non-target (control) (CNB) ISVDs fused to ALB23002 SEQ ID NO: 4 was designated as the tetravalent control construct (IRR00164). All constructs contained a C-terminal A, see Table 9.

[0559] To simulate relevant competition with hIgG, Tg32 mice were pre-loaded with a mixture of purified hIgG (hIVIG; Privigen®). Privigen® was administered intravenously once weekly, with the first administration two days prior to the start of the PK study. A total of three 250 mg / kg injections of Privigen® were administered to generate physiologically relevant serum hIgG concentrations (data not shown) during the study period. Two days after the first Privigen® administration, four to six Tg32 mice per group were intravenously injected via the tail in equimolar amounts (3.5 mg / kg for the tetravalent construct or 2.7 mg / kg for the trivalent construct) (the constructs and building blocks used in these constructs are described in Example 1 above, and see also Table 9 below).

[0560] Blood was collected from two mice at different time points, and serum was prepared. As described in Example 3, the presence of multivalent ISVD constructs in the serum samples was analyzed by ELISA. All in vivo studies were conducted in accordance with Sanofi's institutional animal care policy.

[0561] The results are shown in Figure 2 The half-lives were obtained by estimating the in vivo endosome FcRn affinity in the mechanistic PBPK model, and these half-lives are reported in Table 9.

[0562] In ISVD-containing multivalent constructs containing ISVD domains targeting therapeutic targets (X, Y), the combination of two albumin-binding ISVDs significantly increased the half-life compared to control constructs containing only a single albumin-binding ISVD. The linker length between the two albumin-binding ISVDs had no significant effect on the pharmacokinetic properties (half-life) of the constructs. Furthermore, the half-life was not affected by the presence of hIgG-related levels. For constructs with two albumin-binding ISVDs, the half-life was increased by 1.8 to 2.8 times compared to trivalent and tetravalent control constructs containing only a single albumin-binding ISVD.

[0563] Table 9: Evaluation of the format and calculated half-life of the fusion protein (multivalent ISVD construct) in Tg32 mice.

[0564]

[0565] Example 5: Fusion protein containing one or two serum albumin-binding ISVDs (ALB23002) and additional target-binding ISVD building blocks (X, Y). Pharmacokinetics in cynomolgus monkeys

[0566] Pharmacokinetic studies were conducted on cynomolgus macaques using two of the aforementioned multivalent constructs containing ISVD: F027301987 (a trivalent construct with a single albumin-binding ISVD, Alb223, SEQ ID NO: 18, XY-Alb223) and T026301360 (a tetravalent construct containing two albumin-binding ISVDs, Alb23002, SEQ ID NO: 4 and Alb223, SEQ ID NO: 18, XY-Alb23002-Alb223). Four healthy male cynomolgus macaques (two animals per compound) were administered a single dose of one of the test constructs via intravenous injection at a dose of 10 mg / kg, followed by a two-month observation period. Blood samples were taken at multiple time points (two animals at each time point) for PK analysis, and serum was prepared. The presence of multivalent constructs in the serum samples was analyzed by ELISA as described in Example 3.

[0567] PK parameters were obtained via non-compartmental analysis using the plasma data module in Phoenix WinNonlin® (version 8.2.2.227, Certara). The PK distribution curves for animal 8 (group F027301987) showed a sharp decrease in concentration, typical of antidrug antibody (ADA) interference. ADA analysis confirmed this, and the PK distribution curves were excluded from PK calculations. Comparisons of PK parameters (relative differences compared to F027301978) are reported in Table 10.

[0568] In cynomolgus monkeys, the construct with a double albumin-binding ISVD (T026301360) showed reduced clearance and improved serum half-life (T027301978) compared to the construct with a single albumin-binding ISVD (F027301978). 1 / 2 For T026301360, the relative difference with F027301978 was calculated using the average of two animals (n = 1).

[0569] Table 10: Comparison of PK parameters of multivalent constructs (relative differences compared to F027301978)

[0570]

[0571] Example 6: Generation of multivalent albumin binding expression constructs Protein expression and purification

[0572] A set of constructs containing ISVDs was generated, which typically consist of: (i) one to four albumin-binding ISVDs (ALB23002, SEQ ID NO: 4; HSA006A06, SEQ ID NO: 91; ALBX00002, SEQ ID NO: 54 or T023500029, SEQ ID NO: 20) and (ii) having or not having another albumin-binding construct (DARPIN, SEQ ID NO: 88 or ABD, SEQ ID NO: 90). The different albumin-binding constructs were fused with a 35 GS-linker (as detailed herein, see SEQ ID NO: 52), and if the C-terminal construct was an ISVD, a C-terminal alanine residue was added.

[0573] The multivalent albumin-binding construct (fusion protein or construct) was expressed in CHO and purified from the cell supernatant using a protein A capture step followed by size exclusion chromatography.

[0574] Example 7: Multivalent albumin binding expression construct albumin binding study

[0575] In the first experiment, the affinity of monovalent (ALB00223) and divalent ALB23002 constructs (T032200003) for human and mouse serum albumin (HSA and MSA, respectively) at pH 6.0 and pH 7.4 was determined using a ProteOn XPR36 instrument (Bio-rad Laboratories, Inc.). HSA or MSA (HSA: Sigma-Aldrich-Sigma, catalog number A8763; MSA: Albamin Bioscience, catalog number 2601) was immobilized on a GLC sensor (short matrix, normal capacity) via amine coupling. Constructs were injected at six different concentrations (between 0.95 and 500 nM) and allowed to associate at a rate of 45 µL / min for 120 seconds, followed by dissociation for 600 seconds. The sensor map evaluation was based on a kinetic Langmuir 1:1 (simultaneous ka / kd) fit. The affinity between HSA and MSA is shown in Table 11.

[0576] for Figure 3 Different multivalent albumin-binding constructs shown were evaluated for simultaneous HSA binding at pH 7.4 using SPR.

[0577] Constructs with a C-terminal alanine residue were captured on a chip using an anti-ISVD antibody (CM5 (Stopfan, S series, BR100399)) captured by its Fc chain. Next, in an MCK assay, HSA concentrations were injected at increases from 10 nM to 100 µM over 120 seconds. As a control, a monovalent ALB23002-derived construct (Alb223, SEQ ID NO: 18) was used. The HSA binding levels on constructs containing multiple albumin-binding building blocks were significantly higher than the control. We estimated the amount of HSA molecules bound to the ISVD-containing construct based on the ratio of capture level to binding level and the molecular weights of both the construct and the HSA. In determining the theoretical number of HSA molecules bound, this number increased with increasing HSA concentration until saturation was reached, and all constructs with more than one albumin-binding building block also bound more than one HSA molecule. For constructs containing ALB23002 (or Alb223, which corresponds to ALB23002 with an additional C-terminal A), HSA006A06 (which are variants of the same parent ISVD), or DARPIN, we obtained HSA molecules ranging from 1.6 to 1.7 for bivalent constructs, from 2.4 to 2.5 for trivalent constructs, and 3.2 for tetravalent constructs at the highest HSA concentration (100 µM). These results indicate that simultaneous HSA binding is possible on all albumin-binding constructs contained in the constructs (see Table 12). For bivalent constructs containing ALBX00002, T023500029, or ABD, we calculated that 1.1 HSA molecules bound per construct at the highest HSA concentration (100 µM), which is higher than the 0.8 HSA molecules obtained for ALB23002-A.

[0578] Table 11. Affinity, association rate, and dissociation rate of constructs containing monoalbumin-binding ISVD or dialbumin-binding ISVD to human and mouse serum albumin at pH 6.0 and pH 7.4.

[0579]

[0580] Italics and underlines: Indicative value

[0581]

[0582] The affinity of the purified multivalent albumin-binding construct for HSA at pH 7.4 was determined using a Biacore 8K+ instrument. HSA (Sigma-Aldrich-Sigma, catalog number A8763) was immobilized on the S-series sensor chip C1. Nine different concentrations (between 1.6 and 2500 nM) of the multivalent albumin-binding protein described in Table 12 above were injected, and the proteins were associated at a rate of 30 µL / min for 120 seconds and dissociated for 600 seconds. Sensor plot evaluation was based on fitting of the divalent analyte, and the affinity for HSA is shown in Table 13.

[0583] Table 13. HSA affinity of multivalent albumin binding constructs at pH 7.4

[0584]

[0585] Italics and underlines: Indicative value

[0586] The purified multivalent albumin-binding constructs were also tested in SPR on HSA of varying densities (100 RU to 6700 RU) to determine affinity. Dissociation rates were determined at pH 7.4 and are shown in Table 14. These results indicate that for constructs containing two or more albumin-binding domains, dissociation rates decrease with increasing HSA density, except for TPP-66097 and TPP-66100. Simultaneous HSA binding experiments in Table 13 demonstrate that these constructs can bind more than one HSA molecule at 100 µM HSA.

[0587] Table 14. Dissociation rates of the constructs determined as the density of HSA increases.

[0588]

[0589] ND: Undetermined

[0590] Example 8: Development and Optimization of Mouse PK Assay

[0591] A bottom-up LC-MS2 assay was developed to measure the concentration of constructs containing multiple serum albumin-binding domains in mouse plasma. An internally manufactured fusion Nanobody® construct was used as an internal standard.

[0592] In short, the samples were diluted, reduced, urea-methylated, and the proteins precipitated. The obtained microparticles were digested with trypsin, and the resulting alternative peptides were analyzed in an Agilent 1290 Infinity II UHPLC system connected to a Sciex 6500+ mass spectrometer. For separation, an Aquity UPLC column (Peptide CSH C18 130Å 1.7 µm 50 x 2.1 mm, Waters) was washed at 50°C with stepwise gradients of water / DMSO / formic acid (100 / 1 / 0.5; v / v / v) and acetonitrile / DMSO / formic acid (100 / 1 / 0.5; v / v / v). The mass spectrometer was operated in forward mode according to the manufacturer's instructions. One multiplex reaction was monitored for each construct. The peak area was determined using the AutoPeak algorithm (Sciex OS). Concentrations are determined by using the ratio of the area of ​​the analyte to the area of ​​the internal standard in the same sample and comparing the result with a calibration curve obtained using calibration standards.

[0593] For a construct (T032200003) (ALB23002-35GS-ALB23002, SEQ ID NO: 152) consisting of two ALB23002 building blocks separated by a 35 GS connector, a specific and sensitive ligand binding assay was developed to measure concentrations in mouse serum. Briefly, streptavidin-coated MSD GOLD 96-well SMALLSPOT® plates (Messat) were blocked with a Superblock T20™ (Thermo Scientific) for 30 minutes at room temperature. The plates were then washed and incubated at 600 rpm for 1 hour at room temperature with 2.0 µg / mL of a biotinylated universal mAb targeting the ISVD building block framework. Calibrators and QC were prepared in the pooled mouse serum. After washing the plate, calibrators, QC, and samples were applied to the plate at 0.1% casein PBS with an MRD of 40, and incubated at 600 rpm for 1 hour at room temperature. Following washing, the plate was incubated at 600 rpm for 1 hour at room temperature with 4.0 µg / mL sulfonic acid-labeled mAb targeting another epitope of the ISVD construct. After washing the plate, 2xMSD read buffer (Mesade) was added, and the plate was read using a Quickplex SQ 120 fan-shaped imager (Mesade).

[0594] Example 9: Pharmacokinetic evaluation of a construct containing multiple serum albumin-binding domains in BALB / c mice

[0595] In the first pharmacokinetic study, T032200003, composed of two ALB23002 building blocks separated via a 35GS linker, was evaluated. BALB / c mice (n = 3) were injected with 5 mg / kg of T032200003 (189 nmol / kg), and serum samples were obtained at 0.083, 0.5, 1, 2, 6, 24, 48, 96, and 168 h. Serum samples were analyzed by ligand binding assay.

[0596] A second set of pharmacokinetic studies was performed in BALB / c mice to assess the half-life of a group of broadly multivalent constructs involving serum albumin binding. The constructs were administered intravenously at equimolar doses (approximately 189 nmol / kg) into the tail veins of mice (n = 3 / group), as listed in Table 15. Plasma samples (approximately 10 μL) were obtained serially at 0.25, 1, 4, 8, 24, 48, 72, 168, 336, and 504 h.

[0597] As described in Example 3, the presence of multivalent constructs containing ISVD in plasma samples was analyzed by LC-MS2. All in vivo studies were performed in accordance with Sanofi's institutional animal care policy.

[0598] PK parameters for all constructs were obtained via non-compartmental analysis (NCA) using the plasma data module in Phoenix WinNonlin® (version 8.2.2.227. Certara). Where applicable, sampling times at which suspected ADA effects caused sharp drops in compound concentrations were excluded from the analysis. Results are reported in Table 15.

[0599] In multivalent constructs containing two or more albumin-binding domains, the PK parameter was significantly improved (t) compared to the report value of constructs containing only a single albumin-binding domain. 1 / 2 The half-life ranged from 0.5 to 1.4 days, which corresponds to a half-life of approximately 1.5 days (35 h) for albumin in wild-type mice; Chaudhury et al. 2003, J Exp Med. [Journal of Experimental Medicine] 2003 Feb 3; 197(3):315-22). On average, for multivalent albumin-binding constructs, the half-life was increased to 3 days (range 2.4–3.6), which is approximately 2-fold higher than the reporter values ​​for molecules containing a single albumin-binding domain and our internal experience with ISVDs of extended half-life (via albumin binding).

[0600] Table 15: Evaluation of dosage and pK parameters of the multivalent albumin-binding construct in BALB / c mice

[0601]

[0602] Evaluated in a separate study T032200003.

[0603] This technology project

[0604] 1. A polypeptide comprising:

[0605] (i) specifically binds to at least one immunoglobulin single variable domain (ISVD) of serum albumin; and

[0606] (ii) Specifically binds to serum albumin or at least one additional portion of at least one human serum albumin.

[0607] The at least one ISVD consists essentially of four frame regions (FR1 to FR4) and three complementary determinant regions (CDR1 to CDR3), wherein:

[0608] a) CDR1 contains the amino acid sequence of SEQ ID NO: 1 or differs from SEQ ID NO: 1 by 3, 2 or 1 amino acids, or CDR1 contains the amino acid sequence of SEQ ID NO: 22 or differs from SEQ ID NO: 22 by 3, 2 or 1 amino acids;

[0609] b) CDR2 contains the amino acid sequence of SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 3, 2, or 1 amino acid; and

[0610] c) CDR3 contains the amino acid sequence of SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 3, 2, or 1 amino acid.

[0611] and / or

[0612] a) CDR1 contains the amino acid sequence of SEQ ID NO: 36 or differs from SEQ ID NO: 36 by 3, 2 or 1 amino acids;

[0613] b) CDR2 contains the amino acid sequence of SEQ ID NO: 37 or differs from SEQ ID NO: 37 by 3, 2, or 1 amino acid; and

[0614] c) CDR3 contains the amino acid sequence of SEQ ID NO: 38 or differs from SEQ ID NO: 38 by 3, 2, or 1 amino acid.

[0615] and / or

[0616] a) CDR1 contains the amino acid sequence of SEQ ID NO: 55 or differs from SEQ ID NO: 55 by 3, 2 or 1 amino acids;

[0617] b) CDR2 contains the amino acid sequence of SEQ ID NO: 56 or differs from SEQ ID NO: 56 by 3, 2, or 1 amino acid; and

[0618] c) CDR3 contains the amino acid sequence of SEQ ID NO: 57 or differs from SEQ ID NO: 57 by 3, 2 or 1 amino acids.

[0619] These CDR sequences are determined based on AbM numbering.

[0620] 2. The polypeptide of item 1, wherein at least one ISVD specifically binding to serum albumin comprises: CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 22, CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein these CDR sequences are determined according to AbM numbering.

[0621] 3. The polypeptide as described in any one of items 1 or 2, wherein at least one ISVD specifically binding to serum albumin comprises: CDR1 comprising the amino acid sequence of SEQ ID NO: 36, CDR2 comprising the amino acid sequence of SEQ ID NO: 37; and CDR3 comprising the amino acid sequence of SEQ ID NO: 38, wherein these CDR sequences are determined according to an AbM number, or wherein at least one ISVD specifically binding to serum albumin comprises: CDR1 comprising the amino acid sequence of SEQ ID NO: 55, CDR2 comprising the amino acid sequence of SEQ ID NO: 56; and CDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein these CDR sequences are determined according to an AbM number.

[0622] 4. The polypeptide of any one of items 1 to 3, wherein (i) specifically binds to at least one ISVD of serum albumin:

[0623] a) Having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity with any of the sequences as defined in SEQ ID NO: 4 to 21, 54 or 91 to 93, wherein any C-terminal extensions that may exist and these CDRs are not considered in determining the sequence identity; and / or

[0624] b) Having no more than 7, preferably no more than 5, amino acid differences from any of the sequences as defined in SEQ ID NO: 4 to 21, 54 or 91 to 93, for example having only 3, 2 or 1 amino acid differences.

[0625] 5. The polypeptide of any one of items 1 to 4, wherein at least one ISVD that specifically binds to serum albumin has a sequence selected from SEQ ID NO: 4 to 21, 54 or 91 to 93.

[0626] 6. The polypeptide of any one of items 1 to 5, wherein the amino acid sequence of at least one ISVD of serum albumin specifically binds to the polypeptide has more than 90% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 18.

[0627] 7. The polypeptide of any one of items 1 to 6, wherein the (i) specifically binds to at least one ISVD of serum albumin comprising or consisting of the amino acid sequence of Alb-23002 (SEQ ID NO: 4).

[0628] 8. The polypeptide of any one of items 1 to 7, wherein the (i) specifically binds to at least one ISVD of serum albumin at a concentration between 10 -6 M and 10 -11 M or smaller, preferably between 10 -7 M and 10 -12 The dissociation constant (K) between M and smaller D It specifically binds to serum albumin, as determined using Proteon, Kinexa, BLI, or SPR.

[0629] 9. The polypeptide of any one of items 1 to 8, wherein at least one ISVD contained herein is derived from V H Humanized V H , people V H V HH Humanized V HH or camel-derived V H (Derived from heavy chain ISVD), preferably derived from "V" H ISVD of "Class 3"

[0630] 10. The polypeptide of any one of items 1 to 9, wherein at least one ISVD of serum albumin specifically binds to the polypeptide contains one or more mutations compared to any one of the sequences of SEQ ID NO: 4 to 21, 54 or 91 to 93, which reduce the binding of a pre-existing antibody.

[0631] 11. The polypeptide as described in any one of items 1 to 10, wherein the polypeptide comprises:

[0632] (i) at least one immunoglobulin single variable domain (ISVD) that specifically binds to serum albumin as defined in any one of items 1 to 10; and

[0633] (ii) At least one human serum albumin.

[0634] 12. The polypeptide as described in any one of items 1 to 10, wherein the polypeptide comprises:

[0635] (i) at least one immunoglobulin single variable domain (ISVD) that specifically binds to serum albumin as defined in any one of items 1 to 10; and

[0636] (ii) Specifically binds to at least one additional portion of serum albumin.

[0637] Wherein (ii) specifically binds to at least one additional portion of serum albumin selected from antibodies or fragments thereof, single-chain variable fragments (scFv), preferably immunoglobulin single variable domains (ISVD), DARP-in, affinity molecules, afetin, and albumin-binding domains (ABD).

[0638] 13. The polypeptide of any one of items 1 to 10, wherein (ii) specifically binds to at least one additional portion of serum albumin and is an ISVD, preferably wherein the ISVD is derived from V H or V HH Even more preferably, the ISVD is a single-domain antibody (dAb).

[0639] 14. The polypeptide as described in item 13, wherein (ii) specifically binds to at least one additional portion of serum albumin and comprises an ISVD including three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0640] a) CDR1 contains the amino acid sequence of SEQ ID NO: 1 or differs from SEQ ID NO: 1 by 3, 2 or 1 amino acids, or CDR1 contains the amino acid sequence of SEQ ID NO: 22 or differs from SEQ ID NO: 22 by 3, 2 or 1 amino acids;

[0641] b) CDR2 contains the amino acid sequence of SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 3, 2, or 1 amino acid; and

[0642] c) CDR3 contains the amino acid sequence of SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 3, 2, or 1 amino acid.

[0643] and / or

[0644] a) CDR1 contains the amino acid sequence of SEQ ID NO: 36 or differs from SEQ ID NO: 36 by 3, 2 or 1 amino acids;

[0645] b) CDR2 contains the amino acid sequence of SEQ ID NO: 37 or differs from SEQ ID NO: 37 by 3, 2, or 1 amino acid; and

[0646] c) CDR3 contains the amino acid sequence of SEQ ID NO: 38 or differs from SEQ ID NO: 38 by 3, 2, or 1 amino acid.

[0647] and / or

[0648] a) CDR1 contains the amino acid sequence of SEQ ID NO: 55 or differs from SEQ ID NO: 55 by 3, 2 or 1 amino acids;

[0649] b) CDR2 contains the amino acid sequence of SEQ ID NO: 56 or differs from SEQ ID NO: 56 by 3, 2, or 1 amino acid; and

[0650] c) CDR3 contains the amino acid sequence of SEQ ID NO: 57 or differs from SEQ ID NO: 57 by 3, 2, or 1 amino acid.

[0651] These CDR sequences are determined based on AbM numbering.

[0652] 15. The polypeptide of item 14, wherein the (ii) specifically binds to at least one additional portion of serum albumin as an ISVD comprising: a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 22, a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein these CDR sequences are determined according to AbM numbering.

[0653] 16. The polypeptide of any one of items 14 or 15, wherein the (ii) specifically binds to at least one additional portion of serum albumin as an ISVD comprising: a CDR1 comprising the amino acid sequence of SEQ ID NO: 36, a CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 38, wherein the CDR sequences are determined according to an AbM number, or wherein the (ii) specifically binds to at least one additional portion of serum albumin as an ISVD comprising: a CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the CDR sequences are determined according to an AbM number.

[0654] 17. The polypeptide of any one of items 14 to 16, wherein the (ii) specifically binds to at least one additional portion of serum albumin, the ISVD being...

[0655] a) Having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity with any of the sequences as defined in SEQ ID NO: 4 to 21, 54 or 91 to 93, wherein any C-terminal extensions that may exist and these CDRs are not considered in determining the sequence identity; and / or

[0656] b) Having no more than 7, preferably no more than 5, amino acid differences from any of the sequences as defined in SEQ ID NO: 4 to 21, 54 or 91 to 93, for example having only 3, 2 or 1 amino acid differences.

[0657] 18. The polypeptide of any one of items 14 to 17, wherein the (ii) specifically binds to at least one additional portion of serum albumin is an ISVD having a sequence selected from SEQ ID NO: 4 to 21, 54 or 91 to 93.

[0658] 19. The polypeptide of any one of items 1 to 5, wherein the amino acid sequence of which (ii) specifically binds to at least one additional portion of serum albumin is an ISVD having more than 90% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 18.

[0659] 20. The polypeptide of any one of items 14 to 19, wherein the (ii) specifically binds to at least one additional portion of serum albumin is an ISVD comprising or consisting of the amino acid sequence of Alb-23002 (SEQ ID NO: 4).

[0660] 21. The polypeptide of any one of items 14 to 20, wherein the (ii) specifically binds to at least one additional portion of serum albumin, the ISVD being at a concentration between 10 -6 M and 10 -11 The dissociation constant (K) between M and smaller D It specifically binds to serum albumin, as determined using Proteon, Kinexa, BLI, or SPR.

[0661] 22. The polypeptide of any one of items 14 to 21, wherein the (ii) specifically binds to at least one additional portion of serum albumin is an ISVD containing one or more mutations compared to any one of the sequences of SEQ ID NO: 4 to 21, 54 or 91 to 93, which reduce the binding of a pre-existing antibody.

[0662] 23. The polypeptide of any one of items 1 to 22, wherein the (i) specifically binds to at least one ISVD of serum albumin and the (ii) specifically binds to at least one additional portion of serum albumin or at least one human serum albumin and is suitably linked to each other directly or through one or more suitable linkers or spacers.

[0663] 24. The polypeptide of item 23, wherein the (i) specifically binds to at least one ISVD of serum albumin and the (ii) specifically binds to at least one additional portion of serum albumin or at least one human serum albumin are suitably linked together by a connector selected from SEQ ID NO: 41 to 53, preferably selected from SEQ ID NO: 45 and 52.

[0664] 25. A fusion protein comprising a polypeptide as described in any one of items 1 to 24, wherein the fusion protein further comprises one or more (e.g., one or two) other amino acid sequences, (binding) domains, binding units or other portions or chemical entities.

[0665] 26. The fusion protein of item 25, wherein the polypeptide of any one of items 1 to 24 is directly linked to the at least one other amino acid sequence, (binding) domain, binding unit or other portion or chemical entity, or is linked to the at least one other amino acid sequence, (binding) domain, binding unit or other portion or chemical entity via a linker or spacer.

[0666] 27. The fusion protein as described in any one of items 25 to 26, wherein the fusion protein comprises a polypeptide as described in any one of items 1 to 24, and at least one therapeutic and / or targeted portion.

[0667] 28. The fusion protein as described in any one of items 25 to 27, wherein the at least one other amino acid sequence, (binding) domain, binding unit or other portion or chemical entity comprises an immunoglobulin sequence or a fragment thereof, a single-chain variable fragment (scFv), preferably comprising an immunoglobulin single variable domain (ISVD) such as a (single) domain antibody, V HH Humanized V HH or camel-derived V H .

[0668] 29. The fusion protein as described in any one of items 27 to 28, wherein the therapeutic and / or targeting portion comprises at least one ISVD specifically bound to the therapeutic target.

[0669] 30. The fusion protein as described in item 29, wherein the therapeutic and / or targeting portion comprises a single-chain variable fragment (scFv), preferably comprising an immunoglobulin single variable domain (ISVD) such as a (single) domain antibody, V HH Humanized V HH or camel-derived V H .

[0670] 31. The fusion protein as described in any one of items 25 to 30, wherein the fusion protein further comprises an additional ISVD that specifically binds to (human) serum albumin.

[0671] 32. The polypeptide as described in any one of items 1 to 24 or the fusion protein as described in any one of items 25 to 30, wherein the serum half-life of the polypeptide and / or the fusion protein is at least 2 times, for example at least 5 times, preferably at least 10 times or more than 20 times, for example more than 50 times, more than 100 times, more than 500 times, preferably more than 1000 times, than the half-life of the corresponding other part itself.

[0672] 33. The polypeptide as described in any one of items 1 to 24 or the fusion protein as described in any one of items 25 to 30, wherein the serum half-life of the polypeptide and / or fusion protein in humans is at least 5%, for example at least 10%, at least 25%, at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% of the half-life of serum albumin in humans.

[0673] 34. A composition comprising a polypeptide as described in any one of items 1 to 24 and / or a fusion protein as described in any one of items 25 to 30.

[0674] 35. The composition as described in item 34, wherein the composition is a pharmaceutical composition.

[0675] 36. A method for producing a polypeptide as described in any one of items 1 to 24 and / or a fusion protein as described in any one of items 25 to 30, wherein the method comprises the following steps:

[0676] Express the nucleic acid sequence encoding the polypeptide as described in any one of items 1 to 24 and / or the fusion protein as described in any one of items 25 to 30 in a suitable host cell or host organism or in another suitable expression system; optionally thereafter:

[0677] Isolate and / or purify polypeptides as described in any one of Items 1 to 24 and / or fusion proteins as described in any one of Items 25 to 30.

[0678] 37. A nucleic acid or nucleic acid sequence encoding a polypeptide as described in any one of items 1 to 24 and / or a fusion protein as described in any one of items 25 to 30.

[0679] 38. A vector comprising a nucleic acid or nucleic acid sequence as described in item 37.

[0680] 39. A non-human host or host cell comprising a vector containing a nucleic acid sequence as described in item 38 or expressing a nucleic acid sequence as described in item 37.

[0681] 40. The polypeptide as described in any one of items 1 to 24, the fusion protein as described in any one of items 25 to 30, and / or the composition as described in any one of items 34 to 35, for use as a pharmaceutical agent.

[0682] 41. A kit comprising a polypeptide as described in any one of items 1 to 24, a fusion protein as described in any one of items 25 to 30, a nucleic acid or nucleic acid sequence as described in item 37, a vector as described in item 38, or a host cell as described in item 39.

[0683] 42. The polypeptide of any one of items 1 to 24, wherein the polypeptide comprises or is composed of the following: a sequence selected from SEQ ID NO: 74-81, 87 and 147-154, or a sequence comprising or composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with a sequence selected from SEQ ID NO: 74-81, 87 and 147-154.

[0684] 43. The fusion protein as described in any one of items 25 to 30, wherein the fusion protein comprises or is composed of the following: a sequence selected from SEQ ID NO: 82-86, or a sequence comprising or composed of a sequence having at least 90%, for example at least 95%, or at least 97%, or at least 99% sequence identity with a sequence selected from SEQ ID NO: 82-86.

Claims

1. A polypeptide comprising: Specifically binds to at least one immunoglobulin single variable domain (ISVD) of serum albumin; and specifically binds to at least one additional portion of serum albumin or at least one human serum albumin. The at least one ISVD consists essentially of four frame regions (FR1 to FR4) and three complementary determinant regions (CDR1 to CDR3), wherein: CDR1 contains the amino acid sequence of SEQ ID NO: 1 or differs from SEQ ID NO: 1 by 3, 2 or 1 amino acids, or CDR1 contains the amino acid sequence of SEQ ID NO: 22 or differs from SEQ ID NO: 22 by 3, 2 or 1 amino acids; CDR2 contains the amino acid sequence of SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 3, 2, or 1 amino acid; and CDR3 contains the amino acid sequence of SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 3, 2, or 1 amino acid. and / or CDR1 contains the amino acid sequence of SEQ ID NO: 36 or differs from SEQ ID NO: 36 by 3, 2 or 1 amino acids; CDR2 contains the amino acid sequence of SEQ ID NO: 37 or differs from SEQ ID NO: 37 by 3, 2, or 1 amino acid; and CDR3 contains the amino acid sequence of SEQ ID NO: 38 or differs from SEQ ID NO: 38 by 3, 2, or 1 amino acid. and / or CDR1 contains the amino acid sequence of SEQ ID NO: 55 or differs from SEQ ID NO: 55 by 3, 2 or 1 amino acids; CDR2 contains the amino acid sequence of SEQ ID NO: 56 or differs from SEQ ID NO: 56 by 3, 2, or 1 amino acid; and CDR3 contains the amino acid sequence of SEQ ID NO: 57 or differs from SEQ ID NO: 57 by 3, 2 or 1 amino acids. These CDR sequences are determined based on AbM numbering.

2. The polypeptide of claim 1, wherein (i) at least one ISVD specifically binding to serum albumin comprises: CDR1 containing the amino acid sequence of SEQ ID NO: 1 or 22; CDR2 containing the amino acid sequence of SEQ ID NO: 2; and CDR3 containing the amino acid sequence of SEQ ID NO: 3; or CDR1 containing the amino acid sequence of SEQ ID NO: 36, CDR2 containing the amino acid sequence of SEQ ID NO: 37; and CDR3 containing the amino acid sequence of SEQ ID NO: 38; or CDR1 containing the amino acid sequence of SEQ ID NO: 55, CDR2 containing the amino acid sequence of SEQ ID NO: 56, and CDR3 containing the amino acid sequence of SEQ ID NO: 57; These CDR sequences are determined based on AbM numbering.

3. The polypeptide of any one of claims 1 to 2, wherein (i) specifically binds to at least one ISVD of serum albumin. Having at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% sequence identity with any of the sequences as defined in SEQ ID NO: 4 to 21, 54, or 91 to 93, wherein any C-terminal extensions that may exist and these CDRs are not considered in determining the degree of sequence identity; and / or It has no more than 7, preferably no more than 5, amino acid differences from any of the sequences as defined in SEQ ID NO: 4 to 21, 54 or 91 to 93, for example, only 3, 2 or 1 amino acid differences.

4. The polypeptide according to any one of claims 1 to 3, wherein the (i) at least one ISVD contained therein is derived from V H Humanized V H , people V H V HH Humanized V HH or camel-derived V H (Derived from heavy chain ISVD).

5. The polypeptide according to any one of claims 1 to 4, wherein the polypeptide comprises: At least one immunoglobulin single variable domain (ISVD) that specifically binds to serum albumin as defined in any one of claims 1 to 5, and At least one human serum albumin.

6. The polypeptide of any one of claims 1 to 5, wherein the polypeptide comprises: At least one immunoglobulin single variable domain (ISVD) that specifically binds to serum albumin as defined in any one of claims 1 to 5, and It specifically binds to at least one additional portion of serum albumin. Wherein (ii) specifically binds to at least one additional portion of serum albumin selected from antibodies or fragments thereof, single-chain variable fragments (scFv), preferably immunoglobulin single variable domains (ISVD), DARP-in, affinity molecules, afetin, and albumin-binding domains (ABD).

7. The polypeptide of any one of claims 1 to 6, wherein the (ii) specific binding to at least one additional portion of serum albumin is an ISVD comprising three complementarity-determining regions (CDR1 to CDR3, respectively), wherein: CDR1 contains the amino acid sequence of SEQ ID NO: 1 or differs from SEQ ID NO: 1 by 3, 2 or 1 amino acids, or CDR1 contains the amino acid sequence of SEQ ID NO: 22 or differs from SEQ ID NO: 22 by 3, 2 or 1 amino acids; CDR2 contains the amino acid sequence of SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 3, 2, or 1 amino acid; and CDR3 contains the amino acid sequence of SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 3, 2, or 1 amino acid. and / or CDR1 contains the amino acid sequence of SEQ ID NO: 36 or differs from SEQ ID NO: 36 by 3, 2 or 1 amino acids; CDR2 contains the amino acid sequence of SEQ ID NO: 37 or differs from SEQ ID NO: 37 by 3, 2, or 1 amino acid; and CDR3 contains the amino acid sequence of SEQ ID NO: 38 or differs from SEQ ID NO: 38 by 3, 2, or 1 amino acid. and / or CDR1 contains the amino acid sequence of SEQ ID NO: 55 or differs from SEQ ID NO: 55 by 3, 2 or 1 amino acids; CDR2 contains the amino acid sequence of SEQ ID NO: 56 or differs from SEQ ID NO: 56 by 3, 2, or 1 amino acid; and CDR3 contains the amino acid sequence of SEQ ID NO: 57 or differs from SEQ ID NO: 57 by 3, 2, or 1 amino acid. These CDR sequences are determined based on AbM numbering.

8. The polypeptide of claim 7, wherein (ii) specifically binds to at least one additional portion of serum albumin, the ISVD being... Having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity with any of the sequences as defined in SEQ ID NO: 4 to 21, 54 or 91 to 93, wherein any C-terminal extensions that may exist and these CDRs are not considered in determining the sequence identity. and / or It differs from any of the sequences defined in SEQ ID NO: 4 to 21, 54 or 91 to 93 by no more than 7, preferably no more than 5, amino acids, for example, by only 3, 2 or 1 amino acid differences. Preferably, the additional portion of (ii) that specifically binds to serum albumin is an ISVD having a sequence selected from SEQ ID NO: 4 to 21, 54 or 91 to 93.

9. A fusion protein comprising a polypeptide as described in any one of claims 1 to 8, wherein the fusion protein further comprises one or more (e.g., one or two) other amino acid sequences, (binding) domains, binding units or other portions or chemical entities.

10. The fusion protein of claim 9, wherein the fusion protein comprises a polypeptide as described in any one of claims 1 to 8 and at least one therapeutic and / or targeting portion, optionally wherein the therapeutic and / or targeting portion comprises at least one ISVD specifically bound to a therapeutic target, or wherein the fusion protein further comprises an additional ISVD specifically bound to (human) serum albumin.

11. A composition comprising a polypeptide as described in any one of claims 1 to 8 and / or a fusion protein as described in any one of claims 9 to 10, optionally wherein the composition is a pharmaceutical composition.

12. A nucleic acid or nucleic acid sequence encoding a polypeptide as described in any one of claims 1 to 8 and / or a fusion protein as described in any one of claims 9 to 10.

13. A vector comprising the nucleic acid or nucleic acid sequence as described in claim 12.

14. A non-human host or host cell comprising a vector containing a nucleic acid sequence as described in claim 13 or expressing a nucleic acid sequence as described in claim 12.

15. The polypeptide of any one of claims 1 to 8, the fusion protein of any one of claims 9 to 10, and / or the composition of claim 11, for use as a pharmaceutical agent.

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